Protective Effect of a Herbal Formula Against Carbontetrachloride Induced Hepatotoxicity
This study investigated the protective effects of a
polyherbal formula BCEZ, containing extracts of Bacopa monneiri
Linn. Penn., Curcuma longa Linn., Emblica officinalis Gaertn.
and Zingiber officinale Rosc., on the carbon tetrachloride (CCl4)
induced hepatotoxicity in rats. Hepatic injury was achieved by injecting
0.5 mL kg-1, i.p. of CCl4. The BCEZ at the doses
50, 100 and 250 mg kg-1, p.o. offered significant hepatoprotective
action by reducing the serum marker enzymes like Serum Glutamate Oxaloacetate
Transaminase (SGOT) and Serum Glutamate Pyruvate Transaminase (SGPT).
They also reduced the elevated levels of alkaline phosphatase (ALP). Histopathological
studies further confirmed the hepatoprotective activity of BCEZ when compared
with the CCl4 treated control groups. The results obtained
were compared with silymarin (100 mg kg-1, p.o.), the standard
drug. Thus it can be concluded, BCEZ might be a potential herbal agent
for its hepatoprotective activity.
Liver diseases are world wide problem. Liver is the most important organ,
which plays a pivotal role in regulating various physiological processes
in the body. It has great capacity to detoxicate toxic substances and
synthesize useful principles. The spectrum of its functions include metabolism
and disposition of chemicals (xenobiotics) to which the organ is exposed
directly or indirectly, metabolism of lipids, carbohydrates and proteins,
blood coagulation and immunomodulation. Accordingly, the disorders associated
with this vital organ are numerous and varied. Conventional synthetic
drugs used in the treatment of liver diseases are sometimes inadequate
and can have serious adverse effects. So there is a worldwide trend to
go back to traditional medicinal plants (Venkateswaran et al.,
1997; Mitra et al., 2000; Dhuley and Naik, 1997). Therefore, damage
to the liver inflicted by hepatotoxic agents is of grave consequences.
There is an ever increasing need of an agent which could protect it from
such damage. In view of severe undesirable side effects of synthetic agents,
there is growing focus to follow systematic research methodology and to
evaluate scientific basis for the traditional herbal medicines which are
claimed to possess hepatoprotective activity (Subramoniam et al.,
1998). The development of herbal formulation as naturally occurring inhibitors
of peroxidation and resulting to reduce cell damage without any side effects.
Therefore, lead to important new strategies for disease prevention in
BCEZ is a herbal formula containing whole plant of Bacopa monnieri
Linn. Penn.,fruits of Emblica officinalis Gaertn, rhizome
of Curcuma longa Linn. and Zingiber officinale Rosc. All
the 4 Indian medicinal plants have different biological properties. Curcuma
longa Linn. (Zingiberaceae), commonly known as haridra in Hindi
and turmeric in English is commonly used as an antacid, carminative, stomachic,
blood purifier, wound healing, anti-inflammatory, antibacterial, antiviral
and antioxidant activities (Ammon and Whal, 1991). It is widely used in
the coloring agent in food items and it is a major component of curry
powder. Its medicinal properties have been attributed mainly due to the
presence of curcuminoids. The main component present in the rhizome includes
curcumin, demethoxy curcumin and bisdemetohxy curcumin. Zingiber officinale
Rosc. (Zingiberaceae), is commonly known as sunthi in Hindi and
ginger in English. Ginger taste is sweet, pungent, act as appetizer, an
aphrodisiac and carminative in nature. In Ayurveda, it is considered a
valuable medicine because of its action as rubifacient, antiasthmatic
and stimulant to the gastrointestinal tract. Ginger has been showed the
hypolipidaemic, anti-inflammatory and antihepatotoxic activity (Bhandari
et al., 1998; Penna et al., 2003). The main active ingredients
present in the ginger are gingerols. Emblica officinalis Gaertn.,
(Euphorbiaceae), commonly known as Amla in Hindi. The fruits of the plant
are fleshy with sour, astringent taste and are consumed as raw, cooked,
or even pickled locally. The fruits have been reported to possess antioxidant,
adaptogenic, hepatoprotective, antifungal, antipyretic, analgesic, gastroprotective,
hypolipidaemic, antiulcerogenic activities (Bhattacharya et al.,
1999; Rege et al., 1999; Jose and Kuttan, 2000; Dutta et al.,
1998; Perianayagam et al., 2004; Al-Rehailya et al., 2002;
Mathur et al., 1996; Sairam et al., 2002a, b). Bacopa
monnieri Linn. Penn. (Scrophulariaceae), commonly known as Brahmi,
is reported to be useful in the treatment of insanity, epilepsy and as
an effective drug for nerve tonic and its bacosides are used as a memory
enhancer. Brahmi has been reported to posses antioxidant activity (Tripathi
et al., 1996).
Recently, several traditional formula comprising 4 or more herbs have
been successfully used to treat liver disorders (Achliya et al.,
2004; Mitra et al., 1998). In view of severe undesirable side effects
of synthetic drugs, there is growing focus to follow systematic research
methodology develop and evaluate the traditional herbal formulations which
can be claimed to possess hepatoprotective activity. The aim of present
experimental study on rats, a systematic research was undertaken to evaluate
the possible effect of the formulation on the hepatotoxicity induced by
CCl4 agents and this communication substantiates the therapeutic
utility of the formulation as a hepatoprotective agent.
MATERIALS AND METHODS
Plant material: The plant material used in this study were authenticated
by Dr. H.B. Singh of National Institute of Science Communication and Information
Resources, New Delhi, India. The authenticated sample of plant material
of Bacopa monnieri Linn. Penn. (whole plant, voucher specimen
no. NISCAIR/RHMD/Consult/07-08/882/66/4), Curcuma longa
Linn. (rhizome, voucher specimen No. NISCAIR/RHM/F-3/2006/Consult/723/40),
Emblica officinalis Gaertn. (fruits, voucher specimen No.
NISCAIR/RHMD/Consult/06-07/790/107) and Zingiber officinale Rosc.
(rhizome, voucher specimen no. NISCAIR/RHM/F-3/2004/Consult./495/71) were
collected from Bangalore in the month of June and provided by M/s Natural
Remedies Pvt. Ltd. Bagalore, India.
Preparation and standardization of plant extracts: Coarse powder
of the dried material of Bacopa monnieri, Curcuma longa, Emblica
officinalis and Zingiber officinale was separately extracted
to exhaustion with methanol using a soxhlet apparatus. The methanolic
extract thus obtained was dried separately under reduced pressure at a
room temperature not exceeding 40°C.
Herbal formulation BCEZ: The polyherbal formulation consists of
methanolic extract of Bacopa monnieri, Curcuma longa, Emblica
officinalis and Zingiber officinale in the ratio of 1:1:2:1.
The extracts were mixed properly.
Experimental animals: The study was carried out on mixed sex of
Wistar albino rats (175-210 g) inbred at our animal house. They were fed
with a standard pellet (Golden Feed, New Delhi, India) and water ad
libitum. The rats were kept in standard environmental conditions (temperature
25-28°C and 12 h light/12 h dark cycle) at 37°C and was used for
the estimation of various biochemical parameters.
Treatment: All plant extracts were individually weighed and mixed
properly. The drugs were administered as oral 2% gum acacia suspension.
The group 1 animals of the control group received Vehicle (2% gum acacia
suspension) for seven days. The animal of group 2 also received vehicle
for seven days. The animals of group 3 received the silymarin (100 mg
kg-1 p.o.) for seven days. Animals of group 4, 5 and 6 received
BCEZ at a dose of 50, 100 and 250 mg kg-1 p.o., respectively
for seven days. On 8 day groups 2-6 received carbon-tetra chloride (CCl4,
0.5 mL kg-1, i.p.). The liver was excised out after perfusion,
washed with chilled normal saline solution and 10% w/v liver homogenates
were prepared in ice-cold 0.15 M KCl solution.
Assessment of liver functions: Twenty-four hours after the toxin
administration, the rats of each group were anaesthetized and blood was
collected directly from the heart. The blood samples were allowed to clot
for 20-30 min. Serum was separated by centrifugation at 37°C and used
for estimation of various biochemical parameters.
Assay of serum transaminases: The activities of Serum Glutamate
Oxaloacetate Transaminase (SGOT) and of Serum Glutamate Pyruvate Transaminase
(SGPT) were estimated by using Ecoline kit (E-merck). The enzyme activity
was expressed as mL-1.
Assay of alkaline phosphatase: The activity of serum alkaline
phosphatase (ALP) was estimated by using the Ecoline kit (E-merck). The
enzyme activity was expressed as KA unit.
Estimation of total protein: Total protein (TP) were estimated
by the methods of Lowry et al. (1951). The units were expressed
as g dL-1.
Statistical analysis: Results of the biochemical estimations are
reported as Mean±SD Total variation, present in a set of data was
estimated by one-way analysis of variance (ANOVA) followed by Dunnett`s
test. Minimum level of significance was fixed at p<0.05.
RESULTS AND DISCUSSION
Rats treated with a single dose of CCI4 developed significant
hepatic damage as observed from elevated serum levels of hepatospecific
enzymes as well as severe alterations in different liver parameters (Table
1). Oral administration of BCEZ is seen to lower the levels of marker
enzymes namely SGOT, SGPT and ALP compare to CCl4 treated group
(Table 1). The level of serum proteins was significantly
(p<0.01) increased in rats, which received BCEZ as compared
to CCI4 group (Table 1).
BCEZ demonstrated protective effect in rats against CCl4
induced hepatotoxicity in doses ranging from 50-250 mg kg-1.
The effect of BCEZ seems to be dose dependent. However, the protection
offered by silymarin seemed relatively greater. Figure 1
exhibits the histological section of liver of rats treated with BCEZ.
The normalcy of hepatic cells, central vein and portal triad can be easily
observed. The degree of protection was observed maximally with the highest
dose of the extract.
The present study brings about the potential hepatoprotective activity
of BCEZ and gives insight into its mechanism of action. Liver injury induced
by CCL4 is the best-characterized system of the xenobiotic-induced
hepatotoxicity and is a commonly used model for the screening the anti-hepatotoxic/hepatoprotective
activity of drugs (Brautbar and Williams, 2002; Brent and Rumack, 1993).
In this study, rat treated with single dose of CCl4 developed
a significant hepatic damage, which was observed from a substantial increase
in the activities of serum, SGOT and SGPT. This is indicative of cellular
leakage and loss of functional integrity of cell membrane in liver (Sallie
et al., 1991). Reduction in the levels of SGOT and SGPT towards
the respective normal values by herbal formula of three different doses
(50, 100 and 500 mg kg-1) is an indication of the stabilization
of plasma membranes as well as repair of hepatic tissue damage caused
by CCl4. This effect is in agreement with the commonly accepted
view that serum levels of transaminases return to normal with healing
of hepatic parenchyma and the regeneration of hepatocytes (Maiti et
al., 2005). In the present study, also it was seen that administration
of CCl4 elevates the levels of serum marker enzymes SGPT, SGOT
and ALP and level of total protein is lowered. BCEZ and silymarin treated
groups exhibited lower levels of SGPT, SGOT and ALP as compared to CCl4
treated group. The treatment with BCEZ also significantly elevated
total protein levels. The stabilization of serum SGPT, SGOT and ALP levels
by BCEZ is a clear indication of the improvement of the functional status
of the liver cells. The characteristics feature of experimental hepatic
damage observed is significant decrease in protein level. The rats in
a group which received BCEZ showed rectification of lowered protein levels.
These findings can be further corroborated with histopathological studies.
The histopathological examination clearly reveals that the hepatic cells,
central vein and portal triad are almost normal in BCEZ (250 mg kg-1,
p.o.) group in contrast to group which received CCl4.
||Effects of BCEZ treatment on different biochemical
parameters in the serum of rats
|*p<0.05 and **p<0.01 as compared to control group
by Dunnett`s test
||Effects of BCEZ pretreatment on the CCl4-induced
liver damage in rats. (A) Liver from rat treated with 2% gum acacia
(B) Liver from a rat treated with CCl4, (C) Liver from
a rat treated with BCEZ (50 mg kg-1) plus CCl4,
(D) Liver from a rat treated with BCEZ (100 mg kg-1) plus
CCl4; (E) Liver from a rat treated with BCEZ (250 mg kg-1)
plus CCl4. N; necrosis
Thus, BCEZ can be considered to be an effective hepatoprotective herbal
formula as it ameliorates almost to normalcy the damage caused by CCl4
to hepatic function. It is difficult at this stage to comment on the rational
of inclusion of such herbs together in single formulation for hepatic
protection but the results of this study demonstrated that pretreating
the rats with BCEZ effectively protected the rats against CCl4
induced hepatotoxicity, as evidenced by a significant reduction in the
CCl4 induced rise in SGOT and SGPT levels in rats in a dose-dependent
manner. This phenomenon was also confirmed by histological observation.
The BCEZ formulation used in this study seems to preserve the structural
integrity of the hepatocellular membrane.
We thank to the University Grant Commission (UGC), Government o f India,
for providing fellowship (Grant No. F.10-13/2004 (SA-I)) to first author.
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