Brain KR, Turner TD. The practical evaluation of phytopharmaceuticals. ... Newall CA, Anderson LA, Phillipson JD. Herbal medicines. :a guide for health-care ...
ORIGINAL STUDY
Comparison of the antiinflammatory activity of plant extracts from Cimicifuga racemosa and Mimosa pudica in a rat model R Rathore, A Rahal*, R Mandil, A Prakash and SK Garg *Corresponding author Department of Pharmacology & Toxicology, UP Pt Deen Dayal Upadyay Pashu Chikitsa Vishwavidyalaya Evam Gau Anusandhan Sansthan (DUVASU), Mathura-281001 Uttar Pradesh, INDIA; rahalanu72@gmail@com
Abstract This study examined the phytochemical composition and antiinflammatory activity of methanolic extracts of Cimicifuga racemosa roots and Mimosa pudica seeds. The antiinflammatory effect was assessed using the carrageenan-induced paw oedema model in Wistar rats. Indomethacin (20 mg/kg PO) was used as a positive control and negative controls received no treatment. Both C. racemosa and M. pudica extracts contain flavonoids and other constituents. Both C. racemosa and M. pudica extracts showed an antiinflammatory effect greater than indomethacin, with the effect of M. pudica being more consistent across oral dosing protocols. Based on the conditions of this experimental study, both C. racemosa and M. pudica show some antiinflammatory effects that can be measured by quantifying oedema. Further clarification of this effect and investigation into dosing may be warranted. Aust Vet Pract 2012;42(3):274-278
INTRODUCTION Cimicifuga racemosa (Black cohosh) has been used in Indian medicine for relieving pain during menstruation, childbirth and menopause.1,2 It has been used as a “partus preparatory,” given to women during the last four weeks of pregnancy to aid in childbirth.3 A tincture made from fresh roots was administered per os, believed to reduce the irritability of the uterus, or false labour.3 Mimosa pudica is used in Ayurvedic and Unani medicine to treat fever and nausea, jaundice, leprosy, dysentery, vaginal and uterine complaints, inflammations, burning sensation, fatigue, asthma, leucoderma, leprosy and jaundice.4,5 The roots of M. pudica is used as an antifertility agent in Indian medicine.6 Antiinflammatory effects of M. pudica have been reported.7,8 The flavonoids present in the extract have been reported to block the release of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) as well as significantly suppress leukocytosis, eosinophilia and mucus hypersecretion in asthmatic lung.9 Although there is emerging scientific evidence of the antiinflammatory properties of these plant extracts, much clinical application is anecdotal. This study examined the antiinflammatory activity of extracts from the C. racemosa and M. pudica plants in a controlled, in vivo model. Based on the known effects of likely constituents of C. racemosa and M. pudica, we hypothesized that both would have an antiinflammatory effect beyond that of indomethacin alone.
MATERIALS AND METHODS Preparation of crude extracts The authentic plant materials were procured from Indian Drugs and Botanical Herbs Co, Delhi, air-dried and crushed. The hot methanolic extracts of C. racemosa roots and M. pudica seeds
274 Australian Veterinary Practitioner 42(3) September 2012
were prepared using an extractor at a temperature of 40 ± 5°C and dried at 37°C. Physical characteristics of the extracts were noted and the percent yields calculated.
Phytochemical studies Qualitative phytochemical analysis of the crude hot methanolic extracts were performed as per standard methods10,11 to detect the presence or absence of different constituents including alkaloids, glycosides, flavonoids, resins, tannins, saponins, fixed oils, reducing sugars, proteins and amino acids. Alkaloids (Wagner’s test) - Wagner reagent and the extract were mixed in equal volumes. Appearance of a brown flocculent precipitate indicated the presence of alkaloids. Alkaloids (Dragendroff ’s test) - Dragendroff reagent was sprayed on filter paper and the paper was allowed to dry. The extract solution was applied to the paper impregnated with dragendroff reagent via a capillary tube. Development of an orange-red colour indicated the presence of alkaloids. Amino acids (Ninhydrin test) - Approximately 1-2 mL of 0.5% ninhydrin (in methanol) was added to 1-2 mL of extract. Development of a violet or purple colour indicated the presence of amino acids. Fixed oils and fats (Spot test) - A small quantity of dried extract was pressed between two filter paper sheets. Oil stain on the filter paper indicated the presence of oils and fats. Flavonoids (Shinoda’s test) - Approximately 1 mL of extract was dissolved in 5 mL ethanol and acidified with a few drops of concentrated hydrochloric acid. Half a gram of magnesium turnings were placed in the solution. Development of a bright red colour indicated the presence of flavonoids. Glycosides (Benedict’s test) - Equal volumes of aqueous extract
Nitrates - Approximately 1-4 mL of 5 % salicylic acid (in concentrated sulphuric acid) was added to 1 mL of extract. Development of a yellow colour indicated the presence of nitrates. Nitrite - A few drops of extract was mixed with 2 drops of 0.2 % N-1napthyl ethylene diamine dihydrochloride solution on glass slide. Development of a pink colour indicated the presence of nitrite. Proteins (Biuret test) - One mL of 1% sodium hydroxide was added to 5 mL of extract. Development of a violet colour on addition of 1-2 drops of 1% copper sulphate solution indicated the presence of proteins. Proteins (Xanthoprotein test) - Approximately 0.5 mL of concentrated nitric acid was added to 2 mL of extract in a test tube. Appearance of white or yellow precipitates indicated the presence of proteins. Resins - Approximately 1 gm of crude, dried extract was dissolved in 2 mL methyl alcohol. Development of turbidity on addition of a few drops of distilled water indicated the presence of resins. Saponins (Foam test) - Approximately 0.25-0.5 gm of sodium bicarbonate was added to 5 mL of extract in a test tube and shaken vigorously. Formation of foam indicated the presence of saponins. Tannins (Ferric chloride test) - Three to four drops of 1% ferric chloride solution was added to a small volume of extract in a test tube. Development of a bluish or green colour indicated the presence of tannins.
Antiinflammatory activity Animal model - Seventy two Wistar rats (body weight 200 ± 10 g) were procured from Laboratory Animal Resource Section, Izatnagar, Bareilly. The rats were maintained under standard housing conditions and allowed an acclimatization period of 15 days prior to start of the experiment. The experiment was approved by the Institutional Animal Ethical Committee of DUVASU, Mathura. Experimental design - The antiinflammatory activity of methanolic extracts of C. racemosa (1% in water) and M. pudica (10% in water) was evaluated using the carrageenan-induced rat paw oedema model.12 The study was conducted in two phases. Acute inflammation was induced by injection of 0.1 ml of 1% freshly prepared suspension of carrageenan (Sigma Aldrich, in normal saline) into the plantar aspect of the left hind paw of rats using a microsyringe. In phase I, the rats received a single dose of extract (oral gavage) followed by carrageenan injection administered 30 minutes after treatment. In phase II, the rats received the extract (oral gavage) once daily for seven days followed by carrageenan injection administered 30 minutes after the last treatment. Treatments consisted of C. racemosa (30 mg/kg, 60 mg/kg) and M. pudica (200 mg/kg, 400 mg/kg). There was a single negative control group (n=6, no treatment) and a single positive control group (n=6, indomethacin, one dose at 20 mg/kg). The negative and positive control groups were used for comparison for both phases. Six rats were used in each treatment group for each phase. The paw volume was measured at 0, 0.5, 1, 2, 3, 6, 9, 12 and 24 hours after the carrageenan injection using a plethysmometer
(Ugo-Basile). Briefly, a line was drawn on the tibiotarasal joint of the paw and the paw volume was measured indirectly by immersing the paw to that line in a tank in the plethysmometer. The displaced volume in the tank is considered to be the volume of the paw, influenced by the oedema present.
Statistical analysis The paw volume was expressed as mean ± SE for each treatment group at each time point (n=6). A one way analysis of variance was performed at each time point to determine the fixed effect of treatment. Separate analyses were performed for phase I and phase II. Where there was a significant effect of treatment in each analysis at p0.05, Tukey adjusted). Columns with no superscripts indicate no significant difference.
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