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Hepatoprotective activity of Pterocarpus santalinus L.f.,an endangered medicinal plant

Manjunatha, B

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Indian Journal of Pharmacology

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; Pondicherry Vol. 38, Iss. 1,  (Jan/Feb 2006): 25-28.

Introduction

Pterocarpus santalinus L.f. (Fabaceae) is commonly called as Red sander (English), Kempu honne (Kannada) and Raktachandan (Sanskrit). It is an endangered plant species, endemic to the state of Andrapradesh in India.[1] The plant is renowned for its characteristic timber of exquisite color, beauty and superlative technical qualities and ranks among the finest luxury in Japan.[2] Wood is used as astringent, tonic, as external application for wounds, cuts and inflammations, in treating headache, skin diseases, fever, boils, scorpion sting and to improve sight.[3] The red wood yields a natural dye santalin which is used as a coloring agent in pharmaceutical preparations, food stuffs; fruit extract is used as astringent, diaphoretic, in inflammations, headache, skin diseases, bilious infections and chronic dysentry.[4] Heart wood is known to possess isoflavone glucosides,[5],[6],[7] savinin, calocedrin[8] and triterpene.[9] The lignan isolated from the heartwood is known to inhibit tumor necrosis factor-alpha production and T-cell proliferation.[8] Ethanolic stem bark extract is known to possess antihyperglycemic activity.[10]

The tribal groups of Western Ghats, Shimoga region use stem bark extract of Pterocarpus santalinus in treating diabetes, fever, snake bite and jaundice (About 100 g of powdered stem bark is boiled in 500 ml of water for 3-4 h till the volume is reduced to half, cooled and 10 g of jaggery added to the extract and made into pills. Two to three pills a day for 10 days is administered for acute jaundice).

Review of the literature revealed that this rare medicinal plant remained unexplored for many of its claimed pharmacological activities. In the present study, an effort has been made to evaluate the hepatoprotective activity of Pterocarpus santalinus L.f.

Materials and Methods

Plant material Stem bark of Pterocarpus santalinus was collected during the month of January 2003 from the Ayurvedic medicinal garden at Gajanur, Shimoga district (Forest Department of Shimoga). The voucher specimens (BKM-430, BKM-431) were deposited in the department Herbaria, SRNMN College of Applied Sciences, Shimoga for future reference.

Extraction Stem bark was shade dried for a week and powdered mechanically (Sieve No. 10/44). About 250 g of the powder was extracted with 70% ethanol for 48 h using Soxhlet apparatus. The solvent was distilled off at low temperature under reduced pressure using rotary flash evaporator (Buchi, Flawil, Switzerland). The yield was 21% w/w. Another 250 g of the powdered material was boiled in distilled water for 30 min, kept for 3 days with intermittent shaking, filtered and concentrated using rotary flash evaporator to obtain the aqueous extract. Both the extracts were dried in desiccator. The yield was 16.2% w/w. Both the extracts were subjected to preliminary phytochemical tests.[11]

Drug formulations Oral suspensions containing 45 mg/ml and 30 mg/ml of the aqueous and ethanol stem bark extracts, respectively, were prepared in 1% w/v gum tragacanth. Animals Male Wistar albino rats weighing 150-200 g were procured from the National College of Pharmacy, Shimoga and maintained under standard housing conditions. The animals were fed with commercial diet (Hindustan Lever Ltd., Bangalore) and water ad libitum . The study was permitted by the Institutional Animal Ethical Committee with Reg. No. 144/1999/CPCSEA/SMG.

Acute toxicity studies Acute toxicity study was conducted for both the extracts by stair case method.[12] One tenth of the LD₅₀ doses were selected for the evaluation of hepatoprotective activity.[13]

The animals were divided into five groups of six rats each. Group I served as control and received the vehicle (1 ml/kg/day of 1% w/v gum tragacanth p.o. for 14 days). Group II to V received 0.1 ml/kg/day of CCl₄ i. p. (E-Merck, Mumbai, India) for 14 days. Group III animals received the standard drug silymarin (Ranbaxy Lab, Dewas) in the dose of 100 mg/kg/day, p.o. for 14 days, while the aqueous and ethanol stem bark extracts of P. santalinus were administered to groups IV and V in the dose of 45 mg and 30 mg/kg/day, p.o. respectively for 14 days. The CC₄ and silymarin or the extracts were administered concomitantly to the respective groups.

All the animals were sacrificed on 14th day under light ether anesthesia. The blood sample from each animal was collected separately in sterilized dry centrifuge tubes by carotid bleeding and allowed to coagulate for 30 min at 37°C. The clear serum was separated at 2500 rpm for 10 min and subjected to biochemical investigations viz., total bilirubin.[14] total protein.[15] serum alanine transaminase, aspartate transaminase[16] and alkaline phosphatase.[17]

Results of biochemical estimations are reported as mean±SEM of six animal in each group. The data were subjected to one-way ANOVA followed by Tukey's multiple comparision test. P< 0.001 was considered statistically significant.

Histopathology The liver was excised from the animals and washed with the normal saline. The materials were fixed in 10% buffered neutral formalin for 48 h and then with bovine solution for 6 h and processed for paraffin embedding. Sections of 5m thickness were taken using a microtome, processed in alcohol-xylene series and were stained with alum-haematoxylin and eosin[18] and subjected to histopathological examination.

Results

The LD₅₀ of aqueous and ethanol stem bark extracts were found to be 450 mg/kg, b.w. and 300 mg/kg, b.w, respectively. One tenth of these doses (45 mg/kg, b.w. and 30 mg/kg, b.w.) were selected for the evaluation of hepatoprotective activity. Effect of aqueous and ethanol stem bark extracts of Pterocarpus santalinus on CCl₄ induced liver damage in rats with reference to biochemical changes in serum is shown in [Table 1]. The CCl₄ treated control group showed a significant increase in serum total bilirubin (2.45±0.01), alanine transaminase (1413.00±1.99), aspartate transaminase (2213.50±32.79) and alkaline phosphatase (444.33±1.56) and a decrease in total protein (5.93 ± 0.01) indicating the liver injury caused by CCl₄ . Whereas animals treated with aqueous and ethanol stem bark extracts exhibited a decrease in total bilirubin (0.93±0.02; 0.62±0.01), alanine transaminase (193.36±1.49; 125.06±1.27), aspartate transaminase (242.17±2.02; 221.67±2.59) and alkaline phosphatase (244.30±1.91; 204.43±1.64) along with a significant increase in total protein (7.40±0.02; 8.41±0.02).

Histologically, control animals showed normal hepatic architecture [Figure 1], the group II animals exhibited intense centrilobular necrosis (N), vacuolization and macrovesicular fatty changes (F) [Figure 2]. Silymarin treated animals showed a normal hepatic architecture [Figure 3]. Moderate accumulation of fatty lobules and cellular necrosis [Figure 4] were observed in the animals treated with aqueous extract. However, the ethanol extract treated animals exhibited significant liver protection against CCl₄ induced liver damage, as evident by the presence of normal hepatic cords, absence of necrosis and fatty infiltration [Figure 5].

Discussion

The present investigation indicated that both the extracts of Pterocarpus santalinus provide significant protection against CCl₄ induced hepatotoxicity in rats. CCl₄ is widely used as hepatotoxin in the experimental studies. The CCl₄ is biotransformed by the cytochrome P450 system to produce the trichloromethyl free radicals, which in turn covalently binds to cell membranes and organelles to elicit lipid peroxidation.[19] Several plants viz., Cassia aungustifolia, [20] Wrightia tinctoria, [21]Foeniculum vulgare [22] and Panax notoginseng [23] have been tested for their efficacy in controlling the CCl₄ induced liver damage. Further it has been evident that several phytoconstituents have the ability to induce microsomal enzymes either by accelerating the excretion of CCl₄ or by inhibition of lipid peroxidation induced by CCl₄ .[24] Phytoconstituents like flavonoids,[25] triterpenoids,[26] saponins[27] and alkaloids[28] are known to possess hepatoprotective activity. Phytochemical investigations of aqueous and ethanol extract of stem bark revealed the presence of alkaloids, phenols, saponins, glycosides, flavonoids, triterpenoids, sterols and tannins. The present study revealed that among the two extract tested, ethanol extract of stem bark of P. santalinus found to possess significant protective effect against hepatotoxicity induced by carbon tetrachloride which may be attributed to the individual or combined action of phytoconstituents present in it. The component(s) of the extract responsible for this effect however was not investigated. Further investigations are needed for identification of the active compounds responsible for hepatoprotective activity. The present finding provides scientific evidence to the ethnomedicinal use of this rare plant genetic resource by the tribal group of Western Ghats in treating jaundice.

Acknowledgements

The author is grateful to Sri. Girimaji N. Rajgopal, Secretary, Sri. S.V. Thimmaiah, Joint Secretary, Prof. Darmanada Rao, Registrar, National Education Society, Shimoga, S.M. Vidya, S.R.N.M.N. College of Applied Sciences, Shimoga, Dr.V.Krishna, Dept. of Biotechnology, Kuvempu University and Prof. K.L. Mankani, Prof. Y.N. Manohara and S.D. Jagadeesh Singh, National College of Pharmacy, Shimoga.