ISSN (print) 0975 6221 Vol.2 (2) Sept.2010-March 2011 ISSN ...€¦ · Sandhya S*, Jafferi S.A.H , Vinod K.R , Ottilia Banji , David Banji , Chaitanya R.S.N.A.K.K , Chandrasekhar.J

ISSN (print) 0975 6221 Vol.2 (2) Sept.2010-March 2011 ISSN (Online) 2229 3590

H YG E I A Journal for drugs and medicines

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Hygeia.J.D.Med.Vol.2 (2), Sep.2010-March.2011

• Pharmacological potential of Trichosanthes dioica – an edible plant.

Biren N. Shah* and A. K. Seth

[Abstract][Full Text][Pdf]

• Effect of microwave drying in improving granule cha racteristics in tablets

Deepthi Shyju.*


• Evaluation of In-vitro vector control activity of P hysalis angulata.

Sandhya S*, Jafferi S.A.H , Vinod K.R , Ottilia Banji , David Banji , Chaitanya R.S.N.A.K.K ,

Chandrasekhar.J , Venkataramana.K .


• The Hepatoprotective Effect of The Polyphenolic Com pounds in the Roots of Trichilia connaroides

Wight and Arn

Garima Agarwal* , Anil Kumar Pant , and Subroto Kumar Hore .


• Visible Spectrophotometric method for the estimatio n of Cefepime

Minu Sujith, Sujith Abraham*and Madhu.C.Divakar


• Phytochemical, HPTLC finger printing and antibacter ial activity of Acacia nilotica (L.) Delile

R.Venkataswamy* , A.Doss , H.Muhamed Mubarack , M.Sukumar.


• In vitro evaluation of anthelmintic efficacy of Tri chilia and Ajuga species on Ascaridia galli

G. Agarwal*, A.K. Pant and S.K. Hore


• Anti inflammatory activity of the seed and fruit wa ll extracts of Solanum torvum

M. Rammohan and C.Srinivas Reddy*

[Abstract][Full Text][Pdf] • Pharmaceutical Education

DRUG USAGE IN PREGNANCY SIYAD.A.R [Abstract][Full Text][Pdf]

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Short Review Hygeia.J.D.Med.vol.2 (2), 2010, 1-7 ISSN 0975 6221 ____________________________________________________________________________________________________________ HYGEIA JOURNAL FOR DRUGS AND MEDICINES


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Pharmacological potential of Trichosanthes dioica – an edible plant.

Biren N. Shah*1 and A. K. Seth2 1. Vidyabharti Trust College of Pharmacy, Umrakh, Gujarat, India . 2. Sumandeep Vidyapeeth University, Piperia, Gujarat, India. Article history: Received: 15 May 2010, revised: 16Juner 2010, accepted: 23June 2010, Available online: 20 Sep 2010.

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Abstract

Trichosanthes dioica Roxb. (family: Cucurbitaceae), commonly known as “Sespadula” in English and “Parwal” in Hindi, is widely grown throughout India. Fruits of this plant are used as vegetable in Indian traditional food system from time immemorial. Besides fruits, other parts of the plant, such as the leaves and tender shoots, have also been used in the traditional system of medicine since ancient times. Pointed gourd has been used for overcoming problems like constipation, fever, skin infection, wounds and also improves appetite and digestion. The immature fruits are used as vegetable and as ingredients of soup, stew, curry, sweet, or eaten fried and as dorma with roe stuffing. The present review describes the morphological and pharmacological aspects of Trichosanthes dioica and summarizes the most interesting findings obtained in the preclinical and clinical research related to the plant. Key words: Trichosanthes dioica, Pharmacology, Cucurbitaceae.

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1. Introduction The Indian subcontinent represents one of the richest diverse genetic resources. Of the estimated 250,000 species of flowering plants at global level, about 3000 are regarded as food source; out of which only 200 species have been domesticated. Global diversity in vegetable crops is estimated to be about 400 species of which about 80 species of major and minor vegetables are reported to have originated in India. However, with the advent of cut and burn agriculture, green revolution/commercialized agriculture, the area development projects and the related activities of these diverse resources are declining at a fast pace. Overgrazing, deforestation and over exploitation of native resources under range situations have eroded the biodiversity from this unique ecosystem. ____________________________ * For Correspondence [email protected], Contact: +919978262799 © 2010 Hygeia journal for drugs and medicines, all rights reserved. 0975 6221

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Biren N. Shah et al, Hygeia.J.D.Med.Vol2 (2), 2010,1-7 Moreover, our traditional knowledge about these important indigenous plant species has also decreased in the younger generation influenced by urbanization.Indigenous plant species provide a variety of products like food, medicines, raw materials and are also an important source of renewable energy. The Indian subcontinent had been one of the rich emporia of 2500 plant species used in indigenous treatment and food sources1. Pointed gourd (cucurbitaceae) is a dioecious perennial herbaceous vegetable. The crop is of Indo-Malayan origin and distribution and is extensively grown in eastern India2 and to a lesser extent in other parts of South Asia3. Trichosanthes dioica Roxb. (family: Cucurbitaceae), commonly known as “Sespadula” in English and “Parwal” in Hindi, is widely grown throughout India2. Fruits of this plant are used as vegetable in Indian traditional food system from time immemorial. Besides fruits, other parts of the plant, such as the leaves and tender shoots, have also been used in the traditional system of medicine since ancient times4-6. Some specific medicinal properties have been identified, viz., hypocholesterolemic, hypoglyceridimic, and hypophospholipemic when shade-dried fruits were mixed in the food of nondiabetic animals4, 7. Most recently, its seeds and leaves have also been found as antidiabetic agents by our research group8, 9. It also serves as a rich source of vitamin C4. 2. BOTANY The plant is a perennial, dioecious, and grows as a vine (Fig. 1). Roots are tuberous with long taproot system. Vines are pencil thick in size with dark green cordate simple leaves. Flowers are tubular white

with 16–19 days initiation to anthesis time for pistillate flowers and 10–14 days for staminate flowers. Stigma remains viable for approximately 14 hours and 40–70% of flowers set fruit. Based on shape, size and striation, fruits can be grouped into 4 categories: (1) long, dark green with white stripes, 10–13 cm long, (2) thick, dark green with very pale green stripes, 10–16 cm long, (3) roundish, dark green with white stripe, 5–8 cm long, and (4) tapering, green and striped, 5–8 cm long6.

Figure 1: Trichosanthes dioica plant. 3. PHARMACOLOGICAL PROPERTY 3.a. Anthelmintic activity The in vitro activities of defatted methanol (MeOH) extract of the leaves from Trichosanthes dioica Roxb. (Cucurbitaceae), and its ethyl acetate (EtOAc) and n-butanol (n-BuOH) fractions was evaluated against Pheretima posthuma (Annelida) and Ascaridia galli (Nematoda).

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Biren N. Shah et al, Hygeia.J.D.Med.Vol2 (2), 2010,1-7.

All the extracts demonstrated concentration dependent paralytic and lethal effects on P. posthuma and lethal effects on A. galli. The EtOAc fraction was found to be the most potent followed by the defatted MeOH extract and its n-BuOH fraction. A. galli was found to be more sensitive than P. posthuma against all tests extracts indicating T. dioica as an effective nematocide10. 3. b. Antihyperglycaemic activity The study deals with the effect of a single oral dose of the aqueous extract of Trichosanthes dioica Roxb. (Cucurbitaceae) seeds in different diabetic animal models. Evaluation of the antihyperglycemic effect in normal, sub diabetic, and mild diabetic animal models is based on fasting blood glucose (FBG) and glucose tolerance test (GTT) studies. The graded doses of the extract, viz., 500, 750, 1000, and 1250 mg/kg body weight (b.w.), were administered orally. It was found that the blood glucose concentration decreased in a dose-dependent manner. The dose of 1000 mg/kg b.w. was found to be most effective with a maximum fall of 30.4% at 6 h during FBG studies in normal rats. However, the GTT studies showed the maximum reduction of 26.6% at 5 h in normal rats. Moreover, in case of sub diabetic and mild diabetic rats, the observed reduction in blood glucose levels was 32.8% and 35.9%, respectively, at 3 h during GTT. The data clearly reveal the significant antihyperglycaemic profile of Trichosanthes dioica seeds8. 3. c. Antioxidant activity Antioxidants protect the body against oxidative stress by neutralizing free radicals. Plants contain rich amount of polyphenols which are very potent natural antioxidants. The study was designed to evaluate the relative contribution of different polyphenols such as total phenolics, flavonoids and flavonol contents and their antioxidants activities. For this purpose the total phenolics, flavonoids and flavonol contents of some medicinal plants were determined in the aqueous extracts of leaves of Trichosenthes dioica, fruits of Moringa olifera and Ficus bengalensis as well as seeds of Emblica officinalis. Total antioxidant activity of these extracts was monitored by Free Radical Absorbing Power (FRAP) assay. In this paper, those parts of the plants are used for the analysis of aforesaid parameters which are normally overlooked. The total phenolic content of T. dioica leaves was about two times more than that obtained from the fruits and seeds of M. olifera and E. officinalis, respectively. However, the aerial roots of F. bengalensis registered presence of least phenolic content. The aqueous preparation from E. officinalis exhibited total flavonoid content twice as high as that of the other three plants. The extract from seeds of E. officinalis was found to contain highest antioxidant activity as compared to the preparations from other plants. The high antioxidant activity and flavonoids contents in E. officinalis seeds indicated that it could be exploited as an ingredient in developing a potential antioxidant supplement11.

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Biren N. Shah et al, Hygeia.J.D.Med.Vol2 (2), 2010, 1-7.

In another study, antioxidant activity of fruits of Trichosanthes dioica (Cucurbitaceae) was evaluated and compared with ascorbic acid (Standard). Anti-oxidant activity of aqueous extract of Trichosanthes dioica (TSD) fruits was studied for its free radical scavenging property in different in vitro methods as 1, 1 diphenyl-2- picryl hydrazyl, nitric oxide, reducing power assay and hydrogen peroxide radical method. Different concentrations of aqueous extract of TSD were prepared and evaluated by standard methods. The IC50 values of aqueous extract of TSD were compared with ascorbic acid (Standard) and it was noted that, the extract showed significant concentration dependent free radical scavenging property in all the methods. Results from the study showed that aqueous extract of TSD possess in vitro free radical scavenging activity. The findings could justify the inclusion of this plant in the management of antioxidant activity12. 3. d. Blood Sugar, Serum Lipids, Lipoproteins and Faecal Sterols: Effect of oral administration of 2 ml per day of suspension (in water) of alcoholic extract of whole fruit of Trichosanthes dioica (2%) (= 100 g fresh wt. = 7 g dry wt. = 1/15 g of alcoholic extract) with the help of catheter along with basal diet for four weeks have been studied in the normal albino rabbits. It was observed that this extract lowered the blood sugar, total cholesterol, low density lipoprotein cholesterol and triglyceride levels, and increased the high density lipoprotein cholesterol, phospholipid and faecal sterol levels. Such effects are manifested from the very first week of feeding and are statistically significant13. 3. e. Cholesterol-Lowering Activity This study was to examine the effects of single and repeated oral administration of the aqueous fruit extract of Trichosanthes dioica (TD) at a dose of 50 ml/kg b.w in normal and streptozotocin-induced diabetic rats. The aqueous fruit extracts of TD (50 ml/kg) were administered orally for 15 days, to normal and diabetic rats. The effect of the fruit extracts on cholesterol and triglycerides, were studied. The body weights of the rats were observed. The effect of the fruit extract was compared with vanadate, a reference drug. In normal rats, the aqueous fruit extract of TD induced significant decrease of plasma cholesterol and triglyceride concentrations 6hrs after a single oral administration (P< 0.05), and also in 2 weeks after repeated oral administrations (p< 0.05). TD treatment caused significant decrease of plasma cholesterol levels after a single administration (p<0.01), and after repeated (p<0.01) oral administrations. Significant increase of triglyceride levels was observed 6hrs after a single oral administration of the TD aqueous fruit extract (p< 0.01). One week after repeated oral administration of aqueous extract of TD, the plasma triglyceride levels were significantly decreased (p <0.005). The decreasing trend continued even after 2 weeks (p <0.01). On the other hand, repeated oral administration of TD aqueous fruit extract, caused significant decrease of body weight after 2 weeks of treatment in both normal (p <0.001) and diabetic (p <0.01) rats. The study indicates that the aqueous fruit extract of TD exhibits cholesterol and body weight-lowering activities in both normal and hyperglycemic rats14.

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3. f. Antidiabetic activity In rats with streptozotocin induced severe diabetes mellitus, aqueous extract of Trichosanthes dioica fruits at a dose of 1000mg/kg body weight daily once for 28 days reduced the levels of fasting blood glucose, postprandial glucose, asparate amino transferase, alanine amino transferase, alkaline phosphatase, creatinine, urine sugar and urine protein where as total protein and body weight was increased. No toxic effect was observed during LD5015. The scientific evaluation of the antidiabetic efficacy of aqueous extract of Trichosanthes dioica fruits on streptozotocin-induced diabetic rats is being presented. The graded doses of the extract, viz., 500, 750, 1,000, and 1,250 mg/kg body weight (bw), were administered orally, and it was observed that the blood glucose concentration decreased in a dose-dependent manner. The dose of 1,000 mg/kg bw showed the maximum fall of 23.8% and 19.1% in blood glucose level (BGL) during fasting BGL and glucose tolerance test (GTT) studies, respectively, of nondiabetic rats. Whereas in the case of subdiabetic and mild diabetic models, the same dose showed reduction in BGL of 22.0% and 31.4% during GTT. The study also involves the first use of laser-induced breakdown spectroscopy as a sensitive analytical tool to detect the elemental profile responsible for the antidiabetic activity of aqueous extract of T. dioica fruits that exhibits the antidiabetic activity. High intensities of Ca, Mg, and Fe indicate large concentrations of these elements in the extract, since according to Boltzmann’s distribution law, intensities are directly proportional to concentrations. The higher concentrations of these glycemic elements, viz. Ca, Mg, and Fe, are responsible for the antidiabetic potential of T. dioica as well as other plant already reported by our research group16. 3.g. Antipyretic activity Sudarshan churna is a very potent Ayurvedic preparation, which is used traditionally as antimalarial and antipyretic formulation. Swertia chirata and Trichosanthes dioica is key ingredient in Sudarshan churna. The purpose of study was to evaluate antipyretic activity of Sudharshan churna. Aqueous extracts of Sudarshan churna was evaluated for antipyretic activity using two models including hyperpyrexia-induced in rats by brewer’s yeast and another one hyperpyrexia induced in rabbits by Typhoid-Paratyphoid A, B vaccine. Like Paracetamol (100 mg/kg, p.o.), Sudarshan churna, showed significant reduction in elevated body temperature at 200 mg/kg, p.o. On the basis of study, it was concluded that aqueous extract of Sudarshan churna has shown significant antipyretic activity17. 3. h. Glycemic property This study was to screen the glycemic attributes of an aqueous extract of Trichosanthes dioica leaves in normal as well as various diabetic models. The variable doses of 250, 500, and 750 mg/kg body weight (bw) of the extract were administered orally to normal and streptozotocin (STZ)-induced sub- and mild-diabetic rats in order to define its glycemic potential.

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The dose of 500 mg/kg bw was identified as the most effective dose which brings down the blood glucose level (BGL) by 32.9% (P < 0.001) at 6 h during fasting blood glucose (FBG) studies in normal rats. However, glucose tolerance test (GTT) showed the maximum reduction of 30.9% (P < 0.001) in BGL at 5 h in normal rats with the same dose, whereas the reduction observed was by 40.3% and 88.6% (P < 0.001) in sub- and mild-diabetic rats, respectively, at 3 h of glucose administration only. This evidence clearly indicates that the aqueous extract of Trichosanthes dioica leaves has good hypoglycemic potential along with a high anti-diabetic profile9. 3. i. Burns &Wound Healing The methanolic extract of the plant was selected for assessment of healing potential in the form of simple ointment using full thickness burn wound model in rats. The effect produced by the extract ointment showed significant healing when compared with the control and standard groups. All parameters such as wound contraction, epithelialization period, hydroxyproline content, and histopathological studies were observed significant (P<0.01) in comparison to control group18. 3.j. Hepatoprotective activity The study was carried out to assess the potential of Trichosanthes dioica Roxb. (TD) as a hepatoprotective agent in ferrous sulphate (FeSO4) intoxicated rats. Liver damage was induced in Wistar rats by administering ferrous sulphate (30 mg/kg, p.o) on 10th day. Ethanolic and Aqueous extracts of TD at different doses (100, 200 and 400 mg/kg) and silymarin (100 mg/kg) were administered orally for 10 days. TD-200e showed decrease in the levels of AST (p<0.01), ALT, TB, ALP and increase in TP (p<0.05). TD-200a showed significant decrease in the levels of AST, ALT, TB, ALP and increase in TP levels. The groups treated with 400 mg/kg aqueous and ethanolic extract showed significant (p<0.01) reduction in AST, ALT, ALP, TB and increase in TP level. The pretreatment with TD extracts showed profound histopathological protection to liver cells as evident from histopathological studies. Hence it can be concluded that Trichosanthes dioica Roxb. has significant hepatoprotective activity19. 4. Conclusion Trichosanthes dioica is a well-known plant used in the Indian system of medicine, besides which folklore medicine also claims its uses especially in diabetics and hepatic diseases, etc. Trichosanthes dioica fruit is cultivated in India, Japan, Sri Lanka, China, and Thailand for its vegetable use. Presently there is an increasing interest worldwide in herbal medicines accompanied by increased laboratory investigation into the pharmacological properties of the bioactive ingredients and their ability to treat various diseases.

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Numerous drugs have entered the international market through exploration of ethnopharmacology and traditional medicine. Although scientific studies have been carried out on a large number of Indian botanicals, a considerably smaller number of marketable drugs or phytochemical entities have entered the evidence-based therapeutics. Efforts are therefore needed to establish and validate evidence regarding safety and practices of Ayurvedic medicines.

References 1. M. L. Chadha. Indigenous Vegetables of India with a Potential for Improving Livelihoods. International Symposium on Underutilized

Plants for Food Security, Nutrition, Income and Sustainable Development 2009; 2: 1-8. 2. Chakravarthy, H.M., 1982. Fascicles of flora of India - 11 Cucurbitaceae. Botanical Survey of India, p. 136. 3. J.B. Mythili, Pious Thomas. Micropropagation of pointed gourd (Trichosanthes dioica Roxb.) Scientia Horticulturae 1999; 79: 87-90. 4. Sharma, G., and M.C. Pant. Effects of feeding Trichosanthes dioica (parval) on blood glucose, serum triglyceride, phospholipid,

cholesterol, and high density lipoprotein-cholesterol levels in the normal albino rabbit. Current Sci. 1988; 57:1085–1087. 5. Sharma G, Sarkar A, Pachori SB, Pant MC. Biochemical evaluation of raw Trichosanthes dioica whole fruit and pulp in normal and mild-

diabetic human volunteers in relation to lipid profile. Ind Drug 1989; 27: 24–28. 6. Singh, K. Pointed gourd (Trichosanthes dioica Roxb.). Indian Hort. 1989; 33: 35–38. 7. S.K. Mukharjee, Indian scenario (Abstract-11), 2–4 September, Varanasi, India (1996). 8. Prashant Kumar Rai a; Dolly Jaiswal a; Sandhya Diwakar b; Geeta Watal Antihyperglycemic Profile of Trichosanthes dioica Seeds in

Experimental Models. Pharmaceutical Biology 2008; 46(5): 360–365. 9. Prashant Kumar Rai, Dolly Jaiswal, Rakesh Kumar Singh, Rajesh Kumar Gupta, and Geeta Watal. Glycemic Properties of Trichosanthes

dioica Leaves. Pharmaceutical Biology 2008; 46(12): 894–899. 10. Sanjib Bhattacharya, Pallab Kanti Haldar, Ashoke Kumar Ghosh. In vitro effects of Trichosanthes dioica leaves on annelids and

nematodes. Pharmacologyonline 2009; 2: 242-248. 11. Ratnesh K Sharma, Sanjukta Chatterji, Devendra K Rai, Shikha Mehta, Prashant K Rai, Rakesh K Singh, Geeta Watal and Bechan

Sharma. Antioxidant activities and phenolic contents of the aqueous extracts of some Indian medicinal plants. Journal of Medicinal Plants Research 2009; Vol. 3(11): 944-948.

12. Yogesh Shivhare, Priya Singh, Rajak H., Patil U.K., Pawar R.S. Antioxidant potential of Trichosanthes dioica Roxb (fruits). Pharmacognosy Journal 2009; Vol 1(4): 258-262.

13. Govind Sherma and M.C. Pant. Influence of alcoholic extract of whole fruit of Trichosanthes dioica on blood sugar, serum lipids, lipoproteins and faecal sterols in normal albino rabbits. Indian Journal of Clinical Biochemistry 1992; 7: 53-56.

14. Sharmila Banu G, Kumar G, Rajasekara Pandian M. Cholesterol-Lowering Activity of the Aqueous Fruit Extract of Trichosanthes dioica Roxb (L.) in Normal and Streptozotocin Diabetic Rats. Journal of Clinical and Diagnostic Research. 2007; 1(6): 561-569.

15. Prashant Kumar Rai, Dolly Jaiswal, Devendra K. Rai, Bechan Sharma and Geeta Watal. Effect of water extract of Trichosanthes dioica fruits in streptozotocin induced diabetic rats. Indian Journal of Clinical Biochemistry 2008; 23 (4): 387-390.

16. Prashant Kumar Rai & Sanjukta Chatterji & Nilesh K. Rai & Awadhesh K. Rai & Dane Bicanic & Geeta Watal. The Glycemic Elemental Profile of Trichosanthes dioica: A LIBS-Based Study. Food Biophysics 2010; 5: 17–23.

17. Sushil Bhargava, Paridhi Bhargava, Surendra Saraf, Ravindra Pandey, Shiv Shankar Sukla and Rajesh Garg. Evaluation of antipyretic activity of sudarshan churna: an ayurvedic formulation J. Res. Educ. Indian Med. 2008; 11-14.

18. Yogesh Shivhare, Priya Singh and UK Patil. Healing Potential of Trichosanthes dioica Roxb on Burn Wounds. Research Journal of Pharmacology and Pharmacodynamics 2010; 02(02): 168-171.

19. Ghaisas MM, Tanwar MB, Ninave PB, Navghare VV, Takawale AR, Zope VS, Deshpande AD. Hepatoprotective activity of aqueous and ethanolic extract of Trichosanthes dioica roxb. in ferrous sulphate-induced liver injury. Pharmacologyonline 2008; 3: 127-135.

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SHORT COMMUNICATION

Research article Hygeia.J.D.Med.Vol.2 (2), 2010, 8-13 ISSN 0975 6221 __________________________________________________________________________________________________________________________ HYGEIA JOURNAL FOR DRUGS AND MEDICINES

www.hygeiajournal.com __________________________________________________________________________________

Effect of microwave drying in improving granule characteristics in tablets

Deepthi Shyju.1*

1. Pushpagiri College of Pharmacy Medicity campus, Perumthuruthy (P.O), Tiruvalla, Kerala, India-689107 Article history: Received: 20 June 2009, Revised: 17 January 2010, Accepted: 15 April 2010, Avalable online: 20Sep.2010 ___________________________________________________________________________________________________________________ Abstract

In the present study, paracetamol is used as model drug and the granules were formed by using microwave technique and fluid bed drying technique. The granules prepared by microwave technique and fluid bed drying technique are evaluated for parameters such as amount of fines, drying time, bulk density,compressibility,angle of repose etc.The study indicated that the granules retained their structure in comparison with the conventional drying process. The prepared granules were compressed into tablets and evaluated for hardness, friability, disintegration, and dissolution etc.

Keywords: Paracetamol tablets, Microwave drying, fluid bed drying

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1. Introduction

Major limitations of classical pharmaceutics experiments are longer time, higher cost, longer reaction time and environmental pollution due to the use of large of quantities of solvents/reagents. Since the heating process is very short in microwave procedure, which saves fuel/electricity, chemicals helps to reduce environment pollution. Synthesis of drugs, intermediates, chemicals, activation of chromatographic adsorbents, determination of drug loss on drying ,drying of glasswares,sterilization of glass wares and auxiliaries, drying of granules for the preparation of tablets, enzyme inactivation of food products, hydrolysis of proteins and peptides,saponification of oils etc are a few examples of use of microwave in laboratories.1 The wavelengths of microwaves are in a range of about 1 to 10 mm.In microwave spectroscopy, the source is monochromatic, at a well defined single wavelength which can be rapidly varied. The resolving power is 105 times that of the best infrared grating spectrometer.2 ________________________________________

*For Correspondence: [email protected] Contact: 9447407870 / 0475 2353003 © 2010 HYGEIA journal for drugs and medicines. , All rights reserved. 0975 6221

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DeepthiShyju, et.al, Hygeia.J.D.Med.Vol.2 (2), 8-13

The advantages of microwave drying technique are microwaves systems are morecompact, requiring a smaller equipment space or footprint. Microwaves generate higher power densities, enabling increased production speeds and decreased production costs.3 The aim of the present study was to standardize the drying process for pharmaceutical granulations by microwave technique and compare the present release of drug obtained by microwave technique with other drying technique. 2. Formulation of granules Granules were prepared using paracetamol was used as model drug, starch as binder as well as disintegrate, talc as glidant magnesium stearate was used as lubricant. 2.1. Procedure for Preparation of Granules by Fluidized Bed Drying (FBD): Wet granulation technique was used for the preparation of granules.4The required quantities of drug and other excipients were weighed and passed through British standard sieve no: 60 to get uniform particle size. The powders are then mixed to get uniform blend. The granulating medium was added to the powder blend and mix well until a smooth dough was obtained. The wet granules were passed through sieve no.16 and dried at 60◦c for 1 hour in a fluid bed dryer for a batch. The dried granules were passed through sieve no: 16/22 and the granules which passed through sieve no: 16 but retained on sieve no: 22 were selected. The granules obtained through sieve no.22 were considered as fines. 2.2. Microwave Granulation Procedure: The required quantities of drug and other excipients were weighed and passed through standard sieve no: 60, to get uniform particle size. The powders were then mixed to get a uniform blend. The granulating medium was added to the powder blend and mixed well until smooth dough was obtained. The wet granules were passed through sieve no: 16 and dried at 840 watts in microwave for different time intervals. After every 15 seconds, the granules were observed for dryness and if not dried, the drying process was continued until the granules were completely dried. After complete drying, the dried granules were passed through sieve no: 16/22 and the granules which pass through sieve no: 22 were selected .The granules obtained through sieve no: 22 were considered as fines. 3. Evaluation of granules The granules using both fluid bed and microwave procedure were evaluated for percentage of fines 5, bulk density6, compressibility6 and flow properties using angle of repose6and moisture content determinations. 3.1. Percentage of fines The granules were passed through standard sieve no: 16/22.The material retained on sieve no:22 were collected separately and weighed. From this, the percentage of fines was calculated. 3.2. Moisture content determinations Moisture content (loss on drying) of granules before and after drying was determined

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3.3. Bulk density A given quantity of sample was transferred to a measuring cylinder and was tapped mechanically, using a tapping device till a constant volume was obtained, which referred as bulk volume. The bulk volume was calculated by Bulk volume =mass of sample/bulk volume 3.4. Compressibility The compressibility index of the granules was determined by using loose and tapped bulk densities of granules, according to the equation below; Carrsconsolidationindex= [(Tapped bulkdensity-loosebulk density) x100]/Tapped bulk density 3.5. Flow properties A funnel was fixed at a particular height ‘h’ cm on a burette stand and graph paper was placed below the funnel table.The sample whose angle of repose is to be determined was poured into the funnel by closing the bottom of the funnel.The bottom was opened and sample was allowed to fall onto the paper.The height of the formed pile was measured and the circumference of the pile was drawn with the pencil on the graph sheet.The radius of the pile was noted as ‘r’ cm and the angle of repose was calculated as follows: tanө=h/r or ө= tan-1(h/r) where h=height of the pile,r=radius of pile and ө=angle of repose 4. Preparation of tablets The granules were mixed with glidant and lubricant and compressed using a 16-station rotary tablet machine with 10mm standard concave punches. The batch size was 200 tablets. Two batches of tablets were prepared, corresponding to fluid bed drying granulation procedure and other batch corresponding to microwave drying at 840 watt. The prepared tablets were evaluated for weight variation, hardness, friability, drug content, and disintegration time and invitro dissolution profile. InVitro drug release study: Drug release studies were carried out using USP (XX111) dissolution apparatus following paddle method. Freshly prepared buffer of pH 5.8 (900ml) was placed in the dissolution flask and allowed to attain a temperature of 37±1oC.The tablet was placed at the bottom of the dissolution flask. The paddle was rotated at 50 rpm for 30 minutes. One ml of the sample was withdrawn at different time intervals at 5, 10,15,20,25 and 30 minutes. After each withdrawal, the medium was replaced by equal amount of fresh buffer. The samples were diluted to 10 ml with dissolution medium and used for measurement of absorbance 257nm, in a UV-visible spectrophotometer. Percentage release of drug = Absorbance of sample ×content of standard × Dilution factor/ Absorbance of standard× label claim.

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5. Results & discussion 5.1. Evaluation of Granules: One batch of granules corresponding to fluid bed dried wet granulation and other batch corresponding to microwave drying were prepared and evaluated for percentage of fines, bulk density, compressibility and flow properties using angle of repose. The granule drying time was found to be very less in case of microwave drying. The fluid bed drying method took 60 minutes for complete drying of granules whereas the microwave method took a maximum of 3 minutes at 840 watt. The results of evaluations of granules shown in Table 1 5.2. Evaluation of Tablets: The tablets were evaluated for weight variation, hardness, friability, drug content, disintegration and in Vitro dissolution.. The results of evaluations of tablets shown in Table 2 5.3. Dissolution test From the results, it was found that the tablets prepared by fluid bed dried granulation and those prepared by microwave granulation at an intensity of 840 watt exhibit good release profiles. They released 98-99.5 release in 30 minutes time. From the results, it can be concluded that the batch which were dried at an intensity of 840 watt was ideal batch, and the results were comparable with that of fluid bed dried tablets.Hence, higher intensities can be used for drying of granules in regular classes. The results of in vitro dissolution studies of two batches of tablets were shown in Table 3 and Figure 1 Conclusion It can be concluded that microwave drying effectively improve the characteristics of granules in tablets. It can be stated that the tablet granulation can be dried successfully using a microwave oven. By adopting microwave drying technique, tablets can be prepared in less duration of time, at least 10 times less than fluid bed drying procedure. This can save time, energy and cut down the cost of conducting practical classes.Also, use of such technique can reduce environmental pollution.

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Table 1: Properties of paracetamol granules using Fluid bed and Microwave methods:

Physical properties Microwave dried granules at 840w Fluid bed dried

granules

Amount of fines(%) 13.61 14.10 Bulk density(g/cc) 0.94±0.006 0.94±0.5 Compressibility (%) 6.15± 0.005 6.02 ± 0.003 Angle of repose( 0 ) 15.99± 0.5 14.94 ±0.42 Drying time(min) 2.8± 0.52 60.2± 0.31 Loss on drying (%) 2.5-3.45 3.0-4.5

All the values are represented as mean ± s.d; n=3

Table 2: Properties of paracetamol tablets prepared using fluid bed dried and Microwave dried methods.

All the values are represented as mean± s.d; n=3

Table 3: Cumulative release of drug from two batches of tablets prepared by microwave and fluid bed drying methods

Time(min) Cumulative drug release from Microwave dried tablets at 840 w (%)*

Cumulative drug release from Fluid bed dried tablets (%)*

5 30.15 29.41

10 34.81 31.85

15 49.75 44.59

20 65.93 59.70

25 77.54 74.11

30 99.87 95.54

*Average of three determinations

Evaluation parameters Microwave dried tablets at 840 w Fluid bed dried tablets

Average weight (mg) 660± 0.5 607±0.4 Hardness(kg/cm2) 5.3±0.02 4.56±0.04

Friability (%) 0.109 0.124 Drug content(mg) 508±0.024 506±0.046

Disintegration(sec) 55.02±0.1 44.66±0.4

13

0

20

40

60

80

100

120

0 5 10 15 20 25 30 35 40 45 50

Time (min )

Cu

mu

lati

ve d

rug

rel

ease

(%

)

Percentage ofcumulative drugrelease at 840 w

Percentage ofcumulative drugrelease at FBD


Figure 1 In Vitro dissolution profiles of two batches of tablets prepared by microwave and FBDmethods

References

1. Sharma SV, Sharma GVSR, Suresh B; A ecofriendly technology, Ind. J. Pharm. sci, 2002; jul-aug; 64 (4):337-344.

2. Walter J Moore; Physical chemistry, 5th Edition, 1999, Orient Longman limited, 761.

3 .www.industrialmicrowave.com/faqs.htm

4. Leon Lachman, Liberman HA., KanigL J; Theory and practice of industrial pharmacy, 3rd Edition, Varghese publishing -

House, 1987; 293-345.

5. Lieberman HA, Leon Lachman, Schwartz BJ; Pharmaceutical dosage forms: Tablets, Vol. 2, 2nd Edition, 1989; Replika-

Press, 245-335.

6.Martin A,Bustamanate P,Chun A H C,Physical Pharmacy,4thEdition,Gopsons papers,2003;423-490.

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Research article Hygeia.J.D.Med .vol.2 (2), 2010, 14-21 ISSN 0975 6221 _______________________________________________________________________________________________________

HYGEIA JOURNAL FOR DRUGS AND MEDICINES


____________________________________________________________________________________

Evaluation of In-vitro vector control activity of Physalis angulata.

Sandhya S1*, Jafferi S.A.H1, Vinod K.R1, Ottilia Banji1, David Banji1,

Chaitanya R.S.N.A.K.K1, Chandrasekhar J1, Venkataramana.K2

1. Nalanda College of Pharmacy, Nalgonda, Andhra.Pradesh., India 2. A.S.N Pharmacy College, Tenali, Andhra.Pradesh., India Article history: Received: 15 May 2009, revised: 16December 2009, accepted: 23March 2010 Available online 20 Sep 2010.

__________________________________________________________________________________________________________________

Abstract

The present study was undertaken to evaluate anthelmintic and larvicidal activity of crude ethanolic leaf extract of Physalis angulata belonging to family Solanaceae. Pheretima posthuma was used as the test worms. Various concentrations of ethanolic extracts were tested in the anthelmintic screening, which involved determination of time of paralysis (P) and time of death (D) of the worms. Piperazine citrate was included as standard reference and distilled water as control. In the case of larvicidal activity the study was conducted on Culex quniquefasicatus species of mosquito larvae and the rate of larval mortality was calculated. The results indicated that the crude ethanolic extract significantly demonstrated paralysis and also caused death of the helminthes especially at higher concentration of 50 mg/ml, as compared to standard reference piperazine citrate. Similarly very optimistic results were observed for Culex quniquefasicatus species of mosquito larvae and LC50 value was calculated as 51.8802 mg/l.

Keywords: Anthelmintic, larvicidal, Physalis angulata, Pheretima posthuma, Culex quniquefasicatus

___________________________________________________________________________________________________________________

1. Introduction Ethanopharmacology got its prominence as a science of relationship between primitive society and there environment. Physalis angulata L., Family- Solanaceae commonly known as Cutleaf Ground-Cherry is one such commonly used ethno botanical plant. P.angulata is an annual herb indigenous to many parts of the tropics, including the Amazon. It can be found on most continents in the tropics, including Africa, Asia, and the Americas. It grows up to 1 m high, bears small, cream-colored flowers, and produces small, light yellowish-orange, edible fruit sometimes referred to as cutleaf groundcherry. Fruit is about the size of a cherry tomato, and like tomatoes, it contains many tiny edible seeds inside P.angulata propagate easily from the many seeds the fruit contains; spontaneous clumps of plants can be found along river banks and just about anywhere the soil is disturbed and the canopy is broken. ______________________________________ *For Correspondence: email: [email protected] © 2010 Hygeia journal for drugs and medicines. All rights reserved. 0975 6221:

Contact: +91 9010055004

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Sandhya.S, et al, Hygeia.J.D.Med .vol.2 (2), 2010, 14-21

Organic extracts of the whole plant exhibits Immunomodulatory, Anti-inflammatory, anticancer, antinociceptive, trypanocidal, antimycobial, molluscidal, Antigonorrheal and Antioxidant effects1-9. Substantial scientific evidences are now recognizing multitude of these medicinal uses. Phytochemical investigation of P.angulata has led to elucidation of many novel chemical compounds primarily it constitutes of a seco-steroidal compound Physalin. Purified secosteroids have shown to inhabit lymphocyte function and allogeneic transplant rejection10.

Studies have led to the indication that P.angulata exerts powerful anti-inflammatory by interfering with cyclooxygenase pathway, lymphocyte proliferation, NO, and TGF- beta production11. Anti hepatoma activity of Physalis extracts on apoptosis in human Hep G2 cells was conducted and results conclude that this potent activity is associated with mitochondrial dysfunction12. The extensive survey of literature revealed that P.angulata has diverse pharmacological spectrum which needs further clinical and animal evaluation. P.angulata has all the attributes to be termed as the hidden “Holy Grail of Medicine”.

During the past decade there have been major efforts to plan, implement, and sustain measures for reducing the burden of human disease that accompanies helminth infections. Further impetus was provided at the Fifty-fourth World Health Assembly, when WHO Member States were urged to ensure access to essential anthelminthic drugs in health services located where the parasites - schistosomes, roundworms, hookworms, and whipworms - are endemic.

The Assembly stressed that provision should be made for the regular anthelminthic treatment of school-age children living wherever schistosomes and soil-transmitted nematodes are entrenched. Helminth infections are among the most common infections in man, affecting a large proportion of the world's population.

In developing countries they pose a large threat to public health and contribute to the prevalence of malnutrition, anemia, eosnophilia, and pneumonia. Although the majority of infections due to worms are generally limited to tropical regions, they can occur to travelers who have visited those areas and some of them can develop in temperate climates. Parasitic diseases cause severe morbidity, including lymphatic filariasis (a cause of elephantiasis), onchocerciasis (river blindness), and schistosomiasis. These infections can affect most populations in endemic areas with major economic and social consequences.

Since the discovery of DDT, control of disease-causing mosquito species has been almost completely based on synthetic organic insecticides. Following DDT, conventional pesticides such as malathion and pyrethroids are generally used for mosquito control. But the extensive use of synthetic organic insecticides during the last five decades has resulted in environmental hazards. Besides, this also caused the development of physiological resistance in the major vector species. This has necessitated the need for search and development of environmentally safe, biodegradable, low cost and indigenous methods for vector control, which can be used with minimum care by individual and communities in specific situation (ICMR bulletin, 2003)13.

16


Based of the above findings and traditional claims of the plant an in-vitro anthelmintic and larvicidal assay was conducted to prove them.

2. Materials and methods

Plant collection and authentication: The plant was collected in the month of November and December from the surrounding areas of Nalgonda and Ranga Reddy district ,A.P, India. The plant was identified and authenticated by Department of Botany, Osmania University, Hyderabad. Preparation of Herbarium was submitted and the plant was certified as Physalis angulata L. ; Family – Solanaceae; Voucher no : 00490 (OUAH) 2.1. Collection of worms and larvae: Indian earthworm Pheretima posthuma (Annelida) were collected from the culture environment water logged areas of soil at the Nizam College of science, Osmania University, Hyderabad.The larvae of Cx. quniquefasicatus 3rd & 4th stage instar larvae which were procured from the Dept. of Zoology, Osmania University, Hyderabad. 2.2. Plant extraction14: The leaves of the plant was dried for several days and powdered with the help of an electric grinder and extracted exhaustively with ethanol. The liquid extract was evaporated in vacuum to yield 14.59%w/w. 2.3. Preliminary phytochemical screening The preliminary chemical tests for the ethanolic leaf extract showed presence of steroids, flavonoids , tannins and phenols. 3. Anthelmintic assay15

3.1. Preparation of test sample Samples for in-vitro anthelmintic study were prepared by dissolving and suspending 2.5 g of crude ethanolic extract fractions in 25 ml of distilled water to obtain a stock solution of 100 mg/ml. From this stock solution, different working dilutions were prepared to get concentration range of 10, 25 and 50 mg/ml. The anthelmintic assay was carried as per the method of Ajayieoba E. O. et al with minor modifications. The assay was performed on adult Indian earthworm Pheretima posthuma, due to its anatomical and physiological resemblance with the intestinal roundworm parasites of human beings16-18. Three different concentrations of 10, 25 and 50 mg/ml in distilled water were taken in petriplates and six earth worms of same size were placed in each plate. Time for paralysis was noted when no movement of any sort could be observed except the worms were shaken vigorously. Time for death of worms were recorded after ascertaining that the worms neither moved when shaken vigorously nor when dipped in warm water at 500 C. Piperazine citrate (10 mg/ml) was used as reference standard and distilled water as the control.

17


3.2. Larvicidal assay 19

Drug samples for larvicidal activity were prepared by making a stock solution which was serially diluted in water. Test concentrations are then obtained 25.50, 100.150 and 200 mg/l of the appropriate dilution. The larvicidal assay was carried as per the W.H.O guidelines for larvicidal activity with minor modifications. Batches of approximately 25 third or fourth instar larvae were transferred by means of strainers, screen loops or droppers to small disposable test cups or vessels, each containing 100–200 ml of water along with drug concentrations. The test containers were held at 25–28oC and preferably a photoperiod of 12 h light followed by 12 h dark (12L: 12D). After 24 hr exposure, larval mortality was recorded. Moribund larvae were counted and added to dead larvae for calculating percentage mortality. Dead larvae were those that could not be induced to move when they were probed with a needle in the siphon or the cervical region. Moribund larvae were those incapable of rising to the surface or not showing the characteristic diving reaction when the water was disturbed. The results were recorded where the LC50, LC90 and LC99 values, and slope were also plotted. 4. Data analysis Data from all replicates were pooled for analysis. LC50 and LC90 values were calculated from a log dosage–probit mortality regression line using biological statistical program BIOSATAT 2008, Professional package by Analyst soft .Inc, U.S.A. 5. Results and discussion Tribals of Andhra Pradesh use P.angulata for its anthelmntic properties20. These traditional claims have been proven in this experiment where the plant has shown to exhibit potent anthelmintic activity. It showed a response time of 9 min and 17 min for paralysis and death respectively. The reference drug Piperazine citrate showed the same activity of 19.26 and 63.25 minutes at 10mg/ml respectively.Physalis angulata has exhibited anthelmintic activity in dose dependent manner taking shortest time for paralysis (P) and death (D) with 50mg/ml concentration (Table no.1& fig no.1). As R2 is closer to one the extract shows good co-relation among death time taken at different concentration there by it can be said that the activity is dose dependent in nature. LC50 calculation was done using Probit Analysis. (Biostat 2008 professional software). The end anthelmentic activity of the extract is shown in fig 3. Regarding the larvicidal activity the percentile mortality values of instar larvae treated with different concentration of the leaf extract of P.angulata at the end of 24 hr are represented Table no (2,3,4) for C.quinquefasciatus . The regression equations (based on probit analysis) between the concentration of leaf extract and 24 h per cent mortality of 3rd and 4th instar larvae of C. quinquefasciatus are represented in Fig no 2. The LC50 value was calculated as 51.8802. The end larvicidal activity of the extract is shown in fig 4. 6. Conclusion Phytochemical analysis of crude extract reveled presence of phenols. flavonoids, phenols and steroids.

18


It has been reported that some synthetic Phenols interfere with energy generation in helminth parasites by uncoupling oxidative phosphorilation16. Hence it is possible the extract of P.angualata could also produce similar effects. The control of mosquito-borne diseases can be achieved either by killing, preventing mosquitoes to bite human beings (by using repellents) or by causing larval mortality in a large scale at the breeding centers of the vectors in the environment. The extract of Physalis angualta could be used for spraying in stagnant water bodies which are known to be the breeding grounds for mosquitoes acting as vector for a multitude of infectious diseases. Acknowledgement The authors are grateful to Nalanda College of Pharmacy for providing all necessary information resource, electronic data processing and moral support for the present work. Also they wish to express their thanks to Nizam College of Science and Dept. of Zoology, Osmania University for providing helminth and larvae respectively. Table: 1 Anthelmintic time profile for P.angulata

Results are expressed as Mean±SD from three set of observations.

Fig no : 1 - The line represents death time for P.angulata

Concentration(mg/ml) Time for paralysis(min) Time of Death(min)

10 16.36±0.4 45.04±0.02

25 12.07±0.8 33.37±0.45

50 9.07±0.01 17.05±0.03

Piprazine citrate 19.26±0.62 63.25.±0.58

19


Table no.2 – Efficiency of ethanolic extract of leaf of P.angulata on Cx.quiniquefasicatus

Concentration (mg/l) No. of exposed larvae No. of dead larvae Control 25 2 25 23 5

50 25 13

100 24 17

150 23 20

200 25 23

Table no: 3 – Results of Finneys analysis for the larvicidal activity of P.angulata

Log10[LC50] 1.715

LC50 51.8802

Standard Error LC50 8.0392 LC50 LCL 36.5455

LC50 UCL 66.9256

Log10[LC16] 1.288 LC16 19.4091

LC84 138.6752

LC100 182.0728

Significance Level 0.05 Log10[LC84] 2.142

Beta 2.3419

Alfa 0.9836 Standard Error Beta 0.4197

Standard Error Log10[LC50] 0.067

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Sandhya.S, et al, Hygeia.J.D.Med .vol.2 (2), 2010, 14-21.

Fig: 3: Paralyzed worms in 50mg/ml conc. of in Fig: 4 Image of dead larvae at 24 hrs in Ethanolic extract of P.angulata the ethanolic extract of P.angulata.

Fig no: 2 – Graph for Finneys analysis

References

1. Castro DT, Figurido MB, Immune depression of R. Prolixux by physalins, ‘Journal of experimental Parasitology’, Vol 112(1)2006, 31, 43.

2. Santos A, Cabral TR, Cabral IR, Anti inflammatory effects of P.angulata, ‘Biocell’, vol 6(3), 2008,154. 3. Hwang, J. K, Anticariogenic activity of some tropical medicinal plants against Streptococcus mutans,

‘Fitoterapia’,Vol 75(6), 2004, 596-8. 4. Bastos, G. N., Santos A, Antinociceptive effect of the aqueous extract obtained from roots of Physalis angulata L. on

mice, ‘J. Ethnopharmacol’ ,vol 103(2) , 2006, 241-5. 5. Gracia.MB, Trypanosoma rangeli : Effect of physalin B on the immune reactions of the immune reactions of the

infected larvae of Rhodnius prolixus, ‘Experimental Parasitology’,vol 112(1), 2006, 37,43.

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Sandhya.S, et al, Hygeia.J.D.Med .vol.2 (2), 2010, 14-21.

6. Januario, A. H, Antimycobacterial physalins from Physalis angulata L. (Solanaceae) ‘Phytother Res’, 16(5) 2002, 445-48.

7. Dos JA, Tomassini TC , Molluscidal Activity of P.angulata on Biomphalaria Species, ‘Nuleo-de-Biologica’, vol 1, 2002,192.

8. Choi, E.M, Effect of some medicinal plants on plasma antioxidant system and lipid levels in rats, ‘Phytother. Res.’, Vol 19(5), 2005, 382-6.

9. Soares, M. B, “Physalins B, F and G, seco-steroids purified from Physalis angulata L., inhibit lymphocyte function and allogeneic transplant rejection, ‘Int. Immunopharmacol’, vol 6(3), 2006,408-14.

10. Vieira A.T, Mechanisms of the anti-inflammatory effects of the natural secosteroids physalins in a model of intestinal ischaemia and reperfusion injury, ‘Br. J. Pharmacol.’ Vol 146(2), 2005,244-51.

11. Wu, S. J, Antihepatoma activity of Physalis angulata and P. peruviana extracts and their effects on apoptosis in human Hep G2 cells, ‘Life Sci.’ ,vol 74(16), 2004,2061-73.

12. ICMR Bulletin. Prospects of using herbal products in the control of mosquito vectors, vol 33(1) 2003, 1-10. 13. Khandelwal.K.R,,Practical Pharmacognosy , Nirali prakashan, Pune India, first edn, 2005,27-35. 14. Ajaiyeoba E. O., Onocha P. A and Olarenwaju. In-vitro anthelmintic properties of Buchholzia coiaceae and

Gynandropsis gynandra extract, ‘Pharm. Biol.’ Vol 39(3), 2001, 217-20. 15. Thorn G. W, Adams R. D, E. Braunwald, K. J. Isselbacher and R. G. Petersdorf. Harriasons Principles of Internal

Medicine, McGraw Hill Co., New York, 1997, 1088. 16. Vigar.Z, Atlas of Medical Parasitology. P.G. Publishing House, Singapore, 1984, 216. 17. Chatterjee. K. D,Parasitology. Protozoology and Helminthology, Guha Ray Sree Saraswaty Press Ltd., Calcutta,

1967, 168-169. 18. Guidelines for laboratory and field testing of mosquito larvicides,W.H.O pesticide evaluation scheme 2005, 8-11. 19. Reddy. KN, Patnayak, Reddy CS, Raju VS, Traditional Knowledge on wild food plants in Andhra Pradesh, ‘Indian

Journal of Traditional Knowledge’, Vol 6 (1), 2007, 223.

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Research article Hygeia.J.D.Med.vol.2 (2), 2010, 22-31 ISSN 0975 6221

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The Hepatoprotective Effect of The Polyphenolic Compounds in the Roots of Trichilia connaroides Wight and Arn

Garima Agarwal1*, Anil Kumar Pant1 and Subroto Kumar Hore2

1.Department of Chemistry , G. B. Pant University of Agriculture and Technology, Pantnagar- 263 145, India.

2. Department of Pharmacology and Toxicology, G. B. Pant University of Agricultur and Technology, Pantnagar- 263 145, India. Article history: Received: 15 May 2010, revised: 20 June 2010, accepted: 3 August 2010 Available online: 20 Sep 2010.

________________________________________________________________________________________________________________

Abstract:

Trichilia species are used in the treatment of liver disorders and as a tonic in the traditional medicine. The present study was designed to evaluate the hepatoprotective effects of aqueous extract of T. connaroides roots on carbon tetrachloride induced hepatotoxicity in comparision with the known hepatoprotective agent Liv-52. Carbon tetrachloride induced changes in serum enzymatic levels of aspartate amino transferase , alanine amino transferase , alkaline phosphatase and total protein were restored towards normal levels by the extract. The biochemical observations were supported by the histopathological examination of rat liver sections. The results indicate that the extract offers hepatoprotection in a dose-dependent manner. The effect was comparable to that produced by Liv-52. Simultaneously, in-vitro anti-oxidant activity of the extract as evaluated in terms of reducing power, radical scavenging activity and chelating activity on Fe+2 also support its hepatoprotective action. Phytochemical examination of the extract revealed the presence of antioxidant phenolics. Keywords: Trichilia connaroides, Carbon tetrachloride, Hepatoprotective activity, Antioxidant activity, Histopathology. _____________________________________________________________________________________________________________ 1. Introduction

Liver is a key organ of metabolism and detoxification. Continuous exposure to a variety of environmental toxic agents enhances hepatic injury. A growing interest has emerged around the globe in rediscovering medicinal plants as useful therapeutic agents for the prevention of such injury1. Eventhough modern medicine is advancing at a fast pace no effective drugs are available, to stimulate liver functions and to offer protection to the liver from the damage or help to regenerate hepatic cells 2. Therefore, many folk remedies of plant origin are tested for their potential anti-oxidant and hepatoprotective liver damage in experimental animal model 1. A large number of medicinal preparations are recommended for the treatment of liver disorders due to the lack of reliable liver protective drugs 3.The Meliaceae plant family has long been used in India for its medicinal properties. The genus Trichilia contains 40 genera and 600 species 4,5 distributed in sub-Himalayan tract from Kumaun eastward, Sikkim up to 4000 ft, Khasia Hills, Manipur,E. Ghats in the forests of Godawari and Vizagapatnam up to 4,500 ft. W.Ghats from Poona Southwards through the Nilgiris and Anamalais to Tranvancore, up to 6,000 ft. It is also distributed in Burma, Tonkin, Cambodia, Malay Peninsula and Sumatra6. T. connaroides Wight and Arn. is the only species in the genus to occur in India6. ________________________________________ *For Correspondence: Email: [email protected] Contact: 09986828970 © 2010 Hygeia journal for drugs and medicines. , All rights reserved. 0975 6221

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Garima.A. et al, Hygeia.J.D.Med, vol.2 (2) 2010, 22-31

Roots of T. connaroides are used as a Chinese drug to treat arthritis, pharingitis, tonsillitis and other ailments7. They are also used as tonic in traditional Indian medicine5. A decoction of the leaves is taken in cholera 5. Roots of Trichilia. emetica vahl. syn. T. roka Chiov are used for the treatment of liver disorders in the folk medicine of Mali. So its aqueous root decoction has been studied for the hepatoprotective effects and found to be quite active. The activity was attributed to the presence of the polyphenols 8, 9. Since T. connaroides belongs to the same genus and contains polyphenolic acids hence is was considered as the subject of this study. To date possible anti-oxidant and hepatoprotective activity of T. connaroides has not reported. The present communication investigates the hepatoprotective and in vitro anti-oxidant activities of the aqueous extract of T. connaroides as well as the effects of the phenolic acid components of its root. 2.Materials and Methods 2.1 Plant material and preparation of plant extract Roots of T. connaroides were collected from Kumaun region, India and identified at Forest Research Institute (FRI) Dehradun-Uttaranchal vide herbarium no. M-29. Shade dried and powdered roots (400 g) were extracted with boiling water for three times. The obtained extract (ARE) was then filtered and dried to powder in a freez drier (Labconco Corp.Kansas City, Mo.U.S.A). The dried extract (5g, % yield=1.25) was dissolved in distilled water in the required amount at the time of dosing. 2.2 Chemicals Carbon tetrachloride (CTC), HCl, NaOH, Butylated Hydroxy Toluene (BHT), EDTA, Citric acid, Diethyl ether, Methanol were procured from E-Merk (India) Limited Mumbai. 2, 2-diphenyl-2-picrylhydrazyl (DPPH) radical and phenolic acids standard were procured from the Sigma Aldrich USA. 2.3 Experimental Animals Male albino rats (140-230 g) of Sprague -Dawley strain were procured from the Laboratory Animal Unit, Govind Ballabh.Pant University of Agriculture & Technology., Pantnagar, Uttarakhand and kept in laboratory for one week to acclimatize in the new environment. During this period they were fed with standard rat diet (Lipton, India) and water ad libitum. Lighting was regulated to provide equal hours of light and dark. The study protocol was approved by Committee for the Purpose of Control and Supervision on Experiments on Animals (330/CPCSEA). All the experiments were performed in morning according to current guidelines for the care of the investigation of experimental pain in conscious animals 10. 3. Hepatoprotective Activity Treatment Schedule Carbon tetrachloride (CTC) induced acute toxicity: CTC was diluted with liquid paraffin (1:1) before administration. The animals were divided into six groups of six each. The animals were then subjected to either one of the following treatments for seven days: Group 1: Distilled water (10ml/Kg body weight) Group 2: Distilled water (10 mL/Kg b. wt + CTC (1 mL/Kg b. wt, i.p.) Group 3: ARE [Aqueous root extract of T. connaroides) (100 mg/Kg b. wt) + CTC (1 mL/Kg b. wt, i.p.) Group 4: ARE (200 mg/Kg b. wt) + CTC (1 mL/Kg b. wt, i.p.) Group 5: ARE (400 mg/Kg b. wt) + CTC (1 mL/Kg b. wt, i.p.) Group 6: Liv-52[Himalaya Drug Company, India] (2.5 mL/Kg b. wt) as a standard drug + CTC (1 mL/Kg b. wt, i.p.) The drugs were administered every day in the morning between 8-9. A.M. Weights of all the rats were taken on the first and the final day before feeding. On the 8th day morning, all the rats were anaesthetized with pentobarbital sodium (40 mg/kg b.wt. i.p.) and blood was collected from the orbital sinus through vein puncture.

24

Garima.A. et al, Hygeia.J.D.Med, vol.2 (2) 2010, 22-31 Livers of the sacrificed animals were removed and preserved in 10% formalin solution for histopathological study. General well being and behavior of the animals were observed daily throughout the period. 3.1 Assessment of liver function The hepatoprotective effect of the extract was evaluated by the assay of liver function biochemical parameters (total protein, alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), and alkaline phosphatase (ALP) activities using standard UV-auto-test kits (Span Diagonistic, India) and histopathological studies of the liver. 4. Antioxidant activity The antioxidant activity of ARE(aqueous root extract) was evaluated in terms of following three methods. 4.1 Reducing Power The reducing power of ARE was determined using the earlier reported method 11. 0.5 ml of different concentrations of ARE and standard BHT (5,10,15,20 and 25mg/ml) were mixed separately with 2.5 mL of phosphate buffer (200mM, pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixtures were incubated at 50°C and 2.5 ml of 10% trichloroacetic acid was added to the mixtures, followed by centrifugation 650 x g for 10 min. The upper layer (5mL) was mixed with 5 mL of distilled water and 1mL of 0.1% ferric chloride and the absorbance of the resultant solution were measured at 700 nm using the UV-Visible spectrophotometer. 4.2 Radical Scavenging Activity The scavenging effect on DPPH radical was determined according to the methods developed earlier 11. Various amounts of ARE and standard BHT (5, 10, 15, 20 and 25 mg) were mixed with 5 ml of 0.004% methanol solution of DPPH. Each mixture was incubated for 30min in the dark and the absorbance of the sample was read at 515 nm using the UV-visible spectrophotometer. The DPPH solution was freshly prepared and kept in the dark at 4°C between the measurements. DPPH scavenging activity (%) is calculated as [1 – (At / Ao)] x 100 (Where At is the absorbance of the sample at 515 nm, and Ao is the absorbance of the control at 515 nm). 4.3 Chelating activity The chelating activity of the ARE on ferrous ions Fe+2

was measured according to the method of Decker and Welch 12 .Aliquots of 1ml of different concentrations (5, 10, 15, 20 and 25 mg/ml) of the ARE were mixed with 3.7 mL of deionized water. The mixture was left for reaction with FeCl2 (2 mM, 0.1 ml) and ferrozine (5mM, 0.2 ml) for 10 min at room temperature, and then the absorbance was measured at 562nm. The chelating effect was compared with that of EDTA at a level of 0.01 mM and citric acid at a level of 0.025 M. Chelating activity (%) is calculated as [1 –(At/ Ao)] x 100 (Where At is the absorbance of the sample at 562 nm and Ao is the absorbance of the control at 562 nm). 5. Phytochemical Analysis The dried and coarse powdered roots of T. connaroides were refluxed with 2N HCl to liberate the free phenolics for 2hrs at 70-75°C, cooled to room temperature, centrifuged and then filtered. Filtrate was neutralized with 2N NaOH solution and extracted with diethyl ether. The organic layer was then dried and suspended in water for HPLC analysis. Benzoic, chlorogenic, gallic, ferulic, o-coumaric, p-coumaric, p-hydroxy benzoic, protocatechuic, syringic and vanillic acids were used as phenolic acid standards.13 6. Statistical Analysis Values are given as mean ± S.D. Statistical analysis was done by Student’s t-test in Windows Excel 2003.

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Garima.A. et al, Hygeia.J.D.Med, vol.2 (2) 2010, 22-31 7. Results

7.1 Hepatoprotective Activity (Biochemical examination)

Effect of ARE on CTC-induced hepatotoxicity was evaluated in rats and changes in serum biochemical parameters are presented in Table 1. CTC significantly increased the levels of serum enzymes viz. ALT, AST, ALP and significantly reduced the level of total serum protein in group II rats as compared to group I (normal) indicating the sign of hepatotoxicity. ARE-treatment considerably reduces the level of enzymes in the groups III, IV and V, but statistical significance was not reached but level of serum protein was significantly increased in group IV and V. The results were less significant when compared with the Liv-52. 7.2 Histopathological examination Photomicrographs of haematoxylin-eosin stained liver tissue show the normal liver with normal hepatocytes arranged in hepatic chords (Fig 1-A) Liver sections from CTC treated control revealed massive degeneration and necrotic changes. The degenerative changes consisted of small to large vacuoles in the hepatocytes, nucleus was pushed to one side of the hepatocytes. Mononuclear cellular (lymphocytes) infiltration, deposition of collagen fibers and mild hyperemia was also observed (Fig 1-B). Normal architecture of the liver is restored by higher doses of ARE in group IV and V. Microscopic lesions in the liver of the ARE treated group at a dose rate of 100mg/kg b.wt were almost similar as in the CTC treated group at 8th day post experimentation (Fig 1-C). Microscopic examination of group IV liver section exhibited less intense necrotic changes. Vacuolar degeneration and perivascular infliltration of mononuclear cells was also reduced (Fig 1-D), whereas liver section of Group V rats marked reduction in vacuolar degeneration and necrotic changes and, normal parenchyma is observed as compared to the above mentioned groups (Fig1-E). Reduced deposition of collagen fibers was comparable with Liv-52 treated group (Fig 1-F). 7.3 Effect on liver and body weights in rats The liver weight of CTC treated rats (Group II) increased significantly as compared to normal (Group I) showing the sign of hepatic damage. The liver weight of Liv-52 treated group decreased significantly as compared to CTC treated group showing the protective effect of Liv-52. But the body weight of all the rats remains unaltered (Table 2). 7.4 Antioxidant activity 7.4.1 Reducing Power: ARE exhibited moderate to good reducing power compared to BHT in a dose-dependent manner (Table- 3 and Fig- 2). The reducing power of ARE might be result of their hydrogen- donating ability of its components 11, which reduces the Fe+3 /ferricyanide complex to the ferrous form (Fe+2). The Fe+2 can therefore be monitored by measuring the formation of Perl’s Prussian blue at 700 nm 14. 7.4.2 Radical Scavenging Activity ARE exhibited moderate radical scavenging activities at lower concentration, but at highest concentration its radical scavenging is comparable with the BHT (Table 4 and Fig-3). 7.5. Chelating activity ARE showed chelating activity on Fe+2

in dose dependent manner as illustrated in Table 5, Fig 4. The chelating activity of ARE at all concentration was higher than that of EDTA at 0.01mM and citric acid at 0.025mM (35.01 5% 30.79 % respectively). 7.6. Phytochemical Examination of the T. connaroides extract Phytochemical analysis of ARE reveals the presence of phenolic acids- chlorogenic, ferulic, gallic, p-coumaric, protocatechuic and p-hydroxy benzoic acids.

26

Garima.A. et al, Hygeia.J.D.Med, vol.2 (2) 2010, 22-31 8. Discussion In the present work, aqueous extract of T. connaroides (ARE) was evaluated for hepatoprotective activity against CTC induced liver damage. Hepatotoxic effects of CTC are mainly due to its active metabolite, trichloromethyl radical 15,16 which binds covalently to the macromolecules and induces peroxidative degradation of the membrane lipid of the peroxide and the products like malodialdehyde are formed. This lipid peroxidative damage of the biomembranes is one of the major causes of CTC-induced hepatotoxity 17, 18. AST and ALT are the important indicators of liver damage as they are released into the blood circulation following injury to the liver 19. It had been shown that ARE exerted its action by preserving the structural integrity of the hepatocellular membrane resulting in the reduction of enzymatic level in the blood compared to CTC- treated rats. Significant increase in the level of total serum protein in ARE treated groups is also considered as a sign of hepatoprotection. This stimulation of protein synthesis can be depicted as hepatoprotective mechanism, which accelerates the regeneration process and production of the liver cells20. CTC induced a significant increase in liver weight, which is due to the blocking of secretion of hepatic triglycerides into the plasma 21. ARE at higher doses prevented the increase in the liver weight of pretreated with CTC. Considering all the results, we can confirm that ARE exerted a clear protective action against CTC- induced hepatic damage. Antioxidants are known to interrupt free-radical chain oxidation and to donate hydrogen from phenolic hydroxyl groups, thereby, forming stable free radicals, which do not initiate or propagate further oxidation of lipids 22. The antioxidant activity is evaluated in terms of reducing power, DPPH radical scavenging potential and chelating activity. DPPH has been widely used to evaluate the free radical scavenging capacity of antioxidants 23, 24. The ferrous state of iron accelerates lipid oxidation to reactive free radicals. Fe+2 ion also produces radicals from peroxides 25 and is the most powerful pro-oxidant among various species of metal ions 26. Ferrozine, a chelating agent, was used to indicate the presence of chelator in the reaction system. Ferrozine forms a complex with free Fe+2 but not with Fe+2 bound to extracts. In the presence of chelating agents, the complex formation of ferrous and ferrozine is disrupted, resulting in a decrease in red colour of the complex. Measurement of colour reduction therefore allows estimating the metal chelating activity of the coexisting chelator27. The antioxidant and hepatoprotective activities were studied to correlate each other. The lipid peroxidation is accelerated when free radicals are formed as the result of losing a hydrogen atom from the double bond of the unsaturated fatty acids. Scavenging of free radicals is one of the major anti-oxidant mechanisms to inhibit the chain reaction of lipid peroxidation. The free radical scavenging activity of ARE was evaluated by DPPH assay. DPPH is a well known abstractor of hydrogen. DPPH scavenging activity suggested that ARE contains the free radical scavengers which counter the pathological changes caused by the generated free CCl3 radicals. Scavenging of DPPH radical is related to the inhibition of lipid peroxidation28. Antioxidants are known to interrupt the free-radical chain oxidation and to donate hydrogen from phenolic hydroxyl groups, thereby, forming stable free radicals, which do not initiate or propagate further oxidation of lipids 22. The reducing power of ARE might be due to the presence of compounds having hydrogen- donating ability 11. Antioxidants present in the ARE reduced the Fe+3 /ferricyanide complex to the ferrous form (Fe+2) 14. Reducing power and chelating activity supplemented the radical scavenging effect of ARE. Phenolic groups play an important role in anti-oxidant activity 11 .These polyphenolic compounds in the cell can function as antioxidants and anti-prooxidants by scavenging reactive oxygen species via enzymatic and non-enzymatic reactions 29. It has been reported earlier, that antioxidants are responsible for hepatoprotective action30. Thus our findings suggest that the free radical scavenging and anti-oxidant activities could be the possible mechanism for the hepatoprotective activity of ARE which may be attributed to the presence of phenolic compounds. 9. Conclusion Trichilia connaroides showed hepatoprotective action supported by biochemical parameters, histopathology along with the antioxidant potential. The hepatoprotective effect was found to be comparable to Liv-52 administered as a standard drug. Further studies are in progress for understanding of the mechanism of action.

27


Acknowledgement Research facilities provided by the Govind Ballabh Pant University of Agriculture & Technology Pantnagar and CSIR-UGC fellowship to G. Agarwal are duly acknowledged. Table 1 Effect of aqueous extract of T. connaroides root (ARE) on carbon tetrachloride (CTC, 0.5 ml/kg b.wt.) induced serum biochemical changes in rats. (mean±S.E, n=6) ____________________________________________________________________________________________________

Group

Dose ALP (KA) ALT(IU/L) AST (IU/L) Total serum protein (g/dl)

I Water (10 ml/kg) 8.58 ± 0.54 45.6±5.57 165.83±8.9 8.64 ± 0.414

II CTC (0.5 ml/kg) + water

(10 ml/kg) 24.58 ± 2.5c 329.58±3.8c 294.2±22.9c 3.39 ± 0.12c

III ARE (100 mg/kg) + CTC

(0.5 ml/kg) 22.45 ± 2.7 320.25±15.6 253.5±20.9 3.87 ± 0.29

IV ARE (200 mg/kg) + CTC

(0.5 ml/kg) 20.2 ± 1.96 294.0 ±18.73 226.6±20.2 4.66 ± 0.39x

V ARE (400 mg/kg) + CTC

(0.5 ml/kg) 18.0 ± 3.98 260.25±33.1 241.2±31.5 5.72 ± 0.43y

VI Liv-52 (2.5 ml/kg) + CTC

(0.5 ml/kg) 15.2 ± 4.41 250.0±16.68y 216.55± 26.7y 7.40 ± 0.46z

ALP – Alkaline phosphatase; ALT–Alanine amino transaminase; AST– Aspartate amino transaminase; ARE –Aqueous root extract; CTC- Carbon tetra chloride. Student’s t-test – Pa<0.05, Pb<0.01, Pc<0.001 vs group I and PX<0.05, Py<0.01, Pz<0.001 vs group II Table 2 Effect of aqueous extract of T. connaroides root (ARE) on carbon tetrachloride (CTC, 0.5 ml/kg b.wt.) induced liver and body weights in rats.(mean ± S.E, (n=6).

Group Dose Wet liver weight (g) Body weight(g)

1st day 8th day I

Water (10 ml/kg)

5.78± 0.33

210.83 ± 6.50

203.3 ± 8.75

II CTC (0.5 ml/kg) + water

(10 ml/kg) 7.02 ± 0.58a 188.33 ± 10.38 181.0 ± 0.64

III ARE (100 mg/kg) + CTC

(0.5 ml/kg) 6.37 ± 0.53 200.0 ± 6.454 167.5 ± 5.62

IV ARE (200 mg/kg) + CTC

(0.5 ml/kg) 5.98 ± 1.01 200.8 ± 6.50 176.6 ± 6.29

V ARE (400 mg/kg) + CTC

(0.5 ml/kg) 6.08 ± 0.197 197.5±11.67 168.75 ± 8.8

VI Liv-52 (2.5 ml/kg) + CTC

(0.5 ml/kg) 5.73 ± 0.34x 171.66±11.66 173.0 ± 9.02

Student’s t-test- Pa<0.05 vs. group I and Px vs group II.

28


Table 3

Reducing power of aqueous extract of roots of T. connaroides (ARE) and butylated hydroxy toluene (BHT) at different concentrations.

Weight of ARE (mg/ml)

Reducing power ARE BHT

5

0.833 ± 0.00

0.846 ± 0.30

10 0.92 ± 0.00 1.00 ± 0.00

15 1.00 ± 0.02 1.24 ± 0.01

20 1.25 ± 0.00 1.48 ±0.01

25 1.47 ± 0.00 1.79 ± 0.05

Table 4 Radical scavenging ability of roots of T. connaroides (ARE) and butylated hydroxyl toluene (BHT) at different concentrations.

Weight of ARE (mg) Radical scavenging activity (%) ARE BHT

5 58.3 ± 0.344 91.15±0.38

10 70.24±1.36 91.86±0.07

15 74.91±1.198

92.25±0.08

20 81.62±0.55 92.7±0.09

25 86.27±1.04 93.23±0.14

Table 5 Chelating activity on Fe+2

of aqueous extract of T. connaroides (ARE) at different concentrations.

Weight of ARE (mg/ml) Chelating activity on Fe+2 of ARE (%)

5 58.4 ± 1.47

10 62.92 ± 0.98

15 68.5 ± 1.02

20 76.02 ± 0.76

25 78.62 ± 0.54

29

0

0.5

1

1.5

2

5 10 15 20 25

Concentration of ARE (mg/ml)

Ab

sorb

ance

at

700n

m

ARE

BHT

0102030405060708090

100

0 5 10 15 20 25

Weight of ARE (mg)

Rad

ical

sca

ven

gin

g

acti

vity

(%)

ARE

BHT

0

10

20

30

40

50

60

70

80

90

5 10 15 20 25

Concentration of ARE (mg/ml)

Che

latin

g a

ctiv

ity(%

)


Fig. 1 Photomicrographs of liver section (haematoxylin-eosin)

Group I, x 200; (B) Group II, x 200 (CCl4, 0.5 ml kg-1.); (C) Group III, x 200 (CCl4, 0.5 ml kg-1. + 100 mg kg-1, ARE); (D) Group IV, x 100 (CCl4, 0.5 ml kg-1. + 200 mg kg-1 ARE) ; ( E) Group V, x 100 (CCl4, 0.5 ml kg-1 i.p. + 400 mg kg-1 ARE); (F) Group VI, x 100 (CCl4, 0.5 ml kg-1 + 2.5 ml kg-1 Liv-52)

Fig 2.Reducing power of aqueous extract of root of Tconnaroides (ARE) and butylated hydroxy toluene (BHT) at different concentrate Fig 3. Radical scavenging ability of roots of T. connaroides (ARE) and butylated hydroxy toluene(BHT) at different concentrations.

Fig 4 Chelating activity on Fe+2 of

aqueous extract of T. connaroides (ARE) at different

concentrations.

CV

B

CV

C

D E F

CV

A

CV CV

CV

CV

30

Garima.A. et al, Hygeia.J.D.Med, vol.2 (2) 2010,22-31

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1. Ashan MR, Islam, MK, Bulbul IJ., Musaddik MA, Hauge E. Hepatoprotective activity of methanol extract of some nutritional plants against carbontetrachloride induced hepatotoxicity in rats. Eur. J. Sci. Res 37(2), 2009. 302-310.

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3. Chatterjee TK. Medicinal plants with hepatoprotective properties in herbal opinions, Vol III. Books and Allied (P) Ltd., Calcutta, 2000.135.Kirtikar K R, Basu BD. Indian Medicinal Plant, Vol I. Periodical Experts Book Agency, Delhi, 1993. 553.

4. Kirtikar, KR, Basu, BD, 1993. Indian Medicinal Plant, Vol I. Periodical Experts Book Agency, Delhi, p 553. 5. The Wealth of India, A dictionary of Indian Raw Materials, Vol V. Council of Scientific and Industrial

Research, Delhi 1997, 74. 6. Kirtikar K R, Basu B D . Indian Medicinal Plant, Vol I. Periodical Experts Book Agency, Delhi 1993.p.

555. 7. Zhang H P, Wu S H, Shen Y M, Ma Y B, Wu D G, S Qi S H, X.D Luo . A pentanortriterpenoid with a novel

carbon skeleton and a new pregnane from T. connaroides. Can J Chem 81, 2003. 253. 8. Germano M P, D’Angelo V, Sanogo R, Morabito A, Pergolizzis De, Pasquale R Hepatoprotective activity of

Trichilia roka on CCl4 induced the damage in rats. J Pharm Pharmacol, 53, 2001, 1569-1574. 9. Germano M P, D’Angelo V, Sanogo R, Catania S, Alma R, Pasquale, R.De. Bisignano G .

Hepatoprotective and antibacterial effects of extracts from Trichilia emetica Vahl (Meliaceae). J Ethnopharnacol, 96, 2005, 227-232.

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13. Charpentier, BA, Cowles, JR. Rapid method of analysong phenolic cpompounds in Pinus ellotti using high performance liquid chromatography. J Chromatogr. 208, 1981.132-136.

14. Chung,Y. C, Chang, C.T, Chao, W.W, Lin, C. F, Chou, S.T,. Antioxidant activity and safety of the 50 % ethanolic extract from red fermented by Bacillus subtilis IMR-NRI. Journal of Agric. Food Chem. 50,2002, 2454-2458.

15. Jonston D E, Kroening C. Mechanism of early carbon tetrachloride toxicity in cultured rat hepatocytes. Pharmacol Toxicol, 83, 1998. 231-239.

16. Srivastava S D, Chen N O, Holtzman J L. The in vitro NADPH-dependent inhibition by CCl4 of the ATP-dependent calcium uptake of hepatic microsomes from male rats. Studies on the mechanism of inactivation of hepatic microsomal calcium pump by CCl3 radical. J Bio Chem 265, 1990. 8392-8399.

17. Cotran R S, Kumar V Robbins S L. Cell injury and cellular death. In: Robbins’s Pathological Basis of diseases 5th ed., Prism Book Pvt. Ltd, 1994. p 379.

18. Kaplowitz N, Aw T Y, Simon F R, Stolz A. Drug induced hepatotoxicity. Ann. Intern Med 104, 1986. 826-839.

19. Nelson D L, Cox M M . Lehninger Principles of Biochemistry, Macmillan Worth Publishers, U.S.A. 2000. p 631.

20. Awang D. Milk Thistle. Can Pharm J, 23, 1993. 749-754. 21. Roy C K., Kamath, J V, Asad Mohammed. Hepatoprotective activity of Psidium guajava Linn. leaf extract.

Indian J Exp Biol, 44 ,2006. 305-311. 22. Sherwin E R . Oxidation and antioxidants in fats and oil processing. J Am Oil Chem Soc. 55. 1978. 809-

841. 23. Brand Williams. W, Cuvelier M E, Berset C. Use of a free radical method to evaluate antioxidant activity.

Lebensmittel-Wissenschaft und Technologie 28, 1995. 25-30.

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24. Espin J C, Soler-Rivas, C, Wichers H J. Characteriazation of total free radical scavenger capacity of vegetable oils and oil fraction using 2, 2-diphenyl-picrylhydrazyl radical. J Agric Food Chem, 48, 2000. 648-656.

25. Miller D D. Mineral. In O.R. Fennerna (Ed.), Food Chem. Marcel Deckker, New York. 1996 p 618. 26. Halliwell B, Gutteridge J M C. Oxygen toxicology, oxygen radicals, transition metals and disease. Biochem

J, 219, 1984. 1-4. 27. Yamaguchi F, Agriga, T, Yoshimira Y, Nakazawa H. Antioxidant and anti-glycation of garcinol from

Garcinia indica fruit rind. J Agric Food Chem. 49,2000.180-185. 28. Jain A., Soni M, Deb L, Jain A, Rout S P, Gupta V B, Krishna K L. Antioxidant and hepatoprotective

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29. Madani H., Talebolhosseini M, Asgary, S, Naderi GH. Hepatoprotective Activity of Silybum marianum and Cichorium intybus against thioacetamide in rat. Pak J Nutr, 7, 2008. 172.

30. Mondal S K, Chakraborty G, Gupta M, Mazumder U K. Hepatoprotective activity of Diospyros malabar bark in carbon tetrachloride intoxicated rats. Eur Bull Drug Res, 13, 2005. 25-30.

Research article Hygeia.J.D.Med.vol.2 (2), 2010, 32-37. ISSN 0975 6221 ___________________________________________________________________________________________________________ HYGEIA JOURNAL FOR DRUGS AND MEDICINES


________________________________________________________________________________

Visible Spectrophotometric method for the estimation of Cefepime

Minu Sujith1, Sujith Abraham1*and Madhu.C.Divakar2

1. Nirmala College of Pharmacy, Muvattupuzha, Ernakulam, Kerala- 686661, India. 2. Crescent College of Pharmaceuticalsciences,Payangadi,Kannur,Kerala,India-670358

Article history: received: 10 January2010,, revised:25April2010,accepted:18 june2010 ,available online 31 July 2010. ________________________________________________________________________________ Abstract A simple, accurate and reproducible visible spectrophotometric method was developed for the estimation of cefepime in pure and vial formulation. Cefepime with 1-Chloro-2,4-dinitrobenzene in presence of dimethyl sulfoxide at 45oC have formed a yellow colored complex having maximum absorbance at 420nm.The calibration graph was linear in the range 20µg/ml to50µg/ml. Keywords: Visible spectrophotometry, Cefepime, 1-Chloro-2, 4-dinitrobenzene,Dinitrobenzene.

___________________________________________________________________________________________________________

1. Introduction

Cefepime is 7-(Z)-2-(2-aminothiazol 4-yl)-2-methoxyiminoacetamido)-3-(1-methylpyrrolidino) methyl-3-cephem-4-carboxylate1 is a fourth generation cephalosporin for parental administration. This is mainly used in the treatment of various microbial infections caused by gram+ve and gram-ve microorganisms2,4. Cefepime is official in USP. The USP3 describe a HPLC method for the estimation of cefepime formulations. A review of literature revealed that there are HPLC6-12, U.V Spectrophotometric5 and Colorimetric method13 using Folins-Ciocaltue reagent. Thus the present method aims at developing newer colorimetric method which is rapid, accurate, precise, sensitive and reliable. 2. Experimental 2.1. Instruments used: All colorimetric measurements were done on a Shimadzu Pharmaspeck 1800, Double beam spectrophotometer with 2 quartz cell 1cm path length. The point of maximum absorbance was at 420nm. _________________________________ For correspondence: [email protected] Contact: +91 9447209211 © 2010 Hygeia journal for drugs and medicines. , All rights reserved. 0975 6221

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Minu Sujith et al Hygeia.J.D.Med. vol.2 (2), 2010, 32-37

2.2. Chemicals and reagents: Cefepime was received as a gift sample from Sans Pharmaceutical Ltd, Kerala, India. 1-Chlor-2,4-dinitro benzene and Dimethyl Sulfoxide were of AR grade. This was procured from SD Fine chemicals Mumbai, India. 2.3. Preparation of standard drug solution: The stock solution of cefepime was prepared by dissolving 50mg of the drug in 50ml dimethylsulfoxide.From that 5ml was taken and made up to 50ml. The new concentration was 100µg/ml.To 1ml of the above solution was added 3.6ml of 0.8M 1-Chloro-2,4- dinitro benzene. This was heated at 45oC for ten minutes. The solution was cooled and made up to 5ml with dimethyl sulfoxide. 2.3.1. Optimization of concentration of 1-chloro-2,4-dinitrobenzene: To 1ml drug solution 3.6ml of various concentrations ranging from 0.5 to 1M of 1-chloro-2,4-dinitrobenzene were added and heated at 45oC for 10min. The yellow coloured solution was measured at 420nm. The absorbance was maximum at a concentration 0.8M.Table.1. 2.3.2. Optimization of volume of 0.8M 1-chloro-2,4-dinitrobenzene: Different volumes ranging from 3 to 4ml of 1-chloro-2,4-dinitrobenzene were added into 1ml drug solutions and the same procedure as above was carried out. The maximum absorbance was found to be at 3.6ml; 0.8M.Table.2. 2.3.3. Optimization of temperature: The drug solution along with 3.6ml; 0.8M 1-chloro-2,4-dinitrobenzene were heated for 10min at different temperatures ranging from 30oC to 63oC.The maximum absorbance was found to be at 45oC.Table.3. 2.3.4. Optimization of heating time: After fixing the above parameters, the contents were heated at 45oC at regular intervals of time from 5min to 25min. The absorbance intensity was maximum at 10min.Table.4. 2.3.5. Preparation of calibration graph: From the working standard, concentrations ranging from 10µg/ml to60µg/ml of cefepime were taken. To this 3.6ml; 0.8M 1-chloro-2,4-dinitrobenzene was added. Then the test tubes were heated at 45oC for 10 min.Cooled and made up to 5ml with dimethyl sulfoxide. A blank was also prepared in the same manner without the drug. The absorbance was measured at 420nm.The measured absorbance was plotted against the concentration. From the graph it was found that the Beer’s law concentration range was between 20µg/ml to 50µg/ml.Figure.1.

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Minu Sujith et al Hygeia.J.D.Med. vol.2 (2), 2010, 32-37.

2.4. Analysis of pharmaceutical formulation: An aliquot quantity equivalent to 50mg of cefepime was weighed and transferred to a 50ml standard flask. The contents were dissolved and made up to 50ml with dimethylsulfoxide.From this six individual concentrations were taken. To this was added 3.6ml; 0.8M 1-chloro-2,4-dinitrobenzene and heated at 45oC for 10min.It was cooled and the volumes were made up to 5ml with dimethyl sulfoxide. The absorbance was measured at 420nm. The concentration of the drug was determined by single point standardization. The results are given in Table.5,6 & 7. 2.5. Recovery studies: A known quantity of the standard was added to the pre-analyzed sample formulation and the contents were analyzed by the proposed method. The result of the recovery studies are shown in Table.7. 3. Result and discussion: The proposed colorimetric method for estimation of cefepime in vials by single point standardization was simple, accurate and rapid and can be employed for routine analysis. The cefepime concentration at a range of 20µg/ml to 50µg/ml has followed the Beer Lambert’s law range at 420nm. The percentage deviation from the recovery was at 100% indicating the reproducibility and accuracy of the method. The cefepime content was found to be 95.8553% in vial formulation by the newly developed method. The method was found to be reproducible and can be used for routine estimation of cefepime in bulk and its vial dosage form. 4. Conclusion: A simple, precise, rapid and accurate method was developed for the estimation of cefepime by visible spectrophotometry in vial formulations. The drug sample was heated with 3.6ml;0.8M 1-chloro-2,4-dinitrobenzene for 10minutes at 45oC.A yellow color complex was obtained which showed a λmax at 420nm.The Beer’s-Lamberts range was found to be from 20µg/ml to 50µg/ml.The same method was done in the case of vial formulation and concentration was calculated by single point standardization. The percentage recovery was also calculated. The method gave a satisfactory label claim and recovery value. So this method can be used for the routine analysis of cefepime in bulk and vial formulation.

Table-1: Optimization of concentration of 1-Chloro-2,4-Dinitrobenzene

(M) Absorbance

0.5 0.2903

0.6 0.3412 0.7 0.3904 0.8 0.5008 0.9 0.4813 1 0.4901

35

Minu Sujith et al Hygeia.J.D.Med. vol.2 (2), 2010, 32-37. Table-2: Optimization of volume of 1-Chloro-2,4-Dinitrobenzene

Volume(ml) Absorbance 3 0.5421

3.2 0.5813 3.4 0.6004 3.6 0.6278 3.8 0.6130 4 0.6007

Table-3: Optimization of Temperature Temperature(0C) Absorbance

30 0.6401 33 0.6401 36 0.6401 39 0.6401 42 0.6401 45 0.6649 48 0.6401 51 0.6401 54 0.6401 57 0.6401 60 0.6521 63 0.6312

Table-4: Optimization of heating time

Table-5: Absorbance of Standard solutions at 420nm:

Time(min) Absorbance 5 0.6531 10 0.7049 15 0.6840 20 0.6622 25 0.6130

Absorbance of Standard solutions: Replicate 20µg 25µg 30µg 35µg 40µg 45µg 1 0.0206 0.0250 0.0302 0.0350 0.0403 0.0452 2 0.0204 0.0255 0.0304 0.0354 0.0401 0.0452 3 0.0206 0.0248 0.0298 0.0349 0.0403 0.0456 Mean 0.0205 0.0251 0.0301 0.0351 0.0402 0.0453 SD ±0 .005 ±0.007 ±0.005 ±0.005 ±0.005 ±0.005

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Minu Sujith et al Hygeia.J.D.Med. vol.2 (2), 2010, 32-37.

Table-6: Absorbance of Test solutions at 420nm:

Table-7 Analysis of vial formulation of cefepime at 420nm ________________________________________________________________________________ S.I.No Astd Atest Cstd(µg/ml) C test(µg/ml) Labelled Claim(mg) Amount present(mg) %labelclaim ___________________________________________________________________________________________________________ 1 0.0205 0.0203 20 19.8048 1000 954.9 95.49 2 0.0251 0.0250 25 24.90039 1000 952.838 95.2838 3 0.0301 0.0303 30 30.1993 1000 969.755 96.9755 4 0.0351 0.0350 35 34.90028 1000 956.103 95.6103 5 0.0402 0.0401 40 39.9004 1000 961.91899 96.191899 6 0.0453 0.0451 45 44.801 1000 955.8036 95.58036 ________________________________________________________________________________________________ Mean = 95.8553% S.D = ± 0.393058 Table-8: Recovery studies of cefepime:

Table-9: Recovery studies of cefepime by the new method

_________________________________________________________________________________________________________________________ S.I.No Concentration of sample Amount of standard added Total amount of drug Amount of standard drug %recovery

(mg) (mg) estimated recovered

________________________________________________________________________________ 1 0.0198048 0.010 0.03009967 0.01029487 102.9487 2 0.0301993 0.010 0.0399004 0.097011 97.011 _________________________________________________________________________________

Mean = 99.97985 % S.D = ±0.002078

Absorbance of Test solutions:

Replicate 20µg 25µg 30µg 35µg 40µg 45µg

1 0.0202 0.0249 0.0302 0.0405 0.0450 0.0348

2 0.0202 0.0248 0.0301 0.0399 0.0449 0.0348

3 0.0205 0.0254 0.0307 0.0400 0.0455 0.0355

Mean 0.0203 0.0250 0.0303 0.0401 0.0451 0.0350

SD ±0.005 ±0.005 ±0.005 ±0.005 ±0.005 ±0.005

% Recovery Values at: Replicate 30µg 40µg

1 102.95% 97.010% 2 102.96% 97.018% 3 102.92% 97.006%

Mean 102.94% 97.011% SD ±0.005 ±0.005

37

0

0.01

0.02

0.03

0.04

0.05

0.06

0 5 10 15 20 25 30 35 40 45 50 55

Concentration

Abso

rban

ce

MinuSujith et al Hygeia.J.D.Med. vol.2 (2), 2010,32-37.

Fig.1. Beer Lambert’s concentration range at 420nm.

Acknowledgement: The authors are thankful to Sans Pharmaceutical Limited, Kerala, India for providing gift sample of cefepime and also to Nirmala College of Pharmacy for providing all the facilities to carry out the work.

References

1. Budavari S, Eds. In The Merck Index, 13th Edn. Merck and Co. Inc., Whitehouse Station, N.J.2001; 327. 2. Mishra L, Eds. In Drug Today, Vol.12, No.2.Lorina Publications (India) Inc., Delhi.2004; 283. 3.”TheUnited States pharmacopoeia”,XXVII,NF23,The United States Pharmacopoeial Convention,Inc.,Rockvill,M.D.2005,p358. 4.AHSP: American Society of Health System of Pharmacists;2004;172:8.12.06. 5. Rodenas V, Parra A, Garcia-Villanova j and Gornez MD.J Pharm Biomed Anal.1995;13(9):1095-9. 6.Calahorra B, Campanero MA, Sadaba B and Azanza JR. Biomed Chromatogr.1999;13(4):272-5. 7.Chang YL,Chou MH, LinMF, Chen CF and Tsai TH.J Chromatogr A.2001;914(1- 2):77-82 8.Cherti N, Kinowski JM, Lefrant JY and Bresolle F. J Chromatogr B Biomed Sci Appl.2001;754(2):377-86. 9.Breilh D,Lavallee C, Fratta A,Ducint D, Cony-makhoul P and Saux MC.J Chromatogr B Biomed Sci Appl.1999;721(4):121-7. 10.Valassis IN,Parissi-Poulou M and Macheras P. J Chromatogr B Biomed Sci Appl. 1999;721(2):249-55. 11.Cheunq SW, Chan CY and Chenq AF . J Antimicrob Chemother. 1998;42:121. 12.Elkhaili H, Linger L, Monteil H and Jehl F. J Chromatogr B Biomed Sci Appl.1997; 690(1-2):181-8. 13.Patel S.A, Patel N.M, Patel M.M. J of Pharmaceutical Research. Vol.6,No.3,July 2007:155-157.

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Research article Hygeia.J.D.Med.Vol.2 (2), 2010, 38-42. ISSN 0975 6221 ________________________________________________________________________________________________________ HYGEIA JOURNAL FOR DRUGS AND MEDICINES


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Phytochemical, HPTLC finger printing and antibacterial activity of Acacia nilotica (L.) Delile

R.Venkataswamy1, A.Doss2, H.Muhamed Mubarack2, M.Sukumar1

1. Department of Pharmacognosy, SriRamakrishna College of Pharmacy, Avarampalayam, Coimbatore, TamilNadu, India 2. Department of Microbiology, RVS College of Arts and Science, Sulur, Coimbatore, TamilNadu, India

Article history: Received: 5 April 2010, revised: 16 May 2010, accepted: 22 June 2010, Available online 31 July 2010.

______________________________________________________________________________________________ Abstract The leaves of Acacia nilotica (L.) Delile was extracted with methanol and aqueous medium and studied for in vitro antimicrobial property. The methanol extract was found to be most active against all the bacterial species tested except S.aureus. Active methanol extract was further studied for HPTLC fingerprint and by phytochemical analysis. HPTLC analysis confirmed segregation of four individual compounds with individual Rf values and peak area percentage. The results of phytochemical screening of extract revealed the presence of Tannins, Carbohydrates and Glycosides. This analysis revealed the high antibacterial activity in the methanol extract of Acacia nilotica (L.) Delile

Key words: HPTLC finger printing, methanol, MIC, microorganisms

______________________________________________________________________________________________

1. Introduction

Infectious disease is the number one cause of death accounting for approximately one-half of all deaths in tropical countries. Death from infectious diseases, ranked 5th in 1981, has become the 3 rd leading cause of death in 1992, with an increase of 58% (Arokiyaraj et al., 2009). More than hundreds of plants worldwide are used in traditional medicine as treatments for bacterial infection (Doss et al., 2009). Although many have been treated by conventional pharmaceutical approaches, there is a growing interest in the use of natural products by the general public (Ghosh and Playford, 2003). In addition to the pharmaceutical industry continues to investigate their potential as sources of novel growth factor, immunomodulatory and antimicrobial activity (Sasikumar et al., 2006). Acacia nilotica (L.) Delile commonly known as babul, kikar or Indian gum Arabic tree, has been recognized worldwide as a multipurpose tree. It is widely distributed throughout arid and semi-arid zones of the world. Presently about 20% of the total geographical area of India is wasteland. Growing demand for fuel, fodder, wood and food has extensively depleted or eliminated protective plant cover and exposed soils to processes of degradation resulting in partial to complete loss of soil productivity (Venkataswamy, 2010). A. nilotica species have been reported to possess anti-hyperglycemic (Zourata Lompo-Ouedraogo et al., 2004), antimicrobial (Venkataswamy et al., 2010), antiplasmodial (El-Tahir et al., 1991), anti-inflammatory, analgesic and antipyretic properties (Dafallah and Al-Mustapha, 1996). __________________________________ *For Correspondence: email: [email protected] Contact: +919994997490, © 2010 Hygeia journal for drugs and medicines. All rights reserved. 0975 6221

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R.Venkataswamy et al Hygeia.J.D.Med. vol.2 (2), 2010,38-42

Literature study showed antibacterial activity of this plant, but activity against clinical isolates has not so far been reported. Considering the medicinal value of this plant, we evaluated the antibacterial potential, HPTLC fingerprint and phytochemical analysis of Acacia nilotica (L.) Delile (Leaves). 2. Materials and methods

2.1Plant Collection Leaves of A.nilotica were collected from Ayyasamy Hills, Coimbatore District, Tamilnadu. A voucher specimen was deposited at the department herbarium, Government Arts College, Coimbatore. Collected materials were washed thoroughly, shade dried in open air and grounded into powder.

2.2. Preparation of organic and aqueous extracts

Dried powder of each plant (250g) was placed in 500 ml of water for 24 hours for maceration. After 24 hours the mixture was boiled for 1 hour and then filtered. The filtrate thus obtained was cooled and concentrated to dryness. Soxhelet extractor was used for alcoholic extraction of each drug. 250 gm of dry powder of each drug was packed in a filter paper and inserted into the control tube of the extractor where the solvent got circulated continuously at constant temperature. After 24 hours of extraction the mixture was filtered and the filtrate thus obtained was concentrated to a residue in a distilling unit. The obtained residues were weighed and kept in bottles and were used for further phytochemical and biological screening studies. 2.3. HPTLC Finger Printing Chromatography was performed on 3 × 10 cm HPTLC plates coated with 0.25 mm layer of silica gel 60 F 254 (Merck, Germany). The plate was prewashed with methanol and activated at 110° C for 5 min. The methanol extracted sample was applied as 10 mm bandwidth using a Camag (Muttenz, Switzerland) Linomat IV sample applicator equipped with 100 µl Syringe. A constant application rate of 20 µl/sec was used. Mobile phase was Toluene: Ethyl acetate: Acetic acid (12.5: 7.5: 0.5) and chromatogram were scanned at 365 nm.

2.4. Phytochemical Analysis10

Phytochemical screening of methanol extract was carried out to detect the presence of secondary metabolites, like Alkaloids, Glycosides, Carbohydrates, Saponins, Steroids, Tannins and Proteins.

2.5. Microorganism

Staphylococcus aureus NCIM 5021, Bacillus subtilis NCIM 2010, Escherichia coli NCIM 2118, Pseudomonas aeruginosa NCIM 5029, Klebsiella pneumoniae NCIM 2707, and Proteus vulgaris NCIM 2027 were collected from The National Chemical Laboratory, Pune and stored in the Department of Microbiology, RVS College of Arts and Science, Sulur, Coimbatore, TamilNadu. 2.6. Antibacterial Screening The antibacterial test was performed by following agar disc diffusion method (Bauer et al., 1996; Nair and Chanda, 2005) using Mueller Hinton Agar No. 2 medium for the assay. The zone of inhibition diameters (m.m) were determined for the respective drug groups and tabulated in the table 1 .

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2.7. Minimum Inhibitory Concentration The Minimum Inhibitory Concentration (MIC) was determined by adopting the standard reference method NCCLS (2002). The extracts were dissolved in 2% dimethyl sulfoxide (DMSO). A stock solution of each extract was serially diluted in 96-well microtiter plate with Mueller Hinton broth to obtain a concentration ranging from 8 mg/ml to 0.125 mg/ml. A standardized inoculum for each bacterial strain was prepared so as to give an inoculum size of 105 CFU/ml in each well. Ciprofloxacin was used as a standard antibiotic for comparative analysis with the effectiveness of various extracts against tested clinical isolate and drug resistant bacteria. Microtiter plate was kept at 37°C and incubated for 24 h. Following incubation, the MIC was calculated as lowest concentration of the extracts inhibiting the visual growth of the test cultures on the agar plate. Three replications were maintained. 3. Results and Discussion In the HPTLC fingerprinting of methanol extract gave four spots with the following Rf values: 0.08 (24.74%), 0.21 (22.18%), 0.40 (9.81%), 0.50 (9.33%). Purity of the sample extract was confirmed by comparing the absorption spectra at start, middle and end position of the band. HPTLC is an invaluable quality assessment tool for the evaluation of botanical materials. The HPTLC chromatogram of the methanol extract is presented in Figure 1. The phytochemical analysis of methanol extract showed the presence of glycosides, carbohydrates and tannins (Table 3). Secondary metabolites of plant origin appear to be one of the alternatives for the control of antibiotic resistant human pathogens. The most important bioactive compounds of plants are alkaloids, flavonoids, tannins and phenolic compounds. This antibacterial activity may be due to the presence of secondary metabolites (Arokiyaraj et al., 2009).

The antibacterial activities of extracts of A.nilotica against clinically important microbial pathogens are summarized in Table 1. The MICs of the extracts ranged between 0.125 and 2 mg/ml. Among the two extracts, the methanol was found to be most active against all the tested bacterial species except Staphylococcus aureus [Klebsiella pneumonia (0.125mg/ml), Bacillus subtilis (0.500 mg/ml), E.coli (2.0 mg/ml), Proteus vulgaris (2.0 mg/ml), Pseudomonas aeruginosa (1.0 mg/ml)] (Table 2). Aqueous extract was less active than methanol extract. Compare to standard ciprofloxacin, the extracts showed good activity against all the tested bacteria strains. Several plants which are rich in tannins have been shown to possess antimicrobial activity against a number of microorganisms. The ability of tannin compounds to cause the bacterial colonies to disintegrate probably results from their interference with the bacterial cell wall synthesis, thereby inhibiting the microbial growth (Doss et al., 2009).

Table 1: Antibacterial activity of the leaf extracts of Acacia nilotica (L.) Delile

Extracts Conc.

(mg/ml)

Zone of Inhibition (mm)

Methanol

B.subtilis S.aureus E.coli P.vulgaris K.pneumoniae P.aeruginosa

250

10 - 12 10 15 13

500 11 - 15 12 18 16

Aqueous

250

- - 11 11 12 12

500 - - 13 14 16 14

Standard

(Ciprofloxacin) 28 29 30 22 20 27

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R.Venkataswamy et al, Hygeia.J.D.Med. vol.2 (2), 2010,38-42.

Table 2: Minimum Inhibitory Concentration of the leaf extracts of Acacia nilotica (L.) Delile

Extracts Minimum inhibitory concentration (mg/ml)

B.subtilis S.aureus E.coli P.vulgaris K.pneumoniae P.aeruginosa

Methanol 0.500 - 2.0 0.500 0.125 0.125

Aqueous 2.0 - 2.0 1.0 0.250 0.500

Table 3: Preliminary phytochemical screening of the leaf extracts of Acacia nilotica (L.) Delile S.No. Phytochemicals Tests Results

1 Alkaloids

Dragendroff’s test - Hager’s test - Wagner’s test - Mayer’s test -

2 Glycosides Baljet test + Legal test +

3 Carbohydrates

Molisch’s + Fehling’s + Benedict’s + Barford’s +

4 Saponin Foam test - Blood Haemolysis test -

5 Steroids Libermann Burcherd test - 6 Tannins Ferric chloride test + 7 Proteins Biurette test -

Figure 1: HPTLC fingerprint profile of ethyl acetate extract of Acacia nilotica (L.) Delile

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Venkataswamy et al, Hygeia.J.D.Med. vol.2 (2), 2010,38-42.

Acknowledgement We are extremely thankful to Dr.T.K.Ravi, Principal, SriRamakrishna College of Pharmacy, Avarampalayam, Coimbatore, for providing the necessary facilities for the completion of this work. The authors also wish to thanks Dr.H.Muhamed Mubarack, Advisor, School of Life Sciences, RVS College of Arts and Sciences, Sulur for his generous help.

References

1. Arokiyaraj S, Perinbam, K, Agastian P, Mohan Kumar R. Phytochemical analysis and antibacterial activity of Vitex agnus-castus. International Journal of Green Pharmacy. 3(2):2009, pp: 162-164.

2. Doss A, Dhanabalan R, Sasikumar JM, Palaniswamy M, GeethaV, Ayyappa Das M. Antibacterial activity of twelve medicinal plants from Western Ghats. Pharmacology online. 1: 2009, pp: 83-89.

3. Ghosh S, Playford RJ. Bioactive natural compounds for the treatment of gastrointestinal disorders. Clin Sci (Lond).104: 2003, pp: 547-56.

4. Sasikumar JM, Pichai Anthoni Doss A, Doss A. Antibacterial activity of Eupatorium gladulosum. Fitoterapia. 76(2): 2006, pp: 240-243.

5. Venkataswamy R. Survey of Medicinal plants used by Malasar tribals of Coimbatore District, Tamilnadu, South India and Pharmacognostical, Anatomical and Free Radical Scavenging studies of selected medicinal plants.(Ph.D thesis ; 2010)

6. Zourata Lompo-Ouedraogo, Daan van der Heide, Eline M van der Beek, Hans JM Swarts, John AM Mattheij and Laya

Sawadogo. Effect of aqueous extract of Acacia nilotica ssp adansonii on milk production and prolactin release in the rat. Journal of Endocrinology. 182: 2004, pp: 257–266

7. Venkataswamy R, Mohamed Mubarack H, Doss A, Lakshmi Devi S, Sukumar M. Antimicrobial activity of some

ethnomedicinal plants used by the Malasar tribe of Tamilnadu, South India. Research Journal of Biological Sciences. 2(2): 2010, pp: 25-35.

8. El-Tahir A, Satti GM, Khalid SA. Antiplasmodial activity of selected Sudanese medicinal plants with emphasis on Acacia nilotica. Phytotherapy Research. 13: 1991, pp: 474–478.

9. Dafallah AA, Al-Mustapha Z. Investigation of the anti-inflammatory activity of Acacia nilotica and Hibiscus sabdariffa. American Journal of Chinese Medicine. 24: 1996, pp: 263–269.

10. Kokate CK. Practical Pharmacognosy, (Ed.III). Vallabh Prakashan, Delhi. 1991; pp. 107 & 115 - 125.

11. Bauer AW, Kirby WMM, Sherris JC. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45:1996, pp: 493- 496.

12. Nair R, Chanda S. Anticandidal activity of Punica granatum exhibited in different solvents. Pharm Biol. 43:2005, pp: 21-25.

13. National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard M38-A. Wayne, PA: National Committee for Clinical Laboratory Standards; 2002.

14. Doss A, Mohammed Mubarack H, Dhanabalan R. Antibacterial activity of tannins from the leaves of Solanum trilobatum Linn. Indian Journal of Science and Technology. 2 (2): 2009, pp: 41-43.

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Research article Hygeia.J.D.Med.Vol.2 (2), 2010, 43-53. ISSN 0975 6221

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In vitro evaluation of anthelmintic efficacy of Trichilia and Ajuga species

on Ascaridia galli

G. Agarwal1*, A.K. Pant2 and S.K. Hore1

1. Department of Pharmacology and Toxicology, G. B. Pant University of Agriculture and Technology, Pantnagar, India, 263 145

2.Department of Chemistry, G. B. Pant University of Agriculture and Technology, Pantnagar, India, 263 145

Article history: Received: 15 May 2010, revised: 15 June 2010, accepted: 4 August 2010 Available online 20 Sep 2010.

________________________________________________________________________________________________

Abstract The study was designed to evaluate the effect of the methanol, chloroform, acetone and aqueous extracts of different parts of Trichilia connaroides, Ajuga bracteosa, Ajuga macrosperma, Ajuga parviflora of Indian Himalayan region and a reference drug albendazole on isometric contractions of the poultry worm Ascaridia galli. The frequency and amplitude of spontaneous muscular contractions of A. galli were recorded on the physiograph through force transducer. There was inhibition in amplitude and frequency of the contractile activity as compared to control in dose dependent manner by methanol extracts of seeds (IC50 3.93±0.70), pericarps (IC507.09± 2.64), aqueous extract of roots (IC50 6.40± 4.74) of T. connaroides, methanol extracts of A. parviflora roots (IC50 16.79± 2.93) A. macrosperma roots (IC501.73± 0.02) and A. bracteosa aerial parts (IC50 4.49± 0.72). These observations indicated the paralytic effect of the extracts on A. galli. There was no inhibition contractile activity by chloroform extract of seeds, acetone extract of leaves of T. connaroides and methanol extract of A. bracteosa roots on autorythmicity of A. galli.

Keywords: T. connaroides; Ajuga; A. galli; Albendazole; Autorythmicity. _________________________________________________________________________________________________

Introduction

Helminthiasis or worm infestation is one of the major public health problems in the world. It is responsible for considerable economic losses to the livestock industry in developing countries. Other adverse effects of these parasites include loss of meat, wool and egg production 1. Chemotherapy against helminths involves either selective inactivation of enzymes or blocking of specific receptors in the tissues of the parasites 2. Consequently, there is an urgent need to develop newer, selective and eco-friendly agents to control helminth infections. Plant based anthelmintics can be both sustainable and environmentally acceptable.

Unlike synthetic anthelmintics, plant-based anthelmintics with different modes of action could be of value in preventing the development of resistence. Herbal drugs have been in use since ancient times for the treatment of a variety of acute and chronic parasitic diseases both in humans and in veterinary medicine 3. Traditional system of medicine reports the efficacy of several plant products for eliminating helminths. Several essential oils and plant extracts have been found to possess anthelmintic activity.

_____________________________________________ *For Correspondence: Email: [email protected] Contact: 09986828970© 2010 Hygeia journal for drugs and medicines. All rights reserved. 0975 6221

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Garima.A. et al, Hygeia.J.D.Med, vol.2 (2) 2010,43-53.

Indian system of medicine reports the efficacy of medicinal plants like Chenopodium ambrosioides, Embelia ribes, Carica papaya, Mallotus phillippinensis, Butea frondosa etc. for eliminating helminths 3Himalayan region of India has been considered to be a rich repository of medicinal and aromatic plants since time immemorial. However, very little efforts have been made to study the biodiversity of plant species in this region. This paper presents the anthelmintic activity of Trichiliaconnaroides ,Ajuga bracteosa, Ajuga macrosperma and Ajuga parviflora a of this region on Ascaridia galli which is a common intestinal parasite of the fowl, causing considerable economic losses to poultry farmers. It has many characterstics similar to Ascaris lumbricoides which is pathogenic to human beings 4

Trichilia connaroides:

Trichilia is a genus of trees, rarely shrubs 5. It belongs to the family Meliaceae which contains 40 genera and 600 species 6. Genus Trichilia is important because of medicinally important tetranortriterpenoids or limonoids. Limonoids show a wide range of biological activities including anti-feedant and growth regulatory properties in insects and antifungal, bactericidal and antiviral activities in laboratory animals and humans 7. From the genus Trichilia, only T. connaroides Wight. and Arn. occurs in Indian subcontinent. It is distributed in Sub-Himalayan tract from Kumaun eastward, Sikkim up to 4000 ft, Khasia Hills, Manipur, E. Ghats in the forests of Godawari and Vizagapatnam up to 4,500 ft. W.Ghats from Poona Southwards through the Nilgiris and Anamalais to Tranvancore, up to 6,000 ft. Apart from India it is also distributed in Burma, Tonkin, Cambodia, Malay Peninsula and Sumatra 8. The plant also possesses medicinal value. The bark and leaves possess bitter and tonic properties and a decoction of the leaves is taken in cholera 5. The roots of T. connaroides are used as a Chinese drug to treat arthritis, pharingitis, tonsillitis and other ailments

Ajuga species:

The plants of genus Ajuga belong to the family Lamiaceae. It is a large family comprising about 220 genera and almost 4000 species 10. The increasing number of crops in this family (1959:38, 1986: 129 and 1999:174) reflects the intensification of taxonomical and ethnobotanical research in this field 11. It has worldwide distribution growing under wide variety of soil and climate, but more abundant in Mediterranean regions and in the hills. Genus Ajuga is widely reported to have the clerodane and neo-clerodane diterpenoids with various biological activities 10-11. The plants of this genus are used as folk medicinal plants as antihelmintic, hypoglycemic, antifungal, anti-tumor and antimicrobial agents 12.

A. bracteosa is a perennial herb in the western Himalayas from Kashmir to Nepal. The leaves of this plant are used as stimulant, diuretic, aperient and as a substitute for cinchona. It acts as cardiostimulant in animals and shows anticancer activity in rats and mice 13.

A. macrosperma, found in Himalayan region up to Bhutan, Khasi Mountains, Cittagaon in Burma and China 14. It is traditionally used to alleviate fever and remove phlegm by the Dai minority of Yunnan Province, China 15.

A. parviflora grows in the temperate Kumaon region of the Indian Himalaya at 4000-6000 foot elevations 16. It has found diverse medicinal uses in indigenous systems of medicine. It has been used as an astringent and for the treatment of swollen wounds, diarrhoea, reheumatism, fever, eye trouble and for the diseases of bladder 17-18.

Materials and Methods

Chemicals used- Chloroform, Methanol, Acetone, NaCl, KCl, CaCl2, MgCl2.6H2O, Na2HPO4, Glucose, NaHCO3 were procured from E-Merk (India) Limited Mumbai. Reference drug albendazole (200mg/5 ml) was procured from Lupin Drug Company, India.

45


Collection and identification of plants T. connaroides was collected from Kumaun region, India and identified at Forest Research Institute (FRI) Dehradun vide herbarium no. M-29 .Three species of Ajuga namely A. parviflora, A. bracteosa and A. macrosperma were collected from Kumaun region and identified at Forest Research Institute (FRI) Dehradun and herbariums are deposited at the Department of Chemistry at G.B.P.U.A & T Pantnagar for the future reference. Extraction T. connaroides Seeds and pericarps of T. connaroides were subjected to the repeated soxhlet extraction by hexane followed by extraction with chloroform and methanol separately. Leaves were also subjected to repeated soxhlet extraction by acetone. Roots were extracted in boiling water for three times. Rotary vaccum evaporator concentrated the extracts. The percent yields(w/w) of chloroform extract of seeds, methanol extract of seeds and pericarps, acetone extract of leaves and aqueous extract of roots were 2.2, 1.4, 1.25, 1.0 and 1.25 respectively. Ajuga species A. macrosperma and A. parviflora roots, A. bracteosa roots and aerial parts were subjected to repeated soxhlet extraction by methanol separately. Rotatory vaccum evaporator concentrated the extracts and their percent yields (w/w) were 11.25, 25, 39.47 and 33.2 respectively. A. galli worms were collected from the intestine of freshly slaughtered fowl (Gallus gallus) received for postmortem examination at Animal Disease Diagnostic Centre, Pantnagar. They were stored in Tyrode solution at 40 � 10C. Isometric mounting of A. galli and mechanical recording of the spontaneous muscular activity The worm A. galli was mounted isometrically on a tissue bath of 4 ml capacity in Tyrode solution (Composition in mM: NaCl 136, KCl 5, CaCl2 2, MgCl2.6H2O 0.98, NaH2PO4.2H2O 0.36, Glucose 5.5, NaHCO3 11.9 and PH adjusted to 7.4) maintained at 40 ± 1ºC and allowed to equilibriate for 30 min without any tension. During the equilibration period the bath fluid was changed once every 10 min. Isometric contractions were made after applying a tension of 0.5 g using force transducer (T-301, Pt-1979) and spontaneous muscular activity was recorded using ink writing physiograph (Biodevice, India) at 0.25mm/sec chart speed. Control recordings were made for 15 min before the addition of a drug. Three parameters namely frequency (total no. of contractions in 10 min), amplitude (average of all peaks per 10 min or average tension) of spontaneous muscular contractions and baseline tension (average of all minimum levels of contractions used for measuring amplitude) of the isometrically mounted A. galli were measured. Effect of different extracts and albendazole on autorythmicity of A. galli Cumulative doses of the extracts at a dose rate of 3.125 -100 mg/ml dissolved in Tyrode solution and of albendazole at a dose rate of 0.32-2.56mg/ml were added in the tissue bath containing the isometrically mounted worm. Each dose was allowed to act for 15 min with continuous recording of rhythmic movements of A. galli. The effects of various concentrations of plant extracts on frequency and amplitude of spontaneous muscular contractions and on base line tension of the mounted worm were recorded and compared with the control. Statistical analysis The results are presented as mean ± standard error of the mean. To measure the significance one way T test was applied.

Results and Discussion: The effects of different extracts and the reference drug albendazole on autorythmiity of A. galli are presented in Table 1-11 and Fig 1-3. The acetone extracts of leaves and chloroform extract of seeds of T. connaroides could not alter any of the three parameters of autorythmicity of A. galli, amplitude, frequency and base line tension when used at dose rates of 3.125-50mg/ml bath solution.

46


Methanol extracts of pericarps (MPE) and seeds (MSE) and aqueous extract of roots (ARE) of T. connaroides decreased the amplitude and frequency of autorythmicity of A. galli dose dependently with a highest effect at the concentration of 50mg/ml. MPE and MSE also reduced base line tension of A. galli contractility significantly and dose dependently at the dose rate of 3.125-50mg/ml (Table 1-5 and Fig 1). An inhibition in the amplitude and frequency of rhythmic contractions of A. galli and base line tension was shown by methanolic extract of Ajuga bracteosa aerial part (BAE), Ajuga macrosperma roots (MRE) and Ajuga parviflora roots (PRE). Methanol extract of Ajuga bracteosa roots (BRE) could not alter the contractility of A. galli. The maximum decrease in frequency and amplitude in autorythmicity of A. galli was observed at the cumulative doses of 12.5 mg/ml with BAE, 50 mg/ml with MRE and 100mg/ml with PRE. Among all the above-mentioned extracts, the methanol extract of A. macrosperma roots (MRE) has been found to possess maximum anthelmintic efficacy with the minimum IC50 value (1.73 ± 0.02). Albendazole, a well-proven anthelmintic of benzimidazole group 19 showed comparatively better response for inhibiting amplitude, frequency and base line tension in dose dependent manner. The maximum decrease in frequency, amplitude and base line tension was observed at the dose of 2.56 mg/ml. Albendazole was found to possess lowest IC50 value (0.74± 0.05). The IC50 values of all the extracts are presented in Table 12. Chemotherapeutic agents available for treatment of helminth infection act mainly through three different mechanisms viz, by disruption of the neuromuscular physiology, by blocking the energy metabolism and by disturbing the highly efficient reproductive system of the parasites 20. Several important anthelmintics cause paralysis of helminth parasites by disrupting one or the other aspect of their neuromuscular system. The spontaneous muscular activity was quantified in terms of frequency, amplitude of rhythmic contractions and baseline tension and these parameters were measured before and after drug treatment and values were compared. Rapid and marked change in the spontaneous muscular activity of an isometrically mounted parasite by a drug indicates that the neuromuscular system of the parasites can be used to evaluate anthelmintic activity in vitro 1. Thus it can be concluded that the extracts demonstrate a paralytic effect causing by progressive reduction in spontaneous muscular activity which may be associated with their inhibitory effect on the neuromuscular system of A. galli. The inhibition caused by the T. connaroides extracts i.e. methanolic extract of seeds and pericarps and aqueous extract of roots on the autorythmicity may be attributed to the presence of polyphenols, as on phytochemical examination the plant revealed the presence of phenolic acids 21. Phenolic acids are reported to have the activity against the worm infestation in human beings 22. Moreover, the present study was also supported by the fact that polyphenols like tannins are well researched to produce anthelmintic effect 23. There was no inhibition with chloroform extract of seeds and acetone extract of leaves.The activity found in the extracts of genus Ajuga i.e. methanol extract of A. bracteosa aerial parts, A. macrosperma and A. parviflora roots may be due to the presence of clerodane and neo-clerodane diterpenoids. Clerodane diterpenoids are reported as antifeedant, insecticidal, antiviral, antitumor and anti-microbial agents. They also exhibit the activity against jaundice, urinary diseases and rheumatism 11. Similarly neo-clerodane diterpenoids are found to be antimicrobial, anti-mycobacterial, anti-plasmodial agents. They also have the cancer chemopreventive, hypoglycemic and hypotensive effects 10. Since clerodane and neo-clerodane diterpenoids exhibit various biological activities. Therefore, they may be the active principles responsible for the inhibition of the amplitude and frequency of the autorythmicity of A. galli. Methanol extract of A. bracteosa roots was not effective. Acknowledgements: All the research facilities provided by the Govind Ballabh Pant University of Agriculture & Technology Pantnagar and CSIR-UGC fellowship to G. Agarwal are duly acknowledged.

47


Table 1. Effects of acetone extract of leaves (AEL), chloroform extract of seeds (CES), methanol extracts of pericarps(MEP) and seeds (MES) and aqueous extract of roots (AER) of connaroides on amplitude (mm) of autorythmicity of A. galli ( mean ± S.E, n=6)

Dose mg/mL ALE CSE MPE MSE ARE

0(control) 6.06 ± 0.05 5.44 ± 0.15 12.15±1.37 9.8±1.90 11.82±4.10

3.125 7.80 ± 0.09 z 7.26 ± 0.09z 8.68±0.89 5.4± 1.19x 7.26±2.88

6.25 10.2 ± 0.09 z 9.36 ± 0.07z 5.5±0.71z 3.12 ± 0.64y 4.30±0.73

12.5 12.53 ±0.13 z 10.1 ± 0.03z 3.16 ±0.20z 1.83 ± 0.30z 2.766±0.34

25 13.33 ± 0.26 z 11.36 ± 0.11z 2.3 ± 0.136z 1.11 ± 0.042z 0.626±0.124X

50 13.6 ± 0.22 z 13.26 ± 0.07z 0.59 ± 0.015z 0.55 ± 0.03z

Student’s t-test – Px <0.05, Py <0.01, Pz <0.001 vs. control (0mg/mL)

Table 2. Effects of acetone extract of leaves (AEL), chloroform extract of seeds (CES), methanol extracts of pericarps ( MEP) and seeds (MES) and aqueous extract of roots (AER) of T. connaroides on amplitude (mg) of autorythmicity of A. galli (mean ± S.E, n=6)

Dose(mg/mL) ALE CSE MPE MSE ARE

0(control) 151.6 ± 1.39 136.0 ± 3.93 607.5 ± 68. 94 245.6 ± 47.6 591±187.48

3.125 195 ± 2.41z 181.6± 2.29

z 434.1 ± 44.67

x 135 ± 29.7

x 363.3 ± 131.8

6..25 255.0 ± 2.41z 234.1 ± 1.90

z 275 ± 35.84

z 78.08 ± 16.0

z 215 ± 33.4

12.5 313.3 ± 3.20z 252.5 ± 0.91

z 158.3 ± 10.13

z 45.79 ± 7.58

z 138.3 ± 15.75

x

25 333.3 ± 6.7z 284.1 ± 2.93

z 115 ± 6.83

z 27.75 ± 1.05

z 31.3 ± 5.66

x

50 340.4 ± 5.64z 331.6 ± 1.90

z 29.6 ± 0.785

z 13.8 ± 0.98

z

Student’s t-test – Px<0.05, Py<0.01, Pz<0.001 vs control (0mg/mL)

48


Table 3. Effects of acetone extract of leaves (AEL), chloroform extract of seeds (CES), methanol extracts of pericarps( MEP) and seeds (MES) and aqueous extract of roots (AER) of T. connaroides on % control amplitude of autorythmicity of A. galli ( mean ± S.E, n=6)

Dose

(mg/mL)

ALE CSE MPE MSE ARE

0(control) 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100± 0

3.125 128.6 ± 2.68x 134.30 ±5.39

z 72.47 ± 3.6

z 79.01 ± 28.61 55.93±3.03

z

6.25 168.2 ± 3.07 z 172.6 ± 3.73

z 45.5 ± 3.5

z 40.45 ± 9.76

z 48.27±6.25

z

12.5 206.6 ± 1.8 z 186.24 ± 4.58

z 27.69 ± 3.2

z 23.62 ± 5.35

z 35.63±8.38

z

25 219.0 ± 2.92 z 209.5 ± 4.93

z 20.08 ± 2.2

z 13.8 ± 2.94

z 9.86±3.14

z

50 224.0 ± 2.4 z 244.93 ±8.08

z 5.20 ± 0.59

z 6.92 ± 1.48

z


Table 4. Effects of acetone extract of leaves (AEL), chloroform extract of seeds (CES), methanol extracts of pericarps (MEP) and seeds (MES) and aqueous extract of roots (AER) of T. connaroides on frequeny (/min) of autorythmicity of A. galli ( mean ± S.E, n=6)

Dose (mg/mL) ALE CSE MPE MSE ARE

0(control) 4.75 ± 0.09 2.24 ± 0.11 4.01± 0.61 2.81± 0.20 5.69 ± 0.62

3.125 4.9 ± 0.28 2.53 ± 0.34 2.71 ± 0.65 1.56 ± .04 z 4.53 ± 0.46

6.25 6.35 ± 0.24 z 2.24 ± 0.60 4.13 ± 1.12 0.93 ± 0.13

z 3.08 ± 0.49

z

12.5 6.23 ± 0.29 z 2.93 ± 0.41 3.62 ± 1.01 0.60 ± 0.11

z 1.63 ± 0.07

z

25 6.53 ± 0.24 z 3.05 ± 0.58 2.33 ± 0.61

x 0.66 ± 0.09

z 0.46 ± 0.11

z

50 6.73 ± 0.24 z 2.95 ± 0.74 1.93 ± 0.57

y 0.36 ± 0.09

z


49


Table 5. Effects of acetone extract of leaves (AEL), chloroform extract of seeds (CES), methanol extracts of pericarps( MEP) and seeds (MES) and aqueous extract of roots (AER) of T. connaroides on base line tension (mg) of autorythmicity of A. galli ( mean ± S.E, n=6)

Dose

(mg/mL)

ALE CSE MPE MSE ARE

0(control) 489.4 ±21.9 451.4 ± 0.06 557.0 ± 11.07 563.5 ± 14.59 459.3 ±12.92

3.125 509.0 ±9.82 482.7 ± 0.99 z 285.6 ± 12.3

z 454.0 ±16.84

z 655.8 ± 232.7

6.25 514.0 ±20.3 334.3 ± 2.03 z 119.9 ± 29.83

z 406.1 ±12.2

z 512.3 ±171.3

12.5 556.0±34.30 434.3 ±4.11 z 69.12 ± 14.07

z 437.0± 29.7

z 263.1 ± 3.64

y

25 591.0±45.7x 557.4 ± 8.27

z 21.2 ± 15.8

z 415.0 ± 8.00

z 449.0 ± 18.7

50 590.0 ± 38.1x 625.3 ± 16.6

z 9.47 ± 4.8

z 420.0 ± 7.83

z

Student’s t-test – Px<0.05, P

y<0.01, P

z<0.001 vs control (0mg/mL)

Table 6. Effects of methanol extracts of A. bracteosa aerial part (BAE) and root (BRE), A. macrosperma root (MRE) and A. parviflora root (PRE) on amplitude (mm) of autorythmicity of A. galli (mean±S.E, n=6)

Dose(mg/ml)

BAE

BRE

MRE

PRE

0(control) 7.45 ± 0.81 3.36 ± 0.12 25.43± 0.89 15.24 ± 4.78

3.125 4.72 ± 0.70x 3.68 ± 0.07

x 13.33 ± 0.55

z 13.12 ± 3.43

6.25 2.47 ± 0.22 z 2.877 ± 0.14

x 10.1 ± 0.28

z 11.88 ± 3.90

12.5 0.441 ± 0.022 z 2.81 ± 0.179

x 7.13 ± 0.35

z 9.3 ± 3.48

25 3.01 ± 0.151 4.6 ± 0.29 z 5.11 ± 2.258

x

50 3.09 ± 0.27 1.44 ± 0.100 z 3.13 ± 1.07

x

100 3.42 ± 0.07 0.74 ± 0.028y


y<0.01, P


50


Table 7. Effects of methanol extracts of A. bracteosa aerial part (BAE) and root (BRE), A. macrosperma root (MRE) and A. parviflora root (PRE) on amplitude (mg) of autorythmicity of A. galli (mean±S.E, n=6)

Dose(mg/ml)

BAE

BRE

MRE

PRE

0(control) 372.8 ± 40.9 84.13±3.10 1271.6±44.8 762.03 ± 239.4

3.125 236.1 ± 35.2x 92.2±1.75

x 666.6±27.8

z 656.08 ± 171.9

6.25 123.9 ± 11.13 z 71.93±3.68

x 505±14.49

z 594.16 ± 195.2

12.5 22.0 ± 1.14 z 70.33±4.47

x 356.6±17.59

z 465.0 ± 174.3

25 75.30±3.78 230±14.66 z 255.8 ± 112.9

x

50 77.41±6.75 72.08±5.03 z 156.6 ± 53.61

x

100 85.6±1.87 37.04 ± 1.410y


y<0.01, P


Table 8. Effects of methanol extracts of A. bracteosa aerial part (BAE) and root (BRE), A. macrosperma root (MRE) and A. parviflora root (PRE) on % control amplitude of autorythmicity of A. galli (mean±S.E, n=6)

Dose (mg/ml) BAE BRE MRE PRE

0(control) 100 ± 0 100 ± 0 100 ± 0 100 ± 0

3.125 62.31 ± 4.2 z 110.2 ± 4.11

x 52.36 ± 0.38

z 94.55±10.51

6.25 36.29 ± 6.32 z 86.10±5.31

x 39.80 ± 0.87

z 78.91±13.27

12.5 6.26 ± 0.67 z 83.15± 2.406

y 28.20 ± 1.73

z 56.5±8.65

z

25 89.27 ± 1.41y 18.15 ± 1.19

z 27.04±4.37

z

50 91.25 ± 5.06 5.73 ± 0.52 z 19.96±2.14

z

100 102.96 ± 6.32 7.22±1.556 z


51


Table 9. Effects of methanol extracts of A. bracteosa aerial part (BAE) and root (BRE), A. macrosperma root (MRE) and A. parviflora root (PRE) on frequency (/min) of autorythmicity of A. galli (mean±S.E, n=6)

Dose

(mg/mL)

BAE BRE MRE PRE

0(control) 4.28 ± 0.60 5.65 ± 0.37 3.98 ± 0.51 2.73 ± 0.106

3.125 4.31 ± 0.64 5.99 ± 0.28 3.42 ± 0.23x 2.51 ± 0.91

6.25 2.22 ± 0.57x 6.35 ± 0.14 3.04 ± 0.38 2.22 ± 0.61

12.5 1.46 ± 0.190 z 5.97 ± 0.46 3.38 ± 0.27

x 2.04 ± 0.45

25 6.03 ± 0.164 3.23 ± 0.53 1.69 ± 0.31y

50 6.03 ± 0.12x 2.84 ± 0.56 1.64 ± 0.28

z

100 1.52 ± 0.635x


Table 10. Effects of methanol extracts of A. bracteosa aerial part (BAE) and root (BRE), A. macrosperma root (MRE) and A. parviflora root (PRE) on base line tension (mg) of autorythmicity of A. galli (mean±S.E, n=6)

Dose (mg/mL)

BAE

BRE

MRE

PRE

0(control) 590.2 ± 41.92 528.5 ± 21.06 528.7 ± 25.4 478.7 ± 27.13

3.125 645.25 ± 131.4 500.3 ± 6.42 360.1 ± 42.45 603.5 ± 135.4

6.25 360.0 ± 100.21 510.4 ± 6.32 526.8 ± 8.59 647.6 ± 115.5

12.5 269.3 ± 49.37z 538.6 ± 13.09 643.2 ± 35.31 585.5 ± 83.97

25 577.6 ± 9.27x 476.15 ± 39.39 658.3 ± 145.34

50 629.4 ± 15.54y 488.55 ± 50.97 466.7 ± 155.8

100 640 ± 19.7y 517.2 ± 181.0


Table-11. IC50 values of crude extracts of Trichilia connaroides and some species of genus Ajuga. S.No. Plant Species Extract Plant part used IC50 (mg/ml) 1. T. connaroides MeOH seeds 3.93 ± 0.70 2. T. connaroides MeOH pericarps 7.09 ± 2.67 3. T. connaroides Aqueous roots 6.40 ± 4.74 4. A. parviflora MeOH roots 16.79 ± 2.93 5. A. bracteosa MeOH aerial parts 4.49 ± 0.72 6. A. macrosperma MeOH roots 1.73 ± 0.02 7. Albendazole - - 0.74± 0.05

52

A

0

50

100

150

200

250

300

control 3.125 6.25 12.5 25 50

Dose(mg/ml)

% C

on

tro

l Am

plit

ud

e

(a)

(b)

(c)

(d)

(e)

B

012345678

control 3.125 6.25 12.5 25 50

Dose (mg/ml)

Fre

qu

ency

(/m

in)

(a)

(b)

(c)

(d)

(e)

C

-200

0

200

400

600

800

1000

control 3.125 6.25 12.5 25 50

Dose(mg/ml)

Bas

e lin

e te

nsi

on

(mg

)

(a)

(b)

(c)

(d)

(e)

A

0

20

40

60

80

100

120

control 3.125 6.25 12.5 25 50 100Dose(mg/ml)

% C

on

tro

l Am

plit

ud

e

(a)

(b)

(c)

(d)

B

01

2

34

5

6

78

control 3.125 6.25 12.5 25 50 100

Dose(mg/ml)

Fre

qu

ency

(/m

in) (a)

(b)

(c)

(d)

C

0

200

400

600

800

1000

control 3.125 6.25 12.5 25 50 100

Dose(mg/ml)

Bas

e lin

e te

nsi

on

(mg

)

(a)

(b)

(c)

(d)

A

0

20

40

60

80

100

120

Control 0.32 0.64 1.28 2.56

Dose(mg/ml)

% C

on

tro

l Am

plit

ud

e

B

0

1

2

3

4

5

6

Control 0.32 0.64 1.28 2.56

Dose(mg/ml)

Fre

qu

ency

(/m

in)

C

0

100

200

300

400

500

600

control 0.32 0.64 1.28 2.56

Dose(mg/ml)

Bas

e L

ine

Ten

sio

n (

mg

)

Garima.A. et al, Hygeia.J.D.Med, vol.2 (2) 2010, 43-53.

Fig. 1Effect of (a) Acetone extract of leaves (b) Chloroform extract of seeds (c) Methanol extract of pericarps (d) Methanol extract of seeds (e) Aqueous extract of roots of T. connaroides on (A) % Control amplitude (B) Frequency (C) Base line tension on autorythmicity of A.galli. (n=6)

Fig. 2.Effect of methanol extracts of (a) A. bracteosa aerial parts (b) A. macrosperma roots (c) A. parviflora roots (d) A. bracteosa roots on (A) % control amplitude (B) Frequency (C) Base line tension on autorythmicity of A. galli. (n=6)

Fig. 3.Effect of Albendazole on (A) % Control amplitude (B) Frequency (C) Base line tension on autorythmicity of A. galli. (n=6)

References:

1. Singh T U, Kumar D, Tandan S K. Paralytic effect of alcoholic extract of Allium sativum and Piper longum on liver amphistome, Gigantocotyle explanatum. Indian J Pharmacol, 40, 2008: 64.

2. Chakraborty A K, Mehta R K, Sirivastava P N, Datta I C. Cholinergic effect of tetramisole on Ascaridia galliz. Indian J Pharmacol 10, 1978: 121.

3. Vallachira A, Veterinary Materia Medica (Jaypee Brothers Medical Publishers Ltd, New Delhi) 1998: 142.

4. Khwaja N, Bhargava K P. Kishor K. Neurotransmitters in Ascar idia galli. Indian J Pharmacol 5 ,1973: 346.

5. The Wealth of India, A dictionary of Indian Raw Materials, Vol V. (Council of Scientific and Industrial Research, Delhi) 1997, Vol V, 74.

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Garima.A. et al, Hygeia..J.D.Med, vol.2 (2) 2010, 43-53.

6. Kirtikar, K R, Basu, B D, Indian Medicinal Plant (Periodical Experts Book Agency, Delhi) 1993, Vol I, 553.

7. D E, Koul O, Isman M B, Scudder G G E, Towers G H N. Biological activity of limonoids from rutales. Phytochem, 31, 1992: 377. Champagne.

8. Kirtikar K R, Basu B D. Indian Medicinal Plant (Periodical Experts Book Agency, Delhi) (1993) Vol I ,555.

9. Zhang, H P, Wu, S H, Shen, Y M, Ma, Y B, Wu, D G, Qi, S H, Luo X D, 2003. A pentanortriterpenoid with a novel carbon skeleton and a new pregnane from T. connaroides. Can. J. Chem, 81,2003: 253.

10. Coll J, Tandron Y A. Neo-Clerodane diterpenoids from Ajuga: structural elucidation and biological activity. Phytochem Rev, 7,2008: 25.

11. Merritt A T & Ley S V. Clerodane diterpenoids. Nat. Prod. Rep, 9, 1992: 243.

12. Nawaz H R, Malik A, Khan P M, Saeed A. Ajugin E and F: Two withanolides from Ajuga parviflora. Phytochem, 52, 1999: 1357.

13. Bhakuni D S, Kaul K N. Chemical Examination of Ajuga bracteosa Wall. J Sci Indus Res, 20 B, 1961: 185.

14. Babu C R. Herbaceous Flora of Dehradun. (Publications and information directorate (CSIR) New Delhi) 1977: 405.

15. Shen X Y, Sun H D, Suzuki, A. Two neo-clerodane diterpenoids from Ajuga macrosperma. Phytochem ,33 ,1993: 887.

16. Beauchamp P S, Bottini A T, Caselles M C, Dev V, Hope H, Larter M, Lee G, Mathela C S, Melkani A B, Millar P D, Miyatake M, Pant A.K, Raffle R J, Sharma V K , Wyatt D. Neo-clerodane diterpenoids from Ajuga parviflora. Phytochem, 434, 1996: 827.

17. Manthunath, B L. The Wealth of India.(Council of Scientific and Industrial Research, Delhi)1948 , 1 42.

18. Perry L M, Metzger J. Medicinal Plants of East and Southeast Asia. (The MIT Press Cambridge, London) 1980 ,184.

19. Tracy J W, Webster L T. Drugs used in the chemotherapy of helminthiasis. (J.G. Hardman and L.E. Limbird. (Eds), Goodman & Gilman’s The Pharmacological Basis of Therapeutics, Mc Graw Hill Publication. Phidaelphia) 2001: 1121

20. Geary T. G., Klein R.D., Vanover, L., Bowman, J.W., Tompson D.P. The nervous system of helminths as target for drugs. Journal of Parasitology, 78, 1992, 215-30.

21. Agarwal G, Hore S K, Pant A K. Antioxidant and hepatoprotective effects of Trichilia connaroides Wight and Arn. Roots. Hygiea-journal of Drugs and Medicines. In Press.

22. Shradha V K, Joshi S, Maurya V P, Singh G N, Singh A. Authentication of Kampilaka (Mallotus phillippinensis): An important drug of Ayurveda ( Indian Traditional Medicine). The Internet Journal of Alternative Medicine. 5 2007.

23. Suleiman M M, Mamman M, Aliu Y O, Ajanusi J O. Anthelmintic activity of the crude methanol extract of Xylopia aetiopia against Nippostrongylus brasiliensis in rats. Veterinarski Arhiv. 75, 2005: 487-495.

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Short communication Hygeia.J.D.Med.vol.2 (2), 2010,54-58 ISSN 0975 6221 ________________________________________________________________________________________________________ HYGEIA JOURNAL FOR DRUGS AND MEDICINES


______________________________________________________________________________

ANTI-INFLAMMATORY ACTIVITY OF SEED AND FRUIT WALL EXTRACT OF SOLANUM TORVUM

M. Rammohan1 and C.Srinivas Reddy1* 1, 1* .Vaagdevi College of Pharmacy, Kishanpura, Hanamkonda, Warangal 506001, AP, India,

____________________________________________________________________________________ Abstract

The present study is aimed to evaluate the seed and fruit wall methanol extracts of Solanum torvum for anti-inflammatory activity by Carrageenan induced rat paw edema method by using plethysmometer.all text extracts show significant anti-inflammatory activity, among them seed methanol 500 mg ⁄ kg body weight exhibited superior activity. The anti-inflammatory activity of extract may be presence of flavonoids, sterols and saponins.

Keywords: Solanum torvum, Methanol, Diclofenac sodium, Anti-inflammatory activity

_________________________________________________________________________________

1. Introduction

Inflammation is a non-specific, defensive response of the body to tissue damage The inflammatory response mobilizes the body’s defenses, isolates and destroys microorganisms and other injurious agents and removes foreign materials and damaged cells so that tissue repair can proceed . A variety of chemical agents like histamine (1mg/ml), Carrageenan (1%w/v) and dextran (60mg/ml) have been used to induce edema in the feet of rodents. Anti-inflammatory activity of an extract can be determined by their ability to reduce or prevent edema. Solanum torvum (Solanaceae) popularly know as Sundaikai, Kodusonde in India, is used in Ayurveda and Unani systems of medicine to treat various diseases. The powdered seeds mixed with mustarded oil are used as massage oil for treating skin eruptions.

_______________________________________ For correspondence: [email protected] Contact: +91 9000716969, +91 870 2455111, Fax: +91 870 2460108© 2010 Hygeia journal for drugs and medicines. All rights reserved. 0975 6221

55

M. Rammohan et al ,Hygeia.J.D.Med.Vol.2(2)2010,54-58.

The fumes of burning seeds are inhaled for toothache and the roots are used in the form of a poultice to promote healing of cracks in the feet and anti microbial agent [1, 3 and 4]. Since the anti-inflammatory activity of this plant has not been scientifically evaluated, the present study was undertaken to investigate the effect of methanol extracts of seeds and fruit wall of Solanum torvum for its anti-inflammatory activity

2. MATERIALS AND METHODS

2.1. Plant material

The Fruits of Solanum torvum. (Solanaceae) were collected in November and December 2007 from Thirumala Thirupathi (A.P), India. The plant parts are identified by Dr. Madhavashetty, Taxanomist, Dept of Botany, S.V University, Thirupathi, India and authenticated by comparing with the voucher specimen.

2.2. Extraction

The Seed, fruit wall are separated, dried and powdered and macerated with methanol for 2 days to afford a greenish brown semisolid mass (Seed methanol extract; yield: 6.76% w/w on dried wt, Fruit wall methanol extract; yield:7.12% w/w on dried wt,).

2.3. Preliminary phytochemical studies

Preliminary qualitative phytochemical analysis of Seed methanol extract and Fruit wall methanol extract indicated the presence of spirostanol glycosides, isoflavanoids, alkaloids, tannins and carbohydrates.

2.4. Animals

Wistar rats of either sex, weighing 180-240 gm purchased from NIN, Hyderabad were used. They were housed in standard environmental conditions of temperature, humidity, light and provided with standard rodent food and water ad libitum

2.5 Acute toxicity study

Healthy adult albino mice of either sex, starved over night, were divided into groups (n=6) and were orally fed with increasing doses (250, 500, 750 and 1000 mg/kg body weight) of methanol extracts. The total extracts administered orally in doses up to 1000mg/kg. Animals were observed during first 2hr for gross behavioral changes and once in 30min for next 4hrs and then once in 24hr for next 72hrs to find out percentage mortality.

2.6 Evaluation of Anti-inflammatory Activity

The Wistar rats were divided into six groups of six animals each. Group I served as control and received normal saline orally. Group II served as positive control and received Diclofenac sodium (100 mg/kg).

56


Group III and IV received Seed methanol extract, orally at a dose of 250 and500 mg/kg respectively. Group V and VI received Fruit wall extract, orally at a dose of 250 and 500 mg/kg respectively.

A mark was made on both the hind paws just beyond the tibiotarsal junction, so that every time the paw is dipped in the mercury column up to the marked level to ensure constant paw volume. After 1 hr of administration of the test and standard samples, 0.1 ml of 1% Carrageenan suspension (in normal saline) was injected into dorsal region of sub plantar surface of hind paw of rat subcutaneously with the help of 26 G needle. The initial paw volume of each rat was recorded before drug administration. The paw volumes were measured at the end of 0.5, 1, 2, 3 and 4 hrs using plethysmometer. Any change in paw volume of rats was obtained by subtracting initial paw volume from the paw volumes at different time intervals. The average value of edema was calculated by taking the average of each group at different hours. Percentage inhibition of edema was calculated for each group with respect to its control group.

Percentage(%) inhibition = (A – B) x 100/A

Where A is the mean increase paw volume in rats treated with control and B is the mean increase in paw volume in rats treated with test drug. [2, 8]

3. Statistical analysis

All the results are reported as Mean ± SEM Statistical significance was analyzed employing, one way ANOVA. P-values of 0.05 or less were taken as significant.

4. Results and discussion

The results obtained in this investigation indicate that the percentage protection against edema formation with all extracts was significant. The anti-inflammatory activity was found to be dose dependent up to 3 hrs in case of standard and extracts, followed by a decrease in either case.

From the table it can be observed that the standard drug diclofenac sodium has protected to an extent of 21, 41, 55, 70 and 56% against inflammation induced by Carrageenan at ½, 1, 2, 3 and 4 hrs. Seed methanolic extract at 500 mg ⁄ kg body weight has shown activity comparable to that of diclofenac sodium (100 mg ⁄ kg body weight). This extract has produced more protection then the other extracts and it is less then that of standard.

5. Conclusion: Seed methanolic extract at 500 mg ⁄ kg body weight has shown maximum activity comparable to other extracts. Acknowledgements Authors wish to acknowledge Viswambhara Educational Society and the management, Warangal and, Vaagdevi College of Pharmacy, Warangal (A.P) for the infrastructural facilities and moral support.

57


Table 1.Effect of Seed and fruit wall extracts of Solanum torvum on carrageenan induced at paw edema.

Treatment Dose mg/kg

*MEAN EDEMA VOLUME(ml) and %protection 30 min 1st hr 2nd hr 3rd hr 4th hr

Control - 0.38±0.02

0.65 ±0.003

0.94±0.043

1.14±0.02

0.92±0.001

Standard

100 0.30 ± 0.03 (21)%

0.38±0.009 (41)%

0.42±0.05 (55)%

0.34± 0.02 (70)%

0.40 ± 0.03 (56)%

Seed methanol

250 0.34 ± 0.02 (10)%

0.52 ± 0.06 (20)%

0.68±0.01 (27)%

0.50 ± 0.02 (56)%

0.61 ± 0.04 (33)%

500 0.30± 0.02 (21)%

0.49 ± 0.03 (24.6)%

0.52±0.02 (44)%

0.40 ± 0.03 (65)%

0.48 ± 0.03 (48)%

Fruit wall Methanol

250 0.36± 0.02 (5)%

0.56± 0.01 (16)%

0.70±0.01 (25)%

0.52 ± 0.02 (54)%

0.59 ±0.05 (35)%

500 0.32 ± 0.02 (15.7)%

0.45± 0.02 (30)%

0.51±0.02 (45)%

0.44 ± 0.02 (61)%

0.52 ± 0.02 (43)%

* Edema volume (mean ± SEM)

Table.2 Maximum percentage protection

Treatment Dose mg/kg Maximum percentage protection

P value

Control - -

N.S

Standard

100 70 P<0.001

Seed methanol 250 56 P<0.001 500 65 P<0.01

Fruit wall Methanol 250 54 P<0.001 500 61 P<0.01

References

1. Parotta JA, Healing plants of peninsular India, CABI publishing, London, 2001, 89-90. 2. Turner RA, screening methods in Pharmacology, Academic press, 1971.210-245 3. DumrongkietArthan, PrasatKittakoop, AsimEsen, Jisnuson Svasti, Daraporn), Anti viral iso flavanoid sulphate and

steroidal glycosides from Solanum torvum, Phytochemistry, 59: 2002, 459-463.

58


4. Chah. K.F., Muko. K.N. and Oboegbulem, S.I. Anti microbial activity of methanolic extract of Solanum torvum, Fitoterapia, (7): 2000, 187-189.

5. E. O. Ajaiyeoba (),Comparative phytochemical and Anti microbial studies of Solanum macrocarpum and Solanum torvum, Fitoterapia, (70):1999,184-186

6. Joshi S.D,Kulkari V.D (2006) Journal of natural remedies 6/2 135-140 7. Hukkeri V.I and Savadi R.V), Anti inflammatory activity of leaves of Acacia farneosiana, Indian drugs, 39(12): 2002,

664-666. 8. Handa. S.S, Chawla.A.S, Sharma.A.K. (), Plants with Anti inflammatory activity, Fitoterapia, 63(1). 1992. 9. S.Moses Soundaraaj V.Alagarsamy, G.Senthil Kumar et al Anti inflammatory and Anti microbial activity of Vitex

negunda leaf extracts, Indian drugs, 36(12):1999, 756-758. 10. Joy. B.Mathew S.W. and Gopi T.V Anti inflammatory activity effect of seed oils of some Annona species Indian

drugs, 41(10): (2004), 636-638.

1

DRUGS USAGE IN

PREGNANCY Pharmaceutical education

The use of drugs during pregnancy is a question of fine balance; no harm should be allowed to be fall on the baby because of the drug, and no harm must come to the mother or baby because a disease is being inadequately treated

5.

Knowledge of the harmful effects of drugs on the foetus by the thalidomide disaster was followed by a reduction of the use of drugs by pregnant women. However, several surveys performed in North America and Europe published in the 1990s has found that drug use during pregnancy is still a frequent event, regardless of the country involved.

Siyad A R*,

Hygeia.J.D.Med.vol2 (2),2010.1-15

2010

Siyad A R*,

Department of Pharmacy Practice, Crescent College of Pharmaceutical Sciences,

Kannur, Kerala, India

670358

Hygeia.J.D.Med, vol.2(2), Sep.2010 –Mar.2011 ,1-15.

________________________________________________________________________________________________________

ISSN 0975 6221 HYGEIA JOURNAL FOR DRUGS AND MEDICINES


_____________________________________________________________________________________

DRUG USAGE IN PREGNANCY

Siyad A R*, Department of Pharmacy Practice, Crescent College of Pharmaceutical Sciences, Kannur, Kerala, India,670358

The use of drugs during pregnancy is a question of fine balance; no harm should be allowed to be fall on the baby because of the drug, and no harm must come to the mother or baby because a disease is being inadequately treated5. Knowledge of the harmful effects of drugs on the foetus by the thalidomide disaster was followed by a reduction of the use of drugs by pregnant women6,7. However, several surveys performed in North America and Europe published in the 1990s has found that drug use during pregnancy is still a frequent event, regardless of the country involved8 Placental transport of maternal substrates to the foetus and vice is established at around 5th week of foetal life. The flow across placenta is guided mainly by concentration gradient. It is important to note, that almost every substance used for therapeutic purposes can pass from mother to foetus. But it is the concentration within foetus that may be more important as far as teratogenecity is concerned. One must discard the concept that there is a placental barrier and no drug may be absolutely safe in pregnancy. The exact mechanisms of teratogenecity with various agents are poorly understood, particularly in humans9. The benefit of drug therapy for the mother must thus he weighed against risks for the foetus10,11. Although authorities have argued for therapeutic restraint during pregnancy12, most studies have shown an almost unchanged pattern of drug use among pregnant women over die last two or three decades13,14. In many situations the benefits for the mother may well outweigh a potential risk for the child, example in case where untreated urinary tract infections may result in significant maternal and foetal morbidity and mortality15,16.

PREGNANCY Pregnancy is a sequence of events that begins with fertilization proceeds to implantation, embryonic development and foetal development and normally ends with birth about 38 weeks later or 40 weeks after the last menstrual period(LMP). ______________________________________ *For Correspondence [email protected] Phone- +91-9746718940

Siyad.A.R. et al, Hygeia.J.D.Med, vol.2 (2) 2010,1-15.

Prenatal development is the time from fertilization to birth and includes both embryonic and foetal development. It is divided into 3 periods of 3 calendar months each called trimesters. Accordingly, the nine months of pregnancy is divided into 3 trimesters. The first trimester : is the most critical stage of development during which the rudiments of the all major organ systems appears. The second trimester: is characterized by nearly complete development of organ systems. The third trimester : represents a period of rapid

foetal growth. During the early stages of this period , most of the organ systems are becoming fully functional. 1. Embryonic Period First week of development Include fertilization, cleavage of the zygote, blastocyst formation and implantation. a) Fertilization During fertilization, the genetic material forms a haploid sperm cell (spermatozoon) and a haploid secondary oocyte merges into a single diploid nucleus. It occurs in the uterine tube (Fallopian tube) within 12-4hours after ovulation.

Once a sperm cell enters a secondary oocyte, the oocyte must first complete meiosis-2.It divides into a larger ovum and a smaller second polar body that fragments and disintegrates. The nucleus in the head of sperm develops into the malre pronuclei an the nucleus of the fertilized ovum develops into the female pronuclei. These fuse to produce a single diploid nucleus. The fertilized ovum is called a zygote. b) Cleavage of the zygote After fertilization, rapid mitotic cell divisions of the zygote called cleavage takes place. The first division of zygote begins about 24 hours after fertilization and is completed after 6 hours. By the second day after fertilization, second cleavage is completed and there are 4 cells. By the end of the third day there are 16 cells. The smaller cells formed by cleavage are called blastomeres. Successive cleavages eventually produce a solid sphere of cells called morula.

c) Blastocyst formation By the end of forth day, the number of cells in the morula increases. When the morula enters the uterine cavity, a glycogen rich secretion from the endometrial glands passes into the uterine cavity and enters the morula. At the 32 cell stage, the fluid entered the morula collects between the blastomeres and reorganizes them around a large fluid cavity called the blastocyst cavity.


d) Implantation The blastocyst remains free within the uterine cavity for about 2 days before it attaches to the uterine wall. About 6 days after fertilization, the blastocyst loosely attaches to the endometrium- implantation. About 7 days after fertilization, the blastocyst attaches to the endometrium more firmly. Second week of development a) Development of trophoblast About 8 weeks after fertilization, the trophoblast develops into two layers-synctiotrophoblast and cytotrophoblast. These two layers become part of the chorion(fetal membrane).During implantation, the synctiotrophoblast secretes enzymes that enable the blastocyst to penetrate the uterine lining. Eventually, the blastocyst remains burried in the endometrium. b) Development of the bilaminar embryonic disc Cells of the inner cell mass differentiate into 2 layers around 8 days after fertilization-hypoblast and epiblast. These two cells together form a flat disc-bilaminar embryonic disc. A small cavity appears within the epiblast which enlarges to form the amniotic cavity. c) Development of the amnion As the amniotic cavity enlarges, a thin protective membrane called the amnion develops from the epiblast. As the embryo grows the amnion surrounds the entire embryo, creating the amniotic cavity that becomes filled with amniotic fluid. d) Development of yolk sac On the eighth day after fertilization, cells at the edge of hypoblast migrate and cover the inner surface of the blastocyst wall. These cells form a in membrane called exocoelomic membrane. Together with the hypoblast, the exocoelimic membrane forms the wall of the yolk sac. e) Development of sinusoids On the ninth day after fertilization, the blastocyst becomes completely embedded in the endometrium. The synctiotrophoblast expands into the endometrium and around the yolk sac. Small spaces called lacunae develops within it.By the twelfth day, the lacunae fuse to form larger, interconnected spaces called lacunar networks. The

capillaries around the developing embryo become dilated and are referred as sinusoids. f) Development of extraembryonic coelom About the twelth day after fertilization, the extraembryonic mesoderm develops. Soon a number of large cavities develop in the mesoderm which fuses to form a single large cavity called extraembryonic coelom. g) Development of chorion The extra embryonic mesoderm together with the 2 layers of the trophoblast constitutes the chorion.


Third week of development a. Gastrulation Occurs about 15 days after fertilisation. In this the two layerd embryonic disc transfers in to 3-layered disc

consisting of 3 primary germ layers-ectoderm, mesoderm, endoderm. The first evidence of gastrulation is the formation of the primitive streak (a groove on the dorsal surface of epiblast). The primitive streak establishes the head and tail end of the embryo. At the head end a small group of epiblast cell forms a rounded structure called primitive node. About 16 days after fertilisation, mesodermal cells from the primitive node migrate towards the head end of the embryo and form a hollow tube of cells (notochordal process). By 22 to 24 days, the notochordal process becomes a solid cylinder of cells called notochord (function mesoderm-vertebrae).

b. Neurulation The notochord induces ectoderm cells to form neural plate, neural groove, neural folds and neural tube(develop in to brain and spinal cord). It occurs by the end of third week of neurulation. About 4 weeks, the neural tube develops in to 3 areas-primary brain vescicles-proscencephalon (fore brain), mesencephlon and rhombencephalon.5-weeks-procencephalon develops in to secondary brain vescicle-tolencephalon and diencephalone and the rhombencephalone develops in to secondary brain vescicle called metencephalon and myelencephalone. c. Development of somites Seventeenth day of fertilisation the mesoderm cells the neural tube forms paraxial mesoderm.Paraxial mesoderm segments in to a series of paired cube shaped structures called somites.It differentiate into3regions-myotome (skeletal muscles of neck, trunk, limbs), dermatome (connective tissue –dermis of skin), sclerotome (vertebrae). d. Development of the intraembryonic coelom Small spaces appear in the mesoderm which emerge to form a cavity called intraembryonic coelo Intraembryonic coelom splits to mesoderm. Mesoderm develop in to splanchnic and somatic. Splanchnic-heart, viscera of serous pericardium, blood vessels, smooth muscles, connective tissue of respiratory and digestive organs. Somatic-bones, ligaments and dermis of limbs. e. Development of cardio vascular system

At the beginning of third week, angiogenesis, the formation of blood vessels, in the extraembryonic mesoderm occurs. Blood cells blood plasma develop outside the embryo from the endothelial cells of the yolk sac, allantois and chorion. Blood formation begins within the embryo at fifth week liver and twelfth week in spleen bone marrow and thymus. The heart forms from splachnic mesoderm-cardiogenic area. These mesodermal cells form a pair of endocardial tubes. These fuse to form a single primitive heart tube. f. Development of chorionic villi and placenta By the end of second week, primary chorionic villi develop (projection of chorion).Early in the third week, mesenchyme grows in to the villi forming a core loose connective tissue-secondary chorionic villi. By the end of third week, blood capillaries develop in the villi-chorionic vill Placentation is the process forming placenta. By the beginning of twelfth week, placenta has 2 distinct parts-foetal portions from chorionic villi, chorion and maternal portion formed by deciduas basalis of endometrium. The connection between placenta and embryo is the umbilical cord.

Siyad.A.R. et al, Hygeia.J.D.Med, vol.2 (2) 2010 ,1-15.

D. Fourth week of development The fourth-eighth week of development is very significant because all major organs appears during this time-organogenesis. During fourth week embryo undergo changes in shape and size-tripling its size. A flat 2D-embryonic disc is converted to 3D cylinder-embryonic folding. The cylinder consists of endoderm in the centre(gut),ectoderm outside(epidermis) and mesoderm. E. Fifth- eighth week of development Fifth week- rapid development of brain and growth of head is considerable. By the end of seventh week, the various regions of limbs becomes distinct, heart is 4-chambered. 2. FEATAL PERIOD Tissues and organs developed during embryonic period grow and differentiate. Minor ailments during pregnancy 1. Morning Sickness This usually occurs during the 1st 12 weeks of pregnancy & can be aggravated or even started as a result of seeing, tasting or smelling things which under normal circumstances would have been enjoyable. Suggestions on how to ease morning sickness.

1. Get up slowly after having a warm drink & a day biscuit or juice of toast. 2. Eat small meals at frequent intervals & avoid fatty, spicy food.

3. Drink plenty & don’t go hungry but take meals & fluids slowly. Take drinks between meals rather than with food. 4. Avoid cooking food with strong smells. 5. Some improvement can be seen with the addition of high-dose therapy & the preconceptional use of prenatal vitamins. 6. Attractive therapy such a ginger supplement, acupuncture & acupressure may be beneficial. 2. Heart burn It is a condition in which there is a burning sense & tends to occur in the 2nd half of pregnancy. It can be felt across the lower fort of the chest, in the throat or back of the month or in the stomach. This occurs Broz the value at the top of the stomach opens up slightly & acid rises into the oesophagus. To reduce heartburn

1 Eat smell, frequent, light meals 2 Drink a glass of milk 3 Avoid a totally empty stomach 4 Avoid fatty or empty stomach 5 Bend at your knees rather than at your waist to pick up. 6 Use an extra pillow at night.


Gastric reflex Commonly occurs as a result of things up gastric emptying, interval motility & used lower oesophageal splinter tone.Life style modifies include awareness of portiere, maintaining upright positions (after meals), sleeping in propped poses & directory modifies. 4. Backache

Most women develop backache during pregnancy & it often 1st develops during 5th -7th months. Encourage light exercise & simple analgesia & consider physiotherapy.

5. Breathlessness Feeling of not able to take in enough breath or ‘puffing & panting’ as if done a lot of exercise. These symptoms might also combine with a feeling of dizziness. 6. Fainting In late pregnancy, if lied flat on back, the wt. of the fatty in the uterus way press on some large wins behind the uterus & prevent the blood from the lower part of the body from easily getting back to the heart. This condition is called ‘supine hypertension’ & it reduces blood supply to the placenta. Drugs in pregnancy Drugs used during pregnancy can have temporary or permanent effects on the fetus. Therefore many physicians would prefer not to prescribe for pregnant women, the major concern being over teratogenicity of the drugs. This results in inappropriate treatment of pregnant women. Use of drugs in pregnancy is not always wrong. For example, high fever is harmful for the fetus in the early months. Use of paracetamol is better than no treatment at all. Also, diabetes mellitus during pregnancy may need intensive therapy with insulin. Drugs have been classified into categories A, B, C, D and X based on the Food and Drug Administration (FDA) rating system to provide therapeutic guidance based on potential benefits and fetal risks. Drugs like multivitamins that have demonstrated no fetal risks after controlled studies in humans are classified as Category A. On the other hand drugs like thalidomide with proven fetal risks that outweigh all benefits are classified as Category X. DRUG USE DURING PREGNANCY Category-A Drugs which have been taken by a large no. of pregnant women of childbearing age without any proven increase in the frequency of malformations or other direct/indirect harmful effects on the foetus having been observed. Category-C Drugs which, occurring to their pharmacological effects, have caused or may be suspected of causing harmful effects on the human foetus or neonate without causing malformations. There effects may be reversible.


Category-B1 Drugs which have been taken by only a limited No. of pregnant women & women of childbearing age, without an increase in the frequency of malformation or other direct/indirect harmful effects on the human foetus having been observed.Studies in animals have not shown evidence of an occurrence of foetal damage. Category-B2 Drugs which have been taken by only a limited No. of pregnant women of childbearing age without an in frequency of malformation or other direct/indirect harmful effects on the human foetus having been observed.Studies in animals are inadequate or may be lacking, but available data show no evidence of an occurrence of foetal damage. Category-B3 Studies in animals have shown evidence of an occurrence of foetal damage, the significance of which is considered uncertain in humans. Category-D Drugs which have caused & suspected to have caused or may be expected to cause, an increased incidence of human foetal malformation/irreversible damage. These drugs also have adverse pharmacological effects. Category –X Drugs which have a high risk of causing permanent damage to the fetus that they should not be used in pregnancy when there is a possibility of pregnancy. PREGNANCY RISK FACTORS.

Many factors affect the development of a foetus into a healthy child, some which are beyond control & other that are within control. Common pregnancy risk factors that can be controlled or influenced or 1. Smoking Smoking is not only bad for mother but it is bad for baby. Smoking during pregnancy reduces the amount of oxygen that the baby receives of increase the risk of miscarriage bleeding sickness. It leads to decrease in birth weight, premature birth, increase of SIDS & still birth. 2. Alcohol It can cause foetal alcohol syndrome, including symptoms like low birth weight & behaviour abnormalities. 3. Caffeine This will affect foetal heart rates & awake time (Foetus growth well when sleeping). It will increase risk of stretch marks suddenly guilty coffee intake can cause headache.


4. Nutrition Good nutrition is necessary to developing child lack of folic acid the cell that form the baby’s brain & spine become deficient resulting in open spine (spina bifide) or an undeveloped brain (encephely). Folic acid prevents left palate, low birth weight & help in production of additional red blood cells needed and support growth of foetus. It also prevent stroke of anaemia.Daily requirement in non pregnant is up to 400 mg of in pregnancy 800mg respectively. NUTRITION DO’S AND DONT’S DURING PREGNENCY 1,46

A healthy diet contributes to a successful pregnancy by reducing complication & promoting adequate foetal growth & development. Nutrition is thus as essential component of prenatal care. There are a lot of food to avoid during pregnancy because they may be harmful to mother or future.A well balance diet contributes to normal birth weight for the baby improved fetal brain level decreased chances of pregnancy complication such as morning sickness, fatigue, anaemia & pre – eclampsia. The basis of a well balanced diet should be built around the five big nutrient of pregnancy is calcium, carbohydrate, good fats, iron & protein.Brain products are the main source of carbohydrate complex, carbohydrate provide energy, prevent constipation & nausea & give essential nutrient including fibre, folate, vitamin – B & protein. Fruits & vegetable will provide vitamins mineral as well as fibre to aid digestion vitamin-A derived from green leafy vegetable & yellow fruit are important for the development of baby’s bone, skin, hair & eyes. Vitamin-C is important for growth & tissue repair. Protein is important for the development of foetus. It protect against the development of pre-eclampsia latter in pregnancy. Protein food normally iron rich food is important to keep the blood well oxygenated. Calcium – need for development of teeth, muscles heart, nerve, blood The pregnant women should avoid... a. Processed sandwich meet: Leads to miscarriage or still birth b. Unpasteurized milk or juice: They have the potential to carry listeric which is harmful for mother baby. c. Certain types of Fish: Fishes such as swordfish, Shark, Tuna, Should not given during pregnancy because they contain high level of mercury which is harmful for the unborn baby’s brain. PRESCRIBING IN PREGNANCY

I. Where possible use non drug therapy.

II. Prescribe drugs only when definitely needed. III. Choose the drug having the best safety record. IV. Avoid newer drugs, unless safety is clearly established. V. OTC drugs cannot be assumed to be safe.

VI. Avoid medication in the initial 10 week of gestation. VII. Use the lowest effective dose.

VIII. Use drugs for the shortest period. IX. If possible, give drugs intermittently.


Drug class(condition)

Safety uncertain/unsafe

Safer alternative

1.Anti emetics (Morning sickness and other type of vomiting)

Domperidon(X), Ondansetron Promethazine, Cyclizine, Dicyclomine, Prochlorperazine,metochlopramide, Doxylamine

2.Drug s for peptic ulcer and GERD

Cimitidine, Omeprazole, Pantoprazole, Lansoprazole(X), Cisapride(X), Mosapride.

Ranitidine, Famotidine

3.Laxative (Constipation)

Senna, Bisacodyl, Docusates, Senna, Bisacodyl, Docusates Saline purgatives

Dietary fibre, Ispaghula, Lactulose

4. Anti-diarrhoeals Diphenoxylate-atropine, Loperamide ORS

5.Analgesics (Headache, Bodyache, Joint pain, Visceral pain)

Aspirin, Metamizole, NSAIDs, Codeine, Dextropropoxyphene, Morphine(X), Pethidine, Tramadole

Paracetamol,, Ibuprofen(low dose)

6.Cold-cough remedies Codiene, Dextromethorphan, Bromhexine,Expectorents

Xylometazoline, Oxymetazoline, Budesonide, Cromoglycate (Nasal drops)

7.Anti allergic Cetrizine, Loratadine, Fexofenadine, Astemizole(X)

Chlorpheniramine, Promethazine

8.Anti bacterials(systemic bacterial infections)

Cotrimazole, Fluoroquinolones(X), Tetracycline(X), Doxycycline(X), Chloramphenicol(X), Gentamicin, Streptomycin(X), Kanamycin(X), Tobramycin(X), Clarithromycin, Azithromycin, Clindamycin, Vancomycin, Nitrofurantoin

Penicillin G, Ampicillin, Amoxicillin-Clavulanate, Cloxacillin, Piperacillin, Cephalosporins, Erythromycin

9.Anti tubercular Pyrazinamide, Ethambutol, Streptomycin(X)

Isoniazid, Rifampicin

10.Anti amoebic Metronidazole, Tinidazole(X), Quiniodochlor Diloxanide furoate 11.Anti malarial Quinine(X),Mefloquine, Pyrimethamine ,

Siyad.A.R. et al, Hygeia.J.D.Med, vol.2 (2) 2010

Chloroquine, Proguanil

Sulfadoxine(X), , Primaquine(X)

12.Anthelmintic Albedazole(X), Mebendazole(X), Ivermectin, Pyrantel pamoate, Praziquantel, Diethylcarbamazine(X)

Piperazine, Niclosamide

13.Antifungal (Superficial & deepmycosis)

Amphotericin B(X), Fluconazole, Itraconazole(X), Ketoconazole(X), Griseofulvin(X), Terbinafine

Clotrimazole, Nystanin , Tolnaftate

14.Anti retroviral (HIV-AIDS)

Didanosine, Abacavir, Indinavir, Ritanavir, Efavirenz

Zidovudine, Lamivudine, Nevirapine, Nelfinavir, Saquinavir

15.Anti viral (other than HIV)

Acyclovir, Ganciclovir(X), Foscarnet(X), Amantadine(X), Vidaravine(X), α-IF(X)

-

16.Anti hypertensives ACE Inhibitors(X), Angiotensin antigonist(X), Thiazide diuretics, Furosemide, Propranolol, Nitroprusside

Methyldopa, Hydralazine, Atenolol, Metoprolol, Pindolol, Nifedipine, Prazosin, Clonidine

17.Anti anaemic

Iron salts(oral), Iron dextran(IM), Folic acid, Vitamin B12

18.Anti diabetics

Sulfonylureas(X), Metformin(X), Pioglitazone, Rosiglitazone,

Repaglinide, Nateglinide, Acarbose(X)

Insulin(human)

19.Corticosteroids

Betamethasone, Dexamethasone(high dose & prolonged use)

Inhaled corticosteroids, Topical corticosteroids, Prednisolone oral(low dose)

20.Thyroid hormone - Thyroxine 21.Anti thyroid drugs (thyrotoxicosis)

Cirdimazole, Radioactive iodine(X), Iodide

Propylthiouracil

22.Anti psychotic (schizophrenia)

Chlorpromazine, Fluphenazine(X), Clozapine, Olanzapine, Risperidone

Haloperidol, Trifluperazine

23.Anti manic(bipolar illness)

Lithium carbonate, Valproate, Carbamazepine

-

24.Anti depressants Imipramine(X), Dothiepin(X), Sertraline, Paroxetine, Citalopram, Trazadone, Venlafaxine, Moclobemate

Amitryptyline, Imipramine, Clomipramine, Fluoxetine

25.Anti coagulants (thromboembolism)

Warfarin(X), Acenocoumarol, Thenindione(X) Heparin(unfractionated), Heparin(LMW)

26.Anti asthmatic Theothylline, Ketotifen(X), Monteleukast, Zafirlukast, Systemic Cortico Steroids

Salbumatol/ Salmeterol, Ipratropium bromide, Beclomethasone/ Budesonide, Sodium Cromoglycate [inhaled]


PREGNANCY- THERAPEUTIC CONSIDERATION

Drug use in pregnancy is a critically important topic that is under emphasized in the education of health professionals. This subject encompasses a dichotomous discussion of the benefits of drug therapy for the mother and the potential risk for the embryo foetus. Drug use in pregnancy is a controversial and emotionally charged area because of medico legal and ethical complications. It is the clinician’s responsibility to ensure safe and effective therapy before conception, during pregnancy and after delivery. The patient’s active participation is essential. Both acute and chronic illness must manage during pregnancy and optimal treatments are different from those used in the non pregnant patient. PHYSIOLOGY OF PREGNANCY Maternal pharmacokinetic changes during pregnancy Normal physiologic changes that occur during pregnancy alter medication effects, resulting in the need to monitor and some times adjust therapy. Physiologic changes begin in the 1st trimester and peak during 2nd trimester. During pregnancy, maternal plasma volume, cardiac output and glomerular filtration rate increases by 30-50%, potentially lowering the concentration of renally cleared drugs. As body fat increases during pregnancy, the volume of fat soluble drugs may increases. Plasma albumin concentration decreases, which increase the volume of drugs that are highly protein bound. However, these unbound drugs are more rapidly cleared by the liver and kidney during pregnancy, resulting in little change in concentration. Nausea and vomiting as well as delayed gastric emptying alter the absorption of drugs. Like wise, a pregnancy induced increase in gastric pH may affect absorption of weak acids and bases. Higher level of oestrogen and progesterone alter liver enzyme activity and increase elimination of some drugs but results in accumulation of others. Transplacental drug transfer Placenta is the organ of exchange for a number of substances including drugs, between the mother and foetus. Most drugs move from maternal circulation to foetal circulation by diffusion. Certain chemical properties such as lipid solubility, electrical change, molecular weight and degree of protein binding of medications, may influence the rate of transfer across the placenta Drugs with molecular weight less than 500D readily cross the placenta, where ad larger molecules (600-1000D) cross more slowly. Drugs with molecular weight greater than 1000D such as insulin and heparin do not cross the placenta in significant amounts. Lipophilic drugs such as opiates and antibiotics cross the placenta more easily than do water soluble drugs. Maternal plasma albumin progressively decreases while foetal albumin increase during the

course of pregnancy, which may result in high concentration of certain protein bound drug in the foetus. Foetal

is slightly more acidic than maternal permitting weak bases to more easily cross the placenta. Once in the fetal circulation the molecule becomes more ionized and is less likely to diffuse back in to the maternal circulation. DRUG SELECTION DURING PREGNANCY: Although some drugs have the potential to cause teratogenic effects, most medications required by pregnant woman can be used safely. The overall incidence of congenital malformations is approximately 3-5 percent. Despite, the greater potential of harm with certain drugs, not every exposure results in a birth defect.


Factors such as, the stage of pregnancy when the exposure occurred, the route of administration and the dose can influence outcomes. In the first two weeks after conception, exposure to a teratogen may result in a “all or nothing” effect, which could either destroy the embryo or cause no problems. In the period from 18-60 days, organ systems are developing and teratogenic exposures may result in structural abnormalities. In the reminder of pregnancy, exposure to teratogenic agents may result in retardation of growth, CNS abnormalities or death.Examples of medications associated with teratogenic effects in the period of organogenesis include chemotherapeutic drugs (methotrexate,cyclophosphamide),sex hormones (diethylstilbestrol), lithium, retinoids, thalidomide, certain anti epileptic drugs and coumarin derivatives. Other medications such as NSAIDs and tetracycline derivatives are more likely to exhibit effects in second or third trimester. PREGNANCY INFLUENCED ISSUES Pregnant women commonly experience health issues that are either caused by or exacerbated by the pregnant state. Constipation, gastroesophageal reflux, haemorrhoids and nausea and vomiting affect many women during pregnancy. Gestational diabetes, gestational hypertension & venous thromboembolism have the potential to cause adverse pregnancy consequences. GIT a) Consipation- occurs commonly in pregnancy. Therapy should be instituted first with non drug modalities such as education physical exercise and increased intake of dietary fibre and fluid. Of additional therapy is warranted use of supplementary fibre and a stool softer is appropriate. Lactulose, sorbitoland bisacodyl are acceptable treatments but should not be used routinely. Senna can be used occasionally; castor and mineral oil should be avoided. b) GERD- occurs in up to 80% pregnant women. Therapy includes lifestyle and dietary modifications (small, frequent meals, alcohol and tobacco avoidance, food avoidance prior to bedtime). Drug therapy can be initiated with aluminium, calcium or magnesium antacid preparations, sodium bicarbonate should be avoided. Evidence supports the use of ranitidine and cimetidine, famotidine and nizatidine is limited. c) Nausea and vomiting- affect up to 80% pregnant women. Dietary modifications such as small, frequent meals may be helpful. In pharmacotherapy like pyridoxine and cyanocobalamine have shown efficacy. Antihistamines (including doxylamine), phenothiazines & metoclopramide have been used widely & are considered safe efficacy & safety of ondansetron use are limited. d) Gestational Diabetes- about 40% of pregnant women develop gestational diabetes (defined as glucose interface is identified during pregnancy.)First line therapy for gestational DM includes nutritional intervention for all women and caloric restriction for obese women. Daily self-monitoring of blood glucose is necessary for all women. If nutritional interventions do not result in a fasting plasma glucose concentration<=155 mg/dL or 2-hour post prandial glucose conc.<=130mg/dL, insulin therapy with recombinant human insulin should be instituted . Glyburide can be used as an alternative. e) Hypertension- hypertension in pregnancy includes gestational hypertension (hypertension without proteinuria), preclanysia(hypertension with proteinuria) & chronic hypertension(hypertension diagnosed prior to pregnancy with or without preeclamsia). Blood pressure elevation can become severe (systolic blood pressure 160-170mm haemoglobin or higher or diastolic blood pressure110mm haemoglobin or higher) and result in maternal complications, hospital admission and premature delivery. Preeclampsia can cause eclampsia (seizures in addition to preeclampsia), renal failure, blood caogulation complications for mother, preterm delivery and intrauterine growth limitation for the foetus.


Calcium supplementation help prevent hypertension in pregnancy, supplemental calcium 1g/day decrease the risk of preeclampsia. Prevention of preeclampsia in at-risk women includes daily low dose aspirin therapy after 12 weeks gestation. Commonly used drugs include Labetalol, methyldopa and calcium channel blocker. ACE inhibitors should be avoided throughout pregnancy. Agents to avoid include magnesium sulphate, high-dose diazoxide, nimodipine, chlorpromazine, diazepam and phenytion. f) Thromboembolism: thrap to prevent or treat thromboembolism during pregnancy must not include warfarin because it causes fetal bleeding, malformations of nose and CNS anomalies.For treatment adjused low-dose heparin or unfractionated heparin should be used.

References

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