Hepatoprotective and Antioxidant Potential of Extracts from Whole
Plant of Solanum
surattense Burm
Patil Suhas A.*, Joshi V.G., Sambrekar
S.N. and Desai N. S.
Maratha Mandals College of Pharmacy, Belgaum, India.
ABSTRACT:
Objectives: Alcoholic and aqueous extracts of Solanum surattense Burm were investigated for their Hepatoprotective
and Antioxidant potentials against Liver injury induced by CCl4 in
rats.
Methods: The liver damage was induced in male
albino rats (150-200g) by administering CCl4 (2ml/kg) for 7 days and
extent of damage was studied by assessing biochemical and Antioxidant level in
liver parameters such as aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase
(ALP), total bilirubin, lipid peroxidase
(LPO) with a reduction of total protein, superoxide dismutase (SOD), catalase, and glutathione S-transferase
(GST) in experimental animals. Histopathological
changes of liver sample were compared with respective control.
Results: Treatment of rats with alcoholic and
aqueous plant extract (200mg/kg) for 7 days resulted in significant (P<0.001)
reduction in serum marker enzymes and antioxidant levels to near normal against
CCl4-treated rats. The protection effect was found to be comparable
with Liv52 treated group.
Conclusion: The alcoholic extracts of Solanum surattense Burm have
protected liver from CCl4 damage. The probable mechanism may be due
to Steroid and Tannins known for their Antioxidant activity may
potentially confer protection against oxidative stress induced liver injury.
KEYWORDS: Hepatoprotective,
Antioxidant, Solanum surattense Burm, Carbon tetrachloride, Biochemical parameters,
Histopathology
INTRODUCTION:
Herbal medicines have recently attracted much attention
as alternative medicines useful for treating or preventing life style related
disorders and relatively very little knowledge is available about their mode of
action. There has been growing interest in the analysis of plant products which
has stimulated intense research on their potential health benefits. The liver,
because of its strategic anatomical location, is targeted by many kinds of xenobiotics and therapeutic agents particularly when
administered orally. Moreover, the rapidly increasing morbidity and mortality
rates from liver diseases are largely attributable to the repeated chemical
insult either due to drug use/abuse or from environmental pollution.
Unfortunately so far, in the modern era of medicine there is no specific
treatment to counter the life threatening impact of these dreaded conditions1. Several plants have been investigated and reported to possess
antioxidant property and hepatoprotective activity
eg. Baliospermum montanum2,
Ocimum sanctum3, Tamarindus
indica3 etc. Similarly
traditionally Solanum
surattense burm. family -Solanaceae4,5 is used as Antiasthamatic,
aperients, alterative, astringent, digestive, diuretic, febrifuge and pungent.
Used in bronchitis, cough, constipation and in dropsy6, 7,8,9,10..
It is also used
as anthelmintic, appetizer, carminative, helmenthiasis, flatulence, colic, dyspepsia, anorexia,
leprosy, skin disorders, rheumatoid arthritis, phryngitis,
urolithiasis, amenorrhoea,
lumbago, cardiac disorders7,8,10 stomachic, anti-inflammatory, rejuvenating, epilepsy,7,11 enlargement of liver and
spleen.12 Though the feedback from the patients treated by
traditional healers is quite encouraging, hepatoprotective
activity of Solanum
surattense Burm has not been scientifically investigated. Therefore, the
present study was planned to investigate the effect of extracts of Solanum surattense Burm in CCl4 induced
liver damage in Wistar rats.
MATERIALS AND METHODS:
Preparation
of Solanum surattense burm Extract:
Whole plant of Solanum surattense burm was collected from open field around Belgaum city in the month
of September were identified and authenticated by the taxonomist Dr. Harsha Hegde and the herbarium
(voucher No. RMRC-481) has been preserved at
Regional Medical Research Centre Belgaum, a unit of Indian council for medical
research New Delhi. Dried plants were powdered to moderately coarse grade.
Petroleum ether, chloroform, alcohol and aqueous extracts were obtained by
using soxhlet extractor. The extraction was continued
for 12 cycles or until the solvent in the thimble was clears. After evaporating
the solvent, the dark brown semisolid extract was obtained .The phytochemical analysis was carried out and further extract
was kept in an air tight container at 40c for future use.
Suspensions of each extract were freshly prepared using 0.1% Tween 80, for experimental use.
Phytochemical
screening:
A preliminary phytochemical
screening of Solanum surattense burm was carried out. The phytochemical
profile was performed as described by Wagner et al13. The presence of Glycosides, carbohydrates, fats and
oils alkaloids, proteins steroids and tannins.
Animals:
Swiss albino mice
(2025 g) and male Wistar rats (150200 g) were
procured from Venkatershwara Enterprises, Bangalore,
Karnataka, India, and used throughout the study. They were housed in microlon boxes in a controlled environment (temperature
25±2°C and 12 h dark/light cycle) with standard laboratory diet and water ad
libitum. The study was conducted after obtaining
Institutional Animal Ethical Committee clearance.
Acute toxicity
studies:
Acute oral
toxicity (AOT) of Solanum
surattense
was determined using Swiss albino mice. The animals were fasted for 12 h prior
to the experiment and were administered with single dose of extracts dissolved
in 5% gum acacia and observed for mortality up to 48 hour (short term
toxicity). Based on the short-term toxicity, the dose of next animal was
determined as per OECD guideline 420.
Hepatoprotective
activity:
Rats were divided into five
groups, each group consisting of six animals.
Group I: Controls received the
vehicle of normal saline (2 ml/kg, p.o.).
Group II: Received CCl4 (2 ml/kg, s.c.) at every 72 h for 10 days14.
Group III: Received Liv52 3ml/kg p.o. for
10 days and simultaneously administered CCl4 (2 ml/kg, s.c.)
at every 72 h.
Group IV: Received alcoholic
extract of Solanum
surattense
200 mg/kg p.o. for 10 days and simultaneously
administered CCl4 (2
ml/kg, s.c.) at every 72 h.
Group V: Received aqwueous extract of Solanum surattense 200 mg/kg p.o.
for 10 days and simultaneously administered CCl4 (2 ml/kg, s.c.) at every 72 h.
At the end of experimental period,
all the animals were sacrificed by cervical decapitation. Blood samples were
collected, allowed to clot. Serum was separated by centrifuging at 2500 rpm for
15 min and analyzed for various biochemical parameters.
Assessment of
liver function:
Biochemical
parameters i.e., aspartate amino transferase
(AST)15, alanine amino transferase
(ALT)15 , alkaline phosphatase (ALP)16,
total bilirubin17 and total protein18, were analyzed
according to the reported methods. The liver was removed, morphological changes
were observed. A 10% of liver homogenate was used for antioxidant studies such
as lipid peroxidation (LPO)19, superoxide
dismutase (SOD)20, Catalase21, and glutathione S-transferase (GST)22. A portion of liver was
fixed in 10% formalin for histopathological studies.
Histopathological
studies:
After draining
the blood, liver samples were excised, washed with normal saline and processed
separately for histopathological observation.
Initially the materials were fixed in 10% buffered neutral formalin for 48 hour
and then with bovine solution for 6 hour.
Paraffin sections were taken at 5 mm thickness processed in alcohol-xylene series and was stained with alum hematoxylin
and eosin. The sections were examined microscopically for histopathology
changes.
Statistical
analysis:
The values were expressed as mean
± SEM. Statistical analysis was performed by one way analysis of variance
(ANOVA) followed by Tukey multiple comparison tests.
P values < 0.05 were considered as significant.
RESULTS:
Preliminary phytochemical
studies revealed the presence of Glycosides, carbohydrates, fats and oils
alkaloids, proteins steroids and tannins compounds. For the acute oral toxicity
studies, the extract treated animals were observed for mortality up to 48 h.
Based on the results the extract did not produce any mortality up to 2000 mg/kg
body weight.
Table 1. Effect of of
Solanum surattense on
biochemical parameters in CCl4-induced hepatotoxicity in rats
|
Treatment |
Dose
(mg/kg) |
AST U/L |
ALT U/L |
ALP U/L |
Total
Protein mg% |
Total bilirubin mg% |
|
Control |
|
46.32 ± 0.418 |
92.12
± 0.641 |
122.40
± 0.812 |
9.17
± 0.057 |
0.97
± 0.009 |
|
CCl4 |
2
ml/kg |
294.20
± 0.616a |
483.60
± 0.441a |
259.10
± 1.241a |
5.42
± 0.155 a |
5.617
± 0.124 a |
|
Liv52 |
3ml |
85.08
± 0.596a,d |
128.20
± 0.484a,d |
136.0
± 0.362a,d |
8.22
± 0.131 a,d |
1.16
± 0.162 a,d |
|
Alcoholic extract |
200 |
88.27
± 0.432ad |
156.6
± 0.606a,d |
141.7
± 0.805a,d |
8.1
± 0.133 ad |
1.2
± 0.074a, d |
|
Aqueous extract |
200 |
114.3
± 0.546a,d |
218.5
± 0.352a,d |
164.20
± 0.672a,d |
7.30
± 1.107 a,d |
2.01±
0.085a,d |
N = 6; Values are expressed as
mean ± SEM; a P< 0.001; b P< 0.01; c P< 0.05 Vs Control; d P<
0.001; e P< 0.01; f P< 0.05 Vs
CCl4
Data were analyzed by using one way ANOVA followed by Tukey multiple comparison test.
Table 2. Effect of Solanum surattense on Antioxidant level
in CCl4-induced hepatotoxicity in rat
|
Treatment |
Dose
(mg/kg) |
LPO |
SOD |
CAT |
GST |
|
Control |
|
11.40
± 0.161 |
33.67
± 0.366 |
59.16
± 0.223 |
0.44
± 0.025 |
|
CCl4 |
2
ml/kg |
24.22
± 0.088a |
18.76
± 0.245a |
37.39
± 0.276a |
0.10
± 0.005 a |
|
Liv52 |
3ml |
10.0
± 0.242a,d |
28.36
± 0.309a,d |
48.64
± 0.167a,d |
0.31
± 0.014 a,d |
|
Aqueous extract |
200 |
14.33
± 0.226ad |
25.56
± 0.286a,d |
44.18
± 0.214a,d |
0.18
± 0.012 a,d |
|
Alcoholic
extract |
200 |
11.48
± 0.284a,d |
21.76
± 0.248a,d |
40.30±188a,d |
0.22
± 0.015 ad |
N = 6; Values are expressed as
mean ± SEM; a P< 0.001; b P< 0.01; c P< 0.05 Vs Control; d P<
0.001; e P< 0.01; f P< 0.05 Vs CCl4
Data were analyzed by using one
way ANOVA followed by Tukey multiple comparison test.
LPO = μ moles of MDA/ min/mg
protein; SOD = Units/min/mg protein; CAT = μ mole of H2O2 consumed/ min/mg
protein
GST = μ moles of CDNB
conjugation formed/min /mg protein
The effect of Solanum surattense on serum marker
enzymes is presented in Table 1. The levels of serum AST, ALT, ALP, total bilirubin, were markedly elevated and that of protein
decreased in CCl4 treated animals, indicating liver damage.
Administration of alcoholic and aqueous extract of Solanum surattense at the doses of 200
mg/kg remarkably prevented CCl4-induced hepatotoxicity
in a dose dependent manner. Analysis of LPO levels by thiobarbituric
acid reaction showed a significant (P<0.001) increase in the CCl4
treated rats. Treatment with alcoholic and aqueous extract of Solanum surattense (200
mg/kg) significantly (P<0.001) prevented the increase in LPO level which was
brought to near normal. The effect of Solanum surattense was comparable with that of standard drug Liv52 (Table 2).
CCl4 treatment caused a
significant (P<0.001) decrease in the level of SOD, Catalase
and GST in liver tissue when compared with control group (Table 2). The
treatment of alcoholic and aqueous extract of Solanum surattense at the dose of 200
mg/kg resulted in a significant increase of SOD, Catalase
and GST when compared to CCl4 treated rats. The liver of Liv52
treated animals also showed a significant increase in antioxidant enzymes
levels compared to CCl4 treated rats.
Morphological observations showed
an increased size and enlargement of the liver in CCl4 treated
groups. These changes were reversed by treatment with Liv52 and also extract of Solanum surattense at the
doses tested.
Histopathological studies, showed CCl4 to produce
extensive vascular degenerative changes and centrilobular
necrosis in hepatocytes. Treatment with alcoholic and
aqueous extract of Solanum
surattense.
produced mild degenerative changes and absence of centrilobular
necrosis when compared with control. All these results indicate a hepatoprotective potential of the extract.
DISCUSSION:
Carbon tetrachloride is one of the
most commonly used hepatotoxins in the experimental
study of liver diseases. The hepatotoxic effects of CCl4
are largely due to its active metabolite, trichloromethyl
radica23. These activated radicals bind covalently to the
macromolecules and induce peroxidative degradation of
membrane lipids of endoplasmic reticulum rich in polyunsaturated fatty acids.
This leads to the formation of lipid peroxides. This lipid peroxidative
degradation of biomembranes is one of the principle
causes of hepatotoxicity of CCl424.
This is evidenced by an elevation in the serum marker enzymes namely AST, ALT,
ALP, total bilirubin and decrease in protein.
In the assessment of liver damage
by CCl4 the determination of enzyme levels such as AST, ALT is
largely used. Necrosis or membrane damage releases the enzyme into circulation
and hence it can be measured in the serum. High levels of AST indicates liver
damage, such as that caused by viral hepatitis as well as cardiac infarction
and muscle injury, AST catalyses the conversion of alanine
to pyruvate and glutamate and is released in a
similar manner. Therefore ALT is more specific to the liver, and is thus a
better parameter for detecting liver injury. Elevated levels of serum enzymes
are indicative of cellular leakage and loss of functional integrity of cell
membrane in liver25. Serum ALP, bilirubin
and total protein levels on other hand are related to the function of hepatic
cell. Increase in serum level of ALP is due to increased synthesis, in presence
of increasing biliary pressure26.
Administration of CCl4 caused a significant (P<0.001) elevation
of enzyme levels such as AST, ALT, ALP, total Bilirubin
and decrease in total protein when compared to control. There was a significant
(P<0.001) restoration of these enzyme levels on administration of the leaf
extract in a dose dependent manner and also by Liv52 at a dose of 3 ml/kg. The
reversal of increased serum enzymes in CCl4-induced liver damage by
the extract may be due to the prevention of the leakage of intracellular
enzymes by its membrane stabilizing activity. This is in agreement with the
commonly accepted view that serum levels of transaminases
return to normal with the healing of hepatic parenchyma and the regeneration of
hepatocytes27. Effective control of ALP, bilirubin
and total protein levels points towards an early improvement in the secretary
mechanism of the hepatic cells.
A) Hepatocytes of Control
group showed Normal tubular architecture of liver
B) Hepatocytes of CCl4
treated group showed liver cell necrosis
C) Hepatocytes of Liv52 pretreated group showed normal tubular architecture
D) Hepatocytes of Alcoholic extract pretreated Group showed minimal inflammation with moderate portal triadities and normal tubular architecture
E) Hepatocytes of Aqueous extract pretreated Group
showed minimal inflammation with moderate portal triadities
and normal tubular architecture
The efficacy of any hepatoprotective drug is dependent on its capacity of
either reducing the harmful effect or restoring the normal hepatic physiology
that has been distributed by a hepatotoxin. Both
Liv52 and the plant extract decreased CCl4 induced elevated enzyme
levels in tested groups, indicating the protection of structural integrity of hepatocytic cell membrane or regeneration of damaged liver
cells.
The increase in LPO level in liver induced by CCl4
suggests enhanced lipid peroxidation leading to
tissue damage and failure of antioxidant defense mechanism to prevent formation
of excessive free radicals. Treatment with Solanum surattense significantly reverses
these changes. Hence it is likely that the mechanism of hepatoprotection
of Solanum surattense is due
to its antioxidant effect.
Decrease in enzyme activity of superoxide dismutase
(SOD) is a sensitive index in hepatocellular damage
and is the most sensitive enzymatic index in live injury. Curtis and Mortiz28,
SOD has been reported as one of the most important enzymes in the enzymatic
antioxidant defense system. It scavenges the superoxide anion to form hydrogen
peroxide and thus diminishing the toxic effect caused by this radical. In Solanum surattense causes
a significant increase in hepatic SOD activity and thus reduces reactive free
radical induced oxidative damage to liver.
Catalase (CAT) is an enzymatic antioxidant widely distributed
in all animal tissues, and the highest activity is found in the red cells and
liver. CAT decomposes hydrogen peroxide and protects the tissues from highly
reactive hydroxyl radicals29. Therefore reduction in the activity of
CAT may result in a number of deleterious effects due to the assimilation of
superoxide radical and hydrogen peroxide. A higher dose (500 mg/kg) increases
the level of CAT as produced by Liv 52, the standard hepatoprotective drug.
Glutathione is one of the most abundant tripeptide, non-enzymatic biological antioxidant present in
the liver. It removes free radical species such as hydrogen peroxide,
superoxide radicals and maintains membrane protein thiols.
Decreased level of GSH is associated with an enhanced lipid peroxidation
in CCl4 treated rats. Administration of Solanum surattense significantly
(P<0.001) increased the level of GST in a dose dependent manner.
Extensive vascular degenerative changes and centrilobular necrosis in hepatocytes
was produced by CCl4. Treatment with alcoholic and aqueous extract
of whole plant of Solanum
surattense
produced only mild degenerative changes and absence of centrilobular
necrosis, indicating its hepatoprotective efficiency.
Free radical mediated process has been implicated in
pathogenesis of most of the diseases. The protective effect of Solanum surattense on CCl4
induced hepatotoxicity in rats appears to be related
to inhibition of lipid peroxidation and enhancement
of antioxidant enzyme levels in addition to free radicals scavenging action.
Preliminary phytochemical studies reveal the presence
of steroids and tannins in the extracts of
Solanum surattense.
steroids and tannins are hepatoprotectives30,31. The observed
antioxidant and hepatoprotective activity of Solanum surattense may be
due to the presence of steroids and tannins. Further studies to characterize
the active principles and to elucidate the mechanism are in progress.
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