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Hepatoprotective Effect of Unani Formulation in Rats

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A study of hepatoprotective effect of unani formulation (Qurs Rewand) in rats.

ABSTRACT

Aim of study: To evaluate hepatoprotective effect of Unani formulation (Qurs Rewand).

Materials and Methods: The study was conducted on adult Wistar albino rats of either sex weighing 150-200 g. Animals were divided into five groups of 6 animals each – I (Plain control), II (Negative control–CCl4 treated group), III (Sylimarin treated group), IV and V (UPF treated groups). Hepatotoxicity was induced by single administration of CCl4 (2ml/ kg I.P., 1:1 in liquid paraffin) in group II, III, IV & V on 7th day of treatment. The UPF was administered in a dose of 50 mg/kg and 100 mg/kg, once daily, orally for 7 days in group IV and V respectively. Silymarin was administered orally in the dose of 100 mg/kg body weight, once daily for 7 days in III group and served as standard control. On the 8th day all the animals were sacrificed and the blood was collected. Serum was separated for biochemical estimations. The serum was estimated for ALT, AST, bilirubin, alkaline phosphatase, total protein and TBARS. Histological study of liver was done.

Results: The mean serum ALT, AST, bilirubin, alkaline phosphatase, and TBARS were decreased significantly as compared to CCl4 treated group.While total protein was increased significantly as compound with CCl4 treated group. The histological study showed signs of recovery and regeneration in damaged liver cells as compared to CCl4 treated group.

Conclusion: The study demonstrated significant hepatoprotective activity of Qurs Rewand (Unani Polyherbal formulation).

Key words: Unani Polyherbal formulation, carbon tetrachloride, hepatoprotective activity and Silymarin

Introduction

Qurs-e-Rewand is a Unani polyherbal preparation (Table-1)[1] frequently prescribed by the physicians of Unani medicine in the management of liver diseases such as infective and other hepatitis.[2,3,4] Rubia cordifolia Linn (Rubiaceae) and Agrimonia eupatoria Linn. (Rosaceae) have been scientifically evaluated for their hepatoprotective effect[5], but the compound as a whole has not been studied for its described properties. Therefore present study has been undertaken to investigate its hepatoprotective effect against CCl4 induced hepatotoxicity in albino rats. The damage produced by CCl4 is described to be similar to the pathological changes seen in infective hepatitis and in many other liver diseases.[6] The liver function test was used to assess the extent of liver damage and the protection induced by the test drug. Since CCl­­­­4 is reported to damage the hepatocytes mainly by inducing lipid peroxidation, therefore thiobarbituric acid reactive substance (TBARS) test was employed to study the antioxidant property of the test drug with a view to underline its mechanism of action.[7] Silymarin (100 mg / kg) was used as the standard hepatoprotective agent to confirm the integrity of the test system and also to compare the efficacy of the test drug as, it has been used in the treatment of chronic or acute liver disease, as well as protecting the liver against toxicity.8] The hepatoprotective properties of Silymarin have been related to the inhibition of lipid peroxides formation or scavenging of free radicals.[9] Histological study was conducted to observe the structural status of cell matrix liver.

Materials and Methods

The study was conducted in the Department of Ilmul Advia, Ajmal Khan Tibbiya College, AMU in collaboration with the Departments of Pharmacology and Pathology, Jawaharlal Nehru Medical College, AMU, Aligarh, after obtaining permission from Institutional Ethics Committee.

Test drug procurement and identification:

All the ingredients of UPF were procured from Dawakhana, Tibbiya College, AMU, Aligarh and were identified by comparison for its macroscopic and microscopic characters with authentic specimens of above mentioned at Department of Ilmul Advia, Ajmal Khan Tibya College, A.M.U. Aligarh, U.P. India.(Voucher specimen No. are given below).

Botanical NameVoucher specimen No.

Rheum emodi Wall Sc-0098/09

Rubia cordifolia Linn Sc-0097/09

Creteria lacca Sc-0096/09

Apium graveolens Linn Sc-0095/09

Feoniculum vulgare Mill Sc-0094/09

Agrimonia eupatoria Linn Sc-0093/09

Preparation of extract:

All the ingredients (Table 1) were coarsely powdered before subjection to extraction. The hydro-alcoholic extract of all the ingredients was prepared using by Soxhlet apparatus, in which they were continuously extracted for 6 hours. The extract was filtered by Whatman No. 1 filter paper and evaporated on water bath at 40 - 600C until it dried completely. The prepared extract was stored in the refrigerator for further use.

The dose of the test compound formulation for albino rats were calculated by multiplying its clinical doses described in Unani literature with conversion factor 7.[10]

Drugs and Chemicals

CCl4, n-butanol, acetic acid (Thomas Baker Pvt. Limtd. Mumbai), sodium dodecyle sulphate, thiobarbituric acid (Otto Kemi Mumbai), 1, 1, 3, 3-tetraethoxypropane (Sigma USA), Silymarin (Sigma-Aldrich, Germany), Folin’s reagent (CDH, Mumbai), AST, ALT, Billirubin, Alk.Phos. and Total Protein estimation kits (Span Diagnostic Ltd, Surat).

Animals

Thirty Wistar albino rats of either sex, weighing 150-200 g were divided into five groups of 6 animals each. The animals were kept under standard laboratory conditions. Commercial diet pellets and water were given ad libitum. The room temperature was maintained at 25 ± 10 C.

Treatments

Group I (Plain Control): Distilled water orally in the dose of 1ml /kg, daily for 8 days.

Group II (Negative Control): Distilled water orally in the dose of 1ml /kg, daily for 7 days.

Group III (Standard): Silymarin in dose of 100 mg / kg daily for 7 days.

Group IV & V (Test Groups): Extract of Qurs-e-Rewand in the dose of 50 mg/kg and 100 mg/kg respectively suspended in distilled water daily for 7 days.

On the 7th day the animals of all groups except those in group I were administered carbon tetrachloride i.p. along with their routine treatment and 24 hours later (on 8th day) all the animals including in group I were sacrificed.[11]

Collection of Samples

The blood was collected and was kept for 30 minutes without disturbing. The serum was separated by centrifugation for 15-20 minutes at 5000 rpm. The sera of each animal of all groups were estimated for, ALT & AST [12], bilirubin[13], alkaline phosphatase[14], total protein[15] and TBARS[16], which are index of lipid peroxides.[17]

Histological Examination

The liver of rats of all groups was removed immediately and fixed in 10% formalin.[18] The tissue was processed and sections were cut. The slides were prepared and stained with haematoxyline and eosin stain and the histological changes were observed by photomicroscope under high power magnification.

Statistical analysis

The results are presented as means ± S.E.M. The data were statistically compared for determining significance of difference by one-way ANOVA test, followed by pair-wise comparison of various groups by LSD. The analysis was carried out by using the software of the website, www. analyseit.com. P< 0.05 was considered significant.

Results

Biochemical Parameters

A highly significant increase in levels of serum ALT, AST, bilirubin, alkaline phosphatase was observed in CCl4 treated rats while total protein was found significantly decreased as compared to control group. There was significant reduction in all biochemical parameters and significant increase in total protein after oral administration of test drug at two different doses (P< 0.001). Oral administration of Silymarin also reduced the liver enzyme levels significantly as compared to CCl4 treated group (P< 0.001). But, the liver enzyme levels in silymarin in dose of 100 mg / kg daily and test drug in the dose of 100mg/kg treated groups were statistically similar. It was observed that reduction produced in biochemical parameters of liver was greater in dose of 100 mg/kg than that of 50 mg/kg of test drug, and the difference was found to be significant statistically (p< 0. 05). Table-2

Histological Examination

Group I: There were central blood vessels and radiating cords of hepatocytes as well as the vascular sinusoids with no evidence of fatty changes, necrosis or inflammation. (Fig.1)

Group II: There was centri-lobular (acidophilic) necrosis and vascular congestion. (Fig.2)

Group III: There was mild vascular congestion and peri-vascular infiltrate of mono nuclear cells and fibroblast. No fatty degeneration was observed. (Fig.3)

Group IV (50 mg/kg): There was vascular congestion and peri-lobular hydropic degeneration of hepatocytes (In high magnification only vascular congestion). (Fig.4)

Group V (100mg/kg): The slides showed well preserved hepatic architecture. There was no fatty degeneration, only mild vascular congestion and peri-vascular infiltrate of mono nuclear cells and fibroblast and regenerating hepatocytes were observed. The hepatic architecture was found similar to that observed in group III. (Fig.5)

Discussion

The findings of the present study demonstrated that the test drug (Unani formulation) lowered the biochemical markers of liver function as well as the lipid peroxide towards normal. The histological findings also indicated protective response by bringing the derangement of liver cell matrix to very near to normalcy. Thus the remarkable reduction in CCl4 intoxicated biochemical markers by test drug extract, supplemented by commensurate histopathological findings of rat liver sections indicated hepatoprotective effect of this herbal formulation. The likely mechanism of hepatoprotective response appears to be the anti-oxidant property.

Carbonterachloride has been widely used for inducing hepatic damage due to free radical formation during its metabolism by hepatic microsomes.[19] The clinical features of carbontetrachloride induced hepatic damage resemble that of acute viral hepatitis.[20] The mechanism of producing hepatic damage by CCl4 depends on reductive dehalogenation of CCl4 catalyzed by cytochrome P450 in the liver cell endoplasmic reticulum leading to the generation of unstable complex of CCl4 radical. This trichloromethyl radical reacts rapidly with O2 to yield trichloromethyl peroxy radical which is reported as a highly reactive species. Qurs-e-Rewand appers to exert its hepatoprotective effect by inhibiting lipid peroxidation mediated by CCl4, due to its antioxidant activity as it decreased the lipid peroxide significantly in TBARS test. The test drug further appears to exert hepatoprotective effect due to its effect against cellular leakage and loss of functional integrity of the cell membrane in hepatocytes i.e. they possess membrane stabilizing property, indicated by significant decrease in AST, ALT, Alk. phosphatase and lipid peroxidation.

The biochemical as well as histological observations demonstrated dose dependent protective action of the extract against the liver damage. The extract in dose of 100mg/kg caused greater response. The biochemical markers were found significantly lower and retention of hepatic architecture, reduction in fatty degeneration and necrosis were more marked in this group than the group treated with 50mg/kg. The overall hepatoprotective effect produced by the 100mg/kg was sientifically equal to standard drug Silymarin. Further, quite interestingly the animals treated with Qurs-e-Rewand showed regeneration of hepatocytes, which provide an indication of high clinical and therapeutic value.

The protective effect demonstrated with regard to liver function (Biochemical markers), lipid peroxidation and structure (Histological study) provides conclusive proof that the test drug possesses hepatoprotective activity.

The present study provides scientific support and validation to the Unani claim regarding the hepatoprotective activity of the test drug. The study also provides the interesting information that Qurs-e-Rewand promotes hepatocellular regeneration, so it may be useful in diseases where liver damage is extensive such as chronic hepatitis and cirrhosis.

Conclusion: it can be concluded that both the doses of test drug (Qurs Rewand) possess significant hepatoprotective activity against acute hepatic damage induced by CCl4. Further, the mechanisms and activities of compound drug require more study to understand the hepatoprotective mechanism.

REFERENCES

Table 1: Ingredients of Qurs-e- Rewand

Botanical Name (Family) English Name Quantity (in gm)

Rheum emodi Wall (Polygonaceae) Rhubarb 17.5 gm

Rubia cordifolia Linn (Rubiaceae) Indian Madder 10.5 gm

Creteria lacca (Coccoidea) Lac 10.5 gm

Apium graveolens Linn (Apiaceae) Celery 3.5 gm

Feoniculum vulgare Mill (Umbelliferae) Fennel 3.5 gm

Agrimonia eupatoria Linn (Rosaceae) Agrimony 3.5 gm

Table 2: Effect of test drug (Q.R) and Silymarin on biochemical parameters of liver function in CCl4 induced toxicity.

Groups

(n = 6)

ALT/SGPT (Units/ml)

AST/SGOT (Units/ml)

S. Bilirubin ( mg/dl)

S.Alk. phosphatase (KAU/dl)

Total protein

(gm/100 ml)

Lipid peroxidation

(n mole of MDA/mg of protein)

Group I

25.66 ± 1.30

y3z3a3b3

28.16 ± 1.53

y3z3a3b3

0.83 ± 0.11

y3a2

8.57 ± 0.38

y3z3a3b3

6.81± 0.20

y3z1a3

3.21± 0.35

y3a2

Group II

80.50 ± 3.23

78.00 ± 2.22

2.49 ± 0.16

42.87 ± 1.74

4.14± 0.18

10.67± 0.88

Group III

59.66 ± 2.08

y3a1

60.33 ± 2.38

y3 a2

1.13 ± 0.16

y3

21.30 ± 1.86

y3

5.98 ± 0.16

y3a2

4.51± 0.41

y3

Group IV

67.83 ± 2.24

y3

69.33 ± 2.73

y1

1.58 ± 0.16

y3

24.40 ± 1.59

y3

5.01± 0.32

y1

5.93± 0.61

y3

Group V

57.00 ± 2.76

y3 a2

57.66 ± 2.46

y3a2

1.28 ± 0.23

y3

16.54 ± 1.26

y3z1a3

6.23 ± 0.21

y3a3

4.13± 0.34

y3a2

Values are Mean  SE; n = 6; x = against plain control, y = against CCl4 (2 ml/kg), z = against standard (Silymarin) (100 mg/kg); a = against Q.R. single dose (50 mg/kg), b = against Q.R. double dose (100 mg/kg); 1 = P < 0.05, 2 = P < 0.01, 3 = P< 0.001.

Group I

Fig. 1: Section of rat liver showing central blood vessels and radiating cords of hepatocytes as well as the vascular sinusoids (H & E stain High power)

Group II

Fig. 2: Section of liver showing centrilobular (Acidophilic) necrosis, and vascular congestion and marked congestion of portal vessels (H & E stain, High power)

Group III

Fig. 3: Section of liver showing mild vascular congestion and peri-vascular Infiltrate of mono nuclear cells and fibroblast (H & E stain, High power)

Group IV

Fig. 4: Section of liver showing vascular congestion (H & E stain, High power)

Group V

Fig. 5: Section of liver showing mild vascular congestion and peri-vascular infiltrate of mono nuclear cells and fibroblast and regenerating hepatocytes (H & E stain, High power)


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