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International Journal of Pharmaceutical and Clinical Research 2009; 1(3): 92-94 

Review Article 

Folk Medicinal Value of Some Weeds around Hyderabad 

ISSN 0975 1556 

Krishna CM 1* , Gupta V 1 , Bansal P 1 , Kumar S 1 , Kumar SP 2 , Kumar TP 3 , Sharma S 4 

1 National Institute of Ayurvedic Pharmaceutical Research (NIAPR), Patiala, Punjab, India 

2 Indian Institute of History of Medicine, Osmania Medical College, Hyderabad, India 

3 J. S. S. Ayurveda Medical College, Myorse-15, Karnataka, India 

4 Regional Research Institute, Nagpur, India 

ABSTRACT 

There are many weeds seen which are uprooted to protect the cultivated plants. Some of these plants have good medicinal 

and economic value. Weeds commonly seen in the cultivated lands in and around Hyderabad were collected and identified 

using the standard literature. The medicinal values of these plants have been identified by the folk lore claims and the 

standard literature of the Indian systems of medicine (Ayurveda and Siddha). Most of these weed plants identified are 

being used in the Indian systems of medicine and public domain since many centuries, which have much clinical 

importance. Hence the local people and farmers must be educated regarding the medicinal uses of these weed plants for 

health care and for the generation of revenue through the collection of weeds. 

Keywords: Weed plant, Folk lore, Ayurvedic use, Hyderabad. 

INTRODUCTION 

Plants have been, and still are, a rich source of many natural 

products in major part of India and other countries, most of 

which have been extensively used for traditional human 

health care systems. The vast majority of people in the world 

takes care of themselves and uses healing plants that have 

been used for hundreds of generations. [1-4] India is a country 

of vast biodiversity and traditional knowledge of using herbal 

medicines to cure many ailments. It has nearly 20,000 

species of plants of medicinal and economic importance. 

Indian System of Medicine, which includes Ayurveda and 

Siddha systems of medicine, depends on the medicinal herbs 

for the treatment of various ailments. Estimate of 250,000 

flowering plants in the world [5] , more than 8000 species are 

weeds. [6] The weeds grow along with the crop plants (agroecosystems) 

and are regarded as nuisance for crops. It 

involves lot of money and labour to get rid of these unwanted 

plants by using weedicides and manual eradication. Most of 

the world is ignorant about the positive side of these weeds 

and this unseen part is their medicinal value. Some of weeds 

are the raw materials to the pharmaceutical industries as they 

yield chemicals used in formulation of various drugs, for 

preparing herbal formulations and an important source of 

medicines for indigenous peoples. [7-8] There are a number of 

reasons that the rural communities use weeds as medicine 

found in nearby areas. [9] 

*Corresponding author: Mr. Krishna CM, 

National Institute of Ayurvedic Pharmaceutical Research 

(NIAPR), Patiala, Punjab, India 

E-mail: vikas_4308@rediffmail.com 

The study of medicinally important weeds has not been 

realized as fully as other traditional communities elsewhere 

such as wild plants in forest ecosystems which often exclude 

weed species [10-13] In view of the rapid loss of diversity of 

plants, natural habitats, traditional community life, cultural 

diversity and knowledge of medicinal plants, documentation 

of medicinally important weeds is an urgent matter. 

Secondly, search for new medicines with low cost, more 

potential and without adverse side effect is needed to solve 

the major health problems. [14] 

Hyderabad, the capital city of central Andhra Pradesh, is 

located in Deccan Plateau. It extends on both the banks of 

Musi River up to an area of 250 square km and lies around 

500 meters above the sea level and at 17.366° N latitude & 

78.476° E longitude. It has a forest area of around 5 % and 

around 650 varieties of the plants. Rice, Jowar, Bajra, 

Sesame, Castor, Cotton and other pulses are the important 

plants of cultivation. These plants have a tough competition 

with the weeds and get destroyed. Hence these weed plants 

are removed timely during cultivation. Some of these weed 

plants also have good economic and medicinal value and are 

also used in the tribal medicine for the treatment of various 

ailments. So a folklore survey was taken up to identify the 

weeds of medicinal importance available around Hyderabad 

district of Andhra Pradesh, India. 

A Field survey was conducted in cultivation lands in and 

around Hyderabad and discussion with the agriculturists and 

farmers and information regarding the weeds was recorded. 

The information procured was validated by comparing the 

information given by two or three people. The collected 

weeds were identified by the morphological features using 

92

Krishna et al. / Folk Medicinal Value of Some Weeds.... 

Table 1: LIST OF MEDICINALLY USEFUL WEEDS 

S. 

No 

Botanical name/ Vernacular Name 

(Family) Ayurveda 

Folklore uses 

Siddha 

Useful parts 

1. 

Abutilon indicum/ Tuttiri benda 

(Malvaceae) 

Demulcent, aphrodisiac, laxative, diuretic, 

sedative 

Demulcent, tonic, laxative, diuretic and 

sedative 

Leaves, bark, root, 

seeds 

2. 

Acalypha indica/ Kuppichettu 

Kuppinta 

(Euphrobiaceae) 

Anthelminthic, scabies, arthritis, asthma, 

bronchitis, headache 

Worm infestation, gingivitis, burns, cough, 

constipation, skin eruptions, urinary 

disorders, purgative, emetic 

Root, leaf, whole 

plant 

3. 

Argemone mexicana/ Brahmadandi 

chettu 

(Papaveraceae) 

Diuretic, anodyne, hypnotic, laxative, 

emetic, expectorant, narcotic, sedative 

Leprosy, eczema, cough, leucorrhoea, dental 

diseases 

Root, seed, whole 

plant, latex 

4. 

Aristolochia bracteolate/ Gadide 

gadapa 

(Aristolochiaceae) 

Purgative, emmenagogue, alterative, 

antiperiodic and anthelminthic. 

Intermittent fever, worm infestations, skin 

diseases, eczema, urticaria, leprosy, 

gonorrhoea 

Leaf, seed, root, 

whole plant 

5. 

Boerhavia diffusa/ Atukamamidi 

(Nyctaginaceae) 

Stomachic, laxative, diuretic, expectorant, 

diaphoretic, emetic, edema, anemia, heart 

diseases, dyspnea and eye diseases. 

Nasal disorders, constipation, arthritis, 

dyspnea, jaundice, retention of urine, 

anasarca. 

Whole plant, root 

6. 

Cassia absus/ Chanubala vittulu 

(Caesalpiniaceae) 

Eye diseases 

Skin diseases, eye diseases, constipation, 

ulcers, retention of urine 

Leaf, seeds 

7. 

Cassia occidentalis/ Kasinda 


Purgative, febrifuge, diuretic and 

antiperiodic 

Constipation, rejuvenator. 

Root, leaf, flower, 

seed. 

8. 

Cassia tora/ Tagirisa 


Aperient, germicide, anodyne 

Veneral diseases, glandular swellings, fever, 

skin diseases, uritcaria 

Root, leaf, seeds 

9. 

Cleome viscosa/ Nelavaminta 

Kukkavaminta 

(Capparidaceae) 

Carminative, anthelminthic, antiseptic, 

sudorific, rubefacient, skin diseases, 

polyuria, anemia and gynecological 

problems. 

Indigestion, ear diseases, nasal disorders, 

skin eruptions, fever, worm infestations, 

abdominal disorders, coryza. 

Root, leaf, seed, 

whole plant 

10. 

Cyperus rotundus/ Tunga musta 

(Cyperaceae) 

Stomachic, tonic, demulcent, diuretic, 

anthelminthic, carminative, diaphoretic, 

emmenagogue, vermifuge 

Fever, thirst, worm infestations, dysentery 

and distaste 

Tubers 

11. 

Indigofera tinctoria/ Neeli chettu 

(Fabaceae) 

Alterative, purgative, antiseptic, astringent 

Fever, jaundice, anemia, arthritis, 

leucorrhoea 

Root, leaf 

12. 

Leucas aspera/ Tumiki 

(Labiatae) 

Insecticide, scabies and snake bite Whole plant 

13. 

Oxalis corniculata/ Pulichinta kura 

(Oxalidaceae) 

Antiscorbutic, astringent, appetizing, fevers 

and biliousness 

Fever, headache, abdominal disorders, 

veneral diseases, warts, abscess, digestive 

disorders 

Leaf 

14. 

Phyllanthus amarus/ Nela usiri 

(Euphorbiaceae) 

Diuretic, stomachic, febrifuge, anti-septic. 

Reduces jaundice, ophthalmopathy, fever 

and genitor-urinary disorders 

Jaundice, diabetes, eye diseases, urinary 

disorders, skin diseases, menorrhagia, 

vomiting 

Whole plant 

15. 

Portulaca oleracea/ Gangavavili kura 

(Portulacaceae) 

Diseases of kidney, dysuria, haematuria, 

gonorrhoea, haematemesis and 

haemoptysis 

Eye diseases, amnesia, headache, 

constipation 

Leaf, seed 

16. 

Psoralea corylifolia/ Bavancalu 

(Fabaceae) 

Anthelminthic, diuretic, diaphoretic 

Glandular swellings, skin diseases, skin 

diseases, leprosy, laxative, carminative, tonic 

Seeds 

17. 

Solanum nigrum/ Kamanci chettu 

(Solanaceae) 

Alterative, sedative, diaphoretic, diuretic, 

hydragogue and expectorant 

Ascites, stomatitis, anemia, cough, 

tuberculosis 

Leaf, fruit 

18. 

Solanum surratense/ Vakudu 

(Solanaceae) 

Aperient, digestive, alterative and 

astringent 

Expectorant, diuretic, carminative, cough, 

tuberculosis, fever, hysteria 

Whole plant 

19. 

Sphaeranthus indica/ Bodataramu 

chettu (Asteraceae) 

Stomachic, stimulant, alterative, demulcent 

Eczema, skin diseases, worm infestations, 

piles and aphrodisiac 

Root, leaf, flower, 

seed 

the standard literature and with help of pharmacognosy 

scientist. The medicinal uses of these plants were recorded 

from the folk lore claims and the standard literature of the 

Indian systems of medicine. 

The medicinal uses of the weeds plants used in the Indian 

system of medicine are enumerated as in Table 1. The list of 

the weeds with Botanical name, family, vernacular name, 

medicinal uses (Ayurveda and Siddha) and useful parts is 

mentioned in the alphabetical order of the botanical name of 

the plants. [15-24] As per the collected information it was 

identified that plants belonging to 15 families and 16 genera 

are identified as weeds. Most of the weeds identified were 

herbs or under shrubs and the medicinally useful part for 

most of the plants was whole plant, leaves or seeds. Several 

recent studies have proved the weedy plants contain many 

medically useful active principles (alkaloids, glycosides, 

polyphenolics, steroids, tannins, resins, flavonoids, 

tetraploids and fatty acids) that are able to cure many 

nutritional disorders and diseases [1, 25-35] in the human health 

care system. 

Weeds compete with the cultivated plants and decrease their 

productivity hence these plants are removed for the increased 

productivity of the cultivated plants. Some of these weed 

plants have good medicinal value. These plants form the 

major source of medicine for the tribal people of most of the 

developing countries. So it is important to conserve the 

medicinally useful weeds. Farmers must be educated 

regarding the medicinal uses of the plants and trained to 

preserve the weeds with out affecting the development of the 

cultivated food plants and to collect and process the 

medicinally useful parts of the weed plants. This may help to 

develop additional revenue without disturbing the 

productivity of the commercial plants. 

ACKNOWLEDEMENT 

Authors thank Director General of Central Council for 

Research in Ayurveda and Siddha (CCRAS) for his 

encouragement to complete this work. Authors also express 

their gratitude to the farmers and agriculturists for extending 

their support and giving good information on the weed 

plants. 

IJPCR October-December, 2009, Vol 1, Issue 3 (92-94) 93

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Review Article 

Pharmaceutical Biotechnology in India as a Growing Field 

ISSN 0975 1556 

Bhumika Yogi 1 , Sujeet Gupta 1 , Yogesh Murti 1* , Devender Pathak 1 , Manmohan Singhal 2 , 

Yashwant 2 

1 Rajiv Academy for Pharmacy, Chhatikara, Mathura, Uttar Pradesh, India 

2 School of Pharmaceutical Science, Jaipur National University, Jaipur, Rajasthan, India 

ABSTRACT 

Biotechnology is the science of today and tomorrow. It has application in all major service sectors of the society that is 

health, agriculture, industry and environment. Through the combined efforts of government, academia, research and 

industrial sectors, India is poised to become a major hub and logistics operation centre for R&D, manufacturing, and 

operations in the biotechnology and pharmaceutical industries within the Asia-Pacific region. There are several factors, 

which makes the biotech scenario bright in India. The future of pharmaceutical biotechnology in India holds immense 

potential if the biotechnology firms tap the big growth avenues to develop drugs for rare diseases. 

Keywords: Biotechnology, agriculture, industry. 

INTRODUCTION 

Biotechnology is defined as the use of living organisms or 

parts of organisms to create products, processes and services. 

Four categories of biotechnology have been defined: ‘Green 

Biotechnology’, which concerns the biotechnology of plants; 

‘Red Biotechnology’, related to medical and healthcare 

applications; Blue Biotechnology also known as Marine 

biotechnology, involves a number of technologies that 

transform products from the ocean into new foods, drugs, 

chemicals, and other products and ‘White Biotechnology’ 

also known as grey biotechnology, aimed at industrial uses. 

The development and application of Biotechnology in each 

of these four categories have been touted as catalysts and 

engines of growth for many countries, including developing 

countries. [1] 

Biotechnology can be used to create wealth. In fact, it has 

been identified as a major avenue for the socio-economic 

advancement of developing countries. The scope, scale and 

practice of biotechnology research and development 

currently being carried out in many developing countries 

present a range of entrepreneurial opportunities for 

enhancing technological progress and ultimately a nation’s 

economic and social wealth. 

The development of biotechnology as a pharmaceutical 

industry requires complex multidisciplinary and 

transdisciplinary efforts, including collaboration between 

scientists working in such fields as chemistry, microbiology, 

*Corresponding author: Mr. Yogesh Murti, Lecturer, 

Department of Pharmaceutical Chemistry, Rajiv Academy 

for Pharmacy, Mathura, Uttar Pradesh, India 

Mobile: 09412627553; Fax No.: 05652825050 

E-mail: ymurti@rediffmail.com 

RED BIOTECHNOLOGY 

GREEN BIOTECHNOLOGY 

BIOTECHNOLOGY 

Fig. 1: Showing different categories of biotechnology. 

2 

9% 

3 

7% 

4 

7% 

5 

2% 

BLUE BIOTECHNOLOGY 

WHITE BIOTECHNOLOGY 

1 

75% 

Fig. 2: Pie chart showing percentage of different biotechnology segments 

in India [3] 

molecular biology, pharmacology, physiology, genomics and 

proteomics. Non-science sectors, such as sales and 

marketing, also need to be involved. To develop and 

commercialize a biotech product, therefore, requires a 

synergistic strategy that brings together each of these 

95

components. In much of the developing world, where 

research is carried out mainly in the public sector, there is 

also a need to forge strategic alliances between the public and 

private sectors. 

Indian biotech companies are taking path of global 

collaborations. The biotech industry in India, mainly 

consisting of five distinct segments- BioPharma (1), 

Bioservices (2), BioAgriculture (3), BioIndustrial (4) and 

BioInformatics (5). [2] 

Challenges [4] 

Among the challenges facing the development of 

biotechnology based pharmaceutical industries in India, are: 

� Financing 

� Capacity building 

� Training of entrepreneurial scientists 

� Defining of markets 

� Lack of harmonization of policies 

� Curbing government bureaucracy 

Opportunities [5] 

Despite these challenges, there are several opportunities for 

pharmaceutical biotechnology to grow in India. These 

include: 

� Translation of scientific knowledge into commercial 

businesses. 

� Exploiting strategic advantages such as biodiversity. 

� Developing genetic modification technology. 

� Creating auxiliary services. 

� Trained human resources and knowledge base. 

� Good network of research laboratories. 

� Well developed base industries. 

� Access to intellectual resources of NRI’s in this area. 

� Extensive clinical trials and research access to vast 

and diverse diseases in the huge population. 

� India’s human gene pools and unique plant, animals 

and microbial diversity offer an exciting opportunity 

for genomic research. 

� Several labs have commenced research in stem cell 

and have valuable stem cell lines. 

� Large domestic market and export potential. 

� Low cost research base for international companies 

compared with other countries. 

� Supportive Government policy on embryonic stem 

cells research provides a useful opportunity for 

international companies to pursue such research in 

India. 

Strategies 

Strategies for developing pharmaceutical industrial 

applications of biotechnology in India, therefore, should: 

� Create enterprises 

� Establish and promote close links 

� Tackle intellectual property issues and take advantage 

of local biodiversity, indigenous knowledge and 

science-based innovations 

� Seek involvement in key national economic 

development programmes. 

� Leverage large population bases 

� Maintain long-term vision 

Disadvantages 

� Missing links between research and 

commercialization. 

Murti et al. / Pharmaceutical Biotechnology in India.... 

� Lack of venture capital. 

� Relatively low R&D expenditure by industry. 

� Image of Indian pharma industry - doubts about 

ability of Indian products to meet International 

standards of quality. 

� Inadequate protection of Intellectual Properties Rights 

(IPR), significant improvement remains to be 

undertaken in the areas of implementation and 

enforcement. 

It is indeed conducive for the growth of biotech as Indian 

scientists are gearing up to meet any challenge, especially 

after the completion of the Human Genome Project. There 

are several factors, which makes the biotech scenario bright 

in India. Biotechnology especially, is gaining prominence in 

India & Asia Pacific region with many research institutions, 

university departments and corporate sector companies 

engaging in biotech research activities. In India alone, there 

are 200 research institutes and universities and more than 100 

companies involved in biotech activities. With IPR coming 

into play after 2005, many Indian pharmaceutical companies 

are gearing for the genome battle. India has a large market 

for biotech-based products, most of these are imported. 

Diagnostic market is still untapped with latest cutting edge 

technologies using molecular science. With increased 

funding for biotech activities by foreign investors and large 

Indian corporate and also the growing presence of many 

MNCs in basic research and clinical research like Pfizer, Eli 

Lilly, Quintiles, etc due to cost advantage for new drug 

development, India will be the destiny for larger portion of 

future drug research. 

This is also evidenced by number of collaborations and 

alliances happening with Indian pharma companies. India 

also has manpower cost-advantages and easier scalability to 

higher production levels by increasing the number of people 

at a marginal increase in cost. India has a very good pool of 

scientific talent available at a significantly lower cost. 

There is an enormous potential for the biotech products and 

services. The products include new diagnostic kits to identify 

infectious diseases, reagents to extract nucleic acids and 

several services that can be utilized in drug discovery and 

development. The Indian market is not yet matured for highend 

services like gene expression analysis except for few 

pharmaceutical companies and research institutions. 

However, biotech market sizes are promising in countries 

like Singapore and in the Asia Pacific region. And more and 

more pharma and research institutes from the West are 

looking at India for contract research business as the 

scientific pool is talented and operational and infrastructural 

costs are reasonably low. 

Moreover, India has immense market potential for Custom 

Laboratory Services. Presently, most of the biotech-based 

products are imported. The current Indian market size for 

diagnostics is Rs 6,500 crores. Monoclonal and polyclonal 

antibodies for disease immuno-diagnosis, tissue typing, 

clinical assays and research constitute a huge portion of the 

market. 

THE 7 STEPS TO GLORY [6] 

The Indian biotech industry is at crossroads. On one hand, 

the country is being increasingly seen as a bio-manufacturing 

hub leveraging the advantages that accrue from operating in 

this geography & on the other hand, the domestic industry is 

repeatedly scaling up to participate in the global 

opportunities. 


B 

I 

O 


INDIAN BIOTECH INDUSTRY 7 STEPS TO GLORY 

Capital 

Experience personnel 

Technology secure 

Opportunity addressed 

Identified processes 

Broad based product 

Fig. 3: Showing 7 steps to glory of Indian biotech industry. 

STATUS OF PHARMACEUTICAL 

BIOTECHNOLOGY IN INDIA [7] 

Indian Biotech industry has been a late starter, only a decade 

ago, the contribution of Indian Biotech Sector to global 

biotechnology market was insignificant. There were only a 

handful of Biotech companies in India; many of 

entrepreneurs have made their mark in national as well as 

international arena. An Ernst and Young survey ranks India 

third in the Asia-Pacific region based on the number of 

biotech companies in the country and recognizes India as one 

of the emerging biotech leaders. Not surprisingly, this sector 

has been witnessing heightened activity, having already 

attracted as many as 150 multinationals. Apart from the 

existing talent pool of trained and skilled human resource, 

numerous scientists and regulatory experts, India’s strong 

base for pharmaceutical research and IT services and welleducated 

low-cost English-speaking human capital are 

driving the off-shoring of biotechnology services to India. 

Indian biotech sector is gaining global visibility and being 

tracked for emerging investment opportunities. According to 

international experts, the period 2010-2020 has become 

T 

E 

C 

H 

Harmonize risk and rewards 

Table 1: List of Major Biotech Players in India [8] 

S. No. Company Product Segments URL 

1 

Biocon 

(Bangalore) 

Food and industrial enzymes, Statins, Lovastatin, 

Genomics, Chemical synthesis Molecular Biology 

www.biocon.com 

2 

Panacea Biotech 

(Delhi) 

NDDS for Anti TB combination drug, diabetic, 

asthma, Hep B vaccine 

www.panaceabiotec.com 

3 Wipro Health Science (Bangalore) Diagnostics, DNA sequencing, flow cytometry www.wipro.com 

4 Haffkine Biopharmaceuticals (Mumbai) Vaccines and Diagnostic Kits www.vaccinehaffkine.com 

5 Elli Lilly (Gurgaon) Humlog, Huminsulin for diabetes www.lilly.com 

6 

Nicholas Piramal 

(Mumbai) 

Bioinformatics, genomics (Gen Med) in cardiovascular, 

diabetes and cancer 

www.nicholaspiramal.com 

7 Krebs Biochmicals (Hyderabad) Statin agents for cholesterol www.krebsbiochem.com 

8 Bharat Serums and vaccines (Mumbai) Vaccines, antibody and NDDS www.bharatserums.com 

9 Bio-Rad Lab (Gurgaon) Diagnostics, reagents, protein purification www.biorad.com 

10 

Shantha Biotech 

(Hyderabad) 

Plasminogen activators, interferons, vaccines 

Therapeutic protein through recombinant DNA technology, 

www.shanthabiotech.com 

11 Dr. Reddy’s Lab (Hyderabad) 

cancer vaccine GCSF diagnostic protein for HIV infection, 

therapeutic protein for vaccines, cytokines and antivirals 

www.drreddys.com 

projected as the ‘decade of Asia’ in the field of 

pharmaceutical biotechnology. 

BIOTECHNOLOGY AND ITS IMPACT ON DRUG 

DEVELOPMENT 

Biotechnology finds varied applications such as in medical 

and pharmaceutical sciences, agriculture, food science, 

environmental improvement and in forensic science. But the 

most spectacular and beneficial results for human beings 

which have occurred from the advances made during the last 

two decades by biotechnology are in the area of medicine and 

pharmaceuticals. Biotechnology produced human insulin; 

growth hormone and erythropoietin are the glaring examples 

of the therapeutic agents in use. [9] 

India has a very good tract record of development of 

improved cost beneficial chemical synthesis for various drug 

molecules. With the expertise and experience through 

backward integration of technologies/ products developed 

elsewhere, Indian Pharma is now focusing on in house R&D 

for the development of new technologies and new drugs. 

Another area catching up India is contract research and in the 

area of clinical trials. India with a lot of diversification is 

indeed looked upon as an ideal base for clinical trial. [10] 


BIOTECHNOLOGY SCENARIOS FOR MEDICAL 

APPLICATIONS IN FUTURE [1] 

Scenario BB: Biotech Boost 

After 2005, the biotech market for medical application has 

not only achieved double digit growth rates, it has even 

continued to grow at the astonishing pace of 20 % per year 

on average over the last 10 years since 2005. Growth at such 

a speed has generated a market capitalization of 2,500 billion 

ERU as by now (2015). Because of the continued 

attractiveness of the biotech market, private but also public 

investors were willing to supply enormous funds in venture 

capital and for R&D sponsoring so that nowadays the 

industry is able to serve the bottom of the pyramid. 

Scenario BBM: Biotech Business Matures 

Major players in biotech as well as private and public 

investors in medical biotech research had expected a 

revolution in medical applications, grace to a vastly increased 

knowledge and fundamental research base and thus a vast 

universe of opportunities for new applications with shrinking 

R&D cost. This has not happened. Developing successful 

applications upon the vast knowledge base in biotech turned 

out to be much more difficult and much more expensive than 

expected. The rate of innovation finally didn’t turn out to be 

higher than it was in the years and decades before. 

Scenario BBB: Biotech Bubble Burst 

May be it was the recent memory of the dot.com rise and fall 

that eventually led to the dramatic and turbulent changes in 

the biotech industry. In any case it had many similarities. In 

the early years of the 21 st century, the biotech industry could 

look back on a period of 20 years with growth rates of around 

20 %. The total market capitalization of the Biotech industry 

was more than $300 billion in 2005. So, nobody was really 

prepared for what was to come. 

SCOPE OF CONTRACT RESEARCH IN 

PHARMACEUTICAL BIOTECHNOLOGY IN INDIA 

Biotechnology as an application science has taken firm in 

countries abroad where number of transgenic crops, 

genetically modified food and recombinant therapeutic 

molecules for human and animal health are available in 

market. It is essential to identify different steps involved in 

development of a drug, transgenic or biological and work on 

strategies to involve industries or institutions for specific 

roles. Though some Indian biotech companies have 

undertaken contract research for American and European 

labs and industries. They are doing only a small module for 

them and entirely controlled by them. In such cases, Indian 

laboratories are unable to do any innovative thinking and are 

failing to visualize them as multinational. The purpose of 

contract research should be: 

1. Innovative thinking and competence in molecular 

biology, biotechnology, biochemistry, bio-processing, 

mouse genetic and assay development 

2. Bench top R&D experiments 

3. Discovery of new molecular modifications and 

improvement of existing drugs 

4. High throughput screening for therapeutics 

5. Stability testing; analytical development and 

validation 

6. Provision of testing, facilities including testing in 

human cell lines 

7. Drug delivery research 

8. Clinical trial services 


9. Clinical trials managements, data management, 

biostatistical analysis 

10. Designing of facilities for commercial production 

-cGMP facilities 

-Small to mid-scale manufacturing 

-Pilot scale-up and production scale up 

11. Contract packaging and manufacturing 

12. Providing contractual regulatory affairs services 

13. Industrial training and publishing 

14. Providing pool of trained scientists or lab resources 

Pharmaceutical biotechnology based companies are 

increasingly outsourcing early-stage research and clinical 

studies driven mainly by rising R&D costs and the need to 

improve R&D productivity. With cost saving of around 30- 

50 % compared to Western costs as well as an abundance of 

talented scientist and easy access to raw materials, India is 

seen as a key location for outsourcing research and 

manufacturing in the area of drug discovery and 

development. 

India biotech sector is among the fastest growing knowledge 

based sectors, growing from USD 800 million in 2003 to 

USD 1.45 billion in 2005, and reaching USD 2 billion in 

2008. Over the past few years, the biotech industry has 

invested around 25 % of its revenues with a high proportion 

of the investments going into setting up of infrastructures, 

followed by R&D. 

By decreasing their in-house facilities and staff, and 

outsourcing more of their R&D functions, pharmaceutical 

biotechnology based companies are reshaping the drug 

development services industry. Outsourcing is not a new 

concept to pharmaceutical companies; however, its use 

increased dramatically in the mid-1990s, and it is expected to 

continue to increase going forward. It is estimated by 2020 

nearly 62 % of all pharmaceutical drug development 

expenditures will be committed to outsourcing, as compared 

to the 4 % that was outsourced in the early-1990s. Some 

estimate that there are currently over 1,200 organizations 

involved in the clinical research, including pharmaceutical 

and biotechnology in-house clinical research, site 

management organizations (SMOs), academic medical 

centers, private research sites, and contract research 

organizations (CROs). 

It is imperative that pharmaceutical biotechnology based 

companies pass their product through the testing and 

regulatory process in a rapid, cost effective manner. To 

accomplish this goal, pharmaceutical biotechnology based 

their therapeutics to market faster. These are some factors for 

driving contract research strategy- 

1. Cost Reduction Strategy 

2. Increasing Number of New Chemical Entities 

(NCEs) in Pipeline 

3. Time to Market Pressures 

4. Growing Complexity of Regulatory Requirements 

5. Rapid Access to Patient Recruiting 

Many Indian pharmaceutical biotechnology based companies 

have introduced products of original research through 

transfer from R&D institutions in India in the field of 

vaccines, diagnostics and clinical and contract research and 

trials. Some others have established tie ups and joint venture 

with foreign companies for sourcing technologies, with a 

view to introduce them into the Indian market within the 

framework of Indian laws. Outsourcing of R&D and over 20 

conducting research in specific areas of pharmaceutical 


iotechnology. In addition of these, there are companies in 

Bangalore with excellent technical manpower and worldrenowned 

institutions such as the Indian Institute of Science 

(IISc), the National Centre for Biological Sciences (NCBS), 

Jawahar Lal Nehru Centre for Advanced Scientific Research 

(JNCASR), Center for Cellular and Molecular Biology 

(CCMB), Hyderabad, National Facility for Macromolecular 

Crystallography, BARC, Mumbai, National Facility for High 

Field NMR,TIFR, Mumbai, Central Drug Research Institute, 

Lucknow, National Brain Research Center, New Delhi, all of 

which provide high-quality R&D services to organizations 

worldwide. There are some contract research competitors 

such as Academic Medical Centres and Teaching Hospitals, 

Contract Research Organisations, Preclinical and Clinical 

Laboratory Services and Site Management Organizations. 

INDIAN PHARMACEUTICAL AND BIOTECH 

INDUSTRY DEVELOPMENT STRATEGIES 

Through the combined efforts of government, academia, 

research and industrial sectors, India is poised to become a 

major hub and logistics operation centre for R&D, 

manufacturing, and operations in the biotechnology and 

pharmaceutical industries within the Asia-Pacific region. The 

government’s plans to promote industry include achieving 

targets of NT$150 billon (US$ 4.34 billion) in investments 

and 25 % annual revenue growth by 2010 and five hundred 

biotech-related companies within ten years. Industry, 

institutions and governments bodies all follow development 

guideline as set forth in the Promotion Plan for the 

biotechnology industry. A major aspect of the promotion 

plans is that it is identified specific in need of attention to 

upgrade not only the local biotech industry but also to 

enhance the investment attraction of India to the overseas life 

science community. These five areas of attention are as 

follows: 

1. Related laws and regulations 

2. R&D and application 

3. Technology transfer and commercialization 

4. Investment promotion and cooperation 

5. Marketing information and services 

FUTURE PROSPECTS OF THE PHARMACEUTICAL 

BIOTECHNOLOGY SECTORS IN INDIA 

India is becoming one of the favored destinations for 

pharmaceutical based contract research: 

� The biotech companies are expanding in a big way 

and assuming the global scale. For e.g.: Syngene , one 

of the Biocon groups of companies involved in 

contract research is expanding hugely in terms of 

scientific man power, and it is also acquiring a 

German biotech company as part of its expansion 

programs. 

� Astra Zeneca and Pfizer are the two dominant 

companies in this category and by far the largest. 

Biovitrum focuses on metabolic diseases, obesity 

,type 2 diabetes and oncology. [11] 

� Active Biotech AB (immunology, vaccines ,drugs) 

and SBL Vaccine AB (vaccines) , KaroBio AB, A 

Carlsson Research AB and Medivir AB. [12] 

� Southern India (Hyderabad, Banglore and Chennai) is 

emerging as a hub of novel research in biotech and 

pharma industry. In western India also the growth of 

Biotech sector is seem to be almost in equal measures 

with southern Biotech industries. 


� Two global Biotech companies; Amgen and Biogen 

have set up wholly owned subsidiaries in the country. 

Which means that there is going to be creation of a lot 

of opportunities. 

� As per the latest news, Ranbaxy, one of the leading 

Biotech & Pharma Company had become a part of 

Japanese Biotech Company, with more of expansions 

in its research operation and manufacturing sector 

leading to the creation of opportunities in the next 

years. 

While India represents one of the biggest business 

opportunities for biotechnology in healthcare and 

pharmaceuticals, on account of its huge disease population, it 

is emerging as the bio-power of the world with its qualified 

scientific pool and innovative research and manufacturing 

efforts. The future of pharmaceutical biotechnology in India 

holds immense potential if the biotechnology firms tap the 

big growth avenues to develop drugs for rare diseases. 

CONCLUSION 

Pharmaceutical biotechnology has very good future in India. 

There is a lot that can be done to benefit India, using 

biotechnology. A proper balance between strategic research, 

product planning and effective collaboration will help 

support biotech growth in India. Partnerships with global 

biotech industries have the greatest impact on India’s own 

biobusiness markets. Biotechnology companies are currently 

responsible for a majority of the innovation in the healthcare 

industry. The same is true of the pharmaceutical sector, 

where it is expected that innovations in biotechnology will 

revolutionize the pharmaceutical sector. The Indian Pharma 

Biotech Industry is set for rapid growth fueled by the 

growing market size and number of products as well as 

increased investments. The emergence of Biotech parks and 

state-of-the-art Bio-IT parks will further boost this growth. 

Although we may continue to see some big pharmaceutical 

companies acquiring biotechnology companies, they may 

have to cough up increasing amounts for their smaller 

siblings to clinch the deals. To appreciate the impact of 

biotechnology in pharmaceutical science an understanding of 

some basics of genetics is necessary. 

REFERENCES 

1. Sarkar M., Kumar R. Biotechnology Existing and Future Scenarios. 

Pharma Bio World 2007; 3: 53-65. 

2. Priyanka E.S.L. Indian Biotech Industry Public and Private 

Partnership. The Pharma Review. 2008 (April) 6: 37-39. 

3. Biotech in India cited from http://www.business-inasia.com/countries/biotech_in_india.html 

(accessed 2009 July 20). 

4. Rao M. VC funding: to be or not to be. Pharma Bio World 2006; 4: 

36-39. 

5. Sawant M. SWOT Analysis of Indian Biotechnology market. 

Pharma Bio World 2006; 4: 49-53. 

6. Palnitkar U. Indian Biotech Industry Seven Steps to Glory. Pharma 

Bio World 2007 (January) 5: 82-84. 

7. Tewari R. Indian Biotech Industry: The Leaders in the Marketing. 

Pharma Buzz. 2008 (November) 3: 12-16. 

8. Sahu U. Indian Biotech Industry- A Review. Pharma Bio World 

2005; 4: 92-98. 

9. Kapoor V.K. Biotechnology and its Impact on Drug Development. 

The Pharma Review 2007 (February) 5: 36-40. 

10. Status of Biotechnology in India & Other Countries. The Pharma 

Review 2007 (June) 5: 132-134. 

11. Biotechnology and Pharmaceuticals in Sweden cited from 

http://www.sweden.se/eng/Home/Business/Facts/Biotechnologyand-Pharmaceutical-in-sweden 

(accessed 2009 July 23). 

12. Swedish Biotechnology cited from 

www.iva.se/upload/verksamhed/projekt/suensk%bioteknik/swedish 

%20biotechnology%20%20web-1.pdf (accessed 2009 August 10). 




Research Article 

ISSN 0975 1556 

Anti-inflammatory Activity of Standardised Extracts of Leaves of Three 

Varieties of Ficus deltoidea 

Zunoliza Abdullah 1, 2* , Khalid Hussain 1 , Zhari Ismail 1 , Rasadah Mat Ali 2 

1 School of Pharmaceutical Sciences, University Science of Malaysia, Pulau Pinang 11800, Malaysia 

2 Medicinal Plants Programme, Biotechnology Division, Forest Research Institute Malaysia, 52109 Kepong, Selangor, 

Malaysia 

ABSTRACT 

Present study aimed to evaluate standardised extracts of different verities of Ficus deltoidea, a traditional medicinal plant, 

for anti-inflammatory activity using three in vitro assays, lipoxygenase, hyaluronidase and TPA-induced oedema. 

Methanol and aqueous extracts were standardised by high performance liquid chromatography (HPLC) using two 

pharmacologically active markers, vitexin and isovitexin, which were isolated from methanol extract of the plant having 

code FDT1M. The method was validated and then applied to standardise extracts of the plant. In the extracts, the 

concentration of vitexin and isovitexin varied in the range 2.45 ± 0.00 - 19 ± 0.12 mg/g and 1.58 ± 0.02 - 41.49 ± 0.47 

mg/g, respectively. Different extracts of three varieties of the plant displayed different anti-inflammatory activities (p < 

0.05). The activity of the extracts was found comparable to apigenin, nordihydroguaiaretic acid, indomethacin, which were 

used as control (p < 0.05). The results of this study indicate that extracts of leaves of Ficus deltoidea possess antiinflammatory 

properties. 

Keywords: Ficus deltoidea; Standardization; anti-inflammatory, lipoxygenase; hyaluronidase. 

INTRODUCTION 

Standardization herbal products, consistency in claimed 

efficacy of a product and its batch-to-batch reproducibility, is 

a difficult task and is a main hindrance in their wider 

acceptance. In order to bring these remedies into the 

mainstream pharmaceutical market, solid scientific evidence 

is needed to support the efficacy claims of these products. 

The use of markers, the chemical compounds characteristic 

of a plant, signifies the total active constituents of an extract 

or correlates to pharmacological activity are used to 

standardise herbal products. 

[1-2] Ficus species contain 

compounds such as flavonoids [3-8] , α-tocopherol and its 

derivatives [9] , steroids and triterpenoids [10-15] and alkaloids. 

[11, 16-18] Polyphenolic compounds have shown antibacterial, 

anti-carcinogenic, anti-inflammatory, anti-viral, anti-allergic, 

estrogenic and immune-stimulating properties. [19] Therefore, 

in this study we selected two C-glycosylflavones, vitexin and 

insovitexin, as pharmacologically active analytical markers 

to standardise the extracts of three verities of Ficus deltoidea. 

Markers are in small quantities as compared to the dose used 

hence, it may be beneficial to know about primary 

metabolites of the extract. 

*Corresponding author: Mr. Zunoliza Abdullah, 

Phone and fax +6046563443 

E-mail: a_noliz@hotmail.com; zunoliza@frim.gov.my 

Therefore, the extracts were also analysed for primary 

metabolites such as total proteins and polysaccharides, which 

also contribute in activity. The standardised extracts were 

then evaluated for anti-inflammatory properties. 

Ficus deltoidea Jack (Moraceae) is found in tropical and subtropical 

countries and has several varieties. 

100 

[20] The leaves of 

the plant are used traditionally for treating diabetes, high 

blood pressure, heart problems, gout, diarrhea, pneumonia 

[21] 

and skin diseases. The plant has also exhibited 

antioxidant, hypoglycemic and antinociceptive properties. [22- 

23] [23] 

Zunoliza et al., (2009) reported the presence of high 

content of total polyphenols, flavonoids and tannins in 

extracts of leaves of three verities of the plant which have 

shown good antioxidant activity. Polyphenols have shown 

antibacterial, anti-carcinogenic, anti-inflammatory, anti-viral, 

anti-allergic, estrogenic and immune-stimulating properties. 

[19] 

Therefore, it was hypothesized that extracts of the plant 

having high content of polyphenols might have antiinflammatory 

effects. To test this hypothesis, present study 

aimed to evaluate aqueous and methanol extracts of leaves of 

three verities of the plant for anti-inflammatory activities. 

Inflammation is a complex process involving several 

biochemical changes and mediators. [24] Arachidonic acid, a 

substrate for the biosynthesis of a range of bioactive 

eicosanoids, is metabolised by the cyclooxygenase (COX) 

pathway to prostaglandins and thromboxane A2, or by the

Abdullah et al. / Anti-inflammatory Activity of Standardised Extracts.... 

lipoxygenase (LOX) pathway to hydroperoxyeicosatetraenoic 

acids and leukotrienes, which are important 

mediators in a variety of inflammatory events. [25] Several 

steroidal and non-steroidal anti-inflammatory drugs are based 

on the inhibition of these pathways and are used to treat 

inflammatory conditions. Long-term use of these agents is 

associated with various severe adverse effects. Therefore, 

natural drugs with little side-effects are required to substitute 

these drugs. Keeping it in view, present study aimed to 

evaluate standardised methanol and aqueous extracts of 

leaves of three verities of Ficus deltoidea for antiinflammatory 

activity using different in vitro models. 

MATERIAL AND METHODS 

Chemicals 

Chemical used in this study included: soybean lipoxygenase, 

hyaluronidase, hyaluronic acid, apigenin, 12-Otetradecanoylphorbol-13 

acetate (TPA), indomethacin, 

vitexin (Sigma-Aldrich Chemical Co.), acetonitrile and 

methanol HPLC grade (Fisher) and ortho-phosphoric acid 

(Merck). Isovitexin, previously isolated from the plant, was 

used. 

Plant material 

Three varieties of Ficus deltoidea leaves were selected in this 

study included F. D. var. terengganuensis (2 samples 

encoded as FDT1 and FDT2), F. D. var. angustifolia (FDA) 

and F. D. var. deltoidea (FDD). Sample of FDT1 was 

obtained from Malai Herbal Tea, Malaysia. FDT2 and FDA 

were collected from Terengganu and Selangor, respectively. 

Sample of FDD was purchased from Nutreeherbs Sdn. Bhd. 

Malaysia. All the samples were authenticated by Ms Zainon 

Abu Samah, a botanist, Medicinal Plants Program, 

Biotechnology Division, Forest Research Institute Malaysia 

(FRIM), where voucher specimens having numbers FRI 

48988 for FDT2 and FRI 54761 for FDA were deposited. 

Preparation of extracts 

Dried and ground leaves were extracted with methanol using 

Soxhlet extractor for 17 h to get methanol (M) extracts, 

which were then dried in vacuum at 40°C. To prepare 

aqueous (W) extracts, dried ground and pulverized leaves 

were macerated with water at 50°C for 3 h, the procedure 

was repeated twice and the extracts were filtered and dried in 

freeze dryer. The extracts were encoded as FDT1M, 

FDT1W, FDT2M, FDT2W, FDAM, FDAW, FDDM and 

FDDW, where M and W referred to methanol and water, 

respectively. 

Instrumentation and Chromatographic conditions 

The samples were analysed using HPLC system consisting of 

HP-1100 Agilent Technologies equipped with a quaternary 

pump, on line degasser, auto-sampler, column heater and UV 

detector. Chromatographic separations of the Cglycosylflavones 

were performed on a Lichrospher RP-18 

column (125 mm x 4 mm, 4 μm) which was protected by RP- 

18 guard column (5 μm, 4.0 mm, 3.0 mm i.d.). The 

temperature of the column was maintained at 35°C. A 

gradient elution consisting of solvent A (0.1% orthophosphoric 

acid) and solvent B (acetonitrile) was used at 

flow rate of 1 ml/min as: 0 – 6 min (15 - 25% of A); 6 – 11 

min (25% A); 11 – 14.50 min (25 – 15% A) and 14.50 – 23 

min (15% A). The injection volume was 10 μl and the 

effluent was monitored at 335 nm. 

Preparation of standard solutions 

Stock solutions of vitexin and isovitexin were prepared in 

methanol to get concentration of 500 and 1000 μg/ml, 

respectively. Mix standard stock solution was prepared from 

the stock solutions of vitexin and isovitexin to get the 

concentration of 250 and 500 μg/ml, respectively. Working 

mix standard solutions were prepared by further diluting the 

mix standard stock solution with methanol to a concentration 

of 0.01-250 μg/ml of vitexin and 0.01-500 μg/ml of 

isovitexin. 

Validation of HPLC method 

The linearity of calibration curves was evaluated by linear 

regression analysis and correlation coefficient (R 2 ). The 

limit of detection (LOD) and limit of quantification (LOQ) 

were evaluated by measuring the magnitude of analytical 

background by injecting the blank. In this study, LOD were 

determined by injection a series of solutions until the height 

of the peak signal to baseline noise level ratio (S/N) was 3: 1, 

whilst LOQ values were taken at S/N 10: 1. 

The accuracy and precision of the method were determined 

through within-day and between-day analysis of mix 

standard solutions. A separate standard curve was 

constructed on each day of analysis. The within-day accuracy 

and precision were determined for each compound on three 

concentrations with five replicate on a single day, while the 

between-day accuracy and precision was carried out over five 

consecutive days. The accuracy was expressed as percent of 

true value and respective precision was expressed as relative 

standard deviation (% RSD). 

The accuracy of extraction was evaluated through recovery 

studies by spiking the leave powder with three different 

concentration of standard solution. The accuracy was 

calculated with the value of detected versus added amounts. 

Preparation of samples and their analysis 

Methanol extracts were reconstituted with methanol to a 

concentration of 10 mg/ml, whereas aqueous extracts were 

dissolved in 50 % methanol to produce solutions of 

concentration 10 mg/ml. All the samples were filtered 

through a 0.45 μm filter (Whatman) and kept in HPLC vials. 

All the samples were analysed in triplicate by HPLC using 

analysis conditions mentioned above and markers were 

quantified from the standard curves. 

Determination of Total Protein 

Protein concentration in the extracts was measured according 

to the method of Lowry et al., (1951) [26] using Bovine Serum 

Albumin (BSA) as a standard. Stock solution of BSA was 

prepared in distilled water (500 ppm). A series of dilutions 

(50, 100, 150 and 200 ppm) were prepared at final volume of 

1 ml. Buiret reagent (3 ml) prepared by mixing 50 ml f 2 % 

sodium carbonate in 0.1 N sodium hydroxide and 1 ml of 

0.5% copper sulphate in 1 % potassium tartrate was added to 

standard solution (1 ml) and sample solution 1 ml having 

concentration 500 ppm. The mixture was allowed to stand for 

10 min at room temperature. Then 200 μl Folin-Ciolcateau 

reagent was added and reaction mixture was allowed to 

incubate for 10 min at room temperature, and absorbance was 

measured at 600 nm. Protein content was determined from 

the calibration curves of BSA. 

Determination of Total Polysaccharide 

Total polysaccharide content was determined according to 

the method of Adams and Emerson (1960) using glucose as a 

standard. [27] Briefly described as the extract was washed 

with 80% of hot ethanol by centrifugation at 3000 rpm for 10 

min and the procedure was repeated until the washing did not 

give color with anthrone reagent. The residue was then dried 

over water bath and extracted with 5 ml distilled water and 5 



ml of 25 % HCl for 20 min at 0°C. The tube was then 

centrifuged at 3000 rpm for 10 min and the supernatant was 

transferred to 100 ml volumetric flask. The extraction was 

repeated in the same way and the supernatant was poured 

into the same volumetric flask and the volume was made 100 

ml with distilled water. For analysis, 100 μl of the 

supernatant was taken into a test tube and made up the 

volume 1 ml with distilled water. Glucose stock solution 

(1000 ppm) was prepared in distilled water and a series of 

working standards solutions of concentration 20, 40, 60, 80, 

and 100 ppm were prepared to final volume of 1 ml. 

Anthrone reagent (4 ml) was added to standards and extract 

solutions and the reaction mixture was heated for 8 min in a 

boiling water bath. Test tubes were then cooled rapidly and 

the absorbance was measured at 630 nm against distilled 

water as a blank. Calibration curve of glucose was used to 

determine the total amount of polysaccharide in the samples. 

Anti-inflammatory activity by Lipoxygenase inhibition 

assay 

Lipoxygenase inhibiting activity was measured using 

spectrophotometric method. [28] Test samples and reference 

standards were dissolved in methanol. The reaction mixture 

was prepared in 96-well microplate by adding sodium 

phosphate buffer 160 µl, test sample solution 10 µl and 

soybean lipoxygenase solution 20 µl. Then reaction was 

initiated by adding sodium linoleic acid solution 10 µl, which 

acted as substrate. The enzymatic conversion of linoleic acid 

was measured at 234 nm over a period of 6 min. All the 

reactions were performed in triplicate. 

Anti-inflammatory activity by hyaluronidase inhibition 

assay 

The assay was performed according to Sigma protocol with 

slight modifications. [29] The assay medium consisting of 

1.00-1.67 U hyaluronidase in 100 µl of 20 mM sodium 

phosphate buffer was incubated with 5 µl of the test 

compound (in DMSO) for 10 min at 37 o C. Then the assay 

was commenced by adding 100 µl hyaluronic acid and 

incubated for further 45 min at 37 o C. The undigested 

hyaluronic acid was precipitated with 1 ml acid albumin 

solution. After standing at room temperature for 10 min, the 

absorbance of the reaction mixture was measured at 600 nm. 

The absorbance in the absence of enzyme was used as 

reference value for maximum inhibition. The inhibitory 

activity of the sample(s) was calculated as the percentage 

ratio of the absorbance in the presence of test compound 

versus absorbance in the absence of enzyme. The 

performance of the assay was verified using apigenin as a 

reference under exactly the same experimental conditions. 

Anti-inflammatory activity by 12-Otetradecanoylphorbol 

13-acetate (TPA)-induced ear 

oedema 

TPA induced mouse ear oedema inhibitory assay was used in 

the determination of anti-inflammatory activity. The 

experiment was carried out according to a method described 

by De Young et al., (1989) and Carlson et al., (1985). [30-31] 

The animals were grouped randomly each having 5 animals. 

TPA in acetone (20 µl of 0.05 µg/µl) was applied topically 

on right ear of each mouse. The test sample dissolved in 

acetone was applied topically to the inner surface of the right 

ear (2 mg/ear) about 30 min before each TPA application. 

The sample vehicle in acetone was applied on the other ear, 

which served as control. Indomethacin was used as standards. 

The resulting oedema was measured 6 h after TPA treatment. 

The results were expressed as percentage inhibition till 

complete suppression of erythema. Each value used was the 

mean of individual determinations from 5 mice. Percentage 

inhibition (IE %) of the test sample was calculated as the 

ratio of the weight increase of the ear sections. The study 

protocol was approved by the Animal Ethical Committee of 

the School of Pharmaceutical Science, Universiti Sains 

Malaysia. 

Statistical analysis 

For standardization all the samples were analysed in triplicate 

and results were averaged. Anti-inflammatory activity study 

was determined triplicate, except for TPA assay where 

results are average of 5 replicas. Results of antiinflammatory 

activity of different extracts were analysed by 

one way ANOVA and P < 0.05 was regarded as significant. 

RESULTS AND DISCUSSION 

Vitexin (8-C-glucosyl apigenin) and isovitexin (6-C-glucosyl 

apigenin) have several pharmacological activities such as 

anti-hypertensive, anti-inflammatory, antispasmodic, 

antimicrobial and antioxidant. [32-34] Hence, these compounds 

have been selected as analytical markers to standardise 

aqueous and methanol extracts of leaves of three verities of 

Ficus deltoidea. The chemical structures of the markers are 

presented in Fig. 1. 

The chromatographic conditions were optimized to obtain 

chromatograms with better resolution of adjacent peaks in a 

shorter time. Various mixtures of water and methanol were 

used as mobile phase but the separation was not satisfactory. 

However, when the methanol was replaced by acetonitrile, 

the separation and resolution was improved. Addition of acid 

in mobile phase was found to enhance the resolution and 

eliminate the peak tailing of the target markers. As a result, a 

gradient elution combining acetonitrile and water containing 

0.1 % ortho-phosphoric acid was chosen to get the desired 

separation. The detection was better at 335 nm as compared 

to 270 nm. This optimization produced well-resolved peaks 

of vitexin and isovitexin in a total run of 23 min. The 

chromatograms the mixed standard solution and the extracts 

are given in Fig. 2. 

The method was found linear over the whole range of 

markers investigated; vitexin showed good linearity with a 

regression equation (Y = 19.809X – 26.599 with R 2 = 

0.9997), while isovitexin showed a linearity with regression 

equation (Y = 21.253X – 32.96 with R 2 = 0.9999). For both 

the markers, LOD and LOQ were found to be 1 and 3 μg/ml, 

respectively. For repeatability and reproducibility of the 

method, results indicated that the within-day and betweenday 

RSD values of both the compounds were less than 5.58 

%, which indicated good repeatability and reproducibility. 

The extraction recovery was found to be in the range of 87.29 

– 116.58 % with R.S.D less than 5.02 %. These results 

indicate that the method is repeatable and reproducible 

because results were not compromised in within and between 

day analysis. 

This validated method was applied to standardise methanol 

and water extracts of leaves of three varieties of Ficus 

deltoidea. Peaks were identified by comparing the retention 

times. Methanol and aqueous extracts from Ficus deltoidea 

var. terengganuensis showed several non-identified peaks at 

retention time different from the standards. Both the samples 

of Ficus deltoidea var. terengganuensis showed similar 

HPLC chromatograms but vary in the concentration of 

vitexin and isovitexin. The HPLC profiles of extracts of 



Table 1: Content of markers (vitexin and isovitexin) and total polysaccharide and total protein in the aqueous and methanol extracts of 

Ficus deltoidea, (n = 3) 

Extracts Vitexin (mg/g) Isovitexin (mg/g) Polysaccharides (%) Protein (%) 

FDT1M 4.95 ± 0.12 

7.84 ± 0.18 0.9548 ± 0.06 14.9164 ± 0.88 

FDT1W 2.45 ± 0.00 3.61 ± 0.01 2.1402 ± 0.08 47.9373 ± 1.22 

FDT2M 5.69 ± 0.09 12.78 ± 0.20 0.9657 ± 0.04 35.3691 ± 1.35 

FDT2W 19.00 ± 0.12 20.49 ± 0.09 2.7570 ± 0.27 66.5318 ± 2.74 

FDAM 12.18 ± 0.15 41.49 ± 0.47 0.9984 ± 0.09 18.9127 ± 0.77 

FDAM 6.60 ± 0.09 26.94 ± 0.26 5.1059 ± 0.17 13.6691 ± 0.20 

FDDM 7.30 ± 0.15 1.58 ± 0.02 0.0006 ± 0.00 6.3891 ± 0.26 

FDDW 5.48 ± 0.01 nd 

0.007 ± 0.00 34.6926 ± 0.79 

(FDT1M), (FDT1M), (FDT2M) and (FDT2W) are extracts of Ficus deltoidea var. terengganuensis; (FDAM) and (FDAW) are extracts of Ficus 

deltoidea var. angustifolia; (FDDM) and (FDDW) are extracts of Ficus deltoidea var. Deltoidea, nd (not detected) 

Fig. 1: Chemical structure of two C-glycosylflavones, vitexin (1) and isovitexin (2) 

Fig. 2: Chromatograms of mix standard solution, methanol extracts (M) and aqueous extracts (W) detected at 335 nm, a (mix standard solution); a 

(FDT1M), b (FDT1M), c (FDT2M) and d (FDT2W) are extracts of Ficus deltoidea var. terengganuensis; e (FDAM) and f (FDAW) are extracts of 

Ficus deltoidea var. angustifolia; g (FDDM) and h (FDDW) are extracts of Ficus deltoidea var. deltoidea; (1) vitexin; (2) isovitexin 

Fig. 3: Anti-inflammatory effects of aqueous and methanol extracts and standards on LOX, , HAase and TPA-induced ear oedema, (FDT1M), 

(FDT1M), (FDT2M) and (FDT2W) are extracts of Ficus deltoidea var. terengganuensis; (FDAM) and (FDAW) are extracts of Ficus deltoidea var. 

angustifolia; (FDDM) and (FDDW) are extracts of Ficus deltoidea var. Deltoidea; NDGA ( nordihydroguaiaretic acid); Each value represents mean ± 

SD (n = 3); * P < 0.05 ( a significant difference as compared to nordihydroguaiaretic acid); ** P < 0.05 ( b significant difference as compared to apigenin); 

*** P < 0.05 ( c significant difference as compared to indomethacin) 



Ficus deltoidea var. angustifolia and Ficus deltoidea var. 

deltoidea showed only a few peaks as compared to 

chromatographic profiles of Ficus deltoidea var. 

terengganuensis. From the results, isovitexin was found to 

be main chemical component in Ficus deltoidea var. 

angustifolia, while vitexin was found as the main chemical 

component in Ficus deltoidea var. deltoidea. The content of 

both the markers in different extracts of three verities are 

presented in Table 1. The concentration of vitexin and 

isovitexin in these extracts varies in the range 2.45 ± 0.00 to 

19 ± 0.12 mg/g extracts and 1.58 ± 0.02 to 41.49 ± 0.47 mg/g 

extracts, respectively. 

All aqueous and methanol extracts were evaluated for the 

bioactive compounds, total polysaccharide and protein (Table 

1). These results indicated that percentage of protein in 

water extracts ranged from 13.67 - 66.53 %, while the 

percentage of protein in methanol extracts ranged from 6.39 - 

35.37 %. The percentage polysaccharide in all extracts was 

less than 5.11 %. 

The results of lipoxygenase inhibitory properties of the 

extracts using 15-soybean lipoxygenase are presented in Fig. 

3. It is obvious from the results that all the extracts showed 

low LOX inhibition activity as compared to the standard, 

nordihydroguaiaretic acid (100 % inhibition). Among these 

extracts, aqueous extracts of FDD and methanol extract of 

FDA exhibited zero LOX inhibition activity, whereas 

FDDM, FDT1M and FDT2M exhibited 10.35 ± 0.04 %, 7.55 

± 0.04 % and 7.60 ± 0.01 % inhibition, respectively. These 

results showed that the extracts did not display antiinflammatory 

activity via LOX mechanism. 

According to Guo et al. (2002) [35] antioxidants display antiinflammatory 

effects because the level of free radicals is 

increased in inflammation. Phenolic compounds have the 

potential to block the process of arachidonic acid metabolism 

by inhibiting lipoxygenase activity. However, according to 

Rackova et al. (2007) [36] scavenging effect of free radical 

may not be a critical factor behind the inhibition of LOX 

pathway rather inhibition is suggested to be due to the 

specific interaction of constituents with the enzyme. An 

inhibition of the LOX can be achieved via chelation of its 

non-heme bound iron [37] or by reduction of its ferric form. 

[38-40] It explains the disability of the extracts to inhibit LOX, 

though these extracts have shown antiradical properties. [23] 

An interaction with iron atom at the enzyme catalytic centre 

may be involved in the LOX inhibition mechanism. 

Results of hyaluronidase (HAase) inhibition of methanol and 

aqueous extracts presented in Fig. 3 indicated that the 

extracts exhibited moderate inhibitory activity against 

HAase, which was comparable to that of the standard, 

apigenin (85.60 ± 7.15 %). The data obtained, showed that 

methanol extracts of sample FDT1, FDT2 and FDA have 

slightly higher HAase inhibition activity than the aqueous 

extracts. In contrary, aqueous extracts of FDD exhibited 

slightly higher percentage of HAase inhibition activity as 

compared to methanol extracts. The HAase inhibition 

activity of methanol and aqueous extracts varied from 47.05 - 

62.95 % and 48.97 - 51.0 %, respectively. Except FDDM 

extracts, activity of all the extracts was significantly different 

as compared to standards (P < 0.05). The results indicated 

that these extracts could be considered moderate inhibitor of 

HAase. 

The presence of protein in the assay mixture effect the 

release of HA by HAase and the activity is decreased with 

the increase of protein concentration. [41] The mechanism by 

which low concentration of proteins enhances the enzyme 

activity is unclear but it may be hypothesized that the 

polyanionic HA molecule can bind to the small amounts of 

proteins that facilitate the opening of the HA random coil, 

thus facilitating hyaluronidase accessibility to the HA. Most 

recent study by Descherevel et al. (2008) [42] on the effects of 

added non-catalytic protein, bovine serum albumin (BSA), 

on the HAase activity showed that the addition of BSA 

induces competition with HAase to form non-specific 

complex with HA, thus consequently releases free HAase 

which is catalytically active leading to an increase in 

hydrolysis of HA. The study indicates that the stability of the 

non-specific complex of HA-BSA was higher than that of 

HA-HAase suggesting that as long as the added BSA releases 

HAase by forming non-specific complex with HA, it will 

induce an increase in the hydrolysis of HA that is catalyzed 

by HAase. Under these conditions, BSA acts as activator of 

HAase. However, further addition of BSA increases the 

formation of HA-BSA complexes, which results in lowering 

the initial rate. In this case, the formation of HA-HAase 

complex is hindered, and BSA acts an inhibitor of HAase. 

Therefore, the content of protein in the extracts should be 

considered in the hyaluronidase activity. In this study, the 

total of protein content has been estimated and presented in 

Table 1. As mentioned earlier, the methanol extracts have 

slightly higher percentage of HAase inhibition as compared 

to aqueous extracts. Conversely, the protein content of the 

aqueous extracts is slightly higher than the methanol extracts. 

This may explain the results of moderate percentage of 

HAase inhibition, thus suggesting the involvement or the 

interactions of protein with the other chemical constituents of 

the extracts in this activity. 

Many polyanions, such as glycosaminoglycans (heparin, 

heparin, sulphate, dermatan sulphate), HA derivatives (Osulpfonated 

HA) and synthetic polyanions (polystyrene-4sulfonate) 

are known to inhibit HAase. [42] Formation of HApolycation 

complexes (polycationic polysaccharides) 

indirectly inactivates HAase by hindering HAase 

accessibility to HA. [42] Total polysaccharide content of 

extracts is presented in Table 1. The total polysaccharide 

content in each extracts may affect the HAase activity. 

However, the percentage of total polysaccharides in these 

extracts is quite low as compared to the total protein in 

respective extracts, hence may only contribute to a small 

extenct in HAase activity. 

Results of TPA-induced mice ear oedema shown in Fig. 3 

indicated that methanol extracts exerted noteworthy activity 

as compared to the aqueous extracts. This may indicate the 

presence of active substances endowed with antiinflammatory 

activity. As observed from Fig. 3, FDT1M, 

FDT2M, FDDM and FDAM exhibited moderate to strong 

reduction of oedema (P < 0.05) with percentage inhibition of 

85.46 ± 8 %, 56.56 ± 16 %, 80.46 ± 4 % and 38.74 ± 7 %, 

respectively, and the results were comparable to 

indomethacin, a reference compound that had shown 85.4 ± 

2% inhibition. However, aqueous extracts exhibited low 

inhibition activity with 8.38 ± 10 %, 10.28 ± 4 % and 22.53 ± 

9 % for FDDW, FDT1W, FDAW, respectively, whereas 

FDT2W was found completely inactive in this model. This 

might be due to the high total polyphenols contained in the 

methanol extracts as compared to the aqueous extracts. 

Among aqueous and methanol standardised extracts of leaves 



of three varieties of Ficus deltoidea, methanol extracts 

exhibited potent anti-inflammatory activity in three models; 

lipoxygenase (LOX), hyaluronidase (HAase) and TPAinduced 

ear oedema. Among these models, the antiinflammatory 

activities of methanol extracts were more in 

TPA model. 

ACKNOWLEDGEMENT 

The corresponding author wishes to thank the Forest 

Research Institute of Malaysia for providing financial 

support and Research Assistantship. 

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ISSN 0975 1556 

Evaluation of Medicinal Properties of Hibiscus rosa sinensis in Male 

Swiss Albino Mice 

Nidhi Mishra * , Vijay Lakshmi Tandon, Ashok Munjal 

Department of Bioscience and Biotechnology, Banasthali Vidyapith, Banasthali-304 022 (Rajasthan) INDIA 

ABSTRACT 

The present study was undertaken to evaluate the medicinal properties of Hibiscus rosa sinensis on male mice. For this 

Swiss albino mice were orally administered Hibiscus rosa sinensis (500 mg/kg of body weight) and effect of the treatment 

on, reproductive organ cholesterol level and glucose level. Recovery study was also carried out. The treatment caused 

reduction in the weight of testis, epididymis and evaluation of sperm count indicates that sperm density decreased 

significantly. Histologically, testis in mice treated with the plant extract showed alteration in the seminiferous tubules and 

alteration include decrease in thickness and density of germinal epithelium and hypertrophy in majority of cells moreover 

lumen shows negligible presence of sperms in the treated animal as compared to control. The treatment group had decrease 

in levels of testosterone. Crude extract of blooms of Hibiscus rosa sinensis possess hypoglycemic and hypocholestrolemic 

potentially. The alterations caused in the above endpoints by the plant extract were reversible and by 60 days of the 

treatment withdrawal, the parameters recovered to control levels. The results in Swiss albino mice thus suggest that 

Hibiscus rosa sinensis treatment causes reversible suppression of spermatogenesis, Cholesterol level and glucose level. 

Keywords: Hibiscus rosa sinensis, Seminiferous tubules, Germinal epithelium, Hypertrophy, Testosterone, 

spermatogenesis. 

INTRODUCTION 

In India, the use of different parts of medicinal plant to cure 

specific ailments has been in vogue from ancient times. The 

earliest mention of medicinal use of plants has been noticed 

in “Rig Veda”. These days herbalism is emerging as an 

alternative medicine since it makes healthcare affordable for 

all. It is believed that the mixture of several crude extracts, 

when used in formulation enhances the beneficial effects 

through synergistic amplification and diminishes any 

possible adverse effects. Williamsons further emphasized this 

concept that a whole or partially purified extract of a plant 

offers advantage over a single isolated ingredient. [1] The 

plant Hibiscus rosa sinensis (Malvaceae) commonly known 

as Chinesis Hibiscus or tropica. 

Hibiscus rosa sinesis is a native of china and is a potent 

medicinal plant. It is a common Indian garden perennial 

shrub. [2] And often planted as a hedge or fence plant. 

Hibiscus rosa sinensis flower decoctions are used in India 

and Vanuatu as aphrodisiacs, for menorrhagia, uterine 

haemorrhage and for fertility control. 

[3] It possesses 

anticomplementary, antidiarrhetic and antiphologistic 

activity. 

*Corresponding author: Ms. Nidhi Mishra 

Research Scholar, Department of Bioscience and 

Biotechnology, Banasthali Vidyapith, Banasthali – 304022 

(Rajasthan) INDIA ; Fax: +91-1438-228365 

E-mail: immunesys.nidhi@gmail.com 

Hibiscus rosa sinensis flower showed antispermatogenic 

androgenic antitumor and anticonvulsant activities [4-8] and 

antitumor, antihypertensive, antioxidant, antiammonemic. [9- 

13] [14- 

Leaves and flowers also posses hypoglycemic activity. 

15] 

The present investigation is the first ever study undertaken 

to find the effect of crude extract on reproductive hormone, 

diabetes and on Cholesterol level. 

MATERIAL & METHOD 

Animal-Adult Swiss albino mice of the weight 28-32 g were 

used under investigation. Mice were maintained under 

hygienic condition in well ventilated room with 12 h 

photoperiod (8 am to 8 pm) and were fed pelleted food 

(Hindustan lever); drinking water available ad libitum. 

Animals in each group were housed in polypropylene cages, 

with dry rice husk as the bedding material. General health 

condition and body weight of animals were monitered 

regularly during the entire tenure of the experiment. Animals 

were maintained according to the Guidelines of Institutional 

Animal Ethics Committee. 

Test Material - Crude Extract of the Flower prepared by 

Homogenizing petals in water. 

Drugs and Chemical - All the drugs and chemical used in 

this experiment were purchased from Hi media, Qualigen, 

CDH. The chemicals were of analytical grade. 

Induction of Diabeties 

Animals were injected freshly prepared alloxan monohydrate 

in sterile normal saline at a dose of 150 mg/kg of body 

106

Mishra et al. / Effect of Hibiscus rosa sinensis extract on gonads, glucose, Cholesterol level.... 

weight, intraperitonially. [16] The animals shown blood 

glucose level >200 mg/dl after 48 h were considered as 

diabetic. 

Experimental Design 

The animals were randomly allocated into five groups (10 

mice each) and treated as follows: 

Groups Treatment (Dose) 

Ist Control 

IInd Alloxan Treated (150mg/kg of BW) 

IIIrd Alloxan + H.rosa sinensis Treated (500mg/kg of BW) 

IVth H. rosa sinensis at 500mg/kg of BW for reproductive 

system 

Vth H. rosa sinensis at 500 mg/kg of BW for Cholesterol 

level. 

Aqueous extract of Hibiscus rosa sinensis was suspended in 

sterile distilled water and administered orally, with the help 

of an oral feeding needle. Control received equivalent 

volume of sterile distilled water. The dose of Hibiscus rosa 

sinensis was selected by small experimentation and by 

checking its LD50. Mice were sacrificed in regular interval of 

10 days from the day of commencement of dose till 30 days. 

Then mice were kept for recovery. Blood was collected, and 

serum was prepared and stored at – 20 o C until further use. 

Organ Weight 

Testis and epididymis were dissected out, blotted free of 

blood, adhering tissues and weighed. 

Sperm Count 

Caudal epididymal sperm count was assessed in 

haemocytometer and expressed as 10 6 /ml of suspension. [17] 

Histological Studies 

For Histological studies, testies and epididymus were 

randomly selected from left or right side of mice from fourth 

group and control. Then fixed in Bouin’s fluid, dehydrated in 

graded ethanol series, cleared in benzene and embedded in 

paraffin. Tissue were sectioed at 5μ, and the sections were 

stained with eosin and haemotoxylin. 

Blood Glucose Level 

Blood Glucose level was detected checked by Glucometer. 

Biochemical estimation 

Quantitative Biochemical estimation of cholesterol in blood 

serum. [18] 

Statistical Analysis 

All data were represented in statistical form and student – T 

test analyses were carried out to determine the levels of 

significance between control and experimental groups. 

RESULTS 

BW and Organ weight 

There is no significant difference was observed in BW of 

Hibiscus rosa sinensis control and treated mice and they 

were found healthy in throughout period of investigation. 

Weight of testes and epididymis showed significant reduction 

in comparison to control. After withdrawal of treatment mice 

were kept for recovery period of 2 months and it was found 

there is recovery in weight of testes and epididymis of treated 

mice (Table 1). 

Sperm Density 

Significant reduction in sperm density was found from 10 th 

day onwards and was observed till 30 days and the mice were 

recovered after withdrawal of treatment (Fig 1). 

Histology 

Histological examination of testies of treated and control 

animals indicate that though there is no decrease in diameter 

of seminiferous tubules, in the treated animals, but the 

thickness and compactness of germinal epthilium is 

significantly lost and most of the cells of the germinal 

epithelium have undergone hypertrophy. The process of 

spermatogenesis is highly disrupted and the accumulation of 

sperms within the lumen is almost negligible but after the 

withdrawal of treatment it was found that there is recovery of 

treated mice (Fig 6). While the epididymides of untreated 

control mice showed normal histologic features. 

Hematoxylin stained sections through Days 10, 20, 30 and 90 

of mouse epididymus. After dosing of H.rosa sinensis, 

500mg/kg BW/day showing normal appearance of the 

segment, except that the lumen is empty and stroma appears 

increased after 10 th , 20 th and 30 th day treatment. And after 

recovery of treatment it was found that both control and 

treated mice have normal appearance (Fig 7). 

Testosterone Level 

Assay of testosterone in the serum by ELISA also indicates 

the fall in density of sperms and that of testosterone level are 

correlated to one another (Fig 2). 

Blood Glucose Level 

Blood Glucose level was observed after fasting and PP and it 

was found that it is showing significant decrease after 20 

days treatment in fasting while it is showing significant 

decline after 10 day onwards in PP (Fig 3, 4). 

Cholesterol Level 

Blood Cholesterol Level Decreased from 10 th day only and it 

was showing significant decline till 30 th day treatment. And 

after withdrawal of treatment it was observed that level was 

recovered as of control (Fig 5). 

DISCUSSION 

The result of present study show that Hibiscus rosa sinensis 

treatment did not cause alterations in the body weight but 

weight of testis and epididymis showed a significant 

reduction in treated animals. And there was no decrease in 

diameter of seminiferous tubules. But the thickness and 

compactness of germinal epithelium have undergone 

hypertrophy. The process of spermatogenesis is highly 

disrupted and the accumulation of sperms with in the lumen 

is almost negligible. Flower of Hibiscus rosa sinensis posses 

antifertility property which has been reported long back in 

Ayurvedic text. However, in the last decade it was pointed 

out that the alcoholic extracts of the flowers of Hibiscus rosa 

sinensis decrease spermatogenic elements of testis and 

epidymal sperm count at a dose of 125 and 250 mg/kg of 

BW. [8] The present study also observed similar changes in 

the seminiferous tubules of testis and sperm density though 

the dose was much higher in the present study. Sperm density 

declined significantly. As it was reported in case of 

Bougainvillea spectabilis crude extract showing changes in 

sperm density as well as alterations in seminiferous tubules 

was found. [19] Assay of testosterone in the serum by ELISA 

also indicated the fall in density of sperms and that of 

testosterone level are correlated to one another because 

reduction in the number of spermatozoa in Hibiscus rosa 

sinensis extract treated mice appeared to be due to the 

suppressive effect of the treatment on spermatogenesis as 

sperm number recovered to control level after recovery of the 

process following cessation of the treatment. 

Earlier Kholkute, has described that antifertility action of 

Hibiscus rosa sinensis is season dependent. He also reported 

that it causes significantly inhibitory action on 

spermatogenesis in mediated via pituitary gland without 

affecting pituary adrenal and pituary thyroid function in male 



Table 1: Weight of testis and epididymis in mice after H. rosa sinensis treatment (500 mg/kg of BW) and following recovery from 

treatment. 

Time Interval 

BW(g) 

Control (mg/100gm BW) 

Testies Epididymis BW(g) 

Treated (mg/100 gm BW) 

Testies Epididymis 

10 30.00±00 582.23±3.2 425.47±3.5 30.00±0.81 565.56±1.3 380.29±2.25 

20 30.00±00 597.33±4.7 419.21±1.2 30.00±.40 533.19±1.9* 352.01±3.2 

30 30.00±00 580.21±2.1 415.41±1.8 30.75± .82 510.02±2.5** 341.19±1.5** 

90 a 

30.00±00 585.66±1.51 420±3.7 30.80±.40 493.44±3.65** 325.51±4.33** 

The values are expressed as mean ± SD for 10 animals (n=10) per group. 

a Treatment was discontinued after 30 days, and animals were sacrificed 60 days after withdrawal of treatment. 

* indicates statistically significant at p


A B 

C D 

Fig. 6: Cross Section of Testis of the control and treated mice. (A) Control showing normal appearance of seminiferous tubules. (B) After H.rosa 

sinensis treatment, 500mg/kg BW for 30 days showing Decrease in compactness of germinal epithelium and sperm number was almost negligible in 

Lumen. (C) Control was showing normal appearance after recovery period also. (D) After withdrawal of treatment recovery was observed. 



On 10 th day Control Treated 

20 th days 

30 th day 

90 th day 

Fig. 7: Hematoxylin stained sections through Days 10, 20, 30 and 90 of mouse epididymus .Control mice showing normal histological features. After 

dosing of H.rosa sinensis, 500mg/kg BW/day showing normal appearance of the segment, except that the lumen is empty and stroma appears increased 

after 10 th , 20 th and 30 th day treatment. And after recovery of treatment it was found that both control and treated mice have normal appearance. 

albino rats. [20] 

In second phase of study, deals with the evaluation of 

glucose level in the blood after PP and fasting. From data it is 

clear that crude extract of Hibiscus rosa sinensis blooms 

causes a significant decrease in level of glucose in alloxan 

induced diabetic mice at a dose of 500 mg/kg of BW after 

10 th day treatment as compared to alloxan treated mice. This 

observation supporting the report of Sachdewa and Khemani, 

suggesting that oral intake of Hibiscus rosa sinensis blooms 

causes hypoglycemia even in normal animal. [15] Earlier, it 

has been assigned hypoglycemia property to aqueous extract 

of Hibiscus rosa sinensis concerned, evidence suggest that 

hibiscus mucilage rich in flavonol bioside, in general posses 

antidiabetic property. [14] 

In third phase of study, Cholesterol level of blood 

quantitatively estimated in control as well as treated animals. 

After analysation of data, it is inferred that oral 

administration of Hibiscus rosa sinensis flowers decrease 

blood cholesterol level significantly just after 10 th day. 

Decline was upto 25%. A decline of serum cholesterol and 

triglyceride levels 22% and 32 % in the flower crude extract 

treated animals reported by Sachdewa and Khemani is in 

concordance with the present analysation. [15] Moreover it 

was observed that the mucilage obtained from the plant of 

genus Hibiscus has hypocholesterogenic properties. [21-22] In 

conclusion our result of Swiss albino mice suggests that 

Hibiscus rosa sinensis treatment at doses and durations 

employed in the present study caused marked alteration in 



the male reproductive organs and that alteration are 

reversible after cessation of treatment. Treatment also had a 

reversible effect on Cholesterol level and diabeties. 

ACKNOWLEDGMENT 

This work was supported by fund from Banasthali University 

so I am thankfull to Prof. Aditya Shastri (Vice Chancellor, 

Banasthali University). 

REFERENCE 

1. Williamson: Synergy and other interaction in phytomedicines. 

2001, 8 (5), 401- 409. 

2. Mudgal. Botanical description of Hibiscus rosa sinensis (China rose 

of shoe flower or japakusum). J Res Indian Med. 1974; 9: 105. 

3. Lans: Creole remedies of Trinilad and Tobago. Lulucom, 2006. 

4. Murthy, Reddy, Patil. Effect of benzene extract of Hibiscus rosa 

sinensis on the estrous cycle and ovarian activity in albino mice. 

Biol Pharm Bull. 1997; 20(7): 756-758. 

5. Mathu P, Mathur M. Concentration of Na+ and K+ in serum and 

uterine flushings of ovariectomized, pregnant and cyclic rats when 

treated with extracts of Hibiscus rosa sinensis flowers. Journal of 

Ethnopharmacol. 1990; 28(3): 337-347. 

6. Pakrashi. Flowers of Hibiscus rosa sinensis, a potential source of 

contragestative agent III: Interceptive effect of benzene extract in 

mouse. Contraception 1986; 34(5): 523-536. 

7. Pal, Bhattacharya, Kabir, Pakrashi. Flowers of Hibiscus rosa 

sinensis, a potential source of contragestative agent: II. Possible 

mode of action with reference to anti – implantation effect of the 

benzene extract. Contraception 1985; 32(5): 517-529. 

8. Reddy, Murthy, Patil. Antispermatogenic and androgenic activities 

of various extract of Hibiscus rosa sinensis in albino mice.Indian J 

Exp Biol. 1997; 35(11): 1170-1174. 

9. Hou, Tong, Terahara, Lou, Fujii. Delphenidin 3- sambubioside, a 

Hibiscus anthocyanin, induces apoptosis in human leukemia cells 

through reactive oxygen species -mediated mitochondrial pathway. 

Arch Biochem Biophy. 2005; 440: 101-109. 

10. Hirunpanich, Utaipat, Morales, Bunyapraphatasara, Sato, 

Herunsalee. Hypocholestremic and antioxidant effect of the 

aqueous extracts of Hibiscus sabdariffa Linn. in hypercholestremic 

rats. J Ethanopharmacol. 2006; 103: 252-260. 

11. Chang, Haung, Haung, Wang. Hibiscus anthocyanins- rich extract 

inhibited LDL. Oxidation and oxLDL – mediated macrophages 

apoptosis. Food Chem Toxicol. 2006; 1015-1023. 

12. Herrera, Flores, Chavez-Soto, Tortoriello. Effectiveness and 

tolerability of a standardized extract from Hibiscus sabdariffa in 

patients with mild to moderate hypertension: a controlled and 

randomized clinical trial. Phytomedicine. 2004; 11: 375-382. 

13. Mohamed Essa, Subramanian. Hibiscus sabdariffa affects 

ammonium chloride- induced hyperammonemic rats.In: Evid 

BasedComplement Altern Med. 2007; 4: 321-326. 

14. Sachdewa, Nigam, Khemani. Hypoglycemic effects of Hibiscus 

rosa sinensis L. leaf extract in glucose and streptozotocin induced 

diabetes in rats. Indian J Exp Biol. 2001; 39: 284-286. 

15. Sachdewa, Khemani. Effect of Hibiscus rosa sinensis Linn. ethanol 

flower extract on blood glucose and lipid profile in streptozotocin 

induced diabetes in rats. J Ethanopharmacol. 2003; 89: 61-66. 

16. Aruna, Ramesh, Kartha. Effect of betacarotene on protein 

glycosylation in alloxan induced diabetic rats. Indian J Exp. Biol. 

1999; 37: 399-405. 

17. Prasad, Chinoy, Kadam. Changes in succinic dehydrogenase levels 

in the rat epididymis under normal and altered physiologic 

condition. Fertility Sterility. 1972; 23: 186-190. 

18. Zlatkis, Zak, Boyle, A.J.J. Lab. Clin. Med. 1953; 41: 486. 

19. Mishra, Joshi, Tandon, Munjal. Evaluation of Anti-fertility 

potential of Aqueous extract of Bougainvillea spectabilis leaves in 

Swiss Albino mice. Int J Pharm Sci Drug Res 2009; 1: 19-23. 

20. Khulkute, Udupa. Effect of Hibiscus rosa sinensis on 

spermatogenesis in rats. Planta Medica. 1977; 31: 127- 135. 

21. Woolfe: The effect of okra mucilage (Hibiscus esculentus L.) on the 

plasma cholesterol level in rats. Proc. Nutr. Soc. 1977; 36 (2): 59A. 

22. Sitohy, El-Saadany, labib, El- Massry: Biochmemical dynamics and 

hypocholestrolemic action of Hibiscus sabdariffa (Karkade). 

Nahrung. 1991; 35(6): 567-576. 





ISSN 0975 1556 

Adaptogenic Activity Studies on the Crude Extract of Polyscias 

balfouriana var. Marginata Root and Leaf 

Sandhya S. 1* , Vinod K. R. 1 , Madhu Divakar C. 2 , Nema Rajesh Kumar 3 

1 Nalanda College of Pharmacy, Charlapally, Nalgonda, Andhra Pradesh, India 

2 Cresent College of Pharmacy, Kannur, Kerala, India 

3 Rishiraj College of Pharmacy, Sanwer Road, Indore, Madhya Pradesh, India 

ABSTRACT 

It is a well known fact that stress of any nature produces a non specific state in an organism or a state of ‘stress syndrome’. 

The present study is based on the Adaptogenic activity of Polyscias balfouriana. Studies based on forced locomotor 

activity, behavioral despair test, hypothermia test, hypoxia test and anabolic effect were conducted. The leaf extract was 

found to have better activity than root extract except for the forced locomotor activity. 

Keywords: Adaptogenic Activity, Polyscias Balfouriana. 

INTRODUCTION 

Polyscias balfouriana variety Marginata is an ornamental 

foliage shrub cultivated in gardens. These plants are 

popularly known in trades and horticulture nurseries as 

‘aralias’ [1] since they belong to the family Araliaceae. The 

plant is also known as Scutellarium or dinner plate Aralia or 

Balfour Polyscias. The plant is available through out the 

warmer parts of India, especially kerala and Tamil Nadu. It is 

also available in Irotropical Asia, Malaya. It is a native of 

New Caledonia. The plants coming under the family 

Araliaceae are mainly constituted by triterpenoid saponins 

The chemical studies on the saponins and sapogenins 

revealed that the triterpenoid saponin content in this family 

play an important role in the pharmacological activity like 

stimulation of CNS, anti fatigue and enhancement of nonspecific 

resistance. Ginseng (Panax quinquefolium) a tropical 

plant is one of the few commercially important members of 

this family. [2] The crude extracts as well as its pure glycoside 

(panaxoside) is clinically employed for premature ageing and 

as revitalize. [3] It has anabolic effect, increases immune 

response and physical efficiency in athletes. [4] Hence in the 

present work ginseng was kept as the standard. 

MATERIALS AND METHODS 

The plants Polyscias balfouriana was collected from Tamil 

Nadu agricultural University Coimbatore. The complete 

pharmacological work was explained before the animal 

ethical committee and the written protocol was submitted. 

*Corresponding author: Ms. Sandhya S., 

Nalanda College of Pharmacy, Charlapally, Nalgonda, 

Andhra Pradesh, India 

E-mail: sanpharm@gmail.com 

After the approval the animal experiments commenced. All 

chemicals and reagents used in this work were of analytical 

grade or above. The fresh leaves and roots were extracted for 

72 h with 70 % ethanol by hot continuous extraction using 

soxhlet apparatus. The extracts obtained were concentrated 

under vacuum distillation below 60 o C. They were diluted 

with water and further extracted with chloroform to remove 

the lipid materials. The water extracts left behind were 

extracted with ethyl acetate and then with n-butanol. The nbutanol 

layers were separated and evaporated to dryness to 

give the crude saponin extracts. This was designated as NBS 

extract. In the preliminary chemical tests the NBS extract 

shows highly positive results for triterpenoids and gave good 

colour reactions for Salkowsky test and Liebermann burchard 

test. 

Adaptogenic activity 

The medicinal substances causing a state of non specifically 

increased resistance (SNIR) are named as Adaptogens or 

Athenktropics. [5] Adaptogens are the substances which help 

to increase resistance of the body towards noxious influence, 

including physical, chemical and biological stresses. It was 

proposed that the adaptogens could restore and maintain 

physiological homeostasis irrespective of the direction of the 

physiological perturbation. Panax ginseng is a plant 

adaptogen claimed to be the “elixir of life” in the traditional 

Chinese system of medicine. There after Panax ginseng has 

been extensively investigated experimentally and clinically. 

Ginseng has been claimed to have anti-stress, anti-fatigue, 

mood stabilizing, serenic and cognition–facilitating 

properties. So this plant appears to exert a combination of 

effects on CNS, including arousal facilitation of conditioned 

behavior, augmentation of reflex responses and attenuation of 

fatigue, particularly under the stress conditions. The root 

112

Table 1: Forced locomotor activity [7-12] 

Sandhya et al. / Adaptogenic Activity Studies on the Crude Extract.... 

Animal 

group 

Drug 

Dose 

(mg/kg) Oral 

Fall time in seconds 

Before diazepam administration After diazepam administration 

% decree in fall of 

time in seconds 

Group 1 PBML+diazepam 250mg/kg+5mg/kg 90.5±2.759 50.81±0.3391 28.11%±0.567 

Group 2 PBML+diazepam 500mg/kg+10mg/kg 92.6±0.09685 54.83±2.0479 35.64%±1.261 

Group 3 PBMR+diazepam 250mg/kg+5mg/kg 95.23±2.772 56.66±2.321 42.86%±1.8312 

Group 4 PBMR+diazepam 500mg/kg+10mg/kg 97.84±2.6838 53.36±2.4058 34.54%±2.7312 

Group 5 ginseng+diazepam 250mg/kg+5mg/kg 97.84±2.6838 53.36±2.4058 34.54%±2.7312 

Group 6 ginseng+diazepam 500mg/kg+10mg/kg 101.16±1.602 70.83±1.7448 78.59%±1.1812 

Group 7 1%CMC 2ml 80.83±3.005 47.33±3.0276 19.33%±3.160 

TableNo.2: Behavioral despair test by swim stress induced immobility [7-12] 

Animal group Drug Dose mg/kg oral Duration of immobility in seconds 

Group1 PBML 250mg/kg 191.16 ±12.0196 

Group2 PBML 500mg/kg 211.66±11.5132 

Group3 PBMR 250mg/kg 185.33±6.0736 


Group5 Ginseng 250mg/kg 190.0±7.7028 


Group7 1%CMC 2ml 181.5±7.13326 

[11-12] 

Table No.3: Hypothermia test 

Animal group Drug 

Dose 

mg/kg Oral 

Normal rectal 

temperature 

Rectal temperature after 5 hrs of 

swimming 

Rectal temperature after 30 min after 

the swimming session 

Group1 PBML 250mg/kg 36.4±0.0632 31.5±0.894 33.8±0.894 


Group3 PBMR 250mg/kg 36.6±0.0894 31.4±0.0894 33.8±0.1095 


Group5 1%CMC 2ml 36.5±0.0632 31.3±0.1264 33.3±0.1673 

Table No.4: Hypoxia test [7-12] 

Animal group Drug Dose (mg/kg) oral Survival time in min 







Group7 1%CMC 2ml 30.34±1.287 

[7-12] 

Table No.5: Anabolic effect 

Animal group Drug 

Dose 

(mg/kg) oral 

Initial wt of animal 

(gm) 

Wt of animals after 4 weeks 

(gm) 

Difference in wt after 4 

weeks 





Group5 1%CMC 2ml 74.09±5.3512 92.79±4.2652 18.7±4.8216 

powder of ginseng was used as a standard for the 

Adaptogenic activity studies of the leaves and roots of 

Polyscias Balfouriana. 

The acute toxicity studies of the NBS extracts of the root and 

the leaf were performed by the method of Smith [6] and it was 

proved that the extracts were non toxic and safe up to a dose 

of 2.5 g/Kg body weight. 


The effect of different doses of NBS extracts of the leaves 

and roots of Polyscias balfouriana and root powder of white 

Panax ginseng on the muscle grip strength of mice treated 

with drug diazepam by using Rota rod apparatus was studied 

and observed that both Polyscias balfouriana and Panax 

Ginseng possess anti-depressant and anti-stress activity. The 

root extract at a dose of 500 mg/kg body wt showed better 

anti-depressant and anti-stress activity when compared to the 

either doses of leaf and root extracts of Polyscias 

balfouriana. The effect of different doses of NBS extracts of 

leaves and roots of Polyscias balfouriana and root powder of 

white Panax ginseng on swim stress induced immobility in 

mice was studied and confirmed that both Polyscias 

balfouriana and Panax Ginseng possess anti-stress activity. 

The leaf extract at a dose of 500 mg/kg body weight showed 

better anti-stress activity when compared to other doses of 

root and leaf extracts of Polyscias balfouriana. The effect of 

NBS extracts of leaf and root of Polyscias balfouriana at 

different doses on the hypothermia in mice was studied and 

confirmed that the drug showed anti-stress activity and 

complementary changes in hypothermia. The leaf extract at 

500 mg/kg showed better anti-stress activity when compared 

to other extracts. The effect of NBS extracts of leaf and root 

of Polyscias balfouriana and root powder of white Panax 

ginseng on hypoxia test in mice were studied and confirmed 

that different doses of Polyscias balfouriana and Panax 

ginseng possess anti-stress activity. The leaf extract at 500 

mg/kg showed better anti-stress activity than other doses of 

root and leaf extract. The NBS extracts of root and leaf at 

different doses were studied and confirmed that the leaf 

extract at a dose of 500 mg/kg showed better anabolic effect. 

Adaptogenic activity studies of the NBS extract of leaves and 

root of Polyscias balfouriana were carried out by observing a 


Sandhya et al. / Adaptogenic Activity Studies on the Crude Extract.... 

set of experiments like forced locomotor, behavioral despair 

test by swim stress induced immobility, hypothermia test, 

hypoxia test, anabolic effect. The leaf extract showed at 500 

mg/kg body weight comparable Adaptogenic activity as that 

of white Panax ginseng root extract wherever it was kept as 

the standard. In case of forced locomotor activity the root 

extract at a dose of 500mg/kg body weight showed better 

activity. The present study confirms that the root and leaf 

extracts of Polyscias balfouriana increases the resistance of 

organisms by inducing a state of non-specifically increased 

resistance, irrespective of the nature of the stress. Reactivity 

is the basic feature of living system and ageing is closely 

related to changes in reactivity and adaptation capacity 

resulting from a progressive decrease in self regulatory 

mechanisms. The crude extract of Polyscias balfouriana can 

be employed clinically for premature ageing and as a 

revitalizer. 

REFERENCES 

1. Nayanar MP. Meaning of Indian flowering plant names. 1985, 280. 

2. Brekhman II, Dordymov IV. New substances of plant origin which 

increase non-specific resistance. Ann. Rev. Pharmac.1969; (9): 419. 

3. Popov V, Clinical use of ginseng extract as adjuvant in 

revitalization therapies, Proceedings of international ginseng 

symposium. The central research institute, Republic of Korea, 

1975, 115. 

4. Brekhman II. Panax Ginseng. Med. Sci. Serv. Edn.4, 1967, 17. 

5. Eger W, Med.Exptl. 1961; 4, 251. Quoted from Lazarev N.V and 

Brekhman II, Influence of preparation of Eleutherococcus 

senticosus maxim on neoplastic disease. Med. Sci. Serv. 1967; 4: 9- 

13. 

6. Smith GW. Pharmacological screening tests, Progress in medicinal 

chemistry. Edn.1, Butterworths London, 1960, 1. 

7. Singh N, Nath R, Mishra N, Kohli RP. An experimental evaluation 

of anti-stress effects of Geriforte (An Ayurvedic drug). Quarterly 

journal of crude drug research 1978; 3(16): 125. 

8. Lobo E, Kulkari RD, Desairr. Special Pharmacology of Geriforte. 

Probe 1975, 4, 266. 

9. Singh NK, Agarwal AK, Latha A, Kohli RP. Experimental 

evaluation of Adaptogenic properties of Withania somnifera 

Proceedings of 12 th scientific seminar on Indian medicine B.H.U, 

Varanasi India 1977, 4. 

10. Walter JB, Israel M. General pathology, Edn. 16, Churchill Living 

Stone Pub., New York, 1978, 600, 626, 703. 

11. Selye H, Experimental evidence supporting the conception of 

Adaptation energy, Am. J. Physiol. 1938; 123: 758. 

12. Vagh VT, Kapadia HD, Pavri DN. Clinical Evaluation of an 

indigenous geriatric tonic. Probe, 1975; 4: 292. 





ISSN 0975 1556 

Evaluation of Knowledge towards Breast Cancer among Multiethnic 

Students: A Qualitative Study 

Tahir M. Khan 1, 2* , Mudassir Anwar 3 , Amer H. Khan 1 

1 School of Pharmacy, University Sains Malaysia Pulau Penang 

2 School of Pharmacy, Island College of Technology, Balik Pulau, Penang Malaysia 

3 School of Pharmacy and Health Sciences, International Medical University, Kuala Lumpur, Malaysia 

ABSTRACT 

The main aim of this study was to evaluate and compare the knowledge, attitudes and perception toward breast cancer 

among the students form different ethnic groups. A qualitative study was conducted among the multiethnic students at a 

Malaysian public university in Penang Island. Ethnic groups approached were Malay, Indians, Chinese, Arabs and Thais. 

On an average fifteen students from every group was the part of study. A questionnaire was used to evaluate the 

respondents’ knowledge toward symptoms, causes and treatment of breast cancer. A self designed 15 items questionnaire 

was used to achieve the objective of the study. A total of N=75 students were approached for their participation in the 

study. However, n=51 has shown willingness to be the part of study. Mean age of the respondents were 24 ± 4.46. Overall 

findings demonstrate that the Thai students have comparatively better knowledge than other groups. In term of diagnosis 

the mammography and physical examination were recommended by majority. Surgery and radiation therapy were the 

preferred options to treat breast cancer. Thai students have good knowledge about the symptoms of breast cancer. 

However, knowledge level toward diagnosis was best among Malays and Arabs. Overall findings highlight the need of 

further educational session among all groups for the substitution of negative perceptions with positive and evidence beliefs 

towards the symptoms, causes, prevention diagnosis and treatment of breast cancer. 

Keywords: Breast cancer, Knowledge, symptoms, diagnosis, treatment. 

INTRODUCTION 

Breast cancer is the most common causes of death among 

[1-2] 

Malaysian women. In the year 2000, there were 

approximately one million registered cases of breast cancer 

worldwide with estimated deaths of over three hundred and 

seventy thousand. The incidence of breast cancer was highest 

among the developed countries with an average estimate of 

94.9 per 100,000. However, among less developed countries 

it was about 19.66 per 100,000. [3] The prevalence of breast 

cancer is highest among North American women, 

approximately 10 in hundred at risk of Breast cancer. [3] 

In year 2000 about 3825 new cases of breast cancer were 

reported in Malaysia, with deaths of about one thousand and 

seven hundred. The incidence was estimated to be 34.86 per 

100,000 populations. [3] Still Ministry of health Malaysia is 

unaware of the actual incidence rate of breast cancer. 

Possible factor playing vital role in this regards are lack 

knowledge and awareness towards breast cancer which 

further lead to underreporting. [6] 

*Corresponding author: Mr. Tahir Mehmood Khan, 

Lecturer, School of Pharmacy, Island College of Technology, 

Balik Pulau, Penang Malaysia 

E-mail: tahir.pks@gmail.com 

Moreover, lack of the national breast cancer patients’ registry 

programs is another issue associated with the scarcity of the 

facts. 

In past a high prevalence of breast cancer was seen in the age 

group 45 and over. However, the age of onset is decreasing, 

and more young women than ever are affected. [4] The onset 

of breast cancer is more abrupt among Young women's 

cancers with an aggressive onset resulting in lower survival 

rates. [5] This immediate onset makes it difficult for the 

clinicians’ to diagnose with in time. [5] Keeping in view this 

motivation a preliminary study was conducted among the 

newly registered students at University Sains Malaysia. The 

main aim of this study was to evaluate and compare the 

knowledge, attitudes and perception toward breast cancer 

among the students form different ethnic groups. 

METHODS 

A qualitative study was conducted among the multiethnic 

students at a Malaysian public university in Penang Island. 

Ethnic groups approached were Malay, Indians, Chinese, 

Arabs and Thais. On an average fifteen students from every 

group was the part of study. A questionnaire was used to 

evaluate the respondents’ knowledge toward symptoms, 

causes and treatment of breast cancer. 

Participants 

115

A convenient sampling method was adopted. Five clusters 

were defined on ethnic basis. A total of fifteen students were 

approached from every group. All the new students visited 

the institute of graduate studies for registration of their 

courses on day one [5 th July 2009] were the part of study. 

Study tool 

A self designed 15 items questionnaire was used to achieve 

the objective of the study. Mainly questionnaire comprises of 

five Sections. Some of the items have some sub-items as 

well; the description about the study tool is illustrated as 

follow: 

Section one covers the demographic information of the 

respondents like race, age, marital status, and education level 

and income status. Section Two covers information about 

General knowledge about Breast Cancer. Three questions 

were the part of this section. 

� Have you ever heard about a disease Breast Cancer? 

Y/N 

� Where do you heard about Breast Cancer very first 

time? 

� What do you think Breast cancer is a communicable 

disease? Y/N 

Section Three covers the respondents’ perception about risk 

factors for Breast cancer. Thirteen items were the part of this 

section. Details’ about the items are illustrated in Table 2. 

Section Four comprises of seven items with a main focus on 

how to prevent Breast cancer? Information about the items 

used in this section is mentioned in Table 3. 

Section five was concerned with the knowledge about the 

symptoms, diagnosis and treatment of Breast Cancer. Six 

symptoms of breast cancer were presented to respondents 

Table 4. In addition to symptoms, the knowledge about 

diagnosis and treatment was evaluated by using two 

questions illustrated as follow: 

1-What do you think which is the right way to diagnose 

Breast cancer 

2-What do you think which of the following is the best way 

treat breast cancer? 

Validation and reliability of the questionnaire 

The content validation was conducted by the professionals at 

the department of pharmacy, Island College of technology. 

Fifteen items were finalised, the questionnaire was translated 

in to Malay language in order to make the questionnaire 

easier to understand by the respondents. The translation of 

the questionnaire to Malay language was done by the experts 

at the school of linguistics, USM. The translation was 

rechecked by the professionals at school of pharmacy in 

order to check the appropriateness of the word according the 

study objectives. After this to ensure the face validity of the 

questionnaire a pilot survey conducted among the ICT 

students. A total of twenty students were approached. 

Keeping in view the responses the reliability scale was 

applied and internal consistency of the study tool was 

estimated on the basis of Cronbach's Alpha (α = 0.61). 

Furthermore to assure the validity of the contents factor 

analysis was carried out. The content validity was estimated 

by using Bartlett’s test of sphericity and Kaiser-Mayer-Olkin 

measure of sampling adequacy. The results appear that 

Bartlett’s test of sphericity was significant 0.0000 and 

Kaiser-Mayer-Olkin measure of sampling adequacy was 

0.640. According to Sheridan and Lyndall (2001), a measure 

of more than 0.6 reflects the adequacy of the contents of the 

Khan et al. / Evaluation of Knowledge towards Breast Cancer.... 

questionnaire. [7] Thus these results showed a considerable 

evidence of reliability and validity of the sampling tool. 

Scoring of responses 

The responses about symptoms of Breast cancer were scored 

in order to classify the knowledge in sub-level, this 

classification provide information about the level of 

recognition towards Breast cancer and its symptoms. Six 

items were used to attaint the aim of the study. Every right 

answer adds one score to the respondent’s knowledge level. 

The maximum possible score for theses items was six. For 

the better comparison of knowledge scoring of the response 

were done. Quartiles were applied for the classification of 

knowledge. Classifications of knowledge according to 

quartiles are mentioned as follow: 

Score Knowledge classification 

0-3 Poor 

4 Moderate 

5 Good 

6 Excellent 

Data analysis 

For the purpose of data analysis, the Statistical package for 

social sciences (SPSS13.0®) was used. In order to find out 

the association of knowledge with demographic variables 

chi-square test was applied, where the 20 % cell have 

expected count less than five, Fischer exact statistics was 

applied. However, in order to compare the knowledge level 

among the ethnic groups One way ANOVA was used. 

Moreover, to identify the differences among the different 

racial groups Post-Hoc analysis was conduced. 

Results 

A total of N=75 students were approached for their 

participation in the study. However, n=51 has shown 

willingness to be the part of study. Mean age of the 

respondents were 24 ± 4.46. Details about the demographic 

characteristics of the respondents are illustrated in Table 1. 

Table 1: Demographics of respondents 

Characteristics N % 

Total participants 

Race 

51 

Chinese 

9 

17.6 

Malay 

10 

19.6 

Indian 

10 

19.6 

Arab 

11 

21.6 

Thais 

Age 

(Range 19-37 years) 

(Mean=24± 4.4SD) 

11 

21.6 

19-24 

32 

62.8 

25-29 

9 

17.6 

30-37 

Marital Status 

10 

19.7 

Single 

35 

68.6 

Married 

Educational Level 

16 

31.4 

Secondary 

4 

7.8 

University 

47 

92.2 

General knowledge about breast cancer 

Exploration revealed that majority 49(96.1 %) has heard of a 

disease breast cancer. About 32(62.7 %) of the respondents 

has disclosed that they have heard of breast cancer from the 

television programs. However, 9(17.6 %) disclosed friends 

and family as the source of knowledge. Nearly a half 25(49.0 

%) believes that breast cancer is transmittable disease. 

Further exploration on ethnic ground revealed that majority 

8(15.7 %) among these were Malays followed by Thais 

6(11.8 %), Indians 5(9.8 %) and 3(5.9 %) Chinese and Arabs. 



Table 2: Respondents perception toward risk factors of breast cancer 

Risk Factor 

Malay 

10(19.6%) 

Chinese 

9(17.6%) 

Indian 

10(19.6%) 

Arab 

11(21.6%) 

Thais 

11(21.6%) 

Total 

N(51) 

X 2 

p-value 

Obesity 5 5 6 6 5 27 (52.9%) 0.518 0.993 

Family history of breast cancer 10 8 7 10 9 44(86.3%) 4.264 0.416 

Early Adolescents (< 12 years) 2 4 6 2 3 17(33.3%) 5.818 0.056* 

Late menopause (> 55 years) 3 7 8 3 7 28 (54.9%) 10.861 0.058* 

Smoking can result breast cancer 5 6 7 10 10 38 (74.5%) 8.832 0.006* 

Alcohol Use can result breast cancer 6 6 9 3 6 30 (58.8%) 8.851 0.040* 

Due to pregnancy after 30 years 3 5 6 4 8 26 (50.9%) 5.184 0.054* 

Lack of breast feeding 4 7 7 10 7 35 (68.6%) 6.829 0.331 

Due to use of contraceptive 7 9 7 11 9 43 (84.3%) 6.871 0.532 

Lack of physical activity 5 6 8 2 8 29 (56.9%) 10.566 0.395 

The irritation due to tight bra 7 8 10 8 10 43 (84.3%) 5.030 0.468 

Lack of blood flow to the breast 0 6 10 8 8 32 (62.7%) 23.777 0.016* 

Table 3: Preventive measure for breast cancer 

Malay Chinese Indian Arab Thais Total 

10(19.6%) 9(17.6%) 10(19.6%) 11(21.6%) 11(21.6%) N(51) 

By reducing fatty diet that elevate cholesterol 7 8 6 4 7 32 (62.7%) 

By consuming soy products regularly 3 9 5 6 6 29 (56.9%) 

By Taking Multivitamin 3 7 8 6 8 32 (62.7%) 

Maintain a healthy body weight (BMI less than 25) 

throughout your life 

7 5 7 5 7 31(60.8%) 

By avoid alcohol 7 7 8 4 11 37 (72.55) 

Consume as many fruits and vegetables as possible 9 7 9 11 8 44 (86.2%) 

Breast self examination can be beneficial to prevent 

breast cancer 

10 9 8 10 11 48 (94.1%) 

Table 4: Knowledge about symptoms of breast cancer 

Symptoms of Breast cancer 

Malay 

10(19.6%) 

Chinese 

9(17.6%) 

Indian 

10(19.6%) 

Arab 

11(21.6%) 

Thais 

11(21.6%) 

Total 

N(51) 

A swelling or thickening in or near the breast 10 7 9 11 11 48 

A swelling in the underarm area 10 6 7 11 10 44 

A change in the size or shape of the breast 10 9 6 11 10 46 

Discharge of fluid from Nipple 8 8 7 6 11 40 

The breast skin looks like the skin of an orange 6 3 9 3 9 30 

Nipple looks swollen, red, or scaly 9 9 9 6 11 44 

Table 5: Knowledge difference on ethnic grounds 

Score 

Race 

0-3 

Poor 

4 

Mode 

rate 

5 

Good 

6 

Excel 

lent 

Malay 1 1 2 6 

Chinese 1 2 5 1 

Indian 1 3 3 3 

Arabs 3 4 1 3 

Thais 1 1 2 8 

df F p 

50 

2.691 

0.043* 

One way ANOVA 

Perception regarding risk factors and preventive measure 

for breast cancer 

Findings demonstrated that lack of blood flow to the breast, 

early adolescents (< 12 years), late menopause (> 55 years) 

and pregnancy after 30 years were the mainly identified risk 

factors for breast cancer. Respondent’s perceptions in this 

regard are illustrated in Table 2. Majority of the respondents 

believe that use of vegetable, fruits and by avoiding alcohol 

use can be the preventive measures from breast cancer Table 

3. 

Knowledge about symptoms of breast cancer, diagnosis and 

treatment of breast cancer 

Overall knowledge evaluation revealed that swelling or 

thickening in or near the breast, swelling in the underarm 

area and change in the size or shape of the breast were the 

frequently recognised symptoms of breast cancer (Table 4). 

Findings demonstrate that the Thai students have 

comparatively better knowledge than other groups (Table 5). 

However, further evaluation through post hoc analysis 

revealed significant knowledge difference among Thai and 

Arab student (Table 6). In term of diagnosis the 

mammography and physical examination were recommended 

by majority (Table 7). Surgery and radiation therapy were the 

preferred options to treat breast cancer (Table 8). 

DISCUSSION 

This is a qualitative study that evaluates young women 

knowledge toward symptoms, risk factor, causes and 

diagnosis of breast cancer among different ethic groups. Five 

ethnic groups were part of this study i.e Malay, Chinese, 

Indians, Arabs and Thais. Findings demonstrated a 

significantly (p=0.043) excellent knowledge regarding 

symptoms of breast cancer among Thai students. However, a 

poor knowledge level was observed among the Arab students 

(Table 5). Swelling or thickening of the breast was the most 

commonly recognized symptoms of breast cancer by all 

groups (Table 4). In terms of evaluation of respondents’ 

perception regarding prevention of breast cancer majority 

(94.1%) of the respondent’s belief that breast self 

examination (BSE) can be prevention from breast cancer. 

This statement reflects negative insight of respondents; BSE 

can be helpful in early diagnosis and treatment only. Majority 

of 11(21.65%) of Arab and Indian students have 

recommended excessive use of fruit and vegetables as 

preventive measure for breast cancer. However, the use of 

soya and soya products was highly recommended by the 

Chinese participants. Use of soya and soya products can be a 

preventive measure, high iso-flavone and phytoestrogen 

contents plays a vital role in minimizing the risk of breast 

caner. 


In addition to the knowledge about the symptoms of breast 

cancer, the perception regarding the risk factor is another 

vital issue to be explored. Overall, majority 86.3% of 

respondents stated that family history is the potential most 

risk factor for breast cancer. However, segregation of 

responses on ethnic grounds revealed that majority of the 

Chinese and Arab respondents’ has stated contraceptive use 

as a risk factor for breast cancer. Whilst Indians perceive 

irritation due to tight bra and lack blood flow to breast are the 

factors leads to breast cancer. On other hand Thai students 

hold smoking responsible for the progression of breast 

cancer. Comparatively Malay Chinese and Indian students’ 

were found to have a lack of awareness towards the 

symptoms and risk factors for breast cancer. Public health 

department is playing its part through educational programs 


on media. However, educational session at the schools and 

college levels can be helpful to reduce these knowledge gaps. 

The knowledge about diagnosis and treatment of breast 

cancer are the other two important aspects of the knowledge 

domain. Evaluation of respondents’ knowledge about 

diagnosis revealed that 25 (49.0%) of the respondents have 

recommended mammography followed by physical 

examination. Arabs and Malays were found to have a better 

knowledge regarding diagnosis of breast cancer. However, 

Thai students have poor knowledge regarding diagnosis as 

majority has recommended ultrasound as the diagnostic 

method. In terms of treatment surgery was the recommended 

by the majority followed by radiation therapy. About 4(7.8%) 

of the respondents have recommended the use of herbs to 

treat breast cancer. Majority among these were Malay and 

Indians. 

Table 6: Multiple comparisons among ethnic groups 

Race (I) Race (J) Mean difference (I-J) Sig CI 95% 

Chinese 

.63333 

.673 

-.7169 - 1.9836 

Malay 

Indian 

Arabs 

.60000 

.93636 

.695 

.250 

-.7142 - 1.9142 

-.3477 - 2.2204 

Thais 

-.33636 

.945 

-1.6204 - .9477 

Malay 

-.63333 

.673 

-1.9836 - .7169 

Chinese 

Indian 

Arabs 

-.03333 

.30303 

1.000 

.966 

-1.3836 - 1.3169 

-1.0178 - 1.6239 

Thais 

-.96970 

.244 

-2.2905 - .3512 

Chinese 

.03333 

1.000 

-1.3169 - 1.3836 

Indians 

Malay 

Arabs 

-.60000 

.33636 

.695 

.945 

-1.9142 - .7142 

-.9477 - 1.6204 

Thais 

-.93636 

.250 

-2.2204 - .3477 

Chinese 

-.30303 

966 

-1.6239 - 1.0178 

Arabs 

Malay 

Indians 

-.93636 

-.33636 

.250 

.945 

-2.2204 - .3477 

-1.6204 - .9477 

Thais 

-1.27273(*) 

.045* 

-2.5258 - -.0197 

Chinese 

96970 

.244 

-.3512 - 2.2905 

Thais 

Malay 

Indians 

.33636 

.93636 

.945 

.250 

-.9477- 1.6204 

-.3477 - 2.2204 

Arabs 

1.27273(*) 

.045* 

.0197 - 2.5258 

Table 7: Knowledge about diagnosis of breast cancer 

Race Mammography Ultrasound 

Physical 

examination 

Biopsy 

Chinese 

4 

1 

4 

0 

Malay 

8 

1 

1 

0 

Indian 

3 

1 

5 

1 

Arab 

8 

0 

2 

1 

Thais 

2 

6 

2 

1 

Total 25 9 14 3 

Table 8 Knowledge about treatment of breast cancer 

Race Herbs 

Hormone 

therapy 

Radiation 

therapy 

Surgery 

Chinese 0 

0 

1 

8 

Malay 2 

1 

2 

4 

Indian 2 

0 

3 

5 

Arab 0 

0 

0 

11 

Thais 0 

1 

3 

7 

Total 4 2 9 35 

CONCLUSION 

Thai students have good knowledge about the symptoms of 

breast cancer. However, knowledge level toward diagnosis 

was best among Malays and Arabs. Overall findings 

highlight the need of further educational session among all 

groups for the substitution of negative perceptions with 

positive and evidence beliefs towards the symptoms, causes, 

prevention diagnosis and treatment of breast cancer. 

REFERENCES 

1. Hisham AN, Yip CH. Overview of Breast Cancer in Malaysian 

Women: A Problem with Late Diagnosis. Asian J Surg 2004; 27(2): 

130-3. 

2. Hisham AN, Yip CH. Spectrum of Breast Cancer in Malaysian 

Women: Overview. World J Surg 2003; 27(8): 921-3. 

3. Ferlay F, Bray F, Pisani P, et al. Globocan 2000 (online software). 

Cancer incidence, mortality and prevalence worldwide, Version 1.0. 

IARC Cancer Base No.5. IARC Press, Lyon, France, 2001 

4. Fry RB, Prentice-Dunn S: Effects of a psychosocial intervention on 

breast self-examination attitudes and behaviors. Health Educ Res 

2006; 21: 287-295. 

5. Rosenberg R, Levy- Schwartz R: Breast cancer in women younger 

than 40 years. Int J Fertil Womens Med 2003; 48: 200-205. 

6. Nor Aina E. Cancer Patient Registry-Breast Cancer (NCPR-Breast 

Cancer) Med J Malaysia Vol 63(c), 2008. 

7. Sheridan JC, Lyndall GS. SPSS analysis without anguish version 

10.0 for Windows, Singapore: John Wiley & Sons Australia, Ltd., 

2001. 





ISSN 0975 1556 

Evaluation of Effects of Black Tea and Coffee on Learning Process and 

Memory in Healthy Human Volunteers 

Suraj Baheti, N Saraswathy, Kala Suhas Kulkarni * 

School of Pharmacy and Technology Management, SVKM’S NMIMS University, Mithibai College building, V L Mehta 

road, Vile Parle (west) Mumbai 400056, India 

ABSTRACT 

The study was conducted in healthy human volunteers adhering to good clinical practices and institutional human ethical 

norms. All the volunteers have completed the study. There were no dropouts and no signs and symptoms of any adverse 

effect after consumption of the beverages. This indicates the positive compliance in the study. 

The overall effect observed in the study that the consumption of beverages Black Tea and Coffee increases alertness in the 

volunteers and it helps to facilitate the performance and thus enhance the memory. This is confirmed by the level of 

confidence development in the volunteers to perform the psychopharmacological parameters. The parameters selected in 

the study are the functional characteristics of thought process, learning and memory. The reduction in reaction time after 

the consumption of Black tea and Coffee signifies the effect on learning process. The improvisation of this facilitates the 

memory in the form of retention and its implementation in developing skills. Clinically these results are very promising 

and can be extrapolated for the treatment of neurodegenerative disorders like Alzheimer’s disease, dementia where at least 

partial effect on the process of learning and recent memory. 

Black Tea and Coffee are consumed many times by the individuals in the situations like appearing for examinations where 

the level of performance is of significance to have optimum outcome. These may be recommended for short time use. 

However, the additional effects may be confirmed in larger studies in controlled manner. 

Keywords: Black Tea, Coffee, Memory, Learning, Physiological Performance. 

INTRODUCTION 

Learning is the cognitive process of acquiring skill or 

knowledge or it refers to the acquisition, and transfer to longterm 

memory of experience, Information, and Knowledge 

may subsequently be used for solving problems, making 

decisions, and creating new knowledge for further 

application in daily routines. Learning in general, is the 

change of thought, behavior and perception of the person due 

to his interaction with environment. The theories that analyze 

the nature of learning and its effects are categorized into four 

groups: 

� Behaviorist Orientation 

� Cognitive Orientation 

� Humanistic Orientation 

� Social and circumstantial Orientation 

Behaviorist theory considers that learning is formed by 

change in behavior of a human being with the time as a result 

of stimuli in external environment. It produces behavioral 

change in desired direction to elicit desired response. 

*Corresponding author: Dr. Kala Suhas Kulkarni, 

School of Pharmacy and Technology Management, SVKM’S 

NMIMS University, Mithibai College building, V L Mehta 

road, Vile Parle (west) Mumbai 400056, India 

E-mail: kalakulkarni@gmail.com 

The cognitive orientation indicates learning is a nonobservable 

process. 

It is an internal mental process including insight, information 

processing, memory, perception due to internal cognitive 

structuring. It develops capacity and skills to learn better. 

During Cognitive development, intelligence, learning and 

memory are functions of age, and learning is manifestations 

in adult learning. 

Memory refers to the processes that are used to acquire, 

store, retain and later retrieve information. There are three 

major processes involved in memory: encoding, storage and 

retrieval. Short-term memory is closely related to “working” 

memory. It is very short time that one keeps something in 

mind before rejecting or transferring it to long-term memory. 

Short-term memory is shorter than we think, lasting less than 

a minute. It allows us to remember the first half of a sentence 

we hear or read long enough to make sense of the end of the 

sentence but in order to store that sentence (or thought, fact, 

idea, word, impression, sight etc) for longer than a minute or 

so, it has to be transferred to long-term memory. As we grow 

older our short-term memory span often becomes even 

shorter. This makes us more likely to have trouble keeping 

up with certain tasks. It also gives our brains less time to 

119

Kulkarni et al. / Evaluation of Effects of Black Tea and Coffee on Learning Process.... 

successfully move new information to long-term memory, The volunteers were evaluated for: 

which makes us more likely to forget details of recent events. i. Total time taken to complete the exercise, 

A long-term memory is anything we remember that happened ii. Total correct answers and 

more than a few minutes ago. Long-term memories are not 

all of equal strength. Stronger memories enable us to recall 

iii. Total incorrect answers 

an event, procedure and fact on demand. Long-term memory 

Chart 1: Mathematical Calculation Test 

is not static. We do not imprint a memory and leave it as if 

untouched. Instead, we often revise the memory over time 

perhaps by merging it with another memory or incorporating 

what others tell us about the memory. The process of 

35 

25 

50 

40 

60 

40 

50 

30 

20 

50 

40 

50 

30 

50 

40 

60 

90 

25 

60 

75 

40 

30 

25 

55 

15 

55 

60 

40 

30 

50 

35 

60 

45 

30 

20 

30 

20 

64 

35 

95 

35 

95 

15 

34 

56 

12 

45 

55 

35 

53 

learning and memory develop simultaneously in individuals. 

55 40 30 30 50 40 55 35 43 17 

The learning process begins in infantile stage and is geared 

25 55 20 55 80 45 25 65 17 37 

up during the process of maturity and adolescence age. 

Subsequently the process of learning itself is utilized for 

storage of memory which is recalled as past memory. 

However, the process of recent memory is influenced by 

Total 

20 

30 

25 

60 

15 

75 

80 

50 

80 

35 

60 

20 

40 

45 

25 

45 

35 

20 

15 

65 

30 

30 

20 

25 

50 

38 

12 

13 

27 

20 

many factors which may include repetitive learning 


The present study was carried out with the objective of 

comparison of effect of commonly consumed beverages on 

functional parameters of learning and memory. These 

characteristics are evaluated using psychopharmacological 

tests commonly indicating the process of learning and 

improvising memory. Study was conducted to indicate any 

effects of consumption of normally consumable beverages 

Number Cancellation Test 

In this test, the volunteers were given a table consisting of 

10X10 (100 small squares) and were asked to cancel a 

particular number in a random fashion (Chart – 2). 

The volunteers were evaluated for: 

i. Total time taken to complete the exercise, 

ii. Total number of cancelled number and 

iii. Incorrect number(s) cancelled. 

(Black tea and Coffee) on functional parameters especially 

Chart 2: Number Cancellation 

the learning and memory process in healthy human 6 1 5 7 9 4 3 7 1 2 

volunteers. 

The beverages used were purchased from ready market 

1) Black Tea 

2) Black Coffee 

5 

7 

9 

2 

3 

2 

6 

5 

9 

4 

4 

9 

3 

2 

4 

8 

1 

8 

1 

7 

5 

2 

9 

3 

4 

1 

6 

4 

9 

8 

3 

9 

2 

1 

3 

5 

4 

9 

7 

8 

9 

6 

3 

2 

1 

7 

1 

5 

4 

9 

Preparation of Black Tea 

5 4 3 5 6 2 8 1 3 7 

1. 

2. 

Decoction of raw material was prepared in freshly 

boiled water. 

Decoction was filtered through the sieve. 

8 

6 

4 

5 

1 

5 

9 

7 

5 

4 

3 

2 

1 

4 

5 

3 

1 

9 

6 

7 

4 

7 

5 

6 

6 

4 

2 

3 

9 

4 

3. Sugar was added to the decoction and served to the 

volunteers. 

Preparation of Black Coffee 

1. Coffee powder was added to the freshly boiled 

water. 

2. Decoction was filtered through the sieve. 

3. Sugar was added to the decoction and served to the 

volunteers. 

STUDY PROTOCOL 

This was a human study and hence approval was obtained 

Alphabet Cancellation Test 

In this test, the volunteers were given a table containing 

alphabets in 100 small squares, each alphabet repeated 7 to 8 

times randomly and were asked to cancel a particular 

alphabet (Chart – 3). 

The volunteers were evaluated for: 

i. Total time taken to complete the exercise, 

ii. Total alphabets cancelled and 

iii. Incorrect alphabets(s) cancelled. 

from institutional ethics committee for the conduct of the 

study. The study was conducted adhering to norms of good 

clinical practices and good laboratory practices. No blood 

A 

Q 

D 

G 

Chart 3: Alphabet cancellation Test 

C B F G H M 

S X S B Z X 

B 

A 

V 

D 

samples or any other body fluids were withdrawn during the E T F C F G H B X Z 

study. 

The study was planned to be carried on healthy young 

volunteers. They were divided in two groups each consisting 

of six volunteers. The volunteers of 22-28 years age groups 

S 

K 

J 

K 

F 

L 

J 

E 

J 

S 

A 

H 

H 

H 

B 

G 

D 

J 

D 

L 

J 

A 

D 

A 

K 

K 

G 

D 

G 

A 

A 

S 

A 

S 

G 

S 

X 

D 

X 

D 

N 

V 

C 

V 

X 

Z 

K 

S 

K 

A 

were selected for the study. On detailed discussions with H E S A G J C R Y U 

them regarding the nature and objective of the study, written P O Y E A P O A W R 

informed consent was obtained and enrolled in the study after 

physical evaluation parameters. Subsequently they were Upward Digit Scale Test 

evaluated for the evaluative tests. 

In this test, the volunteers were verbally informed eight 

EVALUATION PARAMETERS 

numbers (10-99) in upward order and were asked to 

The volunteers were evaluated using following parameters. memorize the numbers in the same manner and reproduce in 

Mathematical Calculation test 

the same order (Chart – 4). 

In this test, the volunteers were asked to solve a The volunteers were evaluated for: 

mathematical summation problem (Chart - 1). 

i. Synchrony of numbers 

ii. Missing number(s) 


iii. Incorrect Number(s) 


Chart 4: Upward digit scale 

12 

21 

29 

47 

53 

62 

77 

83 

Fixed Digit Test 

In this test the volunteers were given a fixed double digit and 

were asked to apply mathematical calculation as directed in 

the test chart. Each time the evaluator would give exercise 

the sum as add or cancel. The answer within 10 seconds 

would be given the score of 2, later than 10 second 1 and 

incorrect and missing answer as 0. The total score is 

measured at evaluation each time (Chart- 5). 

The volunteers were evaluated for the following: 

i. Total Score 

ii. Correct answers 

iii. Incorrect answers. 

Chart 5: Fixed Digit Test 

Sr. No. Number Result Score 

1 25 – 15 

2 25 + 45 

3 25 ÷ 5 

4 25 + 40 

5 25 + 7 

6 25 – 11 

7 25 + 50 

8 25 – 13 

Total Score 

Total score was calculated as follows 

Answer Score 

Before 10 sec. 2 

After 10 sec 1 

Incorrect answer 0 

Four Words Test 

In this test the volunteers were given four words with no 

interlink among them and were asked to memorize them in a 

particular sequence they want and to recall the word when 

asked. The words were to be memorized throughout the study 

time. (Chart - 6) 

The volunteers were evaluated for the following 

i. Synchrony of words 

ii. Missing Word(s) 

iii. Incorrect Word(s) 

Chart 6: Four Words Test 

Aircraft Desert 

Needle Lunch 

The study was performed in three steps 

A. Basal Reading: Before administering the 

beverages. 

B. 30 Minutes: Volunteers were evaluated for above 

mentioned tests after 30 minutes of ingestion of 

beverage 

C. 90 Minutes: Volunteers were evaluated for above 

mentioned tests after 90 minutes of ingestion of 

beverage. 

Statistical Evaluation 

The comparative analysis was made using Prism software. 

The values are compared with basal and 30 and 90 minutes. 

The level of significance was defined as p< 0.05 and 0.02. 


Mathematical Calculation Test 

All the volunteers have completed the study. There was no 

dropout from the study volunteers. The basal time taken for 

solving mathematical calculation test was 393.2 and 339.4 

seconds respectively in the groups of Black tea and Black 

Coffee. This time reduced in the same test after 30 minutes in 

both groups to 267.8 & 271 seconds. The reduction in time 

taken to solve the mathematical calculation test indicates the 

influence of beverage on volunteers. This is reflected in the 

form of increasing alertness and to perform better. This effect 

is maintained at 90 minutes evaluation. The number of sum 

correct in this test has been 9.167 and 8.4 at 30 minutes and 

8.33 & 8.8 at 90 minutes in both groups as compared to basal 

which was 8.00 & 8.00. Similar is the result of incorrect 

answers which decreased from 2 to 0.8333 & 1.6 at 30 

minutes (Table 1). 

Table 1: Mathematical Calculation Test 

Beverage 

Total Time Taken 

Incorrect 

Correct Answer 

(Sec.) 

Answer 

30 90 30 90 

Basal 

Basal 

min. min. min. min. Basal 

30 90 

min. min 

Black Coffee 393.2 *267.8 *261.7 8 *9.167 8.333 2 0.8333 0.00 

Black Tea 339.4 *271 365.4 8 8.4 8.8 2 1.6 0.00 

*The level of significance was defined as p< 0.05 

Number Cancellation Test 

The time taken to cancel the given number is decreased after 

Black tea consumption and there is no significant change in 

case of Black Coffee consumption. (Table 2) 

Table 2: Number Cancellation Test 

Total Time Taken Total Number Incorrect Number 

Beverage 

(Sec.) 

Cancelled Cancelled 

Basal 30 min. 90 min. Basal 30 90 

90 

Basal 30 min. 

min. min. min. 

Black 

Coffee 

19.5 *16.83 *16.83 100 97.92 100 0.00 0.00 0.00 

Black 

Tea 

22.8 19.4 20.2 98.33 100 100 0.00 0.00 0.00 


Alphabet Cancellation Test 

The time taken to cancel the given alphabet in random 

manner is decreased after Black tea consumption and there is 

no significant change after Black Coffee consumption. The 

results were uniform at 30 minutes and 90 minutes. (Table 3) 

Table 3: Alphabet Cancellation Test 

Total Time Taken Total Alphabet 

Beverage 

Basal 

(Sec.) 

Cancelled 

30 

30 90 

90 min. Basal 

min. min. min. 

Incorrect Alphabet 

Cancelled 

90 

Basal 30 min. 

min. 

Black 

Coffee 

16.33 16 *15.83 97.92 94.44 100 0.00 0.1667 0.00 

Black Tea 19.4 19.2 16.8 100 100 97.78 0.00 0.00 0.00 


Upward Digit Scale Test 

The analysis of upward digit scale test did not indicate any 

significant difference in memorizing synchronically the 

upward digits, missing numbers and incorrect numbers. 

(Table 4) 



Table 4: Upward Digit Scale Test 

Synchrony Missing Number Incorrect Number 

Beverage 30 

Basal 

min. 

90 

min. Basal 

30 

min. 

90 

90 

Basal 30 min. 

min. min. 

Black 

Coffee 

1.833 2 2.833 0.8333 1.333 1.5 1 0.6667 0.00 

Black Tea 2.4 1.8 1.6 1 0.8 0.4 1.4 1 0.00 

Fixed Digit Test 

The total score obtained in evaluation of fixed digit test 

showed trend towards improvisation of memory. The effects 

were uniform after 30 minutes and 90 minutes evaluation. 

The correct answers were maintained in most volunteers. 

(Table 5) 

Table 5: Fixed Digit Test 

Beverage Basal 

Total Score Correct Answer Incorrect Answer 

30 

min. 

90 min. Basal 

30 

min. 

90 

min. Basal 

30 

min. 

90 

min. 

Black 

coffee 

96.88 100 97.92 7.833 8 7.833 0.2 0.00 0.00 

Black Tea 88.75 *92.5 *93.75 7.2 7.4 7.6 0.8 0.6 0.00 


Four Words Test 

There was ease of memorizing four words each time at 30 

minutes and 90 minutes after the consumption of beverages. 

It was observed that memorizing word, were easier than 

mathematical numbers. (Table 6) 

Table 6: Four Words Test 

Synchrony of Words Missing Words Incorrect Words 

Beverage 

Basal 

30 

min. 

90 

Basal 

min. 

30 

min. 

90 

Basal 

min. 

30 

min. 

90 

min. 

Black 

Coffee 

100 100 100 0.00 0.00 0.00 0.00 0.00 0.00 

Black Tea 100 100 100 0.00 0.00 0.00 0.00 0.00 0.00 

The trend towards making mechanical errors in incorrect 

answers was reduced following consumption of these 

beverages. Although, there was no distinctive difference with 

the individual type of beverages, of them produce alertness, 

concentration and indirectly improved the physiological and 

psychopharmacological performance. 

The level of significance is determined by using students’ 

paired’t-test. The statistical analysis shows very marginal 

significant difference before and after the consumption, 

however, clinically the difference was highly significant. 

(Table 7, 8, 9, 10, 11 & 12) 

Table 7: t-values of Mathematical Calculation Test 

Total Time 

Taken (Sec.) 

Correct Answer 

Incorrect 

Answer 

Beverage 

Basal 

vs. 

30 

min 

Basal 

vs. 

90 

min 

Basal 

vs. 

30 min 

Basal 

vs. 

90 

min 

Basal 

vs. 

30 

min 

Basal 

vs. 

90 

min 

Black Coffee 1.788 2.144 2.445 0.4385 2.445 0.4385 

Black Tea 2.126 0.1928 0.5898 0.7493 0.5898 0.7493 

Table 8: t-values of Number Cancellation Test 

Beverage 

Total Time 

Taken (Sec.) 

Basal 

vs. 

30 min 

Basal 

vs. 

90 min 

Total Number 

Cancelled 

Basal 

vs. 

30 min 

Basal 

vs. 

90 min 

Incorrect 

Number 

Cancelled 

Basal 

vs. 

30 min 

Basal 

vs. 

90 

min 

Black 

Coffee 

1.164 0.9818 1.685 1.122 0.00 0.00 

Black Tea 0.9481 1.812 1.000 1.000 0.00 0.00 

The study protocol and volunteer screening format was 

examined and approved by institutional ethics committee. 

The formal approval was obtained on both protocol and 

volunteer screening form. The study was conducted in 

healthy human volunteers adhering to good clinical practices 

and institutional human ethical norms. All the volunteers 

have completed the study. There were no dropouts and no 

signs and symptoms of any adverse effect after consumption 

of the beverages. This indicates the positive compliance in 

the study. 

Table 9: t-values of Alphabet Cancellation Test 

Total Time Total Alphabet Incorrect Alphabet 

Bevera 

ge 

Taken (Sec.) 

Basal Basal 

vs. vs. 

Cancelled 

Basal Basal 

vs. vs. 

Cancelled 

Basal Basal 

vs. vs. 

30 min 90 min 30 min 90 min 30 min 90 min 

Black 

Coffee 

0.1671 0.6956 0.5502 1.000 0.00 0.00 

Black 

Tea 

0.0982 

9 

1.857 0.00 1.000 0.00 0.00 

Table 10: t- values of Upward Digit Scale Test 

Synchrony Missing Number Incorrect Number 

Bevera Basal Basal Basal Basal Basal Basal 

ge vs. vs. vs. vs. vs. vs. 

30 min 90 min 30 min 90 min 30 min 90 min 

Black 

Coffee 

0.2774 1.225 0.8885 1.581 1.000 0.7906 

Black 

Tea 

0.4966 1.000 0.1728 0.7385 0.5898 0.2500 

Table 11: t- values of Fixed Digit Test 

Bevera 

ge 

Black 

Coffee 

Black 

Tea 

Basal 

vs. 

30 min 

Total Score Correct Answer 

Basal 

vs. 

90 min 

Basal 

vs. 

30 min 

Basal 

vs. 

90 min 

Incorrect 

Answer 

Basal Basal 

vs. vs. 

30 min 90 min 

1.464 1.000 0.00 0.00 1.000 0.00 

0.8018 0.5898 0.00 0.00 0.5345 1.1770 

Table 12: t-values of Four Words Test 

Beverage Basal 

vs. 

30 min 

Black 

Coffee 

Total Score Correct Answer 

Basal 

vs. 

90 min 

Basal 

vs. 

30 min 

Basal 

vs. 

90 min 

Incorrect 

Answer 

Basal Basal 

vs. vs. 

30 min 90 min 

0.00 0.00 0.00 0.00 0.00 0.00 

Black Tea 0.00 0.00 0.00 0.00 0.00 0.00 

It was observed in this study that compliance with both Black 

Tea and Black Coffee tested was excellent. There was no 

drop out from the study indicates the positive compliance. 

All volunteers were co-operative and there were no 

incidences of adverse effects reported during the trial. 

The time taken to complete the mathematical summation and 

other tests like cancellation of random number and random 

alphabet from the chart is reduced, indicating that Black tea 

and Coffee helps to improve alertness and concentration 

while performing the task. During the phase of alertness the 

physiological performance increases which reflects in 

facilitating solving mathematical summation, ability to 

perform faster and better in arithmetic and number 

cancellation and random alphabet cancellation test. The 

influence on performance ability suggests improvement or 

facilitation of learning process with Black tea and Coffee. 

The analysis of results show that the level of statistical 

significance is only marginal, however, this can be improved 



by increasing the number of volunteers and using strict 

adherence to selection criteria of volunteers and evaluation 

conditions.. The large and broad variation in performance 

criteria between volunteers could be one of the factors for 

marginal statistical significance. Black tea ingestion 

produced an increase in alertness and self-reported 

improvements in mood. When taken in regular amounts 

throughout the day, black tea appeared to prevent the diurnal 

pattern of performance reduction. [1] 

An amino acid found in tea called thiamine which could act 

as a neurotransmitter. A study in rats found that thiamine 

modulated serotonin and dopamine levels and appeared to 

improve memory and learning ability. [2] 

Chronic administration of tea polyphenols has shown 

reversal of scopolamine induced retention deficits in passive 

avoidance and spontaneous alertness behavior tasks. [3] 

In a study of two different age groups: 20-25 years and 50-65 

years, it has been observed that the younger participants 

generally performed better than the older on psychomotor 

and cognitive tests. After caffeine, both groups showed an 

improvement in psychomotor and cognitive performance, 

particularly in offsetting the declining performance over time 

in the older participants. [4] 

In another set of experimental studies, the improvement of 

long term memory has been seen in rats. The rats were able 

to find and perform faster after treatment with caffeine 

particularly increase in memory retention. [5] 

In human study it was shown that caffeine possesses 

cognition enhancing property. The result seen in our study on 

volunteers with black coffee consumption shows 

improvement on functional cognitive performance as 

reported by other researchers. [6] 

The study examined the association of caffeinated and 

decaffeinated coffee intake with cognitive function in a 

community-based sample of older adults in 1988–1992. 

Participants were 890 women with a mean age of 72.6 years 

and 638 men with a mean age of 73.3 years from the Rancho 

Bernardo Study. Cognitive function was assessed by 12 

standardized tests, and lifetime consumption and current 

coffee consumption were obtained by questionnaire. After 

adjustment for confounders, higher lifetime coffee 

consumption 

in women was associated with better 

performance on six of 12 tests, with a trend on two other 

cognitive function tests. [7] 

The overall effect observed in the study that the consumption 

of Black Tea and Coffee increases alertness in the volunteers 

and it helps to facilitate the performance and thus enhance 

the memory. This is confirmed by the level of confidence 

development in the volunteers to perform the 

psychopharmacological parameters in subsequent time 

intervals. The parameters selected in the study are the 

functional characteristics of thought process, concentration, 

ability to express and learning and memory. The reduction in 

reaction time in all the tests performed which includes 

mathematical summations, cancellation of numbers and 

alphabets after the consumption of Black tea and Coffee 

signifies the effect on learning process and resultant effects 

on memory. The improvisation of this facilitates the memory 

in the form of retention and its implementation in developing 

skills. Clinically these results are very promising and can be 

extrapolated for the treatment of neurodegenerative disorders 

like Alzheimer’s disease, dementia where at least partial 

effect on the process of learning and recent memory. 

Black Tea and Coffee are consumed many times by the 

individuals in the situations like appearing for examinations 

where the level of performance is of significance to have 

optimum outcome. These may be recommended for short 

time use. However, the additional effects may be confirmed 

in larger studies in controlled manner. 

CONCLUSION 

The overall effect observed in the study that the consumption 

of Black Tea and Coffee increases alertness in the volunteers 

and it helps to facilitate the performance and thus enhance 

the memory. This is confirmed by the level of confidence 

development in the volunteers to perform the 

psychopharmacological parameters in subsequent time 

intervals. The parameters selected in the study are the 

functional characteristics of thought process, concentration, 

ability to express and learning and memory. The reduction in 

reaction time in all the tests performed which includes 

mathematical summations, cancellation of numbers and 

alphabets after the consumption of Black tea and Coffee 

signifies the effect on learning process and resultant effects 

on memory. The improvisation of this facilitates the memory 

in the form of retention and its implementation in developing 

skills. Clinically these results are very promising and can be 

extrapolated for the treatment of neurodegenerative disorders 

like Alzheimer’s disease, dementia where at least partial 

effect on the process of learning and recent memory. 

Black Tea and Coffee are consumed many times by the 

individuals in the situations like appearing for examinations 

where the level of performance is of significance to have 

optimum outcome. These may be recommended for short 

time use. However, the additional effects may be confirmed 

in larger studies in controlled manner. 

REFERENCES 

1. Hindmarch I, Quinlan PT, Moore KL and Parkin C. The effects 

of black tea and other beverages on aspects of cognition and 

psychomotor performance. Psychopharmacology (Berl). 1998; 

139(3): 230-8. 

2. Gardner E.J., Ruxton C.H.S., and Leeds A.R. Black tea–helpful 

or harmful: A review of the evidence. European Journal of 

Clinical Nutrition.2007. 61: 3–18. 

3. Dona M., Dell'Aica, I., Calabrese, F., Benelli, R., Morini, M., 

Albini, A.,Garbisa, S., Neutrophil restraint by green tea: 

Inhibition of inflammation, associated angiogenesis, and 

pulmonary fibrosis, Journal of Immunology, 170 (8), 2003, 

4335–4341. 

4. Rees K, Allen D and Lader M. The influences of age and caffeine 

on psychomotor and cognitive function. Psychopharmacology. 

1999; 145 (2): 181-188. 

5. Angelucei MEM et al. Effects of caffeine on learning and 

memory in rats tested on the Morris water maze. Brazillian 

Journal of Medical and biological research. 2002; 35: 1201-1208. 

6. Riedel W, Hogervorst E, Leboux R, Verhey F, van Praag H, 

Jolles J. Caffeine attenuates scopolamine-induced memory 

impairment in humans. Psychopharmacology (Berl). Department 

of Psychiatry and Neurophysiology, University of Limburg, 

Maastricht, Netherlands. 1995 Nov; 122(2):158-68. 

7. Marilyn Johnson-Kozlow, Donna Kritz-Silverstein, Elizabeth 

Barrett-Connor and Deborah Morton Coffee Consumption and 

Cognitive Function among Older Adults Am J Epidemiol 2002; 

156:842-850. 





ISSN 0975 1556 

Pharmacopoeial Standardization of Hibiscus rosa sinensis Linn. 

Gupta V 1* , Bansal P 1 , Garg A 2 , Meena AK 1 

1 National Institute of Ayurvedic Pharmaceutical Research, Patiala (Punjab), India 

2 Department of Pharmacognosy, S. B. S. College of Pharmacy, Patti, Punjab, India 

ABSTRACT 

The present communication attempts to investigate pharmacognostical and phytochemical details of Hibiscus rosasinensis, 

Linn. (Malvaceae). Results of microscopic studies of leaf show chained midrib of leaf, small and numerous 

epidermal cells, calcium oxalate crystals and absence of trichomes on both upper and lower surface. The Preliminary 

phytochemical analysis revealed presence of carbohydrates, alkaloids, flavonoids, saponins, proteins and amino acids in 

chloroform and alcoholic extract out of six extraction solvents used for these studies. HPTLC studies reveal that alcoholic 

extract gives 8 spots and chloroform extract depicts 5 spots on the TLC plate. Powdered drug analysis after treatment with 

17 different reagents emitted various colour radiations under UV and visible light which may provide a lead in 

identification of the drug in powder form. The study revealed specific identities for Hibiscus rosa- sinensis, Linn which 

may play a key role in identification of plant and can be useful in standardization of the herbal drugs. 

Keywords: Hibiscus rosa-sinensis, pharmacognosy, phytochemical, HPTLC fingerprint, Malvaceae. 

INTRODUCTION 

Medicinal plants have been used in virtually all cultures as a 

source of medicine, since times immemorial. Herbal 

Medicine is still the mainstay of health care in several 

developing countries. The widely used herbal remedies and 

health care preparations as described in ancient texts such as 

the Vedas and the Bible are obtained from commonly used 

traditional herbs and medicinal plants. The medicinal 

properties of these botanicals are being better understood and 

are attributable to the phytochemicals that specific plants 

contain. The efficacy and safety of herbal products therefore 

rely on the quality and proper identification of the raw 

material or the original plant source. One major obstacle that 

might impair the potential use of traditional medicine as 

medicine of choice is the lack of standardization. 

Adulterations and substitutions are common in raw material 

trade of medicinal plants. Unintentional adulterations also 

exist in herbal raw material trade due to various reasons such 

as confusion in vernacular names between indigenous 

systems of medicine and local dialects, lack of knowledge 

about the authentic plant, non-availability of the authentic 

plant, similarity in morphology and /or aroma or careless 

collection. [1] To avoid this accurate authentication is very 

important to prevent the adulteration of target plant with 

other plant species. The techniques used in past for the 

standardization of botanical preparations like HPLC, TLC, 

*Corresponding author: Mr. V. Gupta, 

National Institute of Ayurvedic Pharmaceutical Research, 

Patiala (Punjab), India 

E-mail: vikas_4308@rediffmail.com 

HPTLC, UV spectroscopy, mass spectroscopy, gas 

chromatography, infrared and NMR spectroscopy have 

limitations because the compositions and relative amount of 

chemicals in a species varies with growing conditions, 

harvesting periods, post-harvest processes and storage 

conditions. This can be misleading if the samples are 

deliberately adulterated with a marker compound. Also, it is 

difficult to distinguish closely related species due to similar 

chemical compounds. 

[2] Identification of plants with 

botanical verifications is essential as contamination due to 

misidentification of plant species or parts is common. 

Therefore, it becomes necessary to develop more effective, 

accurate, reliable and sensitive methods for the authentication 

of herbs. In the present study an effort has been made to 

establish physicochemical, pharmacognostic, and 

phytochemical parameters which could be helpful in 

identification of the authentic plant samples and 

differentiating it from adulterants. 

Hibiscus rosa-sinensis, Linn. (Malvaceae) known as China 

rose is an important medicinal plant. [3] It is an evergreen 

woody, glabrous, showy shrub 5-8 feet in height. Leaves are 

bright green, short petiolated, ovate or lanceolate, more or 

less acuminate; irregularly and coarsely serrated towards the 

top, entire near base, glabrous on both sides, a few minute 

stellate hairs on the nerves beneath stipules, lanceolatesubulate 

and glabrous. Pedicels are axillary, solitary, and 

longer than the leaves and joined above the middle. Flowers 

are solitary, axillary, bell shaped, large, 4-6 inches in 

diameter with pistil and stamens projecting from centre. [4] 

Leaves are used as emollient, anodyne, and laxative in 

Ayurveda. [5-6] In South Asian traditional medicine, various 

124

parts of the plant is used in the preparation of a variety of 

foods. [7] The flowers have been reported in the ancient 

Indian medicinal literature to have beneficial effects in heart 

diseases, mainly in ischemic disease [8] and used in folklore 

medicine as demulscent, emollient, refrigerant, aphrodisiac, 

brain tonic and cardio tonic. A decoction of flowers is also 

useful in bronchial catarrh, menorrhagia, and fertility control. 

[9-10] [11] 

The extracts showed hair growth potential , 

anticonvulsive activity [12] and hypoglycemic activity. [13] 

MATERIAL AND METHOD 

Fresh leaves collected from Botanical garden of S.B.S. 

college of Pharmacy, Patti, Punjab were preserved in 70 % 

ethyl alcohol for histological studies. Voucher herbarium 

sample along with the voucher crude drug sample (voucher 

number- 0383/S.R.) is preserved at the herbarium of 

Department of Botanical sciences, GNDU, Amritsar. 

Botanical identification was carried out using local floras. [14- 

15] 

Free hand sections were used for histological studies. 

Quantitative microscopy was done as per the standard 

methods described by TE Wallis. [16] 

Physicochemical studies like total ash, acid insoluble ash, 

water soluble ash, pH of 1 % w/v solution of aqueous extract, 

swelling index and successive extractive values were carried 

out by soxhlet extraction method as per the WHO guidelines. 

[17] 

The fluorescence behavior of the powder drug in the 

visible light and ultraviolet light were carried out by soaking 

the powder in different reagent solutions and viewing under 

the light of required wavelength in a UV chamber. [18-19] 

Preliminary Phytochemical analysis 

Gupta et al. / Pharmacopoeial Standardization of Hibiscus.... 

[18, 20] and high 

performance thin layer chromatography [21-22] were carried 

out from shade dried powder as per standard methods. 

The successive extracts were tested for different constituents. 

The alcoholic and chloroform extracts revealed presence of 

alkaloids, glycosides, flavonoids, proteins and amino acids 

(Table 2). 

Table 1: Physicochemical parameters of leaves of H. rosa-sinensis 

S. No. Parameters Average Values 

1 Total ash (%) 7.75 

2 Acid-insoluble ash (%) 0.75 

3 Water soluble ash (%) 6.32 

4 pH (1% w/v aqueous extract) 7.92 

5 Swelling index 6.33 

6 Loss on drying (%) 4.93 

Extractive value (%) 

1 Petroleum ether 1.45 

2 Benzene 2.60 

3 Chloroform 2.80 

4 Ethyl acetate 3.20 

5 Methanol 15.60 

6 Distilled water 5.30 

Table 2: Preliminary phytochemical Screening of leaf powder of H. 

rosa-sinensis 

RESULT AND DISCUSSION 

Microscopic Characters 

Transverse section of midrib of leaf showed chained, small Table 3: Ultra-violet analysis of leaf powder of H. rosa-sinensis 

and numerous epidermal cells. The mesophyll layer is Treatment Visible Light UV(254 nm) UV (366 nm) 

irregular and comprised of 6-7 layers. Cells of parenchyma 

varied greatly in shape and size and were sometimes, 

elongated or lobed. The xylem vessels were numerous, very 

big in size and circular in shape. Phloem vessels were small 

Drug Powder 

Sulfuric acid 

(conc.) 

Sulfuric acid 

Light green 

Dark green 

Dark green 

Black 

Blackish 

Brown 

Black 

in size, numerous and circular in shape. Calcium oxalate (dilute) 

Light brown Greenish black Black 

crystals were dark stained and numerous in mesophyll 

parenchyma. Trichomes were absent on both upper and lower 

surface. Transverse section of lamina showed cuticle and 

thick walled cells in upper and lower epidermis. Epidermal 

cells were large in size, elongated and compact. Palisade 

parenchyma showed 3 or 4 layers of large, compact and dark 

cells. Dark stained crystals were present in mesophyll layer. 

Hydrochloric 

acid (conc.) 

Hydrochloric 

acid (dilute) 

Acetic acid 

Nitric acid 

(dilute) 

Methanol 

Green 

Brown 

Brown 

Brown 

Green 

Black 

Greenish black 



Dark green 

Black 

Black 

Black 

Black 

Black 

The spongy mesophyll was wider comprising of 6-8 layers of 

lobed tightly interconnected cells. Trichomes were absent on 

both upper and lower surfaces. Vascular bundles had 

Ethanol 

Chloroform 

Petroleum ether 

Brown 

Brown 

Green 

Brownish Black 

Dark green 


Black 

Black 

Black 

compact parallel rows of xylem vessels and fibres. (Fig. 1, 2) Distilled. Water Brown Dark green Black 

Physicochemical parameters 

The percent of loss on drying, total ash, acid insoluble ash, 

water soluble ash, pH of 1 % w/v solution of aqueous extract 

and swelling index has been shown in Table 1. A known 

quantity of dried leaf powder was extracted in a soxhlet 

10% NaOH 

5% Iodine 

Picric Acid 

FeCl3 solution 

Ammonia 

solution 

Brown 

Greenish brown 

Green 

Brown 

Brown 

Green 

Dark green 

Greenish red 

Dark green 

Green 

Black 

Black 

Black 

Black 


apparatus with petroleum ether (60-80ºC), benzene, 

chloroform, ethyl acetate and methanol (95 %) and finally 

macerated with distilled water for 24 hours successively and 

the % of respective extractive values have been shown in 

Table 1. 

Preliminary Phytochemical Screening 

HPTLC study 

The alcohol and chloroform extracts were used to carry out 

HPTLC. For alcohol extract solvent system with toluene: 

ethyl acetate: acetic acid (9.5: 8: 5.2) and for chloroform 

extract toluene: ethyl acetate: acetone (8: 1: 3) was used. 

Alcoholic extract demonstrated 8 spots of Rf value of 0.03, 

IJPCR October-December, 2009, Vol 1, Issue 3 (124-126) 125 

S. 

No 

Test 

Petroleum 

ether 

Benzene 

1. Alkaloids + ve + ve + ve + ve + ve - ve 

2. Quinone - ve - ve - ve - ve - ve - ve 

3. Coumarin - ve - ve - ve - ve - ve - ve 

4. Flavonoids - ve - ve + ve + ve + ve + ve 

5. Steroid + ve - ve - ve - ve - ve - ve 

6. Phenol - ve - ve - ve - ve - ve + ve 

7. Tannins - ve - ve - ve - ve - ve - ve 

8. Glycosides + ve + ve + ve + ve + ve - ve 

9. Terpenoids - ve - ve - ve - ve - ve + ve 

10. Proteins - ve + ve + ve + ve + ve + ve 

11. Amino acids - ve + ve + ve + ve + ve + ve 

Chloroform 

Ethyl acetate 

Methanol 

Water

0.05, 0.12, 0.23, 0.48, 0.59, 0.64, 0.73 and 0.85 a.(Fig. 3) and 

chloroform extract showed 5 spots of Rf value of 0.06, 0.20, 

0.46, 0.80 and 0.93 (Fig. 4). 

Fluorescence Analysis 

Powdered drug under UV and visible light when treated with 

different reagents emitted various colour radiations which 

help in identifying the drug in powder form. (Table 3) 

CONCLUSION 

This study presents a set of diagnostic characters of Hibiscus 

rosa-sinensis, Linn. that will help to identify the drug in 

fragmentary condition as well as in whole form. The results 

of parameters for preliminary phytochemical screening, UV 

analysis and HPTLC studies can act as biomarkers for 

identification and authentification of raw drug samples and 

play an important role in quality control and prevention of 

adulteration. 

Fig. 1: T. S. of midrib of H. rosa-sinensis Leaf 

Fig. 2:T. S. of lamina of H. rosa-sinensis Leaf 

Fig. 3: Alcoholic extract chromatography 

Gupta et al. / Pharmacopoeial Standardization of Hibiscus.... 

Fig. 4:Chloroform extract chromatograph 

REFERENCE 

1. Mitra SK, Kannan R. A Note on Unintentional Adulterations in 

Ayurvedic Herbs. Ethnobotanical Leaflets 2007; 11: 11-15. 

2. Sharma A, Ajay GN, KR Mahadik. Review Article molecular 

Markers: New Prospects in Plant Genome Analysis. 

Pharmacognosy Reviews 2008; 2(3): 21-23. 

3. Anonymous. The Wealth of India-Raw Material. Vol. V, PID, 

Council of Scientific and industrial Research, New Delhi, 1992. 

4. Kiritikar KR, Basu BD. Indian Medicinal Plant. Vol. I, Darshan 

Singh Mahendra Pal Singh, 23-A New Cannaught Place, Dehradun, 

2004. 

5. Chatterzee A, Prakash SC. Encyclopedia of Indian Medicinal 

Plants. PID, Council of Scientific and industrial Research, New 

Delhi, 2001. 

6. Anonymous. The Useful Plants of India. PID, Council of Scientific 

and industrial Research, New Delhi, 1996. 

7. Gilani AH, Bashir S, Janbaz KH, Shah AJ. Presence of cholinergic 

and calcium channel blocking activities explains the traditional use 

of Hibiscus rosa-sinensis in constipation and diarrhea. Journal 

Ethnopharmacology 2005; 102: 94-289. 

8. Gauthaman KK, Saleem MT, Thanislas PT, Prabhu VV, 

Krishnamoorthy KK, Devaraj NS et al. Cardioprotective effect of 

the Hibiscus rosa-sinensis flowers in an oxidative stress model of 

myocardial ischemic reperfusion injury in rat. BMC 

Complementary and Alternative Medicine 2006; 6: 32-39. 

9. Shanmugsundaram ERS, Sundaram P, Srinivas K, 

Shanmugsundaram KR. Double blind cross over study of modified 

Annapavala sindhooram in patients with hyperlipidemia or 

ischemic heart disease. Journal of Ethnopharmacology 1991; 31: 

85-99. 

10. Shrivastav N, Jain SK. Plants bearing antifertility properties. 

Hamdard Medicus 1993; 36: 91-98. 

11. Adhirajan N, Kumar TR, Shanmugasundaram N, Babu M. In-vivo 

and in-vitro evaluation of hair growth of Hibiscus rosa-sinensis. 

Journal of Ethnopharmacology 2003; 88(2-3): 235-239. 

12. Kasture VS, Chopde CT, Deshmukh VK. Anticonvulsive activity of 

Albizzia lebbeck, Hibiscus rosa sinensis and Butea monosperma in 

experimental animals. Journal of Ethnopharmacology 2000; 71(1- 

2): 65-75. 

13. Sachdeva A, Nigam R, Khemani LD. Hypoglycemic effect of 

Hibiscus rosa-sinensis leaf extract in glucose and streptozotocin 

induced hyperglycemic rats. Biol. Pharm. Bulletin. 1997; 

20(7):756-758. 

14. Anonymous. The Ayurvedic Pharmacopoeia of India. Vol. I, 

Ministry of Health and Family Welfare, Department of Yoga & 

Naturopathy, Unani and Siddha, New Delhi, 1996. 

15. Anonymous. Guidelines for the Assessment of Herbal Medicines. 

Vol. II, World Health Organization, Geneva, 1992. 

16. Wallis TE. Textbook of Pharmacognosy. Edn 15, TA Churchill, 

London, 1967. 

17. Anonymous. Quality Control Methods for Medicinal Plant 

Materials. World Health Organization, Geneva, 1998. 

18. Trease GE, Evans WC. Pharmacognosy. Edn 12, Bailliere Tindall, 

London, 1985. 

19. Brain KR, Turner TD. The Practical Evaluation of 

Phytopharmaceuticals. Wright Scientehnica, Bristol, 1975. 

20. Vogel HG. Drug Discovery and Evaluation Pharmacological 

assays. Edn 2, Springer-Verlag, Berlin, Germany, 2002. 

21. Sethi PD. HPTLC Quantative Aanalysis of Pharmaceutical 

Formulation. Edn 1, CBS Publishers, New Delhi, 2001. 

22. Harborne JB. Phytochemical methods – A Guide to Modern 

Techniques of Plants Analysis. Edn 3, Chapman & hall, U.K., 1998. 





ISSN 0975 1556 

Study of Analgesic and Anti-inflammatory Activities of Lagerstroemia 

lanceolata wall (seed) extract 

O. G. Bhusnure 1* , K. R. Alagawadi 2 , P. S. Giram 1 , B. N. Poul 1 

1 Dept. of Pharmaceutical Chemistry, Maharashtra College of Pharmacy, Nilanga- 413521, Dist. Latur (M.S.) India 

2 Dept. of Pharmaceutical Chemistry, K. L. E. Society’s College of Pharmacy, J. N. M. C. Campous,Belgaum 

5900010(K.S.) India 

ABSTRACT 

Analgesic and anti-inflammatory effects were examined in purified light Petroleum ether (40-60°) extract (LLW Oil) of the 

Lagerstroemia Lanceolata wall seed. The analgesic activity effects of graded doses of extract (in LLW10-200 mg/kg p.o.) 

were evaluated in rat against acetic acid induced writhing (chemically induced pain) and hot-plate method (thermally 

induced pain). The analgesia produced by extract was compared with the standard analgesics diclofenac sodium (DIS, 5 

mg/kg p.o.). Acute anti-inflammatory activity of fraction was also evaluated in carrageenan-induced rat paw edema model 

at the doses 50, 100 and 200 mg/kg i.p. and compared with diclofenac sodium (10 mg/kg i.p.). In comparison to control 

group purified Pt. ether extract showed highly significant, dose dependent analgesic activity against thermally as well as 

chemically induced pain (p < 0.001). LLW at the dose of 40 mg/kg has shown highly significant analgesic activity (p < 

0.001) as compared to diclofenac sodium at the doses employed. In comparison to control, LLW at the employed doses 

produced marked acute anti-inflammatory activity in rats (p < 0.001). The results suggest that the Pt. ether extract 

(LLW20-200) of seed has active herbal principles which possess significant analgesic and anti-inflammatory potential as 

reflected by the parameters investigated. Further investigations are, however, necessary to explore mechanism(s) of action 

involved in these pharmacological activities. 

Keywords: Lagerstroemia Lanceolata, Lythraceae, Pt. ether extract, analgesic and anti-inflammatory. 

INTRODUCTION 

The clinically useful drugs against pain and inflammation 

exhibit many adverse effects; this leads to considerable 

interest in search of safer drug for these conditions. 

Lagerstroemia Lanceolata wall is a native Indian tree 

belongs to the family Lythraceae and found from Bombay 

Southwards to Kerala. [1-2] 

The Lagerstroemia Lanceolata wall species has been used in 

the treatment of Asthma, Diabetes Mellitus, Chronic 

Bronchitis, cold, cough and local application for aphthae of 

the mouth. [3] Seeds have been documented for its multiple 

pharmacological activities including narcotic principal. [4] 

The leaves were also evaluated for potent, anti-inflammatory 

and antipyretic activities in the rat. [5] Literature revealed that 

Steroids, Terpenoids,Alkaloids, Antocyanins Ellagic acid and 

tannins, are the major components in the seeds. [6] The 

analgesic and anti-inflammatory activity of the oil and its 

constituents have never been characterized. The present study 

was planned to explore any possible analgesic and anti- 

*Corresponding author: Mr. O. G. Bhusnure, 

Dept. of Pharmaceutical Chemistry, Maharashtra College of 

Pharmacy, Nilanga- 413521, Dist. Latur (M.S.) India 

E-mail: omprakashbhusnure@gmail.com 

inflammatory potential of the light petroleum ether (40-60°) 

extract obtained from the Lagerstroemia Lanceolata seed . 


Plant materials 

The fully mature Lagerstroemia Lanceolata wall seeds (Fig. 

1) were collected from various parts of Belgaum city in the 

state Karnataka, India and the seed was identified and 

authenticated by Dr. Salimath P., Asst. Prof. Dept. Of 

Botany, R. L. Science College, Belgaum, India. The voucher 

specimen (KL 469) was deposited in the K. L. E. Society’s 

College herbarium. 

Fig. 1: Seeds of Lagerstroemia Lanceolata wall 

127

Bhusnure et al. / Study of Analgesic and Anti-inflammatory Activities.... 

Acute toxicity studies 

Acute oral toxicity study [7] was performed as per OECD-423 

guidelines (acute toxic class method). Wistar rats (n = 6) of 

either sex selected by random sampling technique were used 

for the study. The animals were kept fasting for overnight 

providing only water, after which the extracts were 

administered orally at the dose level of 5 mg/kg body weight 

by intragastric tube and observed for 14 days. If mortality 

was observed in 2-3 animals, then the dose administered was 

assigned as toxic dose. If mortality was observed in one 

animal, then the same dose was repeated again to confirm the 

toxic dose. If mortality was not observed, the procedure was 

repeated for further higher dose such as 50, 300 and 2000 

mg/kg body weight. 

Preparation of LLW 

Dried coarsely powdered seeds (500 g) were extracted with 

light Pt. ether (40-60°) using Soxhlet apparatus at 80 ± 2°C 

for 24 h. The Pt. ether extract were dried over anhydrous 

sodium sulphate and solvent was removed in vacuum at 40°C 

to recover the oil by using rotary-evaporator (Rotavapour, 

Buchii, Switzerland). The Pt ether extract thus separated was 

preserved and coded as LLW (yield 10 %). The LLW extract 

was subjected to physicochemical characterization using 

TLC (thin layer chromatography) on pre-coated silica gel-G 

plates using petroleum ether: ether: acetic acid (75: 25: 0.1, 

v/v/v) as mobile phase and iodine as visualizing agent. 

Phytochemical screening was done for detection of alkaloids, 

glycosides, saponins and steroids and oil contents were 

determined for Acid value, Iodine Value, Saponofication 

value, Hydroxy value , Halphen test and Unsaponifiable 

matter in LLW. For pharmacological screening LLW oil was 

emulsified using acacia according to the desired 

concentration (test dose LLW-10, LLW-20, LLW-40, LLW- 

50, LLW-100 and LLW-200) just before use. 

Analgesic studies 

Analgesic activity in mice was assessed in chemically as well 

as thermally induced pain using acetic acid induced writhing 

model [8] and hot plate assay [9] , respectively. 

Acetic acid induced writhing method 

Five groups of mice (n = 6) were randomly formed. The 

groups were treated as control (distilled water, p. o.) and 

standard (DIS 5 mg/kg p.o.) while test groups received 

fraction (LLW) 10, 20 and 40 mg/kg p.o. (LLW-10, LLW-20 

and LLW-30), respectively. Acetic acid solution 0.6 % v/v 

(10 mL/ kg) was injected by intraperitoneal route one hour 

after treatment and number of writhes (i.e. index of pain 

reaction against chemical stimuli characterized by abdominal 

muscle contraction together with turning of trunk and 

extension of hind limbs) was counted over a period of 20 

min. Analgesic activity was expressed as percentage of 

inhibition of writhes with respect to the control group (Table 

1). 

Hot plate method 

Hot plate was maintained at 55 ± 1°C. Albino mice were 

divided in five groups. The animals were placed on the hot 

plate and the basal reaction time taken to cause a discomfort 

(licking of paw or jumping response whichever appeared 

first) was recorded at 0 min. Cut-off period 15 sec. was 

established to prevent damage to the paws. 

Anti-inflammatory studies 

Acute anti-inflammatory activity of LLW at 50,100 and 200 

mg/kg was evaluated using carrageenan induced edema in 

rats described by Winter et al. [10] Five groups of albino rats 

(n = 6) were randomly distributed in control, standard and 

test (LLW-50, LLW-100 and LLW-200) groups. The initial 

paw volumes of each animal were measured by means of a 

mercury plethysmometer. The standard group was treated 

with DIS injection (10 mg/kg, i.p.) while LLW solution and 

distilled water (10 mL/kg, i.p.) were given to the test and 

control groups, respectively. Thirty minutes after treatment 

0.1 mL of 1 % carrageenan solution was injected in the 

plantar region of the left hind paw of rats. Paw volumes were 

again measured 3 h after carrageenan injection. The acute 

difference in edema volume was calculated in each control, 

test and standard group and compared with the control group 

for determination of the percentage of inhibition of the paw 

edema (Table 3). 

Vc-Vt 

Percentage inhibition of oedema = ------------ × 100 

Vc 

Where, Vc is the inflammatory increase in paw volume in 

control group of animals and Vt is the inflammatory decrease 

inpaw volume in drug-treated animals. 


Data obtained from pharmacological experiments are 

expressed as mean ± SEM. Difference between the control 

and the treatments in these experiments were tested for 

significance using ANOVA followed by Tukey’s comparison 

test was applied. P < 0.05 was considered statistically 

significant. 

RESULTS 

Phytochemical screening of the light Pt.ether (40-60°) extract 

(LLW Oil) obtained from Lagerstroemia Lanceolata wall 

seeds was contains glycoside and steroid principals. 

Preliminary screening was carried out in mice for four graded 

doses viz. 5, 10, 20, and 40 mg/kg administered orally as 

well as intraperi-toneally and indicated no gross behavioral 

changes, sedation, morbidity and mortality at these doses. 

Pt.ether (40-60°) extract (LLW Oil)indicated highly 

significant and dose dependent analgesic activity against both 

thermally and chemically induced pain. In acetic acid 

induced writhing method LLW (10, 20, and 40 mg/kg p.o.) 

and standard (DIS 5 mg/kg p.o.) treated animals showed 

significantly reduced number of writhing in 20 min at the rate 

of 28.82 %, 48.58 %, 75.73 % and 63.77 %, respectively, 

when compared to that of control group (p < 0.001). 

Analgesia produced by LLW-40 mg/kg was also found to be 

higher than that of observed for standard diclofenac sodium 

at the employed doses (Table 1). On hot-plate test, LLW Oil 

showed significant elevation in pain threshold in comparison 

to control, as represented in Table 2 and indicated significant 

analgesic activity (p < 0.05) as compared to control at all 

tested doses. 

Acute anti-inflammatory potential of LLW Oil was 

investigated at its minimal dose producing analgesia (i.e. 50, 

100 and 200 mg/ kg, i.p.) against carrageenan induced rat 

paw edema. It was noted that the standard drug diclofenac 

sodium (DIS, 10 mg/ kg i.p.) showed 74.60 % inhibition of 

edema whereas LLW Oil showed 53.70, 59.70 and 62.60 % 

inhibition, at dose LLW-50, LLW-100 and LLW-200 

respectively with respect to control (p < 0.001). In addition, 

the study also demonstrated lower anti-inflammatory 

response of LLW at the used doses in comparison to DIS at 

10 mg/ kg i.p. The results are summarized in Table 3.The 

extracts were found to significantly inhibit the carrageenan- 



Table 1:Analgesic effect of different doses of Pt. ether extract of LLW in acetic acid induced writhings in mice 

Treatment Dose, p. o. (mg/kg) No. of writhes in 20 min The mean ± SE % Reduction 

Control (DW) 10 ml/kg 51.50 ± 0.99 - 

DIS 5 18.66 ± 0.88* 63.77 

LLW-10 10 36.66 ± 0.95* 28.82 

LLW-20 20 26.50 ± 0.84* 48.58 

LLW-40 40 12.50 ± 0.76* 75.73 

One way 

F 309.45 

ANOVA 

p < 0.001 

DIS, diclofenac sodium; DW, distilled water; One way ANOVA followed by multiple Tukeyís comparison test. Values are presented as the mean ± SE 

(standard error); n = 6 for all groups, df = = 4, 25. * p < 0.05 as compared to control; p < 0.05 as compared to diclofenac group. 

Table 2. Analgesic effect of different doses of Pt. ether extract of LLW on hot plate test in mice. 

Treatment Dose, p.o. 

Mean reaction time in seconds 

(mg/kg) 0 min. 30 min. 60 min. 120 min. 

Control (DW) 10mL/kg 3.93 ± 0.04 4.01 ± 0.45 3.96 ± 0.04 3.96 ± 0.04 

DIS 5 3.91 ± 0.05 7.46 ± 0.17* 8.06 ± 0.15* 7.61 ± 0.14* 

LLW-10 10 3.88 ± 0.05 4.32 ± 0.06 4.91 ± 0.07 4.76 ± 0.11 

LLW-20 20 3.91 ± 0.07 4.70 ± 0.09* 5.45 ± 0.08* 5.2 ± 0.06* 

LLW-40 40 3.92 ± 0.05 5.32 ± 0.07* 6.12 ± 0.08* 5.75 ± 0.14* 

One way 

F 0.088 178.89 249.7 155.87 

ANOVA 

p >0.05

REFERENCE 


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York, 1997, pp. 43-86. 

8. Ghosh MN. Fundamentals of Experimental Pharmacology, Ed 2nd, 

Scientific Book Agency, Calcutta, India 1984, pp. 144. 

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111: 544. 

11. Mossa JS, Rafatullah S, Galal AM, Al-Yahya MA., 

Pharmacological studies of Rhus retinorrhaea.,Int. J. 

Pharmacognosy 1995; 166: 96-103. 

12. DiRosa M, Giroud JP, Willoughby DA. Studies of the acute 

inflammatory response induced in rats in different sites by 

carrageenan and turpentine. J. Pathol. 1971; 104: 15-29. 

13. Vinegar R, Schreiber W, Hugo R. Biophasic development of 

carrageenan oedema on rats. J. Pharmacol. Exp. Ther. 1969; 66: 96- 

103. 

14. Crunkhon P, Meacock SER. Mediators of the inflammation induced 

in the rat paw by carrageenan., Br. J. Pharmacol. 1971; 42: 393- 

402. 

15. Deraedt R, Jouquey S., Benzoni J. et al.: Arch. Int. Pharmacodyn. 

Ther. 1976; 224: 30. 

16. Deraedt R, Jouquey S, Delevalee F et al.: Eur. J. Pharmacol. 1980; 

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17. Beirith A, Santos ARS, Rodrigues ALS. et al.: Eur. J. Pharmacol. 

1998; 345: 233. 





ISSN 0975 1556 

In vivo Passive and Iontophoretic Delivery of Lisinopril Using Wistar 

Rat Model 

Abdul Faruk * , Mohan Paul Singh Ishar 

Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar-143005, Punjab, India 

ABSTRACT 

In-vivo studies using animal model were carried out to investigate the pharmacokinetic profile of passive and 

iontophoretically delivered lisinopril. Serum concentration versus time profiles from intravenous (IV), oral and 

iontophoretic routes were analyzed using non-compartmental analysis using TOPFIT ver. 2.0. Pharmacokinetic parameters 

such as AUC0–inf, terminal elimination rate constant (λz), clearance/F and Cmax, were calculated. To validate the calculations 

involved in non-compartmental analysis, the serum profiles were fitted using TOPFIT (ver. 2.0) software to the onecompartmental 

continuous infusion model with zero order absorption. The results of the IV bolus administration of 

lisinopril showed that the pharmacokinetics could be described by a two-compartment model. The bioavailability of 

passive and iontophoretically delivered lisinopril was 62.21 and 82.87 %, respectively which is significantly higher as 

compared to oral route (5.67 %). The corresponding values of Cmax were found to be 87, 95, 15.33 and 25.6 ng/ml, 

respectively for oral, IV bolus, passive and iontophoretically delivered lisinopril. The tmax for oral and IV route was 10 and 

5 min, respectively, while it was 9.0 h both for passive and iontophoretic mediated transport of drug. The simple zero-order 

input rate and clearance effectively defined the delivery pattern of lisinopril from the iontophoretic patch. Good correlation 

was observed between the experimental data and data predicted by the model. Clearance estimated by the model is similar 

to the clearance calculated from intravenous administration, which supports the assumptions in the calculation of dose 

delivered by non-compartmental analysis. 

Keywords: Iontophoresis, Lisinopril, Bioavailability, Transdermal patches, Non-compartmental analysis, TOPFIT, Zero 

order absorption. 

INTRODUCTION 

The skin has been identified as a route of drug administration 

for decades. Several drug delivery systems has been 

developed for utilizing this route and the ultimate goal is to 

ensure that compounds are delivered preferably at a specific 

rate to the systemic circulation. Topical drug delivery system 

has some limitations, arising mainly from excellent barrier 

properties of stratum corneum. Iontophoresis has potential to 

overcome many barriers associated with transdermal delivery 

of drugs and it broadens the spectrum of drugs that can be 

delivered via skin, increases systemic treatment efficacy, 

therefore, it is an alternative to invasive routes of drug 

administration for charged molecules. [1-3] Iontophoresis uses 

a small electrical current to enhance the transport of both 

ionic and nonionic molecules across the skin in controlled 

and programmable manner. [4-5] 

*Corresponding author: Mr. Abdul Faruk, 

Department of Pharmaceutical Sciences, Guru Nanak Dev 

University, Amritsar-143005, Punjab, India 

Tel: +91-183-2258802-08 Extn.3457; 

Fax: +91-183-2258819-20 

E-mail: abdul_faruk@yahoo.com 

The enhancement of drug due to this method results from a 

number of possible mechanisms including the ion-electric 

field interaction (electro repulsion), [5] convective flow 

(electro-osmosis) [6] and current-induced [7] increase in skin 

permeability. The main aim of the present study was to 

evaluate the in vivo delivery of lisinopril using fabricated 

iontophoretic patch to investigate the pharmacokinetic 

parameters such as rate of delivery and to compare the 

pharmacokinetic profiles of iontophoretically delivered 

lisinopril with oral administration, and intravenous injection 

of lisinopril. Lisinopril was selected as a model drug that can 

be extensively administered through this route because it is 

devoid of any pungent skin sensation and burning pain. 

Furthermore, for clinical implications transdermal 

formulation of lisinopril delivery is highly desired. [8] 

Advantages of this route include improved patient 

compliance, avoidance of first pass hepatic metabolism, 

controlled delivery and the possibility to modulate the rate of 

delivery [9-11] which in turn shall lead to patient convenience 

as well as improved compliance. 


The constant current source (0-4 mA) was designed and 

fabricated by University Instrumentation Science Centre 

131

Faruk et al. / In vivo Passive and Iontophoretic Delivery of Lisinopril.... 

(USIC), Guru Nanak Dev University, Amritsar, India, which 

can be operated at a resistive load of 10KΩ and was 

assembled by M/S B. S. Electronics, Amritsar, India. 

Generous gift sample of lisinopril was obtained from 

Ranbaxy Research Laboratory, Gurgaon, India and analytical 

grade chemicals were procured from Qualigens fine 

chemicals, Mumbai. India. Other chemicals and accessories 

used in the experiments like polyvinyl alcohol, electrode gel 

(Electrogel ® ), adhesive tape (Leukoplast ® ) and drug reservoir 

receptacles were obtained from Central Drug House, New 

Delhi, Unichem Laboraties, New Delhi, Beirsdorf (I) Ltd., 

Ponda, Goa, Newsun Plastics, Faridabad, respectively. 

Preparation of lisinopril reservoir gel 

It is reported that when the transdermal iontophoretic 

delivery system is applied in vivo, the semisolid dosage 

formulation may be more appropriate than solution. The gel 

base provides a fast release of drug substance and a high 

degree of clarity in the appearance. [12] Moreover, there is 

always a great volume of water employed in gel formulation, 

which exhibits a high electrical conductivity. [13] Hence the 

transdermal iontophoresis from gel base presently developed 

for in vivo investigation. 

Gel base [14] (100g) were prepared by adding 8 % HPC to the 

solvent mixture (ethanol:propylene glycol: water in the ratio 

of 50:30:20) with constant stirring at 500 rpm for 15 min and 

then allowed to stand in a water bath at normal temperature 

of 25°C for 30 min and set aside for 24 h. [14] Since the 

formulations are to be applied for longer duration of time, 

therefore, it was fabricated in a reservoir of high-density 

polyethylene (HDPE) receptacle (Volume = 5.0 ml). It was 

stuck with an adhesive (Araldite ® ) on to the stripped 

adhesive foam tape of the ECG electrode pad. The 

conductive gel pad of the stripped electrode served as 

indifferent electrode. The additional conductive gel 

(Electrogel ® ) was added to the pad during iontophoresis. 

Additional polymer polyvinylalcohol (PVA) 20 % w/w was 

also used for increasing consistency of the formulation in the 

reservoir. Ingredients of the above prepared gel base plus 

PVA were dissolved in the citrate buffer (77.5 w/w) by little 

warming on the water bath with constant stirring. The 

obtained solution was cooled to a gel of desired consistency. 

Lisinopril (500 μg/ml) was added to 5.0 ml of the gel and 

poured into the reservoir (HDPE receptacle with rubber 

closure) to obtain 2.5 mg of drug. The contents were wrapped 

in aluminum foil and cooled in a refrigerator at 4°C for 24 h. 

Before putting it on the rat skin, the smooth paper over the 

adhesive foam tape was removed and the receptacle placed at 

the iontophoretic site. It was secured by the foam tape and 

additional adhesive tapes (Leukoplast ® ). The rubber closure 

atop the receptacle served as the entry port for Pt wire 

electrodes. 

Preparations of animals for iontophoresis 

Male Wistar rats [15] (10-12 week old) were used in the 

experiments. Food and water were provided ad libitum. The 

average number of replicates for each study was three. Rats 

were anesthetized using intra-peritoneal injection of ketamine 

(75 mg/kg) and xylazine (10 mg/kg). After deep anesthesia 

was induced, hairs were removed from the abdominal area of 

the animal by scissors and care was taken to ensure that no 

abrasion or cuts occur at the selected site. After removal of 

the hair, the site was wiped with a cotton swab soaked in 

benzoin tincture. [16] It was then washed, dried, cleaned with 

an alcohol swab and finally air-dried. Another precaution 

taken was proper maintenance of hygiene in the animal 

housing cage. The puncture spot for blood sampling was 

properly washed with absolute alcohol followed by an 

antiseptic (Dettol ® ) and an antibiotic powder 

(Nitrofurazone ® ) was sprayed after each sampling. The 

experiments involving animals were carried out as per the 

ethical guidelines and housed at appropriate conditions 12 h 

light and 12 h dark side (CPSCA No 226). 

In vivo iontophoretic delivery of lisinopril 

The drug reservoir receptacle was placed at the prepared site 

after removal of the smooth paper covering the foam tape 

adhesive. It was secured at its place by additional adhesive 

tapes. The platinum electrode was inserted through the 

rubber stopper into the reservoir containing lisinopril-PVA 

gel. The indifferent electrode was placed within conducting 

distance from the reservoir electrode. Hairs were clipped off 

at that site and a layer of conductive gel (Electrogel R ) was 

applied at the site. The Pt-electrode was placed in the gel and 

covered with the stripped conductive gel pad ECG electrode. 

It was secured at the place by adhesive tapes. At pH 4.0, 

lisinopril is ionized upto the extent of 85 % [17-18] and was 

thus delivered under the positively charged electrode (anode). 

The ionto-phoretic gel patch was kept at anodal side, while a 

gel formulation containing 0.9 % sodium chloride was used 

at cathodal side. The reservoir gels were applied onto the 

cleaned abdominal area, and the additional batteries and 

resistor were connected in series. 

Presently lisinopril formulation was used at pH 4.0 because 

the net charge would aid delivery of molecule as 

iontophoretic flux by the electro osmosis. [19] 

Experimental protocol 

In iontophoretic studies, the rats were subjected continuously 

for 24 h to a constant current of density 0.1 mA/cm 2 , after 

which the current treatment was stopped. Current flow was 

monitored throughout the patch application period. 

Transdermal administration of drug was accomplished 

through the application of patch (attached on a 2.5 cm dia 

leucoplast adhesive) to the abdominal skin area of the rat. 

Blood samples were collected through either tail or 

sublingual vein at 3.0, 6.0, 9.0 and 24 h. Blood samples were 

allowed to clot and centrifuged at 7200 x g for 10 min and 

serum was collected and stored at –20°C until analyzed for 

serum lisinopril concentrations by HPLC as reported 

previously. [20] 

The current density (0.1 mA/cm 2 ) used is lower than the 

current density reported in the literature, [21] so that longer 

period of iontophoretic treatment would cause no burn or 

harm the skin. It is also established that at a current density 

0.1 mA/cm 2 iontophoresis for longer duration did not 

produce any irritation and emerged as a valuable substitute 

for invasive routes such as injections. 

Intravenous injection and oral administration of lisinopril 

Other routes of administration in rats (n=3) for the purpose of 

comparison were oral solution and an IV bolus injection with 

a 5 days washout period in between treatments. Each rat was 

fasted for 12 h prior to dosing. Male Wistar rats were 

anesthetized as described before. Lisinopril was dissolved in 

water for injection (WFI) and 10 μg/kg dose was given 

intravenously through femoral vein and blood samples were 

collected at 5, 30, 120, 240 and 180 min and evaluated as 

described before. The total amount of blood withdrawn in 

each study was less than 10 % of total blood volume. 



The oral dose (10.8 mg drug in 10 ml given as bolus, and 4 

ml to flush the tube) was administered as a solution of the 

drug in distilled water via a gastric tube. Blood samples were 

withdrawn at 10, 30, 60 and 180 min respectively and 

analyzed by HPLC method. [20] 

Pharmacokinetic data analysis [22] 

Serum concentration versus time profiles from IV, oral and 

iontophoretic routes were analyzed using non-compartmental 

analysis using TOPFIT ver. 2.0. Pharmacokinetic parameters 

such as AUC0–inf, terminal elimination rate constant (λz), 

clearance/F and Cmax, were calculated. Clearance obtained 

from IV data, was used to calculate the dose delivered during 

iontophoresis by the following equation assuming that 

iontophoretic delivery is a zero order infusion: 

F. dose delivered = AUC iontophoretic × clearance IV (1) 

Rate of infusion (R0) at steady state was calculated by the 

following equation: 

R0 = F × dose delivered (2) 

Duration of patch application 

Where, F represents the fraction of dose absorbed into 

systemic circulation. It measures the drug loss in the skin and 

subdermal layers. F × dose delivered is calculated as a single 

function from eq. 1. 

Bioavailability determination 

Absolute availability = [AUC] ev / dose ev (3) 

[AUC] i.v / dose i.v 

AUC was calculated using the trapezoidal rule. 

Pharmacokinetic modeling 

To validate the calculations involved in non-compartmental 

analysis, the serum profiles were fitted using TOPFIT (ver. 

2.0) software to the one-compartmental continuous infusion 

model with zero order absorption as follows: 

Cp =F × dose delivered × (1– e -kt ) If t ≤ T inf (4) 

Cl 

Cp = F × dose delivered×(1– e -kt )×(e -k(t-T inf ) ) If t>T inf (5) 

Cl 

Where, Cp is the serum concentration of lisinopril, k the 

elimination rate constant, Cl the clearance and Tinf is the 

duration of patch application. A number of other pharmacokinetic 

models were also evaluated and include the onecompartment 

model with first-order input and twocompartment 

models with constant and first-order inputs. [23] 


All data are presented as mean ± S.E. Statistical analysis was 

performed using analysis of variance (ANOVA); p < 0.05 

was regarded as significant. 

Table 1: Plasma lisinopril levels following different routes of 

administration (n=3) 

Mean plasma lisinopril level (ng/ml) 

S. 

No. 

Time Oral 

Intaveno 

us bolus a 

Passive a 

Trandsermal 

Iontophoresi 

s a 

1 5.0 min - 95 ± 6.54 - - 

2 10 min 87 ± 5.23 - - - 

3 30 min 72 ± 4.12 44 ± 5.31 - - 

4 1.0 h 43 ± 3.36 - - - 

5 2.0 h - 15 ± 2.33 - - 

6 3.0 h 13 ± 2.15 - 7 ± 1.14 4.3 ± 1.11 

7 4.0 h - 10 ± 1.96 - - 

8 6.0 h - - 12 ± 2.11 22 ± 2.78 

9 9.0 h - - 14.33 ± 2.36 23.60 ± 2.64 

10 24 h - - 15.33 ± 2.59 25.6 ± 1.99 

a All values are expressed as mean ± S.D.; n = 3 

Fig. 1: Plasma drug concentration of lisinopril after IV and oral 

administration in rats 

Fig. 2: Plasma drug concentration of lisinopril after passive and 

iontophoretic applications in rats 

Patches P (for passive delivery) and I (for iontophoretic 

delivery) were selected for in vivo studies. Plasma lisinopril 

levels were measured by the HPLC method. [20] 

The temporal profile of serum lisinopril concentrations after 

oral administration and intravenous injection is shown in Fig. 

1 and pharmacokinetic parameters calculated are given in 

Table 4. The results indicated that iontophoretic device 

delivered lisinopril at an average infusion rate of 198.9 ± 

51.3 ng/min kg and an average steady state concentration of 

23.58 ± 1.24 ng/ml was achieved (Fig. 2). 

The bioavailability of transdermal patches P and I were 

significantly higher as compared to oral route. Oral 

bioavailability was determined to be 5.67 %, while P and I 

showed bioavailability of 62.21 and 82.87 % respectively 

(Table 3). The Cmax was found to be 87 ng/ml for oral, 95 

ng/ml for an IV bolus (Table 8.4), 15.33 ng/ml for P and 25.6 

ng/ml for I (Table 5). The tmax for oral and IV route was 10 

and 5 min, respectively while it was 9.0 h both for P and I 

(Table 2). Transdermal patches demonstrated not only better 

bioavailability but also sustained release property, which can 

be controlled by proper formulation design. 


In vivo studies 



Table 2: Calculation of the total area using trapezoidal rule. 

S. No Time (min) Concentration of drug in plasma (ng/ml) Time interval (min) Average concentration a (ng/ml) Area a (ng. min. ml -1 ) 

Oral (dose: 10.8 mg) 

1 0.0 0.0 - - - 

2 10 87 ± 5.23 10 43.5 ± 5.33 435 ± 12.21 

3 30 72 ± 4.12 20 79.5 ± 6.21 1590 ± 21.21 

4 60 43 ± 3.36 30 57.5 ± 4.24 1725 ± 23.2 

5 180 13 ± 2.15 120 28.0 ± 3.32 3360 ± 25.8 

Total 7110 

IV bolus (dose: 0.528 mg) 

1 0.0 0.0 - - - 

2 5.0 95 ± 6.54 5 47.5 ± 6.54 237.5 ± 15.2 

3 30 44 ± 5.31 25 69.5 ± 6.98 1737.5 ± 34.33 

4 120 15 ± 2.33 90 29.5 ± 3.14 2655.0 ± 36.59 

5 240 10 ± 1.96 120 12.5 ± 1.54 1500.0 ± 39.67 

Total 6130 

Passive delivery (dose: 2.5 mg) 

1 0.0 0.0 - - - 

2 180 7 ± 1.10 180 3.5 ± 0.23 630 ± 16.32 

3 360 12 ± 2.30 180 9.5 ± 1.36 1710 ± 23.47 

4 540 14.33 ± 2.96 180 13.165 ± 1.24 2369.7 ± 25.45 

5 1440 15.33 ± 2.99 900 14.83 ± 2.31 13347 ± 59.84 

Total 18056.7 

Iontophoretic delivery (dose: 2.5 mg) 

1 0.0 0.0 - - - 

2 180 4.3 ± 0.19 180 2.15 ± 0.24 387 ± 14.61 

3 360 22 ± 2.21 180 13.15 ± 1.36 2367 ± 22.36 

4 540 23.6 ± 3.01 180 22.8 ± 2.54 4104 ± 25.33 

5 1440 20.6 ± 1.99 900 22.1 ± 2.61 19890 ± 62.34 

Total 26748 


Table 3: Bioavailability of different formulations 

Formulation Dose AUC (ng. min. ml -1 ) % bioavailability 

Oral Solution 10.8 mg 7110 ± 36.32 5.67 

IV bolus 0.528 mg 6130 ± 32.25 100 

Transdermal Route 

Passive delivery 2.5 mg 18056.7 ± 56.65 62.21 

Iontophoretic delivery 2.5 mg 26748 ± 65.45 82.87 


Table 4: Pharmacokinetic parameters (average ± S.E.) after IV and oral administration of lisinopril in rats. 

Parameters IV Oral 

Cmax (ng/ml) 95 ± 6.54 87 ± 5.23 

Cl (ml/min kg) 25.9 ± 0.92 20.21 ± 0.87 

λz (min -1 ) 0.02 0.03 

Half-life (min) 36.5 ± 3.4 58.2 ± 4.4 

AUC inf (min ng/ml) 6130 ± 36.89 7110 ± 28.30 

Table 5: Pharmacokinetic parameters (average ± S.E) after passive (P) and Iontophoretic transport (I) of lisinopril in rats. 

Parameters Passive delivery (P) Iontophoretic transport (I) 

Cmax (ng/ml) 15.33 ± 2.59 25.6 ± 1.99 

λz (min -1 ) 0.03 0.03 

Half-life (min) 318 ± 26.54 321 ± 23.45 

AUC inf (min ng/ml) 18056.7 26748.0 

Dose delivered (μg/Kg) 21.4 ± 2.6 28.4 ± 2.89 

The results of the IV bolus administration of drug salt 

showed that the pharmacokinetics could be described by a 

two-compartment model (Fig. 1). Results from oral 

administration of drug indicated that drug is rapidly absorbed 

from the GIT and that oral bioavailability was about 5.0 %, 

indicating that extensive loss of drug may be due to 

enzymatic degradation/hepatic first pass metabolism. On the 

other hand transdermal administration resulted in 

bioavailability of over 60 %. Results also indicate that by 

proper formulation design the bioavailability through 

transdermal systems could be greatly enhanced (e.g., for P = 

62.21 % whereas for I it was 82.87 %). This substantial 

increase in bioavailability could be due to the extensive 

vascular system of the rat skin, forced transport of lisinopril 

due to iontophoresis as well as circumventing the ‘first pass’. 

The simple zero-order input rate and clearance effectively 

defined the delivery pattern of lisinopril from the 

iontophoretic patch. Good correlation was observed between 

the experimental data and data predicted by the model. 

Clearance estimated by the model is similar to the clearance 

calculated from intravenous administration, which supports 

the assumptions in the calculation of dose delivered by noncompartmental 

analysis. Singh et al., [24-25] have shown that 

for various drugs the zero-order infusion model defines and 

serves practical purposes of modeling, and less than perfect 

fit may be due to the contribution of electro osmosis during 

iontophoresis. 

CONCLUSION 

The bioavailability of drug can be enhanced nearly 10 folds 

via the transdermal route and can be further enhanced by 

proper formulation design. Passive and iontophoretic 



transdermal patch gave an effective plasma concentration of 

15 and 25 ng/ml, respectively, maintained for about 15 h. 

The iontophoretic patches delivered therapeutically relevant 

concentrations of lisinopril in rats and delivery comparable to 

conventional routes such as intravenous injection was 

achieved. The result confirm that electronic transdermal 

delivery can be a potential mode for systemic delivery of 

therapeutic molecules and present study confirmed the 

viability of lisinopril delivery from designed and fabricated 

patch by iontophoresis, using a convenient device 

configuration. 

REFERNCES 

1. Droog EJ, Sjoberg F. Nonspecific vasodilatation during transdermal 

iontophoresis-The effect of voltage over the skin. Microvasc Res. 

2003; 65(3):172-8. 

2. De Graaff AM, Li GL, van Aelst AC, Bouwstra JA. Combined 

chemical and electrical enhancement modulates stratum corneum 

structure. J Control Rel. 2003; 90(1): 49-58. 

3. Banga AK, Bose S, Ghosh TK. Iontophoresis and electroporation: 

comparisons and contrasts. Int J Pharm. 1999; 179(1):1-19. 

4. Nair VB, Panchagnula R. Effect of iontophoresis and fatty acids on 

permeation of arginine vasopressin through rat skin. Pharmacol 

Res. 2003; 47(6): 563-569. 

5. Kalia YN, Naik A, Garrison J, Guy RH. Iontophoretic drug 

delivery. Adv Drug Deliv Rev. 2004; 56(5): 619-58. 

6. Wang Y, Thakur R, Fan Q, Michniak B. Transdermal 

iontophoresis: combination strategies to improve transdermal 

iontophoretic drug delivery. Eur J Pharm Biopharm. 2005; 60(2): 

179-191. 

7. Pillai O, Nair V, Panchagnula R. Transdermal iontophoresis of 

insulin: IV. Influence of chemical enhancers. Int J Pharm. 2004; 

269(1): 109-120. 

8. Gannu R, Yamsani VV, Palem CR, Yamsani SK, Yamsani MR. 

Iontophoretic delivery of lisinopril: Optimization of process 

variables by Box-Behnken statistical design. Pharm Dev Technol. 

2009; 1: 1-9. 

9. Banga AK. in “Electrically Assisted Transdermal and Topical Drug 

Delivery”, Taylor & Francis, London, UK. 1998. 

10. Banga AK, Bose S, Ghosh TK. Iontophoresis and electroporation: 

comparisons and contrasts. Int J Pharm. 1999; 179: 1-19. 

11. Chang SL, Hofmann GA, Zhang L, Deftos LJ, Banga AK. The 

effect of electroporation on iontophoretic transdermal delivery of 

calcium regulating hormones. J. Control Rel. 2000; 66: 127-133. 

12. Lelawongs P, Liu JC, Siddiqui O, Chien YW. Transdermal 

iontophoretic delivery of arginine-vasopressin (I): Physicochemical 

considerations. Int J Pharm. 1989; 56:13-22. 

13. Phipps, J. B.; Padmanaphan, R. V.; Lattin, G. A. Iontophoretic 

delivery of mode1 inorganic and drug ions. J Pharm Sci. 1989; 78: 

365-369. 

14. Gupta M, Verma PRP, Marwaha RK, Faruk A, Singh G. 

Formulation and evaluation of meloxicam gel. J Pharm Res. 2008; 

7(1): 27-31. 

15. Fang JY, Kuo CT, Huang YB, Wu PC, Tsai YH. Transdermal 

delivery of sodium nonivamide acetate from volatile vehicles: 

effects of polymers. Int J Pharm. 1999; 176(2): 157-167. 

16. Kari B. Control of blood glucose levels in alloxan-diabetic rabbits 

by iontophoresis of insulin. Diabetes. 1986; 35: 217-221. 

17. Neibergall PJ. Ionic Solutions and Electrolytic Equilibrium in 

“Remington’s Pharmaceutical Sciences”, Chap.17, 16th Edn., Osol, 

A. (Eds.), 1980. 

18. Martin A, Swarbrick J, Cammarata A. in “Physical Pharmacy”, 

(IIIrd Eds.), pp. 222, Lea & Febiger, Philadelphia, 1983. 

19. Pikal MJ. The role of electroosmosic flow in transdermal 

iontophoresis. Adv Drug Del Rev. 1992; 9: 201-237. 

20. Tamimi JJ, Salem II, Mahmood AS, Zaman Q, D Ruwayda. 

Bioequivalence evaluation of two brands of lisinopril tablets 

(Lisotec and Zestril) in healthy human volunteers. 

Biopharmaceutics & drug disposition. 2005; 26(8): 335-339. 

21. Santi P, Colombo P, Bettini R, Catellani PL, Minutello A, Volpato 

NM. Drug reservoir composition and transport of salmon calcitonin 

in transdermal iontophoresis. Pharm. Res. 1997; 14: 63-66. 

22. Chaturvedula A, Joshi DP, Anderson C, Morris R, Sembrowich 

WL, Banga AK. In vivo iontophoretic delivery and 

pharmacokinetics of salmon calcitonin. Int J Pharm. 2005; 297: 

190-196. 

23. Ludden TM, Beal SL, Sheiner LB. Comparison of the Akaike 

Information Criterion, the Schwarz criterion and the F test as guides 

to model selection. J Pharmacokinet Biopharm. 1994; 22: 431-445. 

24. Singh P, Maibach HI. Transdermal iontophoresis. Pharmacokinetic 

considerations. Clin Pharmacokinet. 1994; 26: 327-334. 

25. Singh P, Roberts MS, Maibach HI. Modelling of plasma levels of 

drugs following transdermal iontophoresis. J Control Rel. 1995; 33: 

293-298. 





ISSN 0975 1556 

Evaluation of Prescription Pattern and Cost-Effectiveness among 

Patients Associated With Gynaecological Diseases in Government 

Maternity Hospital, Lucknow (U. P.)-India 

Rohit K. Verma * , Kiran Chaudhary, Amita Rai, Ayush Garg, Manisha Pandey, 

Shubhini A Saraf 

Faculty of Pharmacy, Babu Banarasi Das National Institute of Technology & Management, Lucknow, 

Uttar Pradesh, India-227105 

ABSTRACT 

Drug is a very important link between producer and consumer. Thus a study on prescribing pattern and 

pharmacoeconomics helps in accessing the quality of health care services. A retrospective study was undertaken in the 

OBG Department, Maternity Hospital, Lucknow, India. A clinical survey was conducted on 119 gynaecological patients, 

out of which 19 patients were excluded according to exclusion criteria. Audited patients were in the age group of 18 to 60 

years. Clinical data indicated that 22 % of women suffered from irregular menses, leucorrhoea (18 %), vulvitis (16 %), 

osteoporosis (15 %), recurrent bleeding (11 %), uterus inflammation (8 %), endocervitis (2 %), feranculitis (2 %) and 

breast pain (2 %). Clinical survey revealed that 80 % women were prescribed minerals and vitamins, 56 % Analgesics, 47 

% Antibiotics, 42 % Antimicrobials, 39 % Iron tonics, 23 % Steroids, 6 % Anti-allergic, 4 % antispasmodic and 6 % Antiinflammatory 

therapy at the mean age of 38 years. 

It was observed that the drugs prescribed were correct for the current diagnosis and treatment. There were no any earlier 

records pertaining to their medical history and treatment regimen already undertaken. Most of the physicians were very 

much aware about diagnosis and treatment of gynaecological problems. 

Physicians are required to pay more attention while prescribing drugs involved in long term therapy with serious side 

effects such as antibiotics, steroidal drugs etc. Patients on long term drug therapy should undergo proper counseling and 

regular monitoring regarding the use of drugs. Pharmacoeconomical steps taken up by the hospital is highly appreciated. 

But care should be taken that the outcome of the therapy is not compromised while providing cost benefits to the patients. 

Keywords: Gynaecological diseases, Prescription pattern, Pharmacoeconomical evaluation, Patient safety. 

INTRODUCTION 

Irrational prescription of drugs is of common occurrence in 

clinical practice. [1] The study of prescribing pattern is an 

important component of medical audit which helps in 

monitoring, evaluating and making necessary modifications 

in the prescribing practices to achieve a rational and cost 

effective medical care. Auditing of prescriptions forms an 

important part of drug utilization studies. [2] The misuse of 

these agents leads to increased incidence of adverse effects, 

emergence of resistant strains and increase in cost of therapy. 

[3] In India and other Third World countries, women usually 

do not consult physicians or gynaecologists, due to inhibition 

or lack of perception of causes of disease. 

*Corresponding author: Mr. Rohit K. Verma, 

Faculty of Pharmacy, Babu Banarasi Das National Institute 

of Technology & Management, Lucknow, Uttar Pradesh, 

India-227105 Tel: 91-9997856074; 

E-mail: royal_mpcp@yahoo.co.in 

Their only contact with the health care system is through 

health workers for family welfare programs. [4] The lack of 

concern of health care planners for the prevention of 

gynaecological abnormalities can be traced to the lack of 

adequate information about rural health problems. Most data 

on morbidity among nonpregnant women are either hospital 

based [5-6] or selective for a particular disease. [4, 7] Occasional 

studies among the rural population in India have pointed out 

that gynaecological morbidity may be present in 92 % of the 

women. [6] 

Gynaecological health is an important component of any 

woman's health status. Gynaecological disorders can have a 

substantial impact on many aspects of quality of life, 

including reproductive ability, sexual functioning, mental 

health, and the ability to work and to perform routine 

physical activities. [8] 

More than a third of women in the United States will, at 

some point in their lives, develop a gynaecological problem 

that is severe or potentially problematic enough to lead to 

136

Verma et al. / Evaluation of Prescription Pattern and Cost-Effectiveness.... 

hysterectomy. Several studies have been conducted to 

examine gynaecological conditions associated with 

hysterectomy. [9] 

These studies have consistently found that the most common 

principle diagnosis among women having hysterectomy is 

uterine fibroids. Other common indications for hysterectomy 

are uterine prolapsed, endometriosis, menstrual disorder and 

cancer. However, little is currently known about the 

prevalence of these and other gynecological conditions in the 

general population, and about the Impact of these conditions 

on health care system usage. 

Drugs play an important role in protecting, maintaining and 

restoring health. Prescription writing is a science and an art, 

as it conveys the message from the prescriber to the patient. 

The treatment of diseases by the use of essential drugs, 

prescribed by their generic names, has been emphasized by 

the WHO and the National Health Policy of India. [10] The 

International Network for the Rational Use of Drugs 

(INRUD) generated indicators in three main drug use areas 

viz prescribing, patient care and drug systems. [11] 

The cost of drug prescription poses problems in developing 

countries such as India, which allocates only 0.9 % of its 

Gross Domestic Product (GDP), which is Rs. 200 per capita 

to health. [12] The allocation for meeting the cost of the drugs 

is even meager. Moreover the production of pharmaceutical 

preparations in India is grossly imbalanced and there is cut 

throat competition among drug companies, which breeds 

malpractice. Indian markets are flooded with over 70,000 

formulations, as compared to about 350 listed in the WHO 

essential drug list, and pharmaceutical companies encourage 

doctors to prescribe branded medicines, often in exchange for 

favors. This study was therefore undertaken with the 

objective to find out the prescription pattern and cost per 

prescription at different levels of health facilities in the 

public health facilities of Lucknow - the capital city of Uttar 

Pradesh, a state in North India. 

This paper presents prevalence rates for categories of 

gynaecological conditions for women aged 18 to 60, reported 

as part of government maternity hospital. Additionally it 

examines the relationship between the gynecological 

conditions and various other factors, including prescription 

pattern and cost effectiveness among patients associated with 

gynaecological disorders. 

SUBJECTS AND METHODS 

A retrospective study of six months duration was undertaken 

in the department of gynaecology of “Virangna Jhalkaribai 

maternity hospital”, Lucknow (U.P.) and all prescriptions of 

outpatient of the hospital were analyzed for the antibiotics, 

antimicrobials, anti-inflammatory, analgesic, steroids, antiallergic, 

minerals and vitamins prescribed. 

The disease which were included for evaluation of 

prescription pattern were irregular menses, leucorrhea, 

vulvitis ,recurrent bleeding, uterus infection, endocervitis, 

feranculitis, pain in breast, excess cough in pregnancy, 

excess gastric discomfort in pregnancy. Surgical labour, 

Malignancy cases were considered for exclusion criteria. In 

another hand evaluation of cost effectiveness were also 

analyzed according to different categories of medicines. A 

comparative difference between medicine cost in hospital 

and medicine cost in market were analyzed for pharmacoeconomic 

point of view. Audited patient were in age group of 

18 yrs to 60 yrs. The survey was descriptive and data was 

summarized as counts and percentages, some of the questions 

had multiple options to choose from, therefore the sum total 

of percentage is not always 100 %. 


One hundred prescriptions were audited to find out the 

current trend in category of medicines usage in gynecological 

department and cost benefits availability to the patients. 

[1] Prevalence of Gynaecological Diseases /Related 

Complication and Prescribing Pattern in Different 

Disease States. 

(A) Prevalence of gynecological disease 

Out of 100 patients, 22 % were of irregular menses, 18 % of 

leucorrhea, 16 % of vulvitis, 15 % of osteoporosis, 11 % of 

recurrent bleeding, 8 % of uterus inflammation, 2 % of 

endocervitis, 2 % of feranculitis, 2 % of breast pain and 4 % 

of other diseases. (Fig. 1) 

Fig. 1: Prevalence of Gynaecological Diseases / Related Complications 

(B) Prescribing Pattern in Irregular Menses 

For the treatment of irregular menses gynaecologist 

prescribed antibiotics to 36.3 % patients, analgesics to 40.90 

%, anti-inflammatory to 18.18 %, antimicrobials to 22.72 %, 

steroids to 40.90 %, antispasmodic to 18.18 %, iron tonics to 

27.27 % and mineral & vitamins to 68.17 % patients (Fig. 2) 

Fig. 2: Prescribing Pattern in Irregular Menses 

(C) Prescribing Pattern in Leukorrhea 

Maximum patients those who were suffered from leucorrhea 

were prescribed antibiotics to 66.6 % patients, antimicrobials 



to 66.66 %, steroids to 16.66 %, iron tonics to 11.15 % and 

mineral & vitamins to 50 % patients. (Fig. 3) 

Fig. 3: Prescribing Pattern in Leukorrhea 

(D). Prescribing Pattern in Vulvitis 

In the treatment of vulvitis 87.5 % patients treated by 

antibiotics, analgesics were prescribed to 75 %, antimicrobial 

to 12.5 %, Anti-inflammatory 6.25 %, steroids 12.5 %, antiallergic 

6.25 %, iron tonics 12.5 %, minerals & vitamins 

93.75 %. (Fig. 4) 

Fig. 4: Prescribing Pattern in vulvitis 

(E) Prescribing Pattern in Osteoporosis 

For the treatment of osteoporosis, gynecologist prescribed 

antibiotics to 6.66 % patients, analgesics to 60 %, 

antimicrobials to 33.3 %, iron tonics 53.33 % and minerals & 

vitamins to 100 % of patients. (Fig. 5) 

Fig. 5: Prescribing Pattern in Osteoporosis 

(F) Prescribing Pattern in recurrent bleeding 

To treat recurrent bleeding gynaecologists prescribed 

antibiotics to 36.36 %, analgesics to 45.45 %, antimicrobials 

to 36.36 %, steroids 54.54 %, iron tonics 63.63 % and 

minerals& vitamins to 63.63 %. (Fig. 6) 

Fig. 6: Prescribing Pattern in recurrent bleeding 

(G) Prescribing Pattern in Uterus Inflammation 

Prescribing pattern in treatment of uterus swelling were 

antibiotics to 87.5 %, analgesic to 87.5 %, antimicrobials 

62.5 %, steroids 25 %, iron tonics 25 %, mineral &vitamins 

were prescribed to 87.5 % of patients. (Fig. 7) 

Fig. 7: Prescribing Pattern in Uterus Inflammation 

(H)Total Prescription Pattern 

Out of 100 patients, antibiotics were prescribed to 47 %, 

analgesics to 56 %, antimicrobials to 42 %, antiinflammatory 

to 6 %, anti-allergic to 6 %, antispasmodics to 

4 %, steroids to 23 %, minerals and vitamins to 80 % and 

iron tonics to 39 %. (Fig. 8) 

Fig. 8: Total Prescription Pattern 



(I) Visiting pattern and patient response of outpatients 

77 % patients were regularly visit to physician while 23 % 

patients visit occasionally and 79 % of patients were satisfy 

with their medications and 21 % patients responds negatively 

on satisfaction. 

[2] Evaluation of Cost Effectiveness 

(Pharmacoeconomics) In Medicines Provided By 

Maternity Hospitals 

(A) Cost Evaluation of Antibiotics 

Most common antibiotics prescribed by physicians were 

Ampicillin, Ciprofloxacin and Norflox. Medicine provided 

by hospital had better cost benefits compared to market cost. 

Ampicillin, Ciprofloxacin and Norflox medicine provided by 

hospital at 0.29 Rs/unit, 5.32Rs/unit, 02 Rs/unit respectively 

at low cost compare to market costs. (Fig 9) 

Fig. 9: Cost Evaluation of Antibiotics 

(B) Cost Evaluation of Antimicrobials and Antifungal 

drugs 

Antimicrobial prescribed by physician were Metronidazole, 

Fluconazole, Clotrimazole, Soframycin and Betadine had 

been prescribed at cheeper costs. Cost benefits were 0.52 

Rs/unit, 25 Rs/ unit, 24.5 Rs/unit, 2 Rs/ unit and 30.25 % 

Rs/unit respectively. (Fig. 10) 

Fig. 10: Cost Evaluation of Antimicrobials and Antifungals 

(C) Cost Evaluation of Analgesics, Antipyretics, and Antiallergic 

Most common drugs were prescribed in this category were 

Paracetamol, Brufen, Diclofenac, Voveron, Avil, Citrizine at 

cost benefits of 0 Rs/unit, 1 Rs/ unit, 0.09 Rs/ unit, 2 Rs/unit 

respectively. (Fig. 11) 

Fig. 11: Cost Evaluation of Analgesics, Antipyretics, Anti-allergics 

(D) Cost Evaluation of Steroidal Drugs: 

Steroidal medicine used for the treatment of gynecological 

patients were Ethamstylate, Northistorone, Placentrax inj, 

Inferon inj at cost benefits of 10.11 Rs/unit, 4.07 Rs/unit, 

9.18 Rs/unit, 1.75 Rs/ unit respectively. (Fig. 12) 

Fig. 12: Cost Evaluation of Steroidal Drugs 

(E) Cost Evaluation of Minerals & Vitamins 

Common drugs in this umbrella were vitamin E, Calcium 

lactate, B- complexes, Iron tonics and iron tablets at cost 

benefits of 5.04 Rs/unit, 0.85 Rs/unit, 0.33 Rs/unit, 0.37 

Rs/unit respectively. (Fig. 13) 

Fig. 13: Cost Evaluation of Minerals & Vitamins 



CONCLUSION 

It was observed that the drugs prescribed were correct for the 

current diagnosis and treatment. There were no any earlier 

records pertaining to their medical history and treatment 

regimen already undertaken. Most of the physicians were 

very much aware about diagnosis and treatment of 

gynaecological problems. 

Physicians are required to pay more attention while 

prescribing drugs involved in long term therapy such as 

antibiotics, steroidal drugs etc. Patients on long term drug 

therapy should undergo proper counseling and regular 

monitoring regarding the use of drugs. Pharmacoeconomical 

steps taken up by the Government Hospital is highly 

appreciated. But care should be taken that the outcome of the 

therapy is not compromised while providing cost benefits to 

the patients. 

REFERENCES 

1. Ramsay LE. Bridging the gap between clinical pharmacology and 

rational drug prescribing. Br J Clin Pharmac. 1993; 35:575 

2. WHO regional Publications. Studies in drug utilization. European 

Series No. 8. Copenhagen: WHO Regional Publications, 1979. 

3. Soumerai SB, Avorn J, Gortmaker S, Hawley S. Effect of 

government and commercial warning on reducing prescription 

misuse: the case of propoxyphene. Am J Pub Hlth. 1987; 77: 1518. 

4. Bang, R.A. Counselling and choice in family planning. Paper 

presented at international conference on better health for women 

and children through family planning. Nairobi, October 5-9, 1987. 

5. Mali S., Wahi P. N., Luthra U.K. Cancer of the uterine cervix. Ind J 

Cancer. 1968; 26:269-273. 

6. Wahi P.N., Luthra U.K., Mali S. Shamkin M.B. Prevalence and 

distribution of cancer of uterine cervix in Agra district, India. Ind J 

Cancer. 1972; 30:720-725. 

7. Sharma R.S., Dutta K.K., Gupta J.P., Mahelldra Dutta. A 

longitudinal study of morbidity pattern among housewives in rural 

Rajesthan. Indian Journal of Public Health. 1984; 2:28-30. 

8. Carlson KJ, Miller BA, Fowler FJ Jr. The Maine women's health 

study: II. outcomes of nonsurgical management of leiomyomas 

abnormal bleeding and chronic pelvic pain. Obstet Gynecol. 1994; 

83:566-572. 

9. Kjerulff K, Langenberg P, Guzinski G. The socioeconomic 

correlates of hysterectomiesin the United States. Am J Public 

Health. 1993; 83:106-108. 

10. Kishore J. National Health Programs of India. 6th ed. New Delhi: 

Century Publications; 2006; p. 370. 

11. Laing RO. Rational drug use: An unsolved problem. Trop Doct. 

1990; 20:101-3. 

12. Hota PK. New paradigm of Health. NRHM Newsletter 2005; 1:13 





ISSN 0975 1556 

Synthesis, Anti-Viral and Cytotoxic Studies of some 2-phenyl-3substituted 

quinazolin-4-(3H)-ones 

Yuvraj G. 1* , A. Meena 1 , Dillibabu 2 , Gayathiri Shiva Kumar 2 , Makesh Arvind 2 , 

Nema Rajesh Kumar 3 

1 K. K. College of Pharmacy, Department of Pharmaceutical Chemistry & Analysis, Chennai, Tamilnadu, India 

2 Chromosoft research centre, Drug discovery & Medicinal Chemistry Division, Chennai, Tamilnadu, India 

3 Rishiraj College of Pharmacy, Indore, Madhya Pradesh, India 

ABSTRACT 

A Series of Novel 2-Phenyl-3-Substituted Quinazolin-4-(3H) one derivative were synthesized and screened for anti-viral 

activity against panel of human pathogenic viruses. The structures of the synthesized compounds were characterized by 

means of their IR, 1 H-NMR data. The anti-HIV activities of the new compounds were screened in vitro anti-viral activity 

against replications of HIV-1 (IIIB) and HIV-2 (ROD) in MT-4 cells using AZT-as standard. All the compounds displayed 

cytostatic properties in T-lymphocytes MT-4 cells. The compound 4- [4-oxo-2-phenylquinazolin-3(4H)-yl amino] methyl 

amino benzoic acid (QPAB) (CC50=11.90 µg/ml) was found to be more toxic in this series. 2-amino-3-phenyl quinazolin- 

4(3H)-one (BN) exhibited anti-viral activity against Herpes Simplex virus-1,2 and Vaccinia virus in HEL cells at the 

concentration of 10 and 12 µg/ml, whereas cytotoxicity was found to be 100 µg/ml (SI = 10). Among these compounds, 

compounds (QIS and QMB) exhibited anti-viral activity against Vesicular Stomatitis virus in HeLa cells at the 

concentration of 12 µg/ml, whereas cytotoxicity was found to be 100 µg/ml (SI = 9). 

Keywords: Quinazolin derivatives, Anti-Viral, Cytotoxicity Studies. 

INTRODUCTION 

Quinazolin-4-(3H)-One is a versatile lead molecule for the 

design of potential bioactive agents. 2-Phenyl-3-Substituted 

Quinazolin-4-(3H)-ones were reported to have anti-HIV [1- 4] , 

anti-cancer [5-7] and anti-viral [8-13] properties. A large number 

of quinazolines have been synthesized and studied for wide 

range of anti-viral activity, but the anti-viral activities of 

quinazolines against viruses has not been well explored 

[1-7, 9- 

10] . 

Anthranilic acid reacts with benzoylchloride to form 2phenyl-1, 

3-benzoxazin-4-one by N-acylation followed by 

dehydrative cyclisation. 2-phenyl-3-amino quinazolin-4(3H)one 

derivatives were synthesized by condensation of the 

compounds containing hydrazine hydrate with 2- phenyl-1, 

3-benzoxazine-4-one. A series of 2-phenyl-3-substituted 

quinazoline-4(3H)-one derivative were synthesized by 

condensation of the compounds containing primary aromatic 

amino group and formaldehyde with 2-phenyl-3-amino 

quinazolin-4(3H)-one by mannich reaction. 


*Corresponding author: Mr. Yuvraj G., 

Chromosoft research centre, Drug discovery & Medicinal 

Chemistry Division, Chennai, Tamilnadu, India 

E-mail: yuvas2010@chromosoftinfo.com 

SYNTHESIS OF 2-PHENYL-3-SUBSTITUTED 

QUINAZOLIN-4-(3H) - ONE DERIVATIVES 

An equimolar (0.01 mol) mixture of quinazoline, different 

sustituents and formaldehyde was refluxed for 6 h with 10 ml 

of ethanol in acidic condition. The mixture was cooled to 

room temperature and poured into crushed ice, filter and then 

washed with water. The solid thus obtained was 

recrystallised from ethanol. The yield and melting point was 

predicted in Table-1. 

Melting points were determined in open capillary tubes on a 

Thomas-Hoover melting point apparatus and are uncorrected. 

IR spectra were recorded for KBr pellets on a (SHIMADZU- 

8400s) FT-IR spectrophotometer, 1 H-NMR spectra were 

determined BRUKER AMX 400 MHZ with 

tetramethylsilane as an internal standard. The sample is 

dissolved in DMSO-d6 and the 1 H -NMR value is measured 

in δ ppm. 

BN: IR (KBr) : 3212 (NH), 1598 (C=N), 1606 (C=C), 1664 

(C=O); 1 H NMR (DMSO-d6) : 6 – 8.7 (m, 9H, Ar-H), 5.9 (s, 

2H, NH2). 

QSD: IR (KBr) : 3397 (NH), 1508 (C=N), 1603 (C=O), 1080 

(C-Alkyl) , 1432 (SO2) ; 1 H NMR (DMSO-d6) : 6.3 – 8.7 (m, 

13H, Ar-H), 4.7 (s, 1H, NH), 3.4 (s, 2H, -CH2-). 

QPH : IR (KBr) : 3206 (NH), 1528 (C=N), 1729 (C=O), 

1603 (C=C),); 1 H NMR (DMSO-d6) 6.9 – 8.1 (m, 13H, Ar- 

H), 11.2 (s, 1H, NH), 4.4 (s, 2H, -CH2-). 

141

Yuvraj et al. / Synthesis, Anti-Viral and Cytotoxic Studies of some.... 

QBA: IR (KBr) : 3305 (NH), 1723 (C=O), 1573 (C=N), 

1633 (C=C); 1 H NMR (DMSO-d6) : 6.9 – 8.1 (m, 14H, Ar- 

H), 3.2 (s, 2H, -CH2-), 4.9 (s, 1H, NH). 

QNF : IR (KBr) : 1481 (C=N), 1630 (C=C), 1718 (C=O), 

1092 (C-F); 1 H NMR (DMSO-d6) : 6.9 – 8 (m, 11H, Ar-H), 

9.4 (s, 1H, NH), 4.3 (s, 2H, -CH2-), 9 (s, 1H, COOH); 

QBI: IR (KBr) : 3249 (NH), 1591 (C=N), 1676 (C=O), 1591 

(C=C); 1 H NMR (DMSO-d6) : 6.6-8.7 (m, 13H, Ar-H), 4.2 

(s, 2H, -CH2-), 4.8 (s, 1H, NH); 

O 

N 

N 

NH 2 

3-amino-2-phenyl - quinazolin-4-(3H)-one 

O 

N 

QBT: IR (KBr) : 3143 (NH), 1370 (C=N), 1655 (C=O), 1521 

(C=C); 1 H NMR (DMSO-d6) : 6-8.8 (m, 13H, Ar-H), 3.4 (s, 

2H, -CH2-), 4.7 (s, 1H, NH);QNA: IR (KBr) : 3377 (NH), 

1509 (C=N), 1672 (C=O), 1445 (C=C); 1 H NMR (DMSO-d6) 

: 6.5-8.8 (m, 13H, Ar-H), 4.2 (s, 2H, -CH2-), 4.8 (s, 1H, 

NH). 

+ HCHO + 

N 

Reaction Scheme 

HCl / ethanol 

NH CH 2 

H R 

Details of the various synthesized compounds 

CODE R CODE R 

QSM HN SO 2NH 

QSD HN SO 2NH 

QBA HN C 

QCP 

QBT 

N 

O 

N N 

F 

N 

O 

N 

N 

N 

N 

N 

O 

COOH 


R 

QSS HN SO 2NHCOCH 3 

QPH N 

QBI 

QNF 

N 

O 

O 

N 

N N 

F 

O 

N 

COOH 

QPAB HN COOH

QONA 

QIP 

QAA 

HN 


N 

HN 

O 2N 

HOOC 

QSA HN SO 2NH 2 

N 

N 

QIS 

QMB 

QNI 


HN 

O 

C 

N 

N 

N 

N 

O 

O 

SH 

QBR HN Br 

Table 1: Characterization Data of the Synthesized Compounds 

Compound Code Molecular Formula % Yield M.P. (ºC) Rf Value Molecular Weight 

QSM C25H22N6O4S 67.6 180 – 182 0.64 302 

QSS C23H21N5O4SNa 70.7 160 – 164 0.60 486 

QSD C27H25N7O3S 89.5 206 – 210 0.56 527 

QPH C23H16N4O3 57.3 155 – 158 0.44 396 

QBA C22H18N4O2 61.3 120 – 122 0.38 370 

QCF C31H29N6O4F 58.1 221 – 226 0.62 581 

QNF C31H27N6O4F 73.6 210 – 215 0.53 579 

QBI C22H17N5O 80.4 100 – 104 0.47 367 

QBT C21H16N6O 53.4 166 – 170 0.40 368 

QPAB C22H18N4O3 58.2 180 – 184 0.47 366 

QNA C21H17N5O3 53.4 110 – 115 0.84 367 

QIS C23H16N4O3 48.7 140 – 146 0.47 376 

QIP C29H20N6O 55.7 100 – 104 0.76 468 

QMB C22H17N5OS 85.8 170 – 176 0.55 379 

QAA C22H18N4O3 61.3 231 – 235 0.60 366 

QNI C21H17N5O2 62.8 132 – 136 0.49 371 

QSA C21H19N5O3S 53.4 223 – 227 0.63 391 

QBR C21H17N4OBr 55.3 106 – 110 0.86 401 

BIOLOGICAL INVESTIGATION 

Anti-HIV Assay 

Anti HIV assay compounds were tested for their inhibitory 

effects against replication of HIV-1 (IIIB) and HIV-2 (ROD) 

in MT-4 cells. The MT-4 cells were grown and maintained in 

RPMI-1640 DM Medium supplemented with 10 % (v/v) 

heat-inactivated Fetal Calf Serum (FCS), 2 mM-glutamine, 

0.1 % sodium bicarbonate and 20 mcg/ml gentamicin 

(culture medium). Inhibitory effect of test compounds on 

HIV-1 and HIV-2 replications were monitored by inhibition 

of virus-induced cytopathic effect in MT-4 cells and were 

estimated by MTT assay. Briefly, 50 ml of HIV-1 and HIV-2 

(100-300 CCID50) were added to a flat-bottomed microtiter 

tray with 50 mcl of medium containing various 

concentrations of compounds. MT-4 cells were added at a 

final concentration of 6 × 10 5 cells/mL. After 5 days of 

incubation at 37 o C, the number of viable cells were 

determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl 

tetrazolium bromide (MTT) method. Cytotoxicity of test 

compounds against mockinfected MT-4 cells was also 

assessed by the MTT method. Compounds were evaluated 

for their inhibitory effect on the replication of HIV-1 and 

HIV-2 in human MT-4 cells. The anti-HIV and cytotoxicity 

data are presented in Table 2. 

Anti-Viral Assay 

Anti-viral activity and cytotoxicity of the synthesized 

compounds were determined by an in vitro cell culture 

technique. The anti-viral assays were based on inhibition of 

virus-induced cytopathicity in HeLa cells (VSV and RSV), 

HEL cells (HSV-1 and HSV-2), Vero cells (Parainfluenza-3, 

Reovirus-1, Sindbis virus, Coxsackie virus B4 and Punta 

Toro virus). Briefly, confluent cell culture in 96-wells 

microtiter plates were inoculated with 100 CCID50 of virus, 1 

CCID50 being the virus dose required to infect 50 % of the 

cell cultures. After 1 h, virus adsorption period, residual 

virus was removed, and the cell cultures were incubated in 

the presence of varying concentrations (400, 200 and 100 

µg/ml) of the test compounds. Viral cytopathicity was 

recorded as soon as it reached completion in the control 

virus-infected cell cultures that were treated with the test


compounds. The anti-viral and cytotoxicity data are 

presented in Tables 3-5. 

Table 2: Anti-HIV Activity and Cytotoxicity of Synthesized 

Compounds in MT-4 Cells 

Compound code Strain EC50 a (µg/ml) CC50 b (µg/ml) 

QAA 

IIIB 

ROD 

>57.18 

>57.18 

57.18 ± 6.32 

57.18 ± 6.32 

QBA 

IIIB 

ROD 

>106.10 

>106.10 

106.10 ± 11.18 

106.10 ± 11.18 

QBI 

IIIB 

ROD 

>71.85 

>71.85 

71.85 ± 1.16 

71.85 ± 1.16 

QBR 

IIIB 

ROD 

>60.63 

>60.63 

60.63 ± 5.59 

60.63 ± 5.59 

QBT 

IIIB 

ROD 

>61.33 

>61.33 

61.33 ± 4.48 

61.33 ± 4.48 

QCF 

IIIB 

ROD 

>52.48 

>52.48 

52.48 ± 6.75 

52.48 ± 6.75 

QIP 

IIIB 

ROD 

>74.20 

>74.20 

74.20 ± 2.43 

74.20 ± 2.43 

QIS 

IIIB 

ROD 

>27.93 

>27.93 

27.93 ± 25.87 

27.93 ± 25.87 

QNA 

IIIB 

ROD 

>68.70 

>68.70 

68.70 ± 7.95 

68.70 ± 7.95 

QMB 

IIIB 

ROD 

>66.65 

>66.65 

66.65 ± 7.16 

66.65 ± 7.16 

QNF 

IIIB 

ROD 

>61.80 

>61.80 

61.80 ± 3.40 

61.80 ± 3.40 

QNI 

IIIB 

ROD 

>125 

>125 

>125 

>125 

QPAB 

IIIB 

ROD 

>11.90 

>11.90 

11.90 ± 0.42 

11.90 ± 0.42 

QPH 

IIIB 

ROD 

>125 

>125 

>125 

>125 

AZT 

IIIB 

ROD 

0.0012 

0.00062 

65.9 ± 6.1 

65..9 ± 6.1 

a 

Concentrations required to inhibit the cytopathic effect of HIV-1(IIIB) in 

MT-4 cells by 50 %. 

b 

Concentrations required to cause cytotoxicity to 50% of the MT-4 cells 

All the Values are SD of two independent experiments. 

IIIB – HIV-1, ROD – HIV-2 

Table 3: Cytotoxicity and anti-viral activity of compounds in HeLa cell 

cultures 

EC50 b Minimum 

(µg/ml) 

Compound 

Code 

Cytotoxic 

Concentratio 

n a (µg/ml) 

Vesicular 

stomatitis 

virus 

Coxsackie 

virus B4 

Respiratory 

syncytial 

virus 

BN 100 >20 >20 >20 

QAA 100 >20 >20 >20 

QBA ≥100 >100 >100 >100 

QBI ≥20 >20 >20 >20 

QBR 100 >20 >20 >20 

QBT >100 >100 >100 >100 

QCF 100 >20 >20 >20 

QIP ≥20 >20 >20 >20 

QIS 100 12 >20 >20 

QNA 100 >20 >20 >20 

QMB 100 12 >20 >20 

QNF 100 >20 >20 >20 

QNI 100 >20 >20 >20 

QPAB ≥20 >20 >20 >20 

QPH 100 >20 >20 >20 

QSA 100 >20 >20 >20 

QSD 100 >20 >20 >20 

QSM 100 >20 >20 >20 

QSS >100 >100 >100 >100 

DS-5000 >100 4 >100 4 

(S)-DHPA 

(µM) 

>250 112 >250 >250 

Ribavirin 

(µM) 

>250 12 146 10 

a 

Required to cause a microscopically detectable alteration of normal cell 

morphology , b Required to reduce virus-induced cytopathogenicity by 50 

%. 

Table 4: Cytotoxicity and anti-viral activity of compounds in HEL 

cell cultures 

EC50 b (µg/ml) 

Compound 

Code 

Minimum 

cytotoxic 

concentrati 

on a (µg/ml) 

Herpes 

simplex 

virus-1 

(KOS) 

Herpes 

simple 

x 

virus-2 

(G) 

Vacci 

nia 

virus 

Herpes 

simplex 

virus-1 

TK - 

KOS 

ACV r 

BN >100 10 10 12 10 

QAA 100 >20 >20 >20 >20 

QBA >100 >100 >100 >100 >100 

QBI 100 >20 >20 >20 >20 

QBR 100 >20 >20 >20 >20 

QBT >100 >100 >100 >100 >100 

QCF 100 >20 >20 >20 >20 

GIP 100 >20 >20 >20 >20 

QIS 20 >4 >4 >4 >4 

QNA >100 >100 >100 >100 >100 

QMB >100 >100 >100 >100 >100 

QNF 100 >20 >20 >20 >20 

QNI 100 >20 >20 >20 >20 

QPAB 100 >20 >20 >20 >20 

QPH 100 >20 >20 >20 >20 

QSA 100 >20 >20 >20 >20 

QSD 100 >20 >20 >20 >20 

QSM 100 >20 >20 >20 >20 

QSS 100 >20 >20 >20 >20 

Brivudin 

(µM) 

>250 0.04 50 10 >250 

Ribavirin 

(µM) 

>250 >250 >250 >250 >250 

Acyclovir 

(µM) 

>250 2 2 7 2 

Ganciclovir 

(µM) 

>100 0.06 0.1 >100 12 

a 


morphology., b Required to reduce virus-induced 

Table 5: Cytotoxicity and anti-viral activity of compounds in Vero cell 

cultures 

Compound 

Code 

Minimum 

cytotoxic 

concentration a 

(µg/ml) 

Herpes 

simplex 

virus-1 

(KOS) 

EC50 b (µg/ml) 

Herpes 

simplex 

virus-2 

(G) 

Vacci 

nia 

virus 

Herpes 

simplex 

virus-1 

TK - 

KOS 

ACV r 

BN >100 10 10 12 10 

QAA 100 >20 >20 >20 >20 

QBA >100 >100 >100 >100 >100 

QBI 100 >20 >20 >20 >20 

QBR 100 >20 >20 >20 >20 

QBT >100 >100 >100 >100 >100 

QCF 100 >20 >20 >20 >20 

GIP 100 >20 >20 >20 >20 

QIS 20 >4 >4 >4 >4 

QNA >100 >100 >100 >100 >100 

QMB >100 >100 >100 >100 >100 

QNF 100 >20 >20 >20 >20 

QNI 100 >20 >20 >20 >20 

QPAB 100 >20 >20 >20 >20 

QPH 100 >20 >20 >20 >20 

QSA 100 >20 >20 >20 >20 

QSD 100 >20 >20 >20 >20 

QSM 100 >20 >20 >20 >20 

QSS 100 >20 >20 >20 >20 

Brivudin 

(µM) 

>250 0.04 50 10 >250 

Ribavirin 

(µM) 

>250 >250 >250 >250 >250 

Acyclovir 

(µM) 

>250 2 2 7 2 

Ganciclovir 

(µM) 

>100 0.06 0.1 >100 12 

a 


morphology. 

b 

Required to reduce virus-induced cytopathogenicity by 50 %. 




We report our results from a study of 2-phenyl-3-substituted 

quinazoline-4(3H)-one derivative. The inhibitory effect of 

anti-viral drugs on the HIV-induced cytopathic effect (CPE) 

in human lymphocyte MT-4 cell culture was determined by 

the MT-4/MTT-assay. Cytotoxicity of test compounds 

against mock-infected MT-4 cells was also assessed by the 

MTT method. All the compounds displayed cytostatic 

properties in T-lymphocytes MT-4 cells. The compound 4- 

((4-oxo-2-phenylquinazolin-3(4H)-yl amino) methyl amino 

benzoic acid (QPAB) (CC50=11.90 µM) was found to be 

more toxic in this series. The synthesized compounds were 

tested for anti-viral against HeLa Cells (VSV and RSV) HEL 

cells (HSV-1 and HSV-2) and Vero cells (Parainfluenza-3, 

Reovirus-1, Sindbis virus, Coxsackie virus B4 and Punta 

Toro virus. 2-amino-3-phenyl quinazolin-4(3H)-one (BN) 

exhibited anti-viral activity against Herpes Simplex virus-1,2 

and Vaccinia virus in HEL cells at the concentration of 10 

and 12 µg/ml, whereas cytotoxicity was found to be 100 

µg/ml (SI = 10). Among these compounds, compounds (QIS 

and QMB) exhibited anti-viral activity against Vesicular 

Stomatitis virus in HeLa cells at the concentration of 12 

µg/ml, whereas cytotoxicity was found to be 100 µg/ml (SI 

= 9). 

ACKNOWLEDGEMENTS 

The author is grateful to the NMR Research Centre, Indian 

Institute of Science Bangalore for providing the NMR facility 

for this research work. 

REFERENCES 

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2. Alagarsamy V, Pathak US, Pandaya SN, Sriram D, De Clercq E. 

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quinazolin-4(3H) ones. International 

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2-dihydro-2-oxo-3H-indol-3-ylidene) amino]-N-(4, 6-dimethyl-2pyrimidin-2-yl)benzenesulphonamide 

and its derivatives. Antiviral 

Chemistry & Chemotherapy 2006; 17:269-274. 

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Agents and Chemotherapy 2007; 51:3168-3176. 





ISSN 0975 1556 

Embelin - An HPLC Method for Quantitative Estimation in Embelia 

ribes Burm. F. 

Shelar R. 1* , Maurya C. 1 , Tekale P. 1 , Katkar K. 2 , Naik V. 2 , Suthar A. 2 , Chauhan V. S. 1 

1 Department of Chemistry, G. N. Khalsa College of Art, Science & Commerce, Kings Circle, Matunga, Mumbai – 400 019, 

Maharashtra, India. 

2 Piramal Life Sciences Limited, 1, Nirlon Complex, Off. Western Express Highway, Goregaon (East), Mumbai – 400 063, 

Maharashtra, India 

ABSTRACT 

An RP-HPLC method with photodiode array detection was established for the determination of major constituent, Embelin 

in Embelia ribes samples. The Embelin was separated by using isocratic mode consisting of 0.1 % trifluoroacetic acid in 

water and methanol (in proportion of 88:12) at a flow rate of 1.0 mL/min. Under these conditions, a plot of integrated peak 

area versus concentration of Embelin was found to be linear over the range of 5.0-75.0 μg/mL, with a relative standard 

deviation of 0.61-0.96 %. The limit of detection was 20 ng on column and the limit of quantitation was 50 ng on column. 

The determination of the Embelin content in various solvent extracts exhibited a mean content of 0.44-33.0 % w / w. 

Recovery experiments led to a mean recovery rate of 96.49 ± 2.42 %. The proposed method is less time-consuming, 

sensitive and reproducible and is therefore suitable for routine analysis of Embelin in various extracts of E. ribes. 

Keywords: Embelia ribes; Vidang; Pharmaceutical analysis; Embelin; High performance liquid chromatography. 

INTRODUCTION 

The family Myrsinaceae consists of nearly 1000 species of 

trees and shrubs spread over 33 genera including four genera 

namely Myrsine, Maesa, Rapanea and Embelia, which are 

widely used in herbal medicines. [1] Embelia ribes Burm. F. is 

the most correlated species of Vidanga (Sanskrit), a drug 

used in Ayurveda, Siddha as well as in Unani medicine 

system as anthelmintic and to cure skin diseases. [2-3] Embelia 

ribes, a natural source of Embelin has restricted and sporadic 

distribution mainly in the Western Ghats and Eastern 

Himalayas. [4] It is also a critically endangered species of 

conservation importance in India. [5-6] 

Embelia ribes (Embelia fruit) is used in India and in the 

Eastern Colonies for numerous traditional medicinal uses. 

The fruit are used as an anthelmintic, diuretic, carminative, 

contraceptive, anti-bacterial, anti-inflammatory and antiastringent 

as reported in various literatures. [7] Also fruit 

decoction is useful in fevers and diseases of chest and skin. 

Infusion of roots is used for cough and diarrhoea. Aqueous 

extract of the fruits showed antibacterial and antifertility 

activities. 

*Corresponding author: Mr. Rahul Shelar 

Department of Chemistry, 

G. N. Khalsa College of Art, Science & Commerce, 

Kings Circle, Matunga, Mumbai – 400 019, Maharashtra, 

India; Tel: + 91 – 98338 96985 

E-mail: shelar_rahul@yahoo.com 

Seeds were found to possess antibiotic and antitubercular 

properties. A gum obtained from the plant is used as a 

warming remedy in the treatment of dysmenorrhoea. 

Decoction of the leaves is used as a blood purifier. [8] 

Embelin (embelic acid / 2, 5-dihydroxy-3-undecyl-2, 5cyclohexadiene-1, 

4- benzoquinone) has been isolated in 

Embelia ribes Burm. f. (Fig. I) and other species of 

Myrsinaceae family. [9] Embelin revealed antifertility, 

analgesic, anti-inflammatory, antioxidant and antitumour 

properties. [10-11] Embelin as such evaluated against 

Heligmosomoides polygyrus in mice significantly reduced the 

total worm counts. [12] Embelin reported to be a potent oral 

contraceptive. [13] Embelin inhibited pregnancy and also 

possessed anti-estrogenic and weak progestational activity. 

[14] There are no chromatographic methods available for 

quantitation of Embelin in Embelia ribes Burms. Hence, the 

present work focuses on development and validation of highperformance 

liquid chromatographic method with photodiode 

array detection for the determination of this major 

constituent, Embelin, in Embelia ribes extract. 

Fig. 1: Structure of Embelin 

146

Shelar et al. / Embelin - An HPLC Method for Quantitative Estimation.... 

MATERIAL AND METHODS 

Embelin was isolated, purified and structural confirmation 

was carried out. [15] The working standard purity was 

determined to be more than 98.0 % by and hence, was 

considered as working standard for the analysis purpose. 

Chemicals 

HPLC grade Methanol from Merck Specialty Private Ltd 

(Mumbai, India) and Tri-fluoro Acetic acid from 

Spectrochem Private Ltd (Mumbai, India). Deionized water 

was obtained with an in-house Milli-Q Nanopure (Millipore, 

Bedford, MA, USA). 

Collection and authentication of plant 

The fruit of Embelia ribes was purchased from local market 

and botanical authentication was performed at the Botany 

Department of Piramal Life Sciences Ltd, Mumbai. Collected 

plant material (fruits) were dried under shade and grounded 

in to # 18 powder. 

Preparation of the extract 

The 100 g of Embelia ribes fruit dried powder was extracted 

with 500 ml solvent (Hexane, Chloroform, Methanol and 

Water) by stirring at 50°C for 1 h. The filtered extract was 

concentrated under reduced pressure to remove the solvent. 

The extraction carried out for two times with the abovementioned 

protocol. The extract was obtained by drying the 

concentrated pooled extract under vacuum. These extracts 

were used for estimation of Embelin content. 

Equipment 

The HPLC system consisted of an Agilent 1200 series liquid 

chromatograph equipped with an autosampler, a photodiode 

array detector and column (Unisphere Aqua C18, 3 µm, 4.6 × 

150 mm, Agela Technologies, USA). The absorption was 

measured in a full spectrum (200-400 nm) or at 288 nm for 

Embelin. The chromatographic data was recorded and 

processed with EZChrom Elite software. 

Chromatography 

Analyses were carried out at 30°C on a Aqua C18 column (3 

µm, 100 Aº 4.6 × 150 mm, Cat No UA315059-0, Serial No 

M9310515BI0019) (Agela Technologies, USA). The mobile 

phases consisted of Methanol (A) and 0.1 % TFA (B) (in 

proportion of 88:12 v/v) was degassed before used. The flowrate 

was kept at 1.0 ml/min, temperature of column was set at 

30°C±2°C and the injection volume was 10μl. Quantification 

of Embelin was carried out at 288 nm. The peak in the HPLC 

chromatogram of Embelia ribes extract was identified by 

comparing the retention time and UV spectra of Embelin in 

the samples with working standard of Embelin. The peak 

purity was checked by PDA software. 

Standard solution 

Embelin, working standard, in the range of 5 mg was 

accurately weighed or transferred into a 25 ml volumetric 

flask and dissolved in methanol to obtain solution with 0.2 

mg / ml concentration of Embelin. This solution was then 

further diluted to obtain the concentration 0.05 mg/ml of 

Embelin stock solution and stored at −20°C and brought to 

room temperature before use. In the same way, three sets of 

control for Embelin was prepared from a separate stock, so as 

to lie in the lowest, middle and highest regions of the 

calibration curves. Further standard solutions are prepared 

freshly each day by appropriate dilution of stock solution 

with methanol for intraday as well as interday analysis. 

Sample solutions (Evaluation of various extracts) 

Test sample preparation 

Different Embelia ribes extracts (25 mg) were exactly 

weighed into a 25-ml volumetric flask fitted with a glass 

stopper. (Borosil cat. # 14-962-26F) and volume is made by 

methanol and extracted using a sonicator 5 min and allowed 

to stand for 5 min. The mixture was then filtered through 

Whatmann no.42 filter paper and the desired concentration 

(0.5 mg/ml) is obtained. Then 10 μl of the resulting solution 

was subjected to HPLC analysis and the concentration of the 

major constituent, Embelin, in different Embelia ribes 

extracts were calculated based on the equations for the 

calibration curves. 

Linearity and limit of quantitation 

For a long-term use of the analytical method a rigorous 

validation is indicated and requires the following procedures. 

For the preparation of calibration curve the stock solution 

was diluted freshly with methanol to obtain a set of 6 

calibration standards. These standards were measured and the 

integrated peak areas were plotted against the corresponding 

concentrations of the injected standards. The complete 

procedure was repeated on three consecutive days. The so 

obtained three calibration curves were used to calculate a 

mean calibration graph. The limit of quantification was 

defined as that lowest concentration where accuracy better 

than 20.0% was achieved. [16] 

Intraday and interday analysis using Embelin 

Three different concentrations using a different stock 

solution of Embelin were prepared (25.0; 50.0 and 75.0 

µg/mL). For the determination of the intraday precision and 

accuracy three replicates of the standard solution were 

analyzed at the same day. The precision and the accuracy of 

the interday analysis were determined by analyzing the 

standard solution on 3 different days. 

Stability 

Embelin (500 µg) and Embelia ribes methanolic extract (5 

mg) was transferred into a 10-ml volumetric flask and made 

up to volume with methanol. The sample solutions were put 

at 30°C and analyzed on 16 and 24 h to observe the stability 

of sample solutions. 

Robustness and Ruggedness studies 

Robustness and ruggedness parameters were applied by 

making small deliberate changes of the conditions (mobile 

phase composition, column temperature, different lot of 

stationary phase, analyst and equipment) to validate the 

method. 


Chromatography 

Under the current conditions, Embelin along with other 

phytoconstituents of E. ribes extract were eluted within 10 

min. Fig. I & II shows the typical LC chromatograms of 

working standard of Embelin and various extracts of E. ribes 

samples at 288 nm respectively. Fig. 7 shows the UV 

spectrum of working standard Embelin along with its UV 

maxima at 288 nm and peak purity at three different levels of 

peak. Various extracts (Heaxane, Chloroform, Methanl and 

Water extract) of Embelia ribes were analysed by the 

proposed method and the data are recorded in Table I. 

Limit of detection and limit of quantitation 

The limit of detection (LOD) was obtained by successively 

decreasing the concentration of Embelin as long as a signalto-noise 

ratio of 3:1 appeared. The LOD was found to be 20 

ng on column (volume of injection is 10 μL; corresponding 

to a concentration of 2 µg / ml). The limit of quantitation 

(LOQ) was found to be 50 ng on column (volume of 



injection is 10 μL; corresponding to a concentration of 5 

μg/ml) of Embelin. 

Linearity and reproducibility 

The calibration was based on the duplicate analysis of 

calibration working solutions at six concentration levels on 3 

consecutive days for Embelin (5-75 μg/ml) with regression 

(r 2 ) more than 0.9998. (Fig. III) The reproducibility of the 

method was evaluated by analyzing three sets of controls 

(n=3) on 3 separate days (n=3) and calculating the relative 

standard deviation (RSD). As shown in Table II, the RSD 

(%) were founded in the range of 0.49 – 3.61 %. 

mAU 

260 

240 

220 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

-20 

0 1 2 3 4 5 6 7 8 9 10 

Minutes 

Fig. I: LC Chromatogram of working standard Embelin along with its 

UV spectrum 

mAU 

500 

450 

400 

350 

300 

250 

200 

150 

100 

50 

0 

-50 

-50 

0 1 2 3 4 5 6 7 8 9 10 

Minutes 

Fig. II: LC Chromatogram of Methanolic extract of E. ribes 

Table I: Percentage of Embelin in different samples of Embelia 

ribes extracts 

Sr. Sample name 

Concentration of 

test sample 

Embelin content 

(% w/w) 

1 Hexane extract 100 µg / ml 10.10 

2 

Chloroform 

extract 

50 µg / ml 33.34 

3 

Methanol 

extract 

100 µg / ml 14.31 

4 Water extract 500 µg / ml 0.46 

7000000 

6000000 

5000000 

4000000 

3000000 

2000000 

1000000 

mAU 

Embelin @ 3.85 

y = 8527.3x - 250246 

R 2 = 0.9998 

0 

0 100 200 300 400 500 600 700 800 

Fig. III: Calibration curve of Embelin with respect to the area under 

curve at various concentration 

500 

400 

300 

200 

100 

0 

-100 

200 250 300 

nm 

350 

500 

400 

300 

λmax – 

200 

100 288 nm 

-100 

Embelin @ 3.85 min 

0 

mAU 

260 

240 

220 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

-20 

500 

450 

400 

350 

300 

250 

200 

150 

100 

50 

0 

mAU 

mAU 

Table II: Precision and accuracy of the recalculated calibration 

samples 

Given Found, mean ± Precision Accuracy 

(μg/mL) S.D. (μg/mL) (R.S.D., %) (% Deviation) 

25 24.730 ± 0.89 3.6114 - 1.078126 

50 48.249 ± 0.23 0.4911 - 3.501832 

75 71.046 ± 1.27 1.7930 - 5.270758 

Intraday and interday analysis using Embelin 

Furthermore the precision and accuracy of the intraday and 

interday analysis were investigated on the basis of a set of 

standard solution. The results given in Table III stands for a 

quite good trueness of the proposed method particularly 

considering interday and intraday analysis. 

Table III: Intraday and interday precision and accuracy of Embelin 

Intraday Interday 

Given (μg/ml) Precision 

(R.S.D., %) 

Accuracy 

(Percent 

deviation) 

Precision 

(R.S.D., %) 

Accuracy 

(Percent 

deviation) 

25 2.569 - 1.98 2.803 - 2.09 

50 1.096 - 2.60 1.540 - 2.72 

75 1.643 - 3.78 1.884 - 4.06 

Stability 

In the current assay, analyses of stability samples in methanol 

on regular interval (16 and 24 h) revealed that the Embelin, a 

major constituents in the methanolic extract of Embelia ribes 

is stable in solution form with relative standard deviation 

(RSD (%) 1.85 (n = 3) for Embelin at 30°C respectively. 

Robustness and Ruggedness studies 

The method was found to be re-producible from one analyst 

to another. The low values of R.S.D. (1.873 % - 3.219 %) 

obtained after small deliberate changes of the conditions 

(mobile phase composition, column temperature, different lot 

of stationary phase, analyst and equipment) used for the 

method indicated its robustness. 

CONCLUSION 

The need for quality assurance, including confirmation of the 

label strength and content uniformity has long been 

recognized even for herbal medicinal products. A highperformance 

liquid chromatography method has been 

developed for the detection and quantitation of major 

constituents of Embelia ribes extract using a photodiode 

array detector. Analysis of Embelia ribes extract samples 

with the proposed method does assure prolong life of column 

and system due to lower percentage of acid in mobile phase 

and Embelin can be quantitated successfully, using standard 

calibration curve. The method was found to be specific and 

suitable for routine analysis because of its simplicity, and 

reproducibility. The relative standard deviation for the 

investigated Embelia ribes extracts indicates that the method 

is precise and reproducible. 

REFERENCES 

1. Januaro AH, Fatima DM, De Silva F, Viera PC, Fernandes JB, 

Dammarane and cycloartane triterpenoids from three Rapanea 

species. Phytochemistry 1992; 4: 1251-1253. 

2. Githiori JB, Höglund J, Waller PJ, Baker LR, Vet. Parasitol. 2003; 

118 (3-4): 215-226. 

3. Swamy KHM, Krishna V, Shankarmurthy K, Rahiman AB, 

Mankani KL, Mahadevan KM, Harish BG, Naika RH, Wound 

healing activity of Embelin isolated from the ethanol extract of 

leaves of Embelia ribes. J. Ethnopharmacol. 2007; 109 (3): 529- 

534. 



4. Ma OSW, Saunders RMK, Comparative floral ontogeny of Maesa 

(Maesaceae), Aegiceras (Myrsinaceae) and Embelia (Myrsinaceae): 

taxonomic and phylogenetic implications Plant Syst. Evol. 2003; 

243: 39-58. 

5. Ravikumar K, Ved DK, Illustrated field guide: 100 Red Listed 

Medicinal Plants of Conservation Concern in Southern India, 

Foundation for Revitalisation of Local Health Traditions, Bangalore 

2000, 467 

6. Warrier PK, Nambiar VPK, Ganapati PM, Some Important 

Medicinal Plants of the Western Ghats, India: a Profile, Blackwell 

Publishers, New Delhi 2001, 141-156 

7. Seth SD, Johri N and Sundaram KR, Antispermatogenic effect of 

Embelin from Embelia ribes. Ind. J. Pharmac. 1982; 14 (2), 207- 

211. 

8. Anonymous, Wealth of India: Raw Materials, Vol III, CSIR, New 

Delhi, 1952, 167 – 168. 

9. Chauhan SK, Singh BP, Agrawal S, Indian Drugs 1999; 36: 41-43. 

10. Krishnaswamy M, Purushothaman KK. Antifertility properties of 

Embelia ribes: (Embelin). Indian J Exp Biol 1980; 18: 1359. 

11. Chitra M, Sukumar E, Suja V, Shyamala Devi CS. Antitumor, antiinflammatory 

and analgesic property of Embelin, a Plant Product. 

Chemotherapy 1994; 40: 109. 

12. Githiori JB, Hoglund J, Waller PJ, Baker LR, “Evaluation of 

anthelmintic properties of some plants used as livestock dewormers 

against Haemonchus contortus infections in sheep. Parasitol. 2004; 

129 (2): 245-253. 

13. Rathinam K, Santhakumari, Ramiah N, J. Res. Indian Med. Yoga & 

Homeopathy 1976; 11 (4): 84-90. 

14. Li XH, McLaughlin JL, Bioactive Compounds from the Root of 

Myrsine Africana. J Nat. Prod. 1989; 52 (3): 660-662. 

15. Indian Herbal Pharmacopeia Revised edition Published by Indian 

Drug Manufacturer’s Association, Mumbai. 2002, 500 – 501. 

16. Draves AH, Walker SE Determination of Hypericin and 

Pseudohypericin in pharmaceutical preparations by liquid 

chromatography with florescence detection. Journal of 

Chromatography B Biomed. Sci. Appl. 2000; 749 (1): 57-66. 





ISSN 0975 1556 

Synthesis and In-Vitro Anti-Microbial Activity of Some New N-Phenyl 

Acetamide Derivatives 

Singh Pramod a* , Daniel Vivek a , Nema R. K. b , Joshi Ankur b , Bhatt Govinda a 

a Mandsaur Institute of Pharmacy, Mandsaur, Madhya Pradesh, India 

b Rishiraj Institute of Pharmacy, Indore, Madhya Pradesh, India 

ABSTRACT 

The purpose of the research work is to synthesised a new potent antimicrobial derivatives of N-phenyl acetamide. A series 

of new methyl 4-oxo-4-(Piperidine phenylamino) butanoate 4(a) and methyl 4-oxo-4-(Imidazole phenylamino) butanoate 

4(b) were synthesized by treating N -Phenyl acetamide with methyl choloroacetate in the presence of anhydrous potassium 

carbonate to give an intermediate compound (3) which on further treatrment with piperidine and imidazole at 70 o C 

temperature gives the new compounds 4(a) and 4(b) respectively. The structures of newly synthesised derivatives were 

confirmed on the basis of Melting Point, TLC, IR, NMR and spectral data. The anti microbial activity of the synthesized 

compounds was evaluated by Disc diffusion test. 

Keywords: N-phenyl acetamide, methyl chloroacetate, Amines, Antimicrobial activity. 

INTRODUCTION 

An antimicrobial is a substance that kills or inhibits the 

growth of microorganisms such as bacteria, fungi, or 

protozoans, as well as destroying viruses. Antimicrobial 

drugs either kill microbes (microbicidal) or prevent the 

growth of microbes (microbistatic). Disinfectants are 

antimicrobial substances used on non-living objects. [1-4] 

Some N-phenyl acetamide derivatives of N, N-substituted 

acetamides have been found to possess significant 

antimicrobial activity when compared with Ofloxacine. [5-8] 

Based on the structural data some common features can be 

deduced in all the pharmacological divergent classes. [9] A 

basic nitrogen which may be part of heteroaromatic ring or 

cyclic/acyclic system intended to interact electrostatically 

with the appropriate target. [10-12] 

EXPERIMENTAL 

Equipment 

Melting points of all the synthesized compounds were 

determined by Thieles’s tube method and uncorrected by 

melting point determing apparatus (SISCO). The 

intermediate compounds synthesized were confirmed on the 

basis of their melting reported in the literature and the 

functional group tests. All the solvents were used after 

purification 

[13-17] , distillation and dried. Purity of all 

compounds was checked by Silica gel GF254 plates TLC (e- 

Merck and Co.) with Ultra Violet spectroscopy detection 

method. 

*Corresponding author: Mr. Pramod Singh, 

Mandsaur Institute of Pharmacy, Mandsaur, Madhya 

Pradesh, India; E-mail: pramod_kunwar@rediffmail.com 

The FT-IR spectra (KBr cm -1 ) were recorded on the 

Shimadzu Fourier transformed infrared (FT-IR) 

spectrophotometer (spectrum 8400).The physical data of the 

synthesized compounds were presented in Table 1. 

Table 1: Characterization of the compounds 

Compo 

und 

4(a) 

4(b) 

R M.P.(°C) 

N 

H 

N 

H 

N 

128-132 

Yield 

(%) 

47.6% 

M. 

Weight 

298 

150-155 40.2% 300 

Molecula 

r formula 

C16H30N2 

O3 

C15H29N3 

O3 

Preparation of Derivatives 

Preparation of Intermediate methyl 4-oxo-4- 

(phenylamino) butanoate 

Methyl 4-oxo-4-(phenylamino) butanoate {(1) (500 mg} was 

dissolved in ethyl methyl ketone (70 ml) and anhydrous 

potassium carbonate (2.0 g) was added to the solution. The 

reaction mixture was refluxed for 2 h. Then, methyl 

chloroacetate (3 ml) was added, continued refluxing for 6 h 

and reaction was monitored with the help of TLC. [15-16] The 

slurry was filtered and the solvent was removed under 

reduced pressure. The solid was collected & re-crystallized 

from ethanol to yield (M. p. 121-125°C). IR: 3529.49, 

1234.36, 1741.69, 1161.07, 3080.90, 1396.37 cm-1. 1H- 

150

NMR: δ 7.79 (d, J = 7.5, 1H), 7.52 (t, J = 1.5, 1H), 7.06 (d, J 

= 1.5, 1H), 6.94 (dd, J = 1.6, 7.5, 1H), 6.66 (t, J = 1.4, 1H), 

NHCOCH 3 

Starting Material 

(1) 

Singh et al. / Synthesis and In-Vitro Anti-Microbial Activity.... 

+ ClCH 2COOCH 3 

NHCOCH 2CH 2COOCH 3 

Intermediate 

(3) 

Methylchloroacetate 

(2) 

+ R 

Starting material= N-phenyl acetamide 

Intermediate= methyl 4-oxo-4-(phenylamino) butanoate 

R= Amines (Piperidine, imidazole) 

4.92 (s, 2H), 4.70 (s, 1H), 3.78 (s, 3H), 2.15 (s, 2H) (Scheme 

1) 

Ethyl methyl Ketone 

Scheme 1 

Scheme 2 

K 2CO 3 

NHCOCH 2CH 2COOCH 3 

Intermediate 


(3) 

NHCOCH 2CH 2COOCH 2R 

Compound 4 (a-b) 

Table 2: Antimicrobial activities of the compounds 

S. 

No. 

Zone of Inhibition(in mm) 

Compound 

Eschericia coli Pseudomonos aeruginosa Bacillus subtilus Staphylococcus aurens Candida albicans 

01 4a 12 15 18 14 15 

02 4b 13 16 20 16 18 

03 Ofloxacin 25 22 27 24 25 

Table 3: Antimicrobial activities of compounds 

S. No. Microorganism 

Activity Index 

4a 4b 4a 

% Activity 

4b 

01 Escherichia coli 0.48 0.52 48% 52 % 

02 Pseudomonos aeruginosa 0.68 0.72 68% 72 % 

03 Bacillus subtilus 0.66 0.74 66% 74 % 

04 Staphylococcus aureus 0.58 0.66 58% 66 % 

05 Candida albicans 0.59 0.70 59% 70 % 

Preparation of methyl 4-oxo-4-(Piperidine phenylamino) 

butanoate 

Methyl 4-oxo-4-(Piperidine phenylamino) butanoate) (3) 

(100 mg) and Piperidine (2 ml) were stirred magnetically at 

70 o C temperature for 3 h. Crushed ice was added to the 

contents and the solid obtained was crystallized from a 

mixture of acetone and petroleum ether to obtain the target 

compound 4a. (M. P. 128-132°C). IR: 3681.86, 3120.61, 

1261.36, 1666.38, 2852.52, 1165.35 cm-1. 1H-NMR: δ 8.11 

(dd, J = 1.5, 7.5, 1H), 7.89 (dd, J = 1.5, 7.5, 1H), 7.50 (td, J = 

1.5, 7.5, 1H), 7.42 (s, 1H), 7.31 (td, J = 1.5, 7.5, 1H), 5.26 (s, 

2H), 4.92 (s, 2H), 3.78 (ddt, J = 1.0, 2.2, 12.1, 2H), 3.78 (ddt, 

J = 1.0, 2.2, 12.1, 2H), 3.70 – 3.65 (m, 3H), 3.70 – 3.65 (m, 

3H), 3.68 – 3.61 (m, 4H), 3.68 – 3.61 (m, 4H), 3.61 – 3.53 

(m, 2H), 3.61 – 3.53 (m, 2H), 2.45 (s, 2H). (Scheme 2) 

Preparation of methyl 4-oxo-4-(Imidazole phenylamino) 

butanoate

Methyl 4-oxo-4-(Imidazole phenylamino) butanoate) (3) 

(100 mg) and Imidazole ( 2 gm ) were magnetically stirred at 

70 o C temperature for 3 h. Crushed ice was added to the 

contents and the solid obtained was crystallized from a 

mixture of acetone and petroleum ether to obtain the target 

compound 4b. (M. p. 150-155°C). IR: 3616.28, 2856.38, 

2997.54, 1276.79, 1662.52, 1155.38, 1365.51 cm-1. 1H- 

NMR: δ 7.77 (d, J = 7.5, 1H), 7.76 (t, J = 1.5, 1H), 7.30 (d, J 

= 1.5, 1H), 6.98 (dd, J = 1.5, 7.5, 1H), 6.70 (t, J = 1.5, 1H), 

4.91 (s, 1H), 4.70 (s, 1H), 3.52 (q, J = 6.0, 3H), 3.52 (q, J = 

6.0, 3H), 2.15 (s, 1H), 1.27 (t, J = 6.0, 4H), 1.27 (t, J = 6.0, 

4H). (Scheme 2) 

Antimicrobial Activity 

In-vitro antimicrobial activity was carried using Muller 

Hinton agar (Hi-media) paltes at 37°C agar diffusion cup 

plate methods. [18-20] All the compounds were screened for the 

antimicrobial actitvity at 1000 µg/ml (1 mg/ml) concentration 

against the following bacterial stains: Escherichia coli, 

Bacillus subtilis, Staphylococcus aureus, Pseudomonas 

aeruginosa, and Candida albicans. Ofloxacin dissolved in N, 

N – dimethylformide (DMF) was taken as the reference 

standard for comaprision of antimicrobial activity under 

similar conditions. Tested compounds were dissolved in N, N 

-dimethylformide (DMF) to get the a solution of 1mg/ml. 

Inhibition zones were measured in millimeters at the end of 

incubation period of 24 h at 37°C. Paper discs, (3 mm 

diameter), were saturated with the dilutions of and placed on 

the surface of the seeded agar (each disc absorbs 

approximately 0.08 ml of solution). Two discs saturated with 

the reference standard were placed on assay plate opposite 

each other, and other discs of samples were placed in the 

quadrants. All plates were incubated for 24-48 h at 37°C. 

The diameter of zone of inhibition of the reference standard 

discs was measured by the use of millimeter scale. [21-24] 

RESULT AND DISCUSSION 

The aim of the present research work to find out a new 

synthetic potential derivative of New N – Phenyl acetamide 

derivatives with antimicrobial activity. Series of new 

compounds were synthesized and assessed for antimicrobial 

activity. The data reported in Table 2 & 3 shows that effect 

of variation in basic chemical structure of parent compound 

on antimicrobial activity. Substitution of piperdine and 

imidazole to the N-Phenyl acetamide leads to the synthesis of 

4(a) and 4(b) compounds with potent antimicrobial activity. 

CONCLUSION 

Screening and evaluation establised that the new compound 

were Methyl 4-oxo-4-(Piperidine 

phenylamino)butanoate),4(a) and Methyl 4-oxo-4-(Imidazole 

phenylamino) butanoate 4(b) showed a potent antimicrobial 

activity against various Gram positive and Gram negative 

bacteria. Results show that antimicrobial activity is attributed 

due to the substituent piperidine and imidazole ring in the 

parent compound. The data reported in this research article 

may be beneficial as a reference for the medicinal and 

pharmaceutical chemist who is doing research in this field. 

Singh et al. / Synthesis and In-Vitro Anti-Microbial Activity.... 

ACKNOWLEDGEMENT 

The authors are grateful to Department of Biotechnology, 

Mandsaur Institute of Pharmacy and B. R. Nahata college of 

Pharmacy for providing all necessary facilities. 

REFERENCES 

1. http://en.wikipedia.org/wiki/Chemical_synthesis. 

2. http://www.innvista.com/HEALTH/microbes/intro.htm. 

3. Tripathi KD. Antimicrobial drugs. In The Essentials of Medical 

Pharmacology, 5th ed.; Jaypee brothers medical publishers Pvt. 

Ltd.; New Delhi, 2003, pp. 628-629. 

4. http://www.umsl.edu/~microbes/pdf/introductiontobacteria.pdf. 

5. http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/icu8/pdf/introduc 

tion.pdf. 

6. I:\bacterias\Fungal skin infections - athlete's foot, thrush - 

symptoms & treatment.htm. 

7. Projan SJ, Shlaes DM. Antibacterial Drug Discovery: Is it All 

Downhill from here. European Society of Clinical Microbiology 

and Infectious Diseases 2004; 10: 18–22. 

8. I:\tria thia\Dicarboxylic acid - Wikipedia, the free 

encyclopedia.htm. 

9. I:\tria thia\dicarboxylic acids.htm. 

10. I:\tria thia\Heterocyclic compound - Wikipedia, the free 

encyclopedia.htm. 

11. Saag MS, Dismukes WE. Azole Antifungal Agents: Emphasis on 

New Triazoles. Antimicrobial Agents and Chemotherapy 1988; 32: 

1-8. 

12. Gupta RR, Kumar M, Gupta V. Five membered heterocycles with 

more than two heteroatoms. In Heterocyclic Chemistry II, Springer 

(India) Pvt.Ltd., 1999, pp 491-573. 

13. Amudat L, Joshua AO. Synthesis, characterization and antibacterial 

activity of aspirin and paracetamolmetal complexes. Biokemistri 

2007; 19(1): 9-15. 

14. "Antimicrobial Definition from the Merriam Webster Online 

Dictionary". http://www.merriamwebster.com/dictionary/Antimicrobial. 

Retrieved 2009-05-02. 

15. Bauer AW, Kirby WMM, Sherries JC, Turck M. Antibiotic 

susceptibility testing by single disk method. American Journal of 

Clinical Pathology 1966; 45: 493-496. 

16. Kohli D, Hashim SR, Vishal S, Sharma M, Singh AK. Synthesis 

and antibacterial activity of quinazolinone derivatives. International 

Journal of Pharmacy and Pharmaceutical Sciences 2009; Vol. 1, 

Issue 1, July-Sep. 2009. 

17. Butler DE, Leonard JD, Caprathe BW, Italien YJ, Pavia MR, 

Hershenson FM, Poschel PH, Marriot JG, Amnesia-reversal activity 

of a series of cyclic imides. J. Med. Chem. 1987; 30: 498. 

18. Sahm DF, Washington JA. Antibacterial Suspectibility Tests, 

Dilution Methods”, in: A. Balowes, W.J. Hausler, K.L. Hermann, 

H.D. Shadomy (Eds.), fifth ed. American Society for Microbiology, 

Washington, 1991, pp. 1105_/1116. 

19. Fawzy M. Ali Ahmed M. El-Agrody reparation of 4aminophenylacetic 

acid derivatives with promising antimicrobial 

activity. Acta Pharm. 2006; 56: 273-284. 

20. Grover G, Kini SG. Synthesis and evaluation of new quinazolone 

derivatives of nalidixic acid as potential antibacterial and antifungal 

agents, European Journal of Medicinal Chemistry 2006; 41: 256– 

262. 

21. Graul A, Tracy M, Castaner R M, Inhibitory effects of tenilsetam 

on the Maillard reaction. Drugs of the Future 1997; 22: 639. 

22. Gulgun Ayhan-Kılcıgil, Nurten Altanlar. Synthesis and 

antimicrobial activities of some new benzimidazole Derivatives Il. 

Farmaco 2003; 58: 1345-1350. 

23. H. Go¨ker, E. Tebrizli, U. Abbasog˘lu. Synthesis of 1, 2disubstituted 

benzimidazole-5(6)-carboxamides and evaluation of 

their antimicrobial activity. Farmaco 1996; 51: 53-58. 

24. Iizuka H, Oguchi T, Aoki Y, Ohto N, Horikomi K, Miwa T, 

Kamioka T, Kawashima S. Eur. Pat Appl E P, Toatsu Chemicals 

Inc. 1995, 124. 





ISSN 0975 1556 

Formulation and Evaluation of Naproxen Gel Containing Tulsi Oil as 

Penetration Enhancer 

Gupta V 1* , Dwivedi A 1 , Tiwari N 2 , Jain NK 1 , Garud N 3 , Jain DK 1 

1 College of Pharmacy, IPS Academy, Rajendra Nagar, A.B. Road, Indore, Madhya Pradesh, India 

2 Swami Vivekanand College of Pharmacy, Khandwa Road, Indore, Madhya Pradesh, India 

3 Institute of Professional Studies, College of Pharmacy, Shivpuri link road, Gwalior, Madhya Pradesh, India 

ABSTRACT 

The present research has been undertaken with the aim to develop a transdermal gel formulation of naproxen, which would 

attenuate the gastrointestinal related toxicities associated with oral administration. The potential gastrointestinal disorders 

associated with oral administration of naproxen (NSAID’s) can be avoided by delivering the drug to the inflammation site. 

Gel based formulations are better percutaneously absorbed than creams and ointment bases. It is reported that absorption 

profile of naproxen from carbopol gel base is better than other bases. Therefore, naproxen gel formulations were made with 

carbopol 940 having different concentration of tulsi oil as penetration enhancer containing 2 % of naproxen. Permeation 

experiments were performed on excised abdominal rat skin using Keshary Chien diffusion cell. The fixed oil of tulsi 

(ocimum sanctum) is reported to possess significant anti inflammatory activity. Naproxen gels containing various 

concentration of tulsi oil (1 %, 3 %, 5 %, and 7 %) were prepared. To assess the efficacy of formulations in-vitro release, 

rheological properties, drug content and skin irritation studies were performed. The results obtained were encouraging and 

formulation containing naproxen (2 %) and tulsi oil (5 %) found to be better than other formulations. 

Keywords: Gel; Tulsi oil; Naproxen; Penetration enhancer. 

INTRODUCTION 

Naproxen [1] is a potent non-steroidal anti-inflammatory drug 

(NSAID) used for a variety of inflammatory conditions. 

However, its use has been associated with a number of 

gastrointestinal disorders. [2] Like other NSAIDs, the most 

common side effect of peroral naproxen is gastrointestinal 

irritation. These potential side effects may be overcome by 

the topical administration of the drug. Skin is one of the most 

readily accessible organs on human body for topical 

administration and is the main route for topical drug delivery 

system. For a topical formulation to be effective, it must first 

penetrate the skin, only when the drug has entered the lower 

layers of the skin it can be absorbed by blood and transported 

to the site of action, or penetrate deeper into areas where 

inflammation occurs. The stratum corneum provides the 

greatest resistance to penetration, and it is the rate-limiting 

step in percutaneous absorption. The permeation of drugs 

through skin can be enhanced by physical methods such as 

mechanical disruption, electrical disruption, and chemical 

modification or by the use of chemical penetration enhancers. 

The later compounds increase skin permeability by 

increasing the partition coefficient of the drug into the skin 

*Corresponding author: Mr. Vikas Gupta, M. Pharm. 

(Pharmaceutics), COP, IPS Academy, Indore (M.P.)-452012 

E-mail: gupta.vikas.p@gmail.com 

and by increasing the thermodynamic activity of the drug in 

the vehicle. [3] Chemical penetration enhancers modify 

barrier properties of the stratum corneum and hence increase 

drug permeability across skin. Ideally, the effects of the 

penetration enhancer on the skin should be reversible, nontoxic, 

non-allergenic, compatible with drugs and excipients 

and non-irritating. However the synthetic permeation 

enhancers are associated with adverse effect of producing 

irritation and toxicity to the skin. Hence natural products 

have increasingly been used as enhancers due to their better 

safety profile. [4] 

Tulsi is a widely grown, sacred plant of India and it belongs 

to the labiateae family. Leaf contains eugenol (volatile oil), 

ursolic acid (triterpenoid) and rosmarinic acid 

(phenylpropanoid). Other active compounds include 

caryophyllene and oleanolic acid. Seeds contain fixed oils 

having linoleic acid and linolenic acid. It has long history of 

use in ayurvedic system of medicine to treat various ailments 

without any noticeable toxicity. [5] In the present study, 

attempts have been made to explore the penetration 

enhancing activity of tulsi oil. 


Materials 

Naproxen was received as a gift sample from Nicholas 

Piramal, Mumbai, India. Carbopol, propylene glycol, 

153

triethanolamine and other chemicals were of analytical grade 

and used without further purification. 

Method for preparation of gel 

Carbopol 940 (1 %) was finely dispersed in 50:50 propylene 

glycol:water and stirred continuously at 300 rpm for 3 h. 

Then, the naproxen (2 %) passed through 100 mesh was 

finely dispersed in 15 ml of propylene glycol and then added 

to the carbopol mixture. Lastly tulsi oil was added and mixed 

for 1 h. The dispersion was then neutralized and made 

viscous by the addition of triethanolamine. 

[6] 

The 

compositions of different gel formulations are listed in Table 

1. 

Table 1: Composition of different gel formulations 

Item Material name 

F1 

Quantity (%) 

F2 F3 F4 F5 

1. Naproxen 2 2 2 2 2 

2. Carbopol 1 1 1 1 1 

3. Tulsi oil - 1 3 5 7 

4. Propylene glycol 48 46.5 46 45.4 45 

5. Water 48 46.5 46 45.4 45 

Drug Content 

For assay of the drug in gels, naproxen was extracted from 1 

g of each gel formulations with 20 ml of methanol for 30 

min. The resultant mixture was filtered through membrane 

filter (pore size 0.45 mm). The absorbance of the sample was 

determined spectrophotometrically at 272 nm (Shimadzu 

1601 UV-VIS spectrophotometer) using methanol as a blank. 

The concentration of naproxen was estimated from the 

regression equation of the calibration curve. 

Spreadability 

Spreadability was determined by modified wooden block and 

glass slide apparatus. The apparatus consisted of a wooden 

block with fixed glass slide and a pulley. A pan was attached 

to another glass slide (movable) with the help of a string. For 

the determination of spreadability measured amount of gel 

was placed in the fixed glass slide, the movable glass slide 

with a pan attached to it, was placed over the fixed glass 

slide, such that the gel was sandwiched between the two 

slides for 5 min. Now about 50 g of weight was added to the 

pan. [7] Time taken for the slides to separate was noted. 

Spreadability was determined using the following formula: 

S = M.L/T 

Where S is the spreadability in g.cm/s, 

M is the mass in grams and T is the time in seconds. 

Extrudability 

A closed collapsible tube containing gel was pressed firmly 

at the crimped end. When the cap was removed, gel extruded 

until pressure dissipated. Weight in grams required to extrude 

0.5 cm ribbon of gel in 10 seconds was determined. [8] 

Viscosity 

Brookfield digital viscometer (model DV-I+, Brookfield 

Engineering Laboratory, INC., USA) was used to measure 

the viscosity (in cps) of the prepared gel formulations. The 

spindle T-D (spindle code S 94) was rotated at 2.5, 4, 5 and 

10 rpm. The reading, near to 100% torque was noted. 

Samples were measured at 30 ± 1° C. 

Skin irritation study 

Three albino rabbits were selected for the study. 24 h prior to 

the test, the test sites were depilated on both sides of the 

spine and demarcated for the application of the formulation. 

The measured quantity of gel was applied over the respective 

test sites. The test sites were observed for the erythema and 

edema for 48 h after application. 

Gupta et al. / Formulation and Evaluation of Naproxen Gel.... 

In vitro diffusion studies 

Phosphate buffer of pH 6.8 was used for in vitro release as a 

receptor medium. The pretreated skin of albino mice was 

used in keshary - chien diffusion cell. The gel sample was 

applied on the skin and then fixed in between donor and 

receptor compartment of diffusion cell. The receptor 

compartment contained phosphate buffer of pH 6.8. The 

temperature of diffusion medium was thermostatically 

controlled at 37±1ºC by surrounding water in jacket and the 

medium was stirred by magnetic stirrer at 100 rpm. The 

sample at predetermined intervals were withdrawn and 

replaced by equal volume of fresh fluid. The samples 

withdrawn were spectrophotometrically estimated at 262 nm 

using phosphate buffer pH 6.8 as a blank. [9] 


The present investigation aims to develop transdermal gel of 

naproxen containing tulsi oil as a natural penetration 

enhancer for improved penetration of naproxen. The 

mechanism of action of tulsi oil is not well established yet 

but it might be possible that it modifies the barrier properties 

of stratum corneum temporarily to enhance percutaneous 

absorption. Different gel formulations containing naproxen 2 

% (alone) and naproxen 2 % with varying concentrations 1 

%, 3 %, 5 % and 7 % of tulsi oil were prepared and evaluated 

for drug content, pH, spreadability, viscosity, extrudability, 

homogeneity, in vitro drug release, skin irritation and 

stability. 

The drug content of all formulations ranges from 90.5 % - 

92.9 % which shows content uniformity, the pH values of all 

developed gels ranges 6.83-6.89 which lies in the normal pH 

range and would not produce any skin irritation as shown in 

Table 3. The values of spreadability indicate that the gel is 

easily spreadable by small amount of shear. The result of 

spreadability varies from 6.08 to 6.82 g. cm / sec. where as 

the extrudability of gel formulations from the collapsible tube 

varies from 180 to 190 g and the viscosity of formulations 

ranges from 16241 cps to 16896 cps at 10 rpm as shown in 

Table 2. 

Table 2: Rheological data of different formulations 

Formul Spreadability Extruda Viscosity (cps) at rpm 

ations (g.cm/sec) bility (g) 2.5 4 5 10 

F-1 6.08 180 51432 38749 31765 16241 

F-2 6.57 180 51643 38300 31856 16389 

F-3 6.72 185 51954 38452 31521 16427 

F-4 6.79 190 51208 38170 31405 16735 

F-5 6.82 180 51485 38060 31218 16896 

Table 3: Values of evaluation parameters of different formulations 

Formulations pH Drug content (%) Homogeneity 

F-1 6.87 90.5 Good 

F-2 6.89 91.7 Good 

F-3 6.83 92.6 Good 

F-4 6.86 91.8 Good 

F-5 6.88 92.9 Good 

Table 4: In vitro diffusion study of different formulations in PBS (6.8) 

using excised Rat abdominal skin 

Time (hrs) 

F-1 F-2 

% Drug release 

F-3 F-4 F-5 

0.5 18.2 25.8 32.3 38.6 38.7 

1 29.4 33.5 41.9 49.5 50.2 

1.5 38.3 41.6 49.8 55.5 55.8 

2 45.7 50.3 56.4 62.1 62.6 

3 58.5 62.7 68.7 72.2 73.1 

4 70.9 75.4 82.5 85.8 86.4 

5 82.1 85.5 91.5 99.3 99.3 

6 90.6 94.9 96.5 - - 


All developed gels showed good homogeneity with absence 

of lumps. No signs of erythema and edema were found after 

48 h of application in albino rabbits. During the stability 

studies the appearance was clear and no significant variation 

in pH was observed. In vitro drug release study as shown in 

Table 4 that the formulations containing tulsi oil releases the 

drug faster as compared to formulation F- 1 which does not 

contain tulsi oil. It may be concluded from the results that as 

the concentration of tulsi oil increases in the formulations the 

rate of drug release also increases. Percent drug release data 

also shows that there is no any significant difference in the 

amount of drug released from formulation containing 5 % 

and 7 % of tulsi oil. Therefore, formulation containing 5 % of 

tulsi oil was selected as best among all the formulations. It is 

clear that tulsi oil can significantly enhance the penetration of 

naproxen from gel formulation across the skin. 

ACKNOWLEDGEMENTS 

The authors are thankful to Nicholas Primal, Mumbai, India 

for providing gift sample of Naproxen. The authors are 

grateful to College of pharmacy, IPS academy, Indore for 

providing facilities to carry out the investigation. 

Gupta et al. / Formulation and Evaluation of Naproxen Gel.... 

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