Vol 6, Suppl 5, 2013 ISSN - 0974-2441

Research Article
HEPATOPROTECTIVE ACTIVITY OF BERBERISARISTATA ROOT EXTRACT AGAINST CHEMICAL
INDUCED ACUTE HEPATOTOXICITY IN RATS
NAVDEEP DEHAR* 1 , RANI WALIA 2 , R.B VERMA 3 , PINKY PANDEY4
1 MD Assistant Professor, 2 MD Prof. &Head, 3 Professor, Department Of Pharmacology, 4Associate Professor Department of
Pathology M.M. Institute of Medical Sciences and Research, Mullana, Ambala (Haryana, INDIA)
Email: navdeepdehar@gmail.com
Received: 4 September 2013, Revised and Accepted: 26 September 2013
ABSTRACT
Objective: To study the effects of root extract of Berberis aristata in rat model of acute hepatotoxicity induced by carbon tetrachloride (CCl 4). CCL4 is
commonly used hepatotoxin in the experimental studies of liver diseases. Liver damage induced by CCL 4 involves biotransformation of free radicals
derivatives, increased lipid peroxidation and excessive cell death. Berberis aristata root extract, “berberine chloride” is known to possess multiple
pharmacological activities including anti-microbial, antiviral, anti-inflammatory, cholesterol lowering, anti cancer and anti-oxidant effects. The
present study was conducted to evaluate the hepatoprotective activity of berberine in chemical induced hepatotoxicity in rats.
Material & Methods: The experimental protocol was approved be the IAEC. Adult wistar rats aged 7-9 weeks were injected intraperitoneally with
50% CCl4 as 1:1 mixture in liquid paraffin. Berberine was administered i/p before or after CCl 4 treatment in various groups. Twenty-four hours after
CCl4 injection, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase(ALP) activities, total serum bilirubin
levels and liver weight were measured. Histological changes of liver were examined with microscopy.
Results: Serum ALT, AST, ALP activities significantly decreased in a dose-dependent manner in both pre-treatment and post-treatment groups with
berberine. Histological examination showed lowered liver damage in berberine-treated groups.
Conclusion: The present study demonstrates that berberine possesses hepatoprotective effects against CCl4-induced hepatotoxicity and that the
effects are both preventive and curative. Berberine should have potential for developing a new drug to treat liver toxicity.
Keywords: CCl4, Berberine, hepatoprotective activity, antioxidant
INTRODUCTION
Liver is a vital organ of metabolism and excretes virtually every drug habitat dependent variations in the content of berberine in the roots
and toxin introduced in the body. The global burden of liver and stem-bark of Berberis species [13]. To confirm the nature and
disorders amount to more than 30,000 deaths annually extent of action of berberine in Berberis asistataroot extract found in
[1].Hepatotoxicity is one of the main reasons behind withdrawal of a the Himalayan Region in India, it was necessary to do a more
drug from the market. 50% of all acute liver failures and 5% of all systematic and comprehensive study of hepatoprotective effects of
hospital admissions are associated with drug-induced hepatotoxicity berberine in CCl4 induced acute liver toxicity in rats. The present
[2]. Numerous medicinal plants are being used for the treatment of study aimed at exploring the preventive and curative effects of
liver disorders. In spite of tremendous strides in modern medicine, berberine on liver tissue injury, liver enzymes, total bilirubin and
there is hardly any drug that offers protection to the liver from drug liver weight.
induced damage or help in regeneration of hepatic cells [3]. Many
polyherbal formulations reputed to have hepatoprotective action are MATERIAL AND METHODS
available in the market for liver disorders but treating a liver STUDY CONDUCT
disorder by using a precise herbal drug is still an intriguing problem
[4, 5]. The study was conducted at the Department of Pharmacology in
collaboration with Department of Pathology and Biochemistry,
CCl4 is the most commonly used hepatotoxin in the experimental MMInstitute of Medical Sciences &Research, Mullana, India. The
studies of liver diseases. Liver cell injury induced by CCl 4 involves experimental protocol was reviewed and approved by Institutional
the metabolism of CCl4 to trichloromethyl free radical by the mixed Animal Ethics Committee. (Ref. no.: 101/IAEC/MMIMSR/2011)
function oxidase system. This leads to wide spread disturbances in
hepatocyte function like increased lipid peroxidation and excessive DRUGS AND CHEMICAL REAGENTS
cell death [6,7]. This model of CCl4 liver induced injury has been
widely used in new drug development for liver diseases.Berberis Berberine chloride, CCl4, Heparin, Thiopentone sodium,
aristata is an edible plant in South Asian traditional medicine. Phenobarbitone and Liquid paraffin were obtained from Hindustan
Particularly its fruit, root and stem are rich in berberine, which is Pharmaceuticals, Amritsar, India. ALT, AST, ALP and Serum bilirubin
being used as a tonic remedy for liver and heart.It is known to kits were purchased from Sigma Aldrich, Bangalore, India.
possess multiple pharmacological activities like antimicrobial,
ANIMALS
antiviral, anti-inflammatory, cholesterol lowering, anticancer and
antioxidant activity [8]. The drugs having antioxidant activity are Healthy albino rats (Wistar strain) of either sex, aged 7 weeks,
effective in treating CCl4 induced toxicity weighing 130-250 gm were obtained from Central Animal House of
[9,10].Coptidisrhizoma(Huanglin), a popular Chinese herb, rich in MMIMSR,Mullana. The animals were housed in the groups of 6-8 per
berberine has exhibited hepatoprotective effects on CCl 4 induced cage for a minimum of 5 days prior to pharmacological experiments
injury in a study conducted by Feng et al, 2010 [11]. Antioxidant with free access to standard rodent diet with water and maintained
effects of berberine against LDL oxidation [12] and inflammation on 12 hr light/ dark cycle and temperature
have also been reported previously. Janbaz&Gilani,2000 found that (22o±3oC).Animalhousing, handling techniques and experiment
berberine has no curative action on CCl 4 induced liver injury at low
protocols (drug treatment and sacrifice) were complied with the
doses; however, levels of ALT and AST were ameliorated after
guidelines of CPCSEA, India.
berberine treatment [8]. There have been reports suggesting the
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Asian J Pharm Clin Res, Vol 6 Suppl 5, 2013, 53-56

ACUTE TOXICITY STUDIES Table:1 Effects of Berberine chloride on Chemical induced Acute
Hepatotoxicity in rats
The acute oral toxicity studies were performed for the root extract of
Treatment Group
Berberis aristata, berberine chloride, according to the OPPTS (Office (n=6)
Biochemical parameters (mean S.E.M)

of Prevention, Pesticide and Toxic Substance) Conventional Acute AST(IU/L) ALT(IU/L) ALP(IU/L) T.Bil(IU/L) Liver Weight
Toxicity Test [14]. NEGATIVE
* 42.773 * 34.17 * 47.012 * 0.2733 * 1.95
CONTROL
(L.paraffin) 13.10 4.84 6.71 0.05 0.05
Animals were divided into 6 groups with 6 animals in each group: TOXIC CONTROL 529.39 502.56 *293.84 0.81 2.54
(CCl4) 86.13 47.03 32.37 0.06 0.11
Group 1- Negative control (liquid paraffin, 1ml/kg, i/p) * 343.11 * 250.65 * 200.83 * 0.74 2.38
BC5mg+CCl4
Group 2 - CCl4Toxic control(50% CCl4 in liquid paraffin, i/p) 175.70 25.34 32.20 0.09 0.08

Group 3- CCl4 + Berberine 5mg/kg, i/p (post treatment) BC10mg+CCl4 * 70.27 * 65.11 * 81.28 * * 0.38 * 2.22
20.33 7.63 15.03 0.10 0.015
Group 4- CCl4 + Berberine 10mg/kg, i/p (post treatment)
* 37.277 * 41.3586 * 48.886 * 0.23 * 2.0329
Group5- CCl4 + Berberine 20mg/kg, i/p (post treatment) BC20mg+CCl4
16.90 6.47 6.04 0.03 0.05
Group 6- CCl4 + Berberine 10mg/kg, i/p (pre treatment) PRE BC10+CCl4 * 57.61 * 52.3267 * 62.695 * 0.2883 2.2717
18.80 6.69 13.07 0.06 0.13
Rats from the group 2-6 were injected with CCl4 at a dose of 1ml/kg, * p-value <0.05 significant in comparison to Control Group(Fisher LSD)
i/p as a 50% liquid paraffin solution while group 1 served as a F=39.438 F=72.524 F=25.178 F=15.544 F=5.092
One -way ANOVA Df 30,5 Df 30,5 Df 30,5 Df 30,5 Df 30,5
negative control receiving 1ml/kg, i/p of liquid paraffin only. P=.000* P=.000* P=.000* P=.000* P=.002*
Berberine was suspended in distilled water at concentration of 5,10
and 20 mg/kg and administered i/p to rats in groups 3-5 Values are expressed as mean ± SEM for albino rats in each
respectively after 6 hours of CCl4 treatment. Rats in group 6 were group. P values: *<0.05, **<0.01, when compared with control
administered berberine 10mg/kg i/p twice daily for 2 days before group
CCl4 treatment. The CCl4 control group (group 2) was only The serum levels of AST, ALT, ALP and T. bilirubin were significantly
administered with distilled water of equal volume after CCl 4 toxicity. increased (p<0.01) in CCl4 treated group 2 rats. Group 3-5 rats
BLOOD SAMPLE COLLECTION treatedwith CCl4 followed by berberine chloride at doses of 5, 10 and
20 mg/kg, i/p respectively showed significantdecrease (p<0.05) in
24 hours after CCl4 treatment, the animals were anaesthetized using AST, ALT, ALP and T. bilirubin levels when compared to group 2 rats.
thiopentone sodium, 40mg/kg, i/p. Blood samples were collected by The CCl4 (1ml/kg, i/p) intoxication elevated levels of serum
cardiac puncture in sterilized dry centrifuge tube and allowed to biochemical parameters: AST(529.39±86.13), ALT(502.56±47.03),
coagulate for 30 mins at 37 ºC. The serum was separated at 2500 ALP (293.84±32.37), T. bilirubin (0.81±0.06). The total liver weight
rpm (micro centrifuge) for 10 mins and subjected to biochemical was also increased significantly (2.54±0.11, p<0.05) indicating acute
investigations viz. aspartate aminotransferase (AST), alanine hepatocellular damage and fatty degeneration.
aminotransferase (ALT), alkaline phosphatase (ALP) and total
bilirubin (TB). When compared with CCl4 toxic control group, the groups post
treated with berberine chloride at doses of 5,10 and 20 mg/kg, i/p in
ASSESSMENT OF LIVER FUNCTION CCl4 intoxicated rats exhibited significant reduction of
AST(343.11±175.70,70.27±20.33, 37.27±16.90), ALT
The AST, ALT, ALP activities in serum sample were analyzed by
(250.65±25.34,65.11±7.63,41.35±6.47),
using Chemistry Auto Analyzer (RT 1904C). The values were
ALP(200.83±32.20,81.28±15.03,48.88±6.04) and T.bilirubin
expressed in terms ofInternational Units/litre (IU/L).
(0.74±0.09,0.38±0.10, 0.23±0.03) levels and liver weight
HISTOPATHOLOGICAL ANALYSIS (2.38±0.08,2.22±0.01,2.03±0.05), (p<0.005). Hence, the activities of
the liver enzymes in berberine post treated groups of low dose,
Immediately after blood collection the animals were sacrificed by an medium dose and high dose decreased significantly in a dose
overdose of phenobarbitone 200 mg/kg, i/p. The liver of each rat dependent manner(AST:F=39.43, p<0.05;ALT:F=72.52, p<0.05;ALP:
was promptly removed and fixed in 10% formaldehyde solution for F=25.17, p<0.05) when compared with CCl 4 toxic control group. The
at least 24 hours before histopathological study. Samples were then high dose (20mg/kg) of berberine suppressed the AST,ALT, ALP and
embedded in paraffin wax and thin sections were stained with T.bilirubin levels to lower than normal levels as in group 1 (table1).
hematoxylin and eosin (H&E) and mounted on glass slides. The The effects of Berberine pretreatment on enzyme levels,T. bilirubin
degree of liver damage was estimated under light microscope and and liver weight show that, at dose of 10 mg/kg, the extract could
images were captured with Nikon Pentahead Microscope (ECLIPSE offer significant degree of protection against CCl 4 induced
80i, Y-THR-L, Japan) at original magnification of 210 X. hepatotoxicity. The serum levels of AST,ALT,ALP and T. bilirubin
decreased to 57.61±18.80,52.32±6.69,62.69±13.07 and 0.28±0.06
STATISTICAL ANALYSIS
respectively as compared to CCl4 toxic control group. The liver
The data were analyzed with the help of computer software SPSS weight also remained with in the normal limits indicating the
version 16 for windows. All data are expressed as mean ± SEM. The preventive action of Berberine.The results shown by the pre
results were inferred by one-way analysis of variance (ANOVA) test treatment group are comparable to the results with the medium
followed by histopathological analysis. For multiple group dose of Berberine post treatment group and are statistically
comparison, Fisher’s LSD test was used. P values (two tailed) <0.05* significant (p<0.05 in both groups).
&<0.001** were considered statistically significant and highly
The efficacy of extract was tested for its hepatoprotective activity,
significant respectively.
the relationship between dose and percentage reduction in each
RESULTS case has been shown in Fig 2a as below:

Effects of berberine on AST, ALT, ALP and T. bilirubin levels in rats

from various treatment groups are shown in Table 1 as below:

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Asian J Pharm Clin Res, Vol 6 Suppl 5, 2013, 53-56

The results from the histopathological studies of liver sections in

various groups are in agreement with the observed serum levels of
biochemical parameters.
Group 1-(Liquid paraffin- control, Fig 1) Section shows central veins
with radiating chords of hepatocytes (normal architecture)
Group 2-(CCl4 Toxic control, Fig 2) Section shows macro vesicular
fatty change around central vein and large areas of necrosis with
inflammation.
Group 3-(Berberine chloride-5 mg/kg +CCl4, Fig 3) section
showsleaky hepatocytes, macrovesicular fatty change
Group 4-(Berberine chloride-10 mg/kg +CCl4, Fig 4) section
showslymphomononuclear cell infiltrate in periportal and
centrilobular areas as well as scattered in hepatic parenchyma with
Figure 2a: shows effect of Berberine chloride on biochemical few cells showing feathery degeneration with significantly dilated
estimation of AST, ALT, ALP, T. bilirubin & liver weight and and congested sinusoids.
percentage reduction in various parameters after treatment
with different doses Group 5-(Berberine chloride-20mg/kg +CCl4, Fig 5) section
showspresence of normal hepatic cords, absence of necrosis and
The percentage reduction of various biochemical parameters after macrovesicular fatty change (near normal architecture)
treatment with berberine also showed significant results (p<0.05) in
AST (35%, 86%, 93%), ALT (50%, 87%, 91%), ALP (32%, 72%, 83), Group 6-(Pre- Berberine chloride 10 mg+CCl4, Fig 6) section shows
T. bilirubin (90%, 53%, 72%) and liver weight reduced to 6%, normal hepatic architecture, nearly similar to that of healthy control
12%and 20%. with very mild hepatic steatosis.

HISTOPATHOLOGY

Figure 1: Negative control- Liquid Paraffin Figure 2: CCl4 Toxic Control

Figure 3: Berberine chloride(5 mg/kg) + CCl4 Figure 4: Berberine chloride(10 mg/kg) + CCL4

Figure 5: Berberine chloride(20 mg/kg) + CCL4 Figure 6: Pre-Berberine chloride(10 mg/kg) + CCL4

Figure 1-6: show representative photographs of histopathological changes showing the effects of berberine chloride at different doses on
liver tissue of CCL4 intoxicated rats
Hepatic tissue changes were improved in the histopathology produced by the pre-treatment dose of 10mg/kg were similar to that
sections in berberine post and pre treated rats as compared to the produced by post treatment 10mg/kg dose as evidenced by the
toxic control group.The above results indicate the curative and decreased serum enzyme levels and T. bilirubin (Tab 1, Fig 1).These
preventive effect of berberine against CCl4 induced acute liver results were confirmed by the histopathological changes in the
damage in dose dependent manner. section of the liver (Fig 2 to Fig 6).Drugs are an important cause of
liver injury and account for 75% of the liver disease induced
DISCUSSION mortality and morbidity. The drug-induced liver injures range from
The aqueous root extract of Berberis aristata showed good asymptomatic elevation of liver enzymes to necrosis, fatty
hepatoprotective activity when administered at doses of 10mg/kg degeneration and fulminant hepatic failure [1,2]. Drugs having anti
and 20 mg/kg; i/p and the effects were dose dependent. The effects oxidant activity are effective in treating CCl 4 induced hepatotoxicity

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Asian J Pharm Clin Res, Vol 6 Suppl 5, 2013, 53-56

[15]. The root extract of Berberis aristatais known to have anti 3. Chatterjee TK. Medicinal Plants with Hepatoprotective
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There are reports about the habitat dependent variations in the Hepatoprotective effects of Coptidis rhizome aqueous
Berberine content of Berberis species found in the North Himalayan extract on carbon tetra chloride-induced acute liver
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In conclusion, the root extract of Berberis aristata showed good Protective effects of berberine against Low-Density
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The effects are both preventive and curative in nature and were cytotoxicity on endothelial cells. J Agne hood Chem.
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13. Andola HC, Gaira KS, Rawal RS, Rawat MS, Bhatt ID: Habitat-
ACKNOWLEDGEMENT
dependent variations in berberine content of Berberis
The authors are immensely grateful to Mr. Parmod Kumar for his asiaticaRoxb. ex. DC. In Kumaon, Western Himalayan. Chem
constant support throughout this research work. Biodivers.2010; 7(2):415-20.
14. Health Effect Test Guidelines: Acute Oral Toxicity (Computer
CONFLICT OF INTEREST Program) OPPTS 870,1100. United States Environmental
Protection Agency. December 2002;(7101):1-37.
The authors declare no conflict of interest.
15. Shenoy KA, Somayaji SN, Bairy KL: Hepatoprotective effect of
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