Methanol extract of Oxalis corniculata reported that relaxant activity on isolated rabbit ileum which was dose dependent and also showed cardio relaxant activity on isolated rabbit hear.¹²Water extracts of Oxalis corniculata showed antifungal activity. ¹³ It is also reported to be used in oral health care.¹⁴

MATERIAL AND METHOD:

Plant Material

Procurement of drugs: The Oxalis corniculata leaves were collected from Dharwad district of Karnataka in southern India.

Authentication: Oxalis corniculata Linn. Oxalidaceae were authenticated at s SSVPS science college, Dhule (Maharastra) by Dr. D. A. Patil.

Drying and size reduction: In the present study, Oxalis corniculata leaves was reduced to coarse powder (# 40 size mesh) using mechanical grinder.

Extraction: The powdered material was subjected for extraction by maceration by chloroform water and continuous hot extraction by petroleum ether (40- 60^oC), chloroform and alcohol. After the complete extraction, the solvents were distilled off and concentrated on a water bath to get a dry residue. Some part of the total extracts was reserved for phytochemical investigation and assessment of hepatoprotective activity.

Phytochemical screening: Identification of chemical constituents was carried out on the same extracts used in pharmacological tests according to the methodology¹⁵.

Materials used: CCl₄was obtained from Sigma-Aldrich. All other chemicals used were of analytical grade.

Assessment of Hepatoprotective activity:

Animal selection: Female Albino Wistar strain rats weighing between 120-150 gm were used for hepatoprotective model. Rats were kept in polypropylene cages and led on standard diet and water. The registration number of animal ethical committee is CPCSEA 221 resolution No.12. The animals were exposed to 12 hours of darkness and light each. The bedding materials of cages were changed everyday. Rats were divided into groups of six.

LD₅₀ study It was carried out by administering large doses of extracts from 500 mg/Kg to 5000 mg/Kg. At the dose of 5000 mg/Kg half the population of animals was found to be dead for every extract. Thus, this dose was considered as LD₅₀. Therefore the dose 500 mg/kg was used in this study.

The method according to Handa SS and Sharma A¹⁶ has been used in this study. The above extracts were administered on group of 6 female albino Wistar rats, weighing about 120-150g, for recording enzymatic levels and histopathology during the evaluation. Animals were administered with carbon tetrachloride (2 ml/kg) i.p. to induce hepatotoxicity. Marked increased in the serum level or SGOT, SGPT, SALP, and Serum Bilirubin was taken as indication of hepatotoxicity.

The procedure consists of:

· Group A – Served as Control and received single daily dose of 1 ml/kg i.p. of sucrose solution for 4 days along with 1 ml/kg s. c. of olive oil on 2^nd and 3^rd days.

· Group B – Also received single daily dose of 1 ml/kg i. p. aqueous sucrose solution for 4 days with 2 ml/kg of Carbon tetrachloride by subcutaneous route dissolved in an equal volume of olive oil on 2^nd and 3^rd days.

· Group C– Received standard drug Liv-52 as a single daily dose of 5 ml/kg of oral route for 4 days with 2 ml/kg of carbon tetrachloride by subcutaneous route on 2^nd and 3^rd days.

· Group D, E, F and G received single daily dose of 500 mg/kg of extracts by oral route for 4 days respectively, with 2 ml/kg of carbon tetrachloride by subcutaneous route on 2^nd and 3^rd days.

All the rats in all the groups were sacrificed on 5^th day under light anesthetic ether. Blood from each rat was collected through cardiac puncture under ether anesthesia for biochemical investigation like SGOT, SGPT, SALP, and serum bilirubin estimation. Blood was allowed to coagulate at 37⁰C for 30 min and the serum was separated by centrifugation at 2500 rpm for 10 minutes. The liver of all the experimental animals were removed and processed immediately for histological investigation.

Estimation of Serum Glutamate Pyruvate Transaminase (SGPT):

SGPT or ALT is located in the cytosol of the liver cell. During liver cell inflammation, they are released into circulation due to increased permeability of cell membrane break down of liver cells. Hence, determination of SGPT as index of the extent of liver damage.

Diagnostic reagent kit was used for determination of SGPT also called as "Alanine amino transaminase" (ALT) activity by method of Reitman and Frankel.¹⁷

Estimation of SGOT:

SGOT (AST) is located on the cytosol of liver cell. In addition, it is also found in the mitochondria. It is also found in many tissue such as heart, liver, skeletal muscle and kidney which rich source of SGOT in that order, liver are being the second richest source of SGOT the importance of SGOT levels in hepatic damage of hepatic cells leads to increased levels of SGOT in blood serum.

SGOT Kit is based on Reitman and Frankel's method.¹⁷SGOT catalyzes the transfer of the amino group of L-aspartate (ASP) to a-ketoglutarate of the (a-KG) resulting in the formation of oxaloacetate (OAA) and L-glutamate (L-Glu). The oxaloacetate so formed, is allowed to react with 2, 4-DNPH to form 2, 4 dinitrophenyl hydrazone derivative which is brown coloured in alkaline medium. The hydrazone derivative of oxaloacetate similar to pyruvate is considerably more chromogenic than that of a-KG. The final colour developed does not obey Beer's law.

Estimation of SALP / ALP:

SALP Kit is based on Kind and King Method.¹⁸Alkaline phosphatase (ALP) at an alkaline pH hydrolyses di Sodium Phenylphosphate to form phenol. The Phenol formed reacts with 4 -Aminoantipyrine in the presence of Potassium Ferricyanide, as an oxidizing agent, to form a red colored complex. The intensity of the colour formed is directly proportional to the activity of ALP present in the sample.

Table 1: Showing enzymatic SGOT level (IU/ L). (Oxalis corniculata)

Sr. No	control	CCl₄	Standard	Methanol	Pet-ether	Chloroform	Aqueous
1	47	150	56	92	80	93	57
2	43	153	50	97	65	94	52
3	44	147	63	93	72	82	67
4	44	153	68	103	71	103	61
5	45	159	64	82	65	92	66
6	43	160	61	94	68	97	62
Mean	44.5	153.7	60.33	93.5	70.17	93.5	60.83
SD	1.51	5.04	6.41	6.89	5.63	6.89	5.63
SE	0.61	2.06	2.61	2.81	2.30	2.81	2.3
F ratio	309
P value	-	-	P<0.01	P<0.05	P<0.01	P< 0.05	P< 0.01

P<0.05 is significant

Table 2: Table 5 showing enzymatic SGPT level (IU/ L). (Oxalis corniculata)

Sr. No	Control	CCl₄	Standard	Methanol	Pet-ether	Chloroform	Aqueous
1	40	133	35	80	53	62	43
2	38	137	39	65	54	63	42
3	39	141	43	72	56	68	47
4	33	143	47	71	58	70	49
5	35	144	45	65	61	77	51
6	36	151	52	68	58	71	54
Mean	36.8	141.5	43.5	70.17	56.67	68.5	47.67
SD	2.63	6.18	5.99	5.63	2.94	5.54	4.63
SE	1.07	2.52	2.44	2.30	1.2	2.26	1.89
F ratio	379.6
P value	-	-	P<0.01	P<0.05	P<0.01	P<0.05	P<0.01

P<0.01 is significant

Table 3: Showing enzymatic SALP level (IU/ L). (Oxalis corniculata)

Sr. No	control	CCl₄	Standard	Methanol	Pet-ether	Chloroform	Aqueous
1	27.6	81.7	41.2	74	58.1	75	41.4
2	24.2	92.8	45.8	85	38.2	82	46.1
3	25.3	100.1	44.3	89	61.8	74	44.5
4	24.8	81.1	42.3	75	63.7	81	42.6
5	25.2	93.6	45.9	81	64.8	89	46.1
6	25.8	89.6	41.1	82	56.7	85	42.2
Mean	25.4	89.82	43.43	81	57.15	81	43.82
SD	1.16	7.36	2.19	5.76	9.77	5.76	2.04
SE	0.47	3.00	0.9	2.35	3.92	2.35	0.83
F ratio	11.8
P value	-	-	P<0.01	P<0.05	P<0.01	P<0.05	P<0.01

P<0.05 is significant

Table 4: Showing Serum Bilirubin level (IU/ L). (Oxalis corniculata)

Sr, No	Control	CCl₄		Standard	Methanol		Pet-ether	Chloroform		Aqueous
1	2.31	9.3	3.22		8.6	3.6		11.02	3.3
2	2.21	12.4	4.23		10.8	5.1		11	4.4
3	2.11	13.8	6.11		11.3	7.0		11.5	6.7
4	2.03	12.6	5.25		11.2	6.8		8.9	5.8
5	2.08	13.8	6.9		12.4	7.3		12.2	7.0
6	2.03	13.2	5.8		11.8	6.1		11.5	5.9
Mean	2.12	12.52	5.25		11.02	5.98		11.02	5.51
SD	0.11	1.68	1.33		1.30	1.40		1.30	1.41
SE	0.04	0.68	0.45		0.53	0.57		0.53	0.57
F ratio	53.16
P value			P<0.01		P<0.05	P<0.01		P<0.05	P<0.01

Statistical analysis: All the values were expressed as mean ± S.E. Stastical significance was determined using ANOVA followed by Dunnets test at a probability level of P ≤ 0.05. (Table No. 1-4; graph 1-4).

RESULT AND DISCUSSION:

Phytochemical screening of the solvent ether, chloroform, ethanol extracts that were used in the pharmacological test has revealed the presence of steroids, glycoside, alkaloids, tannins and phenolic acids as major chemical constituents and in aqueous extract shows presence of flavonoids, phenolic compounds and glycosides.

In our experiments it is observed that the lipid peroxidation levels in the CCl₄ group is increased. This clearly indicates that there is a significant hepatic damage due to CCl₄ and this is further evident from the fact that there is elevation in the levels of various markers of hepatic damage like SGOT, SGPT and ALP. Treatment with Oxalis corniculata leaves extract has decreased the levels of lipid peroxidation and the elevated levels of above mentioned biochemical markers to the near normal levels. It may be concluded that the hepatoprotective effect of Oxalis corniculata is due to the prevention of the depletion in the tissue GSH levels. Phytochemical investigation of leaves of Oxalis corniculata contains phenolic compound and flavanoids which are present in the methanol extract. Therefore Oxalis corniculata extract possess hepatoprotective activity (Table No. 1-4; Graph No.1-4).

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Received on 15.12.2009

Accepted on 10.03.2010

Research Journal of Pharmacognosy and Phytochemistry. 2(2): March -April 2010, 136-139