Hepatoprotective Activity of Oxalis corniculata Leaves against Carbon Tetrachloride Induced Hepatotoxicity in Female Albino Wistar Rats

 

Jadhav Anil G*1, Patil Mrutyunjay B1, Narkhede Sachin B2 and G Vidya Sagar3

1 KLES College of Pharmacy, Belgaum, Karnataka, India.

2 Smt.BNB Swaminarayan Pharmacy College,Salvav (Vapi).

3Dean,Veerayatan institute of pharmacy,kutch-370 460,gujarat

 

ABSTRACT:

In recent times lot of interest has been generated to find out a natural remedy for hepatic disorders caused by toxins like alcohol and hepatitis virus.  The present study is designed and investigate the hepatoprotective activity of Oxalis corniculata leaves against carbon tetrachloride (CCl4) induced hepatotoxicity in female Albino Wistar rats. Forty two Albino Wistar female rats were divided in to seven equal groups each containing six animals. Four groups received the extracts of Oxalis corniculata leaves and intraperitoneal (i.p.) CCl4 (2 ml/kg) with administration of extracts. One group was control, one treated with CCl4 and one with normal saline. The hepatotoxicity was assessing by plasma concentration of serum bilirubin and enzymes activities. The aqueous extract (500mg/kg b.w) of Oxalis corniculata  significantly reduced carbon tetrachloride induce damages in the liver which is measured by SGOT, SGPT, SALP and serum bilirubin levels in blood. The attempt has been made to extract the active constituent and identified by phytochemical investigation and hepatoprotective activity of leaves of Oxalis corniculata.

 

KEYWORDS: Oxalis corniculata, carbon tetrachloride, hepatoprotective.

 

INTRODUCTION:

Oxalis corniculata Linn. Family Oxalidaceae1 It is a diffuse annual or perennial, procumbent or more or the less erect acidic herb, 6 – 25 cms high.2, 3 It is found mostly in all the regions throughout the warmer parts of India, Sri Lanka and other countries.4 Growing in various regions up to altitude of nearly 8000 ft. in Himalaya. It occurs as a common herb specially in shaded localities, ravines, river banks, warm margins, gardens, fields, roadsides, forest edges and scrub jungles, ground patches.5

 

Leaves palmately 3-foliolate; petioles 3.8 – 9 cm long very slender, pubescent; stipules small, oblong, adnate to the petiole; leaflets 1.2 to 2.5 cm. long, abcordate, cuneate at base, subsessile, glabrous or with a few appressed hairs and with ciliate margins. 6

 

The leaves of Oxalis corniculata Linn. are used in scurvy, fever, inflammation7, pain, indigestion, warts, corns, dyspepsia, bilious headache,8 heart disease, piles, haemorrhage,5 also useful in anaemia, tympanitis, inflamed ulcers, cephalagia, dysmenorrhoea, amenorrhoea, hepatopathy and also in Datura poisoning as antidote.9 Leaves acts as diuretic febrifuge, antibacterial, vermifuge, emmenagogue, antiseptic,8 refrigerant,5 antiscorbutic.10 The paste of top shoots along with a few fruits of black pepper is applied to boils, abscess, wound and weeping eczema.10 In unani formulation (Kushta Tamysar) Oxalis corniculata Linn it is used as stomachic, liver tonic, aphrodisiac and used in paralysis, facial paralysis, rheumatism, gout and catarrh.11


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- 60oC), 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: CCl4 was 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 2nd and 3rd 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 2nd and 3rd 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 2nd and 3rd 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 2nd and 3rd days.

 

All the rats in all the groups were sacrificed on 5th 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. 17 SGOT 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.18 Alkaline 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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2.     Vaidyaratnam P. S., Varier’s. Indian Medicinal Plants – A compendium of 500 species, 1994; Vol. IV:  202.

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13.   Iqbal, M.C.M., S. Meiyalaghan, K.B. Wijesekara and K.P. Abeyratne. Antifungal activity from water extracts of some common weeds, Pakistan Journal of Biological sciences2001; 4, 7, 843-845.

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17.   Reitman, S. and Frankel AS. A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. ;1967, 26. p. 56.

18.   Kind PR and King EJ. Estimation of plasma phosphatase by determination of  hydrolyzed phenol with antipyrine. J. Clin. Pathol; 1954, 7 .p.322          

 

 

Received on 15.12.2009

Accepted on 10.03.2010   

© A&V Publication all right reserved

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