Author: Ternikar, S G; Alagawadi, K R; Pasha, Ismail; Dwivedi, S; Rafi, M Ahammed; Sharma, T
Date published: December 1, 2010
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In India Sida cordifolia Linn. (Family: Malvaceae) is popularly known as "Countrymallow" and "Bala" found along roadsides throughout the tropical and subtropical plains of India and Sri Lanka . It is reported to possess analgesic, anti-inflammatory , anticancer , diuretic, laxative, hypoglycemic  and hepatoprotective activities . Further, studies showed that aqueous fraction of hydroalcoholic extract of leaves induces vasodilatation , hypotension and bradycardia . Furthermore, it possess antioxidant property  and the leaves of Sida cordifolia were used as antioxidant in myocardial injury .
In spite of all above extensive studies, there is no information about the anti-microbial and antiinflammatory properties of seed oil of Sida cordifolia [9,10]. Hence, the present study was undertaken. The anti-microbial activity of seed oil was assessed using Staphylococcus aureus, E. coli, Candida albicans and Aspergillus niger. Likewise, the acute anti-inflammatory activity of oilwas analyzed against carrageenan induced paw edema in albino rats with reference to standard drug diclofenac sodium respectively.
MATERIALS AND METHODS
Experimental animals: Albino rats (150-200 gm) and albino mice (20-30gm) were purchased from Sri Venkateshwara Traders; Bangalore. The experimental protocolwas approved by the institutional animal ethics committee (IAEC) and care of laboratory animals was taken as per CPCSEA guidelines (Registration No. CPC237). The animals were housed in polypropylene cages in the adequately ventilated room. The rats were fed standard feed and water ad libitumthroughout the course of the study. Lightning was natural sequence being 12 hours light and 12 hours dark.
Plant material and preparation of extract: Seeds of Sida cordifolia were collected from Chennai and identified in Department of Botany, RLS College, Belgaum. The seeds were collected and dried under the shade in laboratory for 7-12 days.After complete drying, powdered and extracted thoroughly with light petroleum ether (40-60 °C) in a soxhlet extractor for 24 - 48 hours. Aqueous extract was prepared by maceration with distilled water for 7 days and the filtrate was concentrated using rotary evaporator (rota-vapour bucchi, Switzerland) and dried over anhydrous sodium sulphate and solvents were removed in vacuum at 40°C by using rotary evaporator to recover oil. The seed oil was filtered through whattman filter paper No. 1 to remove any foreign particles and stored properly. The petroleum ether (40-60°C), chloroform, alcohol and aqueous extract were first subjected for the preliminary investigation and then it was subjected to acute toxicity studies, antimicrobial and anti-inflammatory activity .
Drugs: For antimicrobial activity standard drugs used were norfloxacin and griseofulvin of 50 µg/ml. Therapeutic dose of petroleum ether extract of seeds of Sida cordiofolia were taken as 100 µg/ml and 300 µg/ml. Diclofenac sodium100mg/kg served as a standard drug for carrageenan induced paw edema in albino rats, therapeutic dose of petroleum ether extract of seeds were taken as 400 mg/kg body weight.
Acute toxicity studies: The acute oral toxicity was carried out as per the guidelines set by organization for economic cooperation and development . The albino micewere divided into three groups of six each. The test seed oil of Sida cordifolia was administered orally to different group in increasing dose level of 50mg/kg body weight and continued upto 4000 mg/kg and observed continuously for 1 hour and then frequently for 24 hour and thereafter daily for 14 days. During this period the animals were observed for gross behavioral morphological profiles .
Anti bacterial activity: Anti-bacterial activity were assayed against two pathogenic microorganisms viz. E .coli (NCTC 10418), Staphylococcus aureus (NCTC 6571). The nutrient agar brothwas prepared by the usual method. Nutrient agar was spread in a Petri dish (13 cm in diameter) and allowed to set for 30minute.About 5ml of nutrient agar was inoculated aseptically in a test tube with 0.2 ml of 24 hour old bacterial subculture at 40-50 °C, mix well by gentle shaking and spread over the previously settled layer of nutrient agar in the Petri dish. The cups (6 mm in diameter) were formed with the help of borer and filled with 0.05 ml solution of sample in DMF.Along with the test solution in each Petri dish one cup was filled with solvent which act as the control. The plate was incubated at 37± 1°C for 24 hour and the resultant zones of inhibition of bacterial growth weremeasured in mm and are recorded.
Antifungal activity: Aspergillus niger and Candida albicans were employed for testing antifungal activity using the cup-plate method. The culture was maintained on sabouraud's agar slants. 15 ml of sabouraud's agar medium was spread in petri dish (13 cmin diameter) and allow to set for 30 minute. 5 ml of sterilized sabouraud's agar medium was inoculatedwith 72 hour old 0.2 ml suspension of fungal spores in a test-tube and spread over the previously settled layer of sabouraud's agar medium in the Petri dish. The cups (6 mm diameter) were punched in a Petri dish and filled with 0.05 ml of a solution of the sample in DMF. The plates were incubated at 30 °C for 48 hour. After the completion of incubation period the zone of inhibition of growth in millimeter were measured. Along with the test solution in each petri dish one cup was filled up with solvent, which act as control. The zone of inhibition was recorded [14,15].
Edema induce by carrageenan: Carrageenaninduced hind paw edema model was used for the assessment of anti-inflammatory effect . Rats of the either sex ranging from 150-200 gm where randomly selected and fasted for 18 hours before the experiment, but had free access to water. The animals were divided into three groups of 6 rats each. Standard drug diclofenac sodium (100mg/kg p.o.) seed extract (400mg /kg, orally) and control saline (1mg/kg, orally) were given prior to 60 min. Then acute inflammation was induced by injecting carrageenan (0.1 ml of 0.2% carrageenan) in subplantar region of the right hind paw of the rat and the paw volume was measured at 0, 30, 60, 180 & 300 minutes with the help of Plethysmometer (Model 7159, UGO Basile, Italy) . The difference between one reading and one of the subsequent readings provides the actual edema volume at that time. The mean paw volume at different time was calculated and compared with the control and standard. The percentage inhibition of inflammation was calculated by using the following formula prided by Vogel .
Antimicrobial activity: As illustrated in table 1 chloroform and petroleum ether extract of seed were resistant at 100µg/ml concentration, whereas at 300 µg/ml concentration shownmoderate activity against S. aureus in comparison with norfloxacin disc. Against E. coli organism, these extracts were resistant at 100 µg/ml concentration, where as they showed moderate activity at 300 µg/ml concentration. However, at 300µg/ml the activity was comparable as that of standard drug norfloxacin, except the chloroform extract of seed which was resistant.
Both the seed extracts showed moderate anti fungal activity against A. niger at 100 µg/ml concentration. However at 300 µg/ml the activity was significant as compare to standard drug griseofulvin.
Against C. albicans seed extracts were resistant at 100µg/ml concentration, whereas the same extracts at 300µg/ml concentration showed promising activity and comparable to standard drug griseofulvin.
Acute anti-inflammatory activity: The acute toxicity study of the seed oil up to a dose level of 2000 mg/Kg b.w. showed nomortality. The petroleum ether extract of seeds displayed significant antiinflammatory activity at a dose of 400 mg/Kg b.w. As illustrated in table 2, the inflammationwas reduced to 1.10 (P< 0.05) at 30 minutes from 0.350 at 0 minutes, 1.00 (p< 0.01) at 60minutes, 0.650 (p< 0.01) at 180 minutes and 0.375 (p< 0.01) at 300 minutes, against carrageenan induced rat paw edema. After comparing with control group the value of seed extract was significant. While diclofenac sodium as reference drug showed significant inhibition at 0.68 (P < 0.01) at 30minutes, 0.55 (P < 0.01) at 60minutes, 0.35 (P < 0.01) at 90 minutes and 0.30 (P < 0.01) at 180 minutes.
Norfloxacin, a fluoroquinolone, is a synthetic, broad spectrumantibiotic. It inhibits deoxyribonucleic acid synthesis intracellularly in bacteria. It also alters the structure of the enzyme resulting in abnormal changes in polypeptide production [19,20]. Griseofulvin is a metabolic product of Penicillium species. It inhibits fungal cell mitosis and nuclear acid synthesis, by interference with the function of microtubules .
Acute inflammatory response, characterized by erythema, edema and pain, is a complex phenomenon involving various chemical mediators such as histamine, 5-HT, various chemotactic factors, bradykinin, leukotrienes, and prostaglandins. Calcium is responsible for the release of these mediators. The calcium is also involved in the action of these mediators on vascular endothelium to induce vasodilation and enhance permeability, resulting in characteristic feature of inflammation [22,23].
Many mediator appears to be involved in the carrageenan induced inflammation including histamine, 5- HT, kinins and prostaglandins . The early phase (1-2 hours) of the carrageenan model is mainly due to histamine, serotonin, and increased synthesis of prostaglandins in the surrounding of damaged tissue. The late phase is sustainedmediated by prostaglandin release as well as by bradykinin and leukotrienes [24,25].
In present investigation the petroleum ether extract of seeds of Sida cordifolia was screened for acute toxicity test to establish effective dose. The tests were performed in albino mice and the extract was administrated orally in the form of suspension by triturating with water and 0.5% gum acacia CMC.
Study shows that the seed extract significantly reduced carrageenan induced paw edema at 0, 30, 60, 180, 300 minutes. The observation suggested that the test compound may act in both early and late phase of carrageenan induced acute inflammation. It is concluded from this study that seed extract possess both anti-microbial and anti-inflammatory properties.
We would like to thank, Dr. F.V, Manvi, Principal and Dr Alagwadi of KLEs University College of Pharmacy, Belgaum. Mr Ismail Pasha - Research Scientist, RanbaxyResearch Laboratories, Gurgaon.
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TERNIKAR, S. G.,1 ALAGAWADI, K. R.,1 ISMAILPASHA,2* DWIVEDI, S.,3 MAHAMMED RAFI3 AND SHARMA, T.4
1 Department of Pharma-chemistry, KLE'S University College of Pharmacy, Belgaum; 2 Department of MACR,
Ranbaxy Research Laboratories, Gurgaon; 3 Department of Pharmacology, KLE'S University College of
Pharmacy, Belgaum; 4 Department of Pharmacognosy, KLE'S University College of Pharmacy,
Belgaum. E. mail: firstname.lastname@example.org
Received: November 11, 2010;Accepted:November 29, 2010