Abstract: Centella asiatica L. (Apiaceae) is highly valued for treatment of leprosy, tuberculosis, wound healing, stomach aches, arthritis, varicose veins, high blood pressure and as a memory enhancer. Centella asiatica being rich in saponins is also reported to possess antioxidant, anticancer and antiulcer properties.
Triterpenoid saponins, which represent a highly diverse class of secondary metabolites, display gastroprotective and antiulcer activity and/or useful for modulation of peptic ulcers associated with Helicobacter pylori.
The major objective of the current research work was to evaluate the peptic ulcer healing activity of powder of Centella asiatica whole plant and its total saponin rich-extract and to compare their efficacy with the modern drug, misoprostol. Ulceration in Albino wistar rats was induced with Indomethacin (30 mg/kg, p. o.). The first dose of Centella asiatica, its saponin rich-extract and modern drug treatment (p. o.) were given six hours after the indomethacin administration and the treatment continued for the subsequent five days. Gastric volume, gastric pH, free and total acidity, ulcer index and histopathological parameters were studied in control and treated groups. The reduction of ulcer index as well as gastric acid output in treated animals was found to be statistically significant with respect to control group of animals. In the present research work, a suitable method for optimum extraction of saponin rich-extract from Centella asiatica has been developed. Also, a simple, rapid and sensitive UV/Visible spectrophotometric method for the determination of total saponin content has been developed and validated. Asiatic acid was used as standard for spectrophotometric measurement and total saponin content were expressed in equivalents (mg/g).
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Keywords: Peptic ulcer healing, UV/Visible spectrophotometer, Indomethacin, Centella asiatica, Asiatic acid, Misoprostol.
Centella asiatica (syn. Hydrocotyle asiatica), a plant of the family Umbelliferae is a tropical medicinal plant with a long history of therapeutic use occurring in parts of India, Sri Lanka, China, Indonesia, Malaysia, Australia and Southern and Central Africa. Clinical trials have shown that extracts of C. asiatica heal wounds, burns and ulcerous abnormalities of the skin, cure stomach and duodenal ulcers, and are effective in the treatment of leprosy, lupus, scleroderma and diseases of the veins. (Verma et al., 1999; Kai et al., 2008).
Centella asiatica accumulates large quantities of pentacyclic triterpenoid saponins, The biological activity of saponins has been attributed to these characteristics. Major bioactive constituents among the triterpenoid saponins include madecassoside and asiaticoside, and their aglycones viz., asiatic acid and madecassic acid (Tiwari et al., 2010). There are many existing biological and phytochemical saponin screening methods available including the vanillin-sulfuric acid (Hiai et al.,1976, Shiau et al., 2009) and antimony pentachloride colorimetric assays (Hanzas and Barr, 1969; Shiau et al., 2009).In the present research work, a suitable method for optimum extraction of Total Triterpenoid saponin (TTP) from Centellaasiaticahas been developed and TTP content determined as Asiatic acid equivalents (mg/g) by UV-Visible spectrophotometer.
The major objective of the current research work was to evaluate the peptic ulcer healing activity of whole plant powder of Centella asiatica, its TTP rich-extract and to compare the efficacy with the modern drug, Misoprostol on Albino wistarrats against indomethacin induced gastric ulceration in female albino wistar rats.
MATERIAL AND METHODS
Plant Material: Centella asiatica plant materials were collected in the month of February, 2010 from perumbavoor, Kerala. Herbarium of the plant was authenticated from Agarkar Institute, Pune. The dried plant materials were ground in a mechanical grinder and sieved through BSS sieve number 85.
Preparation of TTP Rich extracts: The Centella asiatica plant powder (50 g) was soaked in Ethanol: Distilled water (500 mL) for 1 day. The extracts were filtered using Whatmann Filter paper no. 41 and evaporated on a water bath. The crude extract was stored in a dessicator prior to use.
Vanillin-Sulfuric acid assay: For quantification of TTP from plant extracts, the procedure used by Shiau et al. (2009) was employed. To 0.25 mL of plant extract, 0.25 mL vanillin solution (10%) and 2.5 mL of sulphuric acid (72% w/v) were added and thoroughly mixed in an ice water bath. The mixture was then warmed in a water bath at 600C for 10 min and then cooled in ice-cold water bath. Absorbance at 535 nm was recorded against the blank using a Shimadzu UV/Visible (160) spectrophotometer.
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Toxicity study: Toxicity study for Centella asiatica plant powder and its TTP rich extract was performed on female albino swiss mice as per the OECD guideline (OECD 420, 2001).
Experimental protocol for induction of gastric ulcers: The experimental protocol was similar to the one adopted by banerjee D. et al., 2008. Female albino wistar rats weighing 180-200 g each were used in this study. The rats were given a balanced laboratory diet and given tap water ad libitum. They were kept at 20 ± 2°C, 65-70% humidity, and day/ night cycle (12 h/12 h). The animals were fasted for 24 h prior to their use in the experiments. Ulceration in the rat was induced by administrating indomethacin (18 mg/kg p.o.) suspended in 0.5% CMC in a single dose. The animals were deprived of food for next 6 h after ulcer induction but had free access to tap water. The first dose of centella asiatica plant powder, its TTP Rich extract and misoprostol were given 6 h after the indomethacin administration. In the subsequent six days, the test samples were given at 9 AM on each day. The rats were sacrificed on the 1st, 3rd and 7th days, 4 hrs after administering of the last dose.
Determination of gastric juice volume and pH: The gastric juice volume and pH was determined as described by Zaman R. et al.
Macroscopic valuation and gastric ulcer index: The gastric ulcer index was calculated as described by Dokmeci D. et al.
TPP Rich extract: The optimal conditions for the extraction of Total triterpenoid saponins contained in Centella asiatica plant powder include the following parameters:
The volume fraction of Ethanol :50%
Extraction time :12 hrs
Solvents to raw material ratio 20:1.
Under these conditions, the experimental yield was 15.74 mg/g
pH of Gastric Content
Total Acidity (mEq/L/100g)
6.23 ± 0.22
2.30 ± 0.05
121.67 ± 4.55
20.10 ± 1.82
2.62 ± 0.31*
4.32 ± 0.61*
27.50 ± 0.82*
3.02 ± 0.99*
3. 75 ± 0.24*
2.82 ± 0.08
65.60 ± 1.12*
12.00 ± 0.66*
TPP Extract of
3.83 ± 0.52*
2.90 ± 0.74
60.10 ± 0.72*
11.70 ± 0.42*
*p<0.05 vs. control; values are in Mean ± SEM
Overall, the present study established that Centella asiatica and its TPP rich extract possesses significant healing property against indomethacin induced stomach ulceration in rats. These results along with its non-toxicity suggested PK as a promising anti-ulcerogenic formulation for further evaluation.