ISSN: 2455-7080 - Albert Science International Organization

ISSN: 2455-7080

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ASIO Journal of Experimental Pharmacology & Clinical Research (ASIO-JEPCR)

Volume 1, Issue 1, 2016, 16-22

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CHENOPODIUM ALBUM LINN. LEAVES PREVENT CARBON TETRACHLORIDE-INDUCED LIVER FIBROSIS IN RATS

Manish M. Wanjari1†, Vanika Bajpai2, Yadu Nandan Dey1, Sudesh N. Gaidhani3, Gajji Babu4

1Department of Pharmacology, National Research Institute for Ayurveda-Siddha Human Resource Development, Gwalior – 474 009,

India

2Department of Biochemistry, BIMR College of Professional Studies, Gwalior – 474 011, India

3Department of Pharmacology, Central Council for Research in Ayurvedic Sciences, New Delhi – 110 058, India

4Department of Kayachikitsa, Ayurveda Contraceptive Drug Research Institute, Ahmedabad – 380016, India

ARTICLE INFO ABSTRACT

Research Article

Received: 10 Dec., 2015 Accepted: 12 Jan., 2016

Corresponding Author:

Manish M. Wanjari

Department of Pharmacology, National Research Institute for Ayurveda-Siddha Human Resource Development, Gwalior – 474 009, India.

The leaves of Chenopodium album Linn. are traditionally used in treatment of jaundice and spleen enlargement. The present work investigated the effect of aqueous extract of leaves of Chenopodium album on experimentally induced hepatotoxicity in rats to substantiate the traditional use in liver diseases. The hepatotoxicity was induced in rats by carbon tetrachloride (CCl4) treatment and in addition, vehicle or aqueous extract of the leaves of Chenopodium album (CAE) (100, 200 and 400 mg/kg) or silymarin (25 mg/kg) were administered daily orally for seven days. The hepatotoxicity was assessed by estimating the activities of marker enzymes and by histological studies. The oxidative stress was assessed by measuring lipid peroxidation and activities of superoxide dismutase and catalase in liver. The CAE treatment significantly prevented the CCl4-induced elevations the serum levels of glutmate oxaloacatate transaminase, pyruvate oxaloacatate transaminase, alkaline phosphatase, bilirubin, lactate dehydrogenase and triglycerides while decreased the total protein. Treatment with CAE attenuated the CCl4-induced fibrosis in liver tissue and also decreased elevated lipid peroxidation levels and inhibited decrease in activities of superoxide dismutase and catalase enzymes in liver. The effects of the CAE were comparable to that of standard antioxidant hepatoprotective agent, silymarin. These findings indicate the CAE exhibited hepatoprotective effect against CCl4-induced liver fibrosis which may be attributed to antioxidant action of CAE due to presence of antioxidant phytochemicals like flavonoids and other phenolic compounds. It indicates the therapeutic potential of Chenopodium album leaves and validates its traditional medicinal use in liver disorders.

Keywords: Bathua; Hepatotoxicity; Silymarin; Oxidative stress, Masson’s trichrome

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1. INTRODUCTION

The liver is constantly exposed to many kinds of xenobiotics and therapeutic agents. It is involved in the metabolism and many other biochemical functions. As the liver deals with many complex molecules and carries out biotransformation of same, it is always at the risk of detrimental physiological and pathological alterations. Such alterations and subsequent impaired functions of the liver are characterized as liver diseases which include cirrhosis, jaundice, tumors, metabolic and degenerative lesions, liver cell necrosis, hepatitis, etc [1]. Steroids, vaccines and anti-viral drugs which are used as hepatotherapeutic agents, have potential side effects especially when administered for long period [2].

Hence, hepatoprotective agents of plant origin have attracted special interest, and numerous medicinal plants and their formulations are developed for liver disorders in view of their ethnomedicinal use [3]. Chenopodium album Linn. (family: Chenopodiaceae) is herbaceous vegetable plant locally known as Bathua. It is cultivated as pot-herb and usually grown in gardens. The plant is extensively cultivated and consumed in Northern India as a food crop. The plant is traditionally used for its anthelmintic, laxative, hepatoprotective, sedative, diuretic, aphrodisiac action and to improve the appetite [4]. It is also used in abdominal pains, eye disease, throat troubles, piles, diseases of the blood, heart and spleen and biliousness [5, 6].

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The medicinal property of this plant mainly resides in leaves and seeds. The leaves and other aerial parts of this plant contain flavonoids viz. quercetin, kaempferol, etc [7, 8]. The plant is recommended by Hindu physicians for correction of hepatic disorders and splenic enlargement [9]. Ethnobotanical studies of Chikar and its allied areas of district Muzzafarabad (Pakistan) reported the folk medicinal uses of seeds and leaves of Chenopodium in hepatic disorder and enlarged spleen and decoction of leaves is given orally against jaundice [10]. Further, Chenopodium album is also used as folk medicine in the area of Chhachh (Distt. Attock) Punjab, Pakistan for the treatment of liver diseases and jaundice [11]. It is one of the important constituent of Unani preparation, Kabdeen that is used in viral hepatitis [12]. The survey report on herbal hepatoprotective formulations used to treat acute and chronic hepatic disorders in Lucknow, Uttar Pradesh (India) has enlisted Chenopodium album as an important hepatoprotective plant in demand [13]. Despite immense ethnomedicinal use of leaves of Chenopodium album in the treatment of hepatic disorders and splenic enlargement, no studies were undertaken to observe their effects on liver fibrosis. Hence, the present investigations demonstrate the effect of aqueous extract of leaves of Chenopodium album on carbon tetrachloride (CCl4) induced hepatic damage and liver fibrosis in rats.

2. MATERIAL AND METHODS 2.1. Plant material

The leaves of Chenopodium album were collected from the local market of Gwalior in February 2009. The plant was identified and authenticated by Dr. N.K. Pandey, Research Officer (Botany), National Research Institute for Ayurveda-Siddha Human Resource Development, Gwalior. A voucher specimen (Accession no. 1299) has been deposited in the herbarium of the Institute.

2.2. Drugs and chemicals

Carbon tetra chloride was purchased from Qualigens Fine Chemicals, Mumbai, India. Olive oil (Figaro) was purchased from local market of Gwalior. Thiobarbituric acid, and pyrogallol were procured from Sigma-Aldrich, USA. Glutamate pyruvate transaminase, glutamate oxaloacetate transaminase, and alkaline phosphatase estimation kits were procured from Merck Specialties Private Limited, Mumbai while total protein, bilirubin and triglycerides estimation kits were procured from Span Diagnostic Ltd., Surat, India. LDH estimation kit was procured from Reckon Diagnostics Pvt. Ltd. Baroda, India. Sylimarin (Limarin) was obtained as suspension from the market. All remaining chemicals used in the experiment were of the highest grade commercially available.

2.3. Preparation of aqueous extract of leaves of Chenopodium album (CAE)

The leaves were shade dried and subjected to size reduction to a fine powder by using dry grinder. This

powder (100 g) was soaked in 1000 ml purified water, mixed and kept in dark and dry place for 48 h. Chloroform was added in quantity of 1% total mixture to avoid the microbial growth. After 48 h, solution was filtered by Whatman filter paper No. 1. The filtered extract was dried in rotary evaporator. After drying, dark brown extract was obtained (20% w/w). The process of extraction was carried out thrice with fresh 100 g powder of the leaves to get sufficient aqueous extract for studies.

2.4. Preliminary phytochemical screening

Preliminary phytochemical screening of CAE was carried out to detect the presence of various phytochemicals by standard procedures [14].

2.5. Animals

Healthy adult Wistar rats of either sex weighing about 200-250 g, between 2–3 months of age were used in the study. They were housed in groups in polypropylene cages, under standard conditions (12:12 h light: dark cycle; temperature 22±3°C; 40–60 % relative humidity) and had free access to standard rat pellet diet (Ashirwad brand, Chandigarh, India) and filter water, ad libitum. The experiments were carried out in accordance with guidelines prescribed by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the use of animals was approved by the Institutional Animal Ethics Committee of the Institute (Proposal No. CRI-GWL/IAEC/2009/04).

2.6. Acute toxicity study

Healthy Wistar rats, starved overnight, were subjected to acute toxicity studies to determine non-observable adverse effect dose level (NOAEL) by acute toxic class method of oral toxicity as per Organization for Economic Co-operation and Development (OECD) 423 guidelines [15].

The rats (n=3) were orally administered CAE in the limit test dose of 2000 mg/kg orally and observed continuously for behavioral, neurological and autonomic profiles for 2 h and after a period of 24, 72 h and thereafter up to 14 days for any lethality, moribund state or death. The limit test was repeated in another group of rats (n=3) for confirmation and approximate LD50 determination.

2.7. Experimental induction of hepatotoxicity

Hepatotoxicity was induced in Wistar rats by intraperitoneal (i.p.) administration of carbon tetrachloride (CCl4) (1:1: CCl4: Olive oil in the dose of 1 ml/kg) for two continuous days as described previously with modifications [16, 17]. After 48 hours of last dose of CCl4, hepatotoxicity was confirmed be carrying out various biochemical and histological parameters.

2.8. Grouping and treatments

The rats were divided into six groups (n=5). Group I received only olive oil (1 ml/kg, i.p.), and remaining groups (group II, III, IV and V) received 1 ml/kg, i.p. CCl4

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in olive oil for two continuous days. Group II (control) received the vehicle of the extract (5 ml/kg, distilled water, orally), group III, IV and V received CAE (100, 200 and 400 mg/kg respectively, orally). Group VI received silymarin suspension (SIL) (25 mg/kg, orally), a known antioxidant and hepatoprotective agent [18, 19].

The vehicle/drugs were administered daily orally for seven days and CCl4 administration was done on day 5 and 6 of vehicle/drug treatments.

2.9. Assessment of liver function test and hepatic damage

On day 8 of the experiment, blood was withdrawn by micro-capillary technique from the retro-orbital plexus [20] under light ether anesthesia and collected in 1.5 ml Eppendorff tubes. It was centrifuged at 3000 rpm to obtain serum, which was used to assess following liver function parameters using semi-autoanalyser (Microlab 300, Merck Specialities Pvt. Ltd. New Delhi). I) Glutamate oxaloacetate transaminase (SGOT) [21] II) Glutamate pyruvate transaminase (SGPT) [21] III) Alkaline phosphatase (ALP) [22] IV) Bilirubin (total) [23] V) Total protein (24) VI) Lactate dehydrogenase (LDH) [25] VII) Triglycerides (STG) [26]

2.10. Histological studies

After the withdrawal of blood, the animals were sacrificed by cervical dislocation. Abdomen was cut opened and aorta was cut to washout the blood from tissues. The liver was dissected out. A piece of liver was fixed in 10% neutral buffered formalin. It was further processed by conventional method to obtain thin sections of 4 µm thickness which were subsequently stained with haematoxylin and eosin, Masson’s trichrome (Accustain Trichrome Stains, Sigma-Aldrich, USA). Staining was done as per manufacturer’s protocol. The sections were studied under microscope.

2.11. Assessment of oxidative stress in liver The piece of liver isolated above was thoroughly washed with ice-cold 0.1 M phosphate buffered saline (pH 7.4). It was blotted dry and homogenized in 1.15% KCl to prepare a 10% w/v suspension. This suspension was centrifuged at 16000×g for 1 h in a cooling centrifuge at 0C. The supernatant was then employed for further assessment of lipid peroxidation and enzyme activities. Lipid peroxidation (LPO) was assessed by a previously reported method of Ohkawa et al. [27] with some modifications. The results are expressed as nM MDA/ mg protein. The activities of superoxide dismutase (SOD) [28] and catalase (CAT) [29] were assessed as described previously. The activity of SOD and CAT is expressed as units/mg protein. The protein content of the liver tissue was determined by Biuret method [24].

2.12. Statistical analysis

The data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. A statistical

difference of P<0.05 was considered significant in all cases.

3. RESULTS

3.1. Phytochemical screening of CAE

The qualitative tests for identifying the nature of phytochemicals in CAE revealed the presence of carbohydrates, proteins, amino acids, sterols, volatile oils, flavonoids, glycosides and phenolic compounds.

3.2. Acute toxicity study of CAE

Acute toxicity studies revealed that the CAE was safe up to a dose level of 2000 mg/kg of body weight (limit test) and non-observable adverse effect level (NOAEL) dose is more than 2000 mg/kg. No lethality or any toxic reactions or moribund state were observed up to the end of the observation period of 14 days.

3.3. Effect of CCl4 treatment on liver function test and marker of hepatic damage

One-way ANOVA showed that the CCl4 treatment (1 ml/kg, i.p. on two continuous days) has significant influence on liver functions parameters (P<0.0001 in all cases). Post hoc test indicated CCl4 treatment significantly (P<0.001 in all cases) elevated the levels of SGOT, SGPT, ALP, total bilirubin, STG while decreased the total protein as compared to olive oil control. LDH levels, marker of hepatic damage, were found significantly (P<0.001) increased as compared to the olive oil control (Table 1).

3.4. Effect of CAE treatment on liver function test and marker of hepatic damage One-way ANOVA showed that CAE (200, 400 mg/kg/day, orally) or SIL (25 mg/kg/day, orally) treatment for seven days has significantly influence on liver functions parameters (P<0.0001) in CCl4 treated rats. The CAE or SIL significantly (P<0.05-0.001, wherever applicable) attenuated the elevation in levels of SGOT, SGPT, ALP, total bilirubin, STG and LDH, and decrease in total protein (Table 1).However, lower dose of CAE 100 mg/kg did not significantly (P>0.05) influence any of the liver function markers. The effect of CAE was comparable to that of SIL (Table 1).

3.5. Effect of CAE treatment on histology of liver of CCl4 treated rats Carbon tetrachloride treated vehicle group exhibited marked liver damage and fibrosis characterized by degeneration of liver parenchyma and marked presence of collagen tissue stained blue with Masson’s trichrome stain in the portal triad [Figure 1B]. Liver section from olive oil treated animals [Figure 1A] showed normal hepatic architecture with central canal having radiating hepatocytes and presence of minimal amount of collagen tissue (arrow) stained blue with Masson’s stain was evident in the portal triad. Silymarin [Figure 1C] or CAE treatment (400 mg/kg) [Figure 1D] showed significant attenuation of CCl4-induced hepatic damage as indicated by presence of lesser amount of collagen tissue as compared to control [Figure 1B].

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Table 1: Effect of CAE on liver function parameters

Rats were treated for 7 days with vehicle or CAE (100, 200 and 400 mg/kg/day, orally) or silymarin (SIL) (25mg/kg/day, orally) along

with olive oil or CCl4 in olive oil (1 ml/kg, i.p.) treatment on day 5 and 6 liver functions markers viz. SGOT, SGPT, ALP, total bilirubin,

total protein, LDH and STG were assessed on day 8. Results are expressed as mean SEM (n=5) *P<0.001 vs. olive oil or ##P<0.05,

#P<0.01, **P<0.001 vs. CCl4 treated vehicle control (One-way ANOVA followed by Tukey’s multi-comparison post hoc test).

Figure 1: Effect of CAE on histopathology of liver

Histological sections of liver from olive oil treated control rats (A) showing normal parenchymal architecture of the interconnecting sheets or plate like arrangement of cells, radiating outward from the central vein with minimal presence of collagen tissue (blue color). Liver section from CCl4 treated rats (B) that received vehicle shows extensive loss of hepatic lobules with clear indications of marked degeneration and fibrosis characterized by the presence of high amount of collagen tissue. Sections of liver of CCl4 treated rat which concurrently received SIL (25mg/kg/day, orally) (C) and CAE (400 mg/kg/day, orally) (D) show a lesser degree of vascular degeneration and fibrosis compared to alone CCl4 treated control rats (B).

Treatments Liver Function Parameters

SGOT

(U/l)

SGPT

(U/l)

ALP

(U/l)

Total

Bilirubin

(mg/dl)

Total Protein

(g/dl)

LDH

(IU/l)

STG

(mg/dl)

Olive oil 47.33

±4.77

55.50

±2.51

127.2

±4.43

0.68

±0.09

7.87

±0.28

678.80

±81.97

66.50

±4.493

CCl4 + Vehicle 130.00

±10.10*

119.80

±8.40*

243.8

±1.92*

1.57

±0.06*

5.15

±0.26*

1856.00

±127.60*

135.70

±14.120*

CCl4 + CAE 100 113.80

±10.54

94.67

±10.16

232.00

±5.48

1.48

±0.08

5.80

±0.23

1592.00

±121.90

99.33

±3.836

CCl4 + CAE 200 84.17

±9.21#

75.17

±7.73**

209.2

±7.42#

1.13

±0.05#

6.82

±0.32##

976.60

±63.81**

79.17

±4.430**

CCl4 + CAE 400 65.33

±6.30**

67.83

±4.56**

164.3

±11.30**

0.86

±0.08**

7.32

±0.33#

680.00

±86.59**

55.00

±6.496**

CCl4 + SIL 58.67

±9.49**

41.00

±4.09**

151.8

±10.58**

0.67

±0.09**

7.35

±0.59#

626.20

±31.90**

56.17

±4.826**

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3.6. Effect of CAE treatment on antioxidant status of liver

One-way ANOVA indicated that CCl4 significantly induced changes in oxidative stress parameters (P<0.0001) and CAE (200 or 400 mg/kg/day, orally) or SIL (25 mg/kg/day, orally) treatment for seven days has significant (P<0.0001) ameliorating effect on same. CAE or SIL treatment decreased (P<0.001) elevated lipid peroxidation levels in

CCl4 treated rats (Fig. 2A). Furthermore, CAE treatment prevented (P<0.05-0.001) the reductions in the activities of superoxide dismutase and catalase [Fig. 2 (B and C)] due to CCl4 treatment. The effects of CAE were much significant at 400 mg/kg dose and comparable to that of SIL, standard antioxidant hepatoprotective drug. The lower dose of CAE (100 mg/kg) influenced neither (P>0.05) lipid peroxidation nor the activities of antioxidant enzymes.

Figure 2: Effect of CAE on oxidative stress markers in liver

Rats were treated for 7 days with vehicle or CAE (100, 200 and 400 mg/kg/day, orally) or silymarin (SIL) (25mg/kg/day, orally) along with olive oil or CCl4 in olive oil (1 ml/kg, i.p.) treatment on day 5 and 6 and oxidative stress markers viz. LPO (A), SOD (B) and CAT (C) were assessed in liver on day 8. Results are expressed as mean SEM (n=5) *P<0.001 vs. olive oil or $P<0.05, #P<0.01, @P<0.001 vs. CCl4 treated vehicle control (One- way ANOVA followed by Tukey’s multi-comparison post hoc test).

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4. DISCUSSION

Acute toxicity study of the CAE (2000 mg/kg, orally) revealed that there was no toxicity of any nature or moribund stage during the observation period. This illustrates that the non observable adverse effect dose level (NOAEL) of CAE is more than 2000 mg/kg. Based on this, the CAE was administered in the dose range of 200 mg/kg (one tenth of the limit test dose level). The previous studies have also employed extract of Chenopodium album in the similar dose level [30].

The present investigations revealed that administration of CCl4 caused a marked impairment in the liver function, as indicated by significant increase in serum levels of marker enzymes; and produced extensive histological damage to the liver as evidenced by fibrotic changes. This is in concordance with the earlier reports [31-33]. CCl4

undergoes metabolism in liver to form trichloromethyl peroxyl (CCl3O2

•) radical [34]. The free radicals are known to oxidize the essential macromolecular structures viz. DNA, proteins and lipids and eventually produce cytotoxicity [35, 36]. The higher levels of lipid peroxidation are clinically evident in liver disorders (37) and the antioxidant therapy was found to ameliorate the same [38]. In present study, concurrent with the hepatic damage, CCl4 has elevated the LPO levels and decreased the antioxidant status, as evidenced by reduced activities of SOD and CAT in liver tissues and confirms the oxidative damage by CCl4 .

It was observed that treatment with CAE not only ameliorated the CCl4-induced impairment in the liver functions but also prevented the cytotoxic damage (fibrosis) to the liver. These findings confer the hepatoprotective effect to CAE. In addition, CAE treatment also attenuated the CCl4-induced rise in LPO levels and the decrease in SOD and CAT activity suggesting in vivo antioxidant action of CAE against CCl4-induced free radicals. The observed in vivo antioxidant activity of CAE is in concordance with the recently reported in vitro antioxidant activity [39, 40]. Thus, it can be contemplated that the observed hepatoprotection offered by CAE may be ascribed to its in vivo antioxidant activity. Furthermore, such delineation was substantiated by the attenuation of CCl4-induced changes in the levels of hepatic function markers and reduction in the antioxidant status of liver by silymarin, a well proven hepatoprotective and antioxidant drug [18, 19]. The observed antioxidant and hepatoprotective effects of CAE were comparable to that of silymarin.

The phytochemical analysis of CAE revealed the presence of fair amount of antioxidant phytochemicals flavonoids and phenolic compounds as reported earlier [7, 8]. As the antioxidant activities of the flavonoids are well demonstrated and they are often found effective in hepatic disorders [41, 42], it is possible that the observed hepatoprotection by CAE may be subsequent to the antioxidant effect shown by the flavonoids or other phenolic compounds present in CAE. The screening in other models of hepatotoxicity would only reveal the exact mechanism and activity-guided phytochemical investigations would delineate exact phytochemicals responsible for the in vivo antioxidant activity and hepatoprotection.

5. CONCLUSION

In conclusion, the aqueous extract of the leaves of Chenopodium album ameliorated the hepatotoxicity and liver fibrosis produced by CCl4 and attenuated the oxidative stress in liver tissue. The hepatoprotective activity of CAE may be subsequent to its antioxidant effect. The study scientifically validates of ethnomedicinal use of leaves of Chenopodium album in jaundice and advocates its effectiveness in liver fibrosis.

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