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| Year : 2011 | Volume
: 5
| Issue : 1 | Page : 69-74 |
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| Effect of time on extraction of Ashwagandha in various Hydroalcoholic compositions and their anti-inflammatory activity |
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Anupama Singh1, Vikas Anand Saharan2, Rajesh Garg3, VB Gupta3
1 Department of Pharmacognosy, Jodhpur National University, Boranada, Jodhpur, Rajasthan, India
2 Seth G. L. Bihani S. D. College of Technical Education, Sri Ganganagar, Rajasthan, India
3 Department of Pharmacognosy, B. R. Nahata College of Pharmacy, Mhow-Neemuch Road, Mandsaur, Madhya Pradesh, India
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| Date of Submission | 13-Feb-2011 |
| Date of Acceptance | 03-Mar-2011 |
| Date of Web Publication | 15-Jun-2011 |
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 Abstract | | |
Ashwagandha, is a plant of Solanaceae or nightshade family which have botanical name Withania somnifera. It is mainly cultivated in Madhya Pradesh, Rajasthan, Uttar Pradesh and many other states of India. Among all states Madhya Pradesh (Neemuch-Mandsaur region) is the major producer of Ashwagandha. Major phytoconstituents are withanolides and alkaloids. These phytoconstituents have many pharmacological activities such as anti-cancer, diuretic, immunomodulatory, anti-inflammatory, anti-stress. The yield of active constituents may vary with time, temperature, number of extractions, drug and solvent ratio. Extracts were prepared in different water and alcohol compositions at different time intervals. These prepared extracts were chromatographed and number of phytoconstituents was present. Some of the extracts were used for performing anti-inflammatory activity. The activity was performed by carragenean-induced paw oedema method in rats. A few extracts were found effective reducing the oedema. Extract obtained at 15 h was found superior in anti-inflammatory activity which is proposed due to some additional phytoconstituents extracted at that point of time. Keywords: Anti-inflammatory activity, Ashwagandha, Time
How to cite this article:
Singh A, Saharan VA, Garg R, Gupta V B. Effect of time on extraction of Ashwagandha in various Hydroalcoholic compositions and their anti-inflammatory activity. Int J Green Pharm 2011;5:69-74 |
How to cite this URL:
Singh A, Saharan VA, Garg R, Gupta V B. Effect of time on extraction of Ashwagandha in various Hydroalcoholic compositions and their anti-inflammatory activity. Int J Green Pharm [serial online] 2011 [cited 2013 May 19];5:69-74. Available from: http://www.greenpharmacy.info/text.asp?2011/5/1/69/82098 |
| Introduction | |  |
Withania somnifera, also known as Ashwagandha, is a plant in Solanaceae or nightshade family. Indian medicinal plant Withania somnifera have been used in the Ayurvedic system of medicine and have anti-arthritic, anti-bacterial, anti-oxidant, anti-diabetic, anti-tumour, [1],[2],[3],[4],[5],[6] anti-inflammatory activity, [7] immunomodulatory activity [8] and analgesic activity, [9] anti-sertogenic activity, anabolic activity and anti-stress activity. [10] It also possesses adaptogenic, cardiotropic, cardioprotective and anticoagulant properties. [11] Many withanolides have been isolated from Withania somnifera which have important role in treatment of various disorders. Withaferin A and 3-b-hydroxy-2, 3- dihydrowithanolide F have shown promising antibacterial, anti-tumour, immunomodulating and anti-inflammatory properties. [12] The aerial parts contain 5-dehydroxywithanolide-R and withasomniferin-A. [13] Withacoagin, coagulin, and withasomidienone together with other withanolides and Withaferin-A showed significant anti-inflammatory activity. [14]
Various factors affects yield of active constituents such as time, temperature, number of extractions, drug and solvent ratio. But till date no such study on effect of factors has been reported with extracts of Ashwagandha. The present study aimed to prepare extracts of Ashwagandha at different time durations with diferent water and alcohol compositions, collected from Neemuch, Mandsaur region, Madhya Pradesh, to perform anti-inflammatory activity.
| Materials and Methods | | |
Drug and its Authentification
Ashwagandha roots were collected from Manasa Madhya Pradesh in the month of March. The crude drug was authenticated at KNK College of horticulture, Mandsaur Madhya Pradesh and assigned the voucher number BRNCP/W/003/2007/Ashwagandha.
Extraction
Drug was extracted using water and alcohol in different compositions and at different time intervals using soxhletion. The powdered drug was weighed and filled in the soxhlet apparatus. The soxhlet was fitted to the round bottom flask, and the assembly was attached to the condenser. The paraffin wax was used to seal the assembly joints. The solvent (100% water, 75% water, 50% water, 25% water, 100% alcohol) for extraction was filled in round bottom flask and extraction was carried out at different time intervals and 40 extracts were prepared. After completion of the extraction procedure the extract was taken out and its amount in percentage was determined. On the basis of extracted amount and solvent used for extraction eight extracts were further selected from these 40 extracts for anti-inflammatory activity.
Different extracts taken are as follows:
- A:100% Water 0% Alcohol (15 hours)
- B:100% Water 0% Alcohol (24 hours)
- C:75% Water 25% Alcohol (15 hours)
- D:75% Water 25% Alcohol (24 hours)
- E:50% Water 50% Alcohol (15 hours)
- F:50% Water 50% Alcohol (24 hours)
- G:25% Water 75%Alcohol (15 hours)
- H:25% Water 75%Alcohol (24 hours)
Chromatographic Profile of Different Extracts
Thin layer chromatography (TLC) was performed to identify the different constituents present in different extracts. Different solvent systems such as acetonitrile: water (75:25) and toluene: ethyl-acetate: acetic acid (65:33:2). Iodine chamber was used as a detecting reagent for prepared TLCs.
Anti-inflammatory Activity
Both male and female albino rats weighing, 100-200 g, were divided into 11 different consisting of five rats in each group (one standard, two control groups (simple control and model control) and eight test groups). All the rats were kept for overnight fasting. Simple control received the vehicle (2%, w/w gum acacia dispersion in distilled water) and model control received the carrageenan (1%, w/w dispersion in distilled water). Test groups received carrageenan (1%, w/w dispersion in distilled water) and the extract (1 g/kg of body weight) and standard (10 mg/kg of body weight) group was administered indomethacin. The carrageenan (0.05 ml of 1%, w/v concentration) was administered by subcutaneous injection into the plantar side of the left hind paw. Carrageenan-induced inflammation in half an hour. Extracts were administered orally to test groups. The left hind paw was marked with ink at the level of the lateral malleolus and immersed in mercury up to this mark. The paw volume was measured with plethysmometer immediately after oral administration of extracts, 3 and 6 and 24 h from oral administration. [15]
| Results and Discussion | | |
Extraction
Ashwagandha was extracted using different solvent compositions at different time intervals. Extracts were dried and results in (%, w/w) are presented in [Table 1]. On the basis of extracted amount and solvent (cost factor) used for extraction, eight extracts were further selected from those 40 extracts. As presented in [Table 1] extracted amount increased up to 15 th h. Further it decreased after 15 th h than again it increased at 24 th h. The fluctuation was caused in extracted amount, the decrease in overall extractive was probably due to degradation and increase at 24 h was anticipated as a result of appearance of plant parts in the extract or appearance of any other constituent. Therefore, selected extracts were subjected to chemoprofiling and bioprofiling.
Chromatographic Profile of Different Extracts
Thin layer chromatography (TLC) was performed to identify the different constituents present in different extracts. Results are shown in [Table 2], [Table 3], [Table 4] [Table 5] and [Table 6] and [Figure 1]. Phytoconstituents having R f values 0.18, 0.24, 0.35, 0.40, 0.47, 0.55, 0.61, 0.72, 0.73, 0.80, 0.88, were present in the solvent system (toluene: ethyl-acetate: acetic acid:65:33:2). [16]  | Figure 1: Chromatographic profile in (a) acetonitrile: water (75:25); (b) toluene: ethylacetate: acetic acid (65:33:2) of 50% alcoholic extract
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 | Table 2: Comparative Rf values of extracts in water at different time intervals
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 | Table 3: Comparative Rf values of extracts in hydroalcoholic compositions (75% water:25% alcohol) at different time intervals
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 | Table 4: Comparative Rf values of extracts in hydroalcoholic compositions (50% water:50% alcohol) at different time intervals
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 | Table 5: Comparative Rf values of extracts in hydroalcoholic compositions (25% water:75% alcohol) at different time intervals
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 | Table 6: Comparative Rf values of extracts in alcohol at different time intervals
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Anti-inflammatory Activity
The animal studies were conducted as per the approved protocol of institutional animal ethical committee of B. R. Nahata College of Pharmacy, Mandsaur, Madhya Pradesh, India.
It was performed by carragenean induced paw oedema method results are shown in [Figure 2], [Figure 3] and [Figure 4] and [Table 7]. When the selected extracts were administered to test groups significance difference in inflamed paw volume was observed in all the test groups but the level of significance varied among different groups. At 0 h, no significant difference in the paw volume was observed of the inflamed rats, yet the groups C, D and G showed significant difference (P<0.01) with respect to (w.r.t) model control and standard while H showed significant difference (P<0.001) w.r.t model control after 3 h of treatment. When the treatment was continued to 6 h, showed significant decrease of inflammation in groups C and E rats (P<0.05) was observed and in group G rats significance difference (P<0.001) w.r.t model control. [17] | Figure 2: Paw volume of control, model control and treated rats at 0 h after carragenan administration
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 | Figure 3: Paw volume of control, model control and treated rats at 3 h after carragenan administration
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 | Figure 4: Paw volume of control, model control and treated rats at 6 h after carragenan administration
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| Conclusion | | |
The present study provides an insight that how phytoconstituents in plants may vary with geographical regions, solvents, methods of extraction and time duration of extraction. TLCs performed showed presence of 4-5 different phytoconstituents in different extracts.
Further, animal studies revealed that sample C, D, E, G and H were effective in controlling inflammation. However, extracts C, D, G and H showed anti-inflammatory activity after 3 h and extract C, E and G showed anti-inflammatory activity after 6 hr. Considering the economic point of view (because less alcohol is being consumed in the process), extractive amount (42%, w/w) and animal activity extract C is more recommended.
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Correspondence Address:
Anupama Singh
Department of Pharmacognosy, Jodhpur National University, Boranada, Jodhpur, Rajasthan
India
DOI: 10.4103/0973-8258.82098
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7] |
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