Antioxidant and Hepatoprotective Potential of Isolated Fraction of Revia hypocrateriformis
Successive Ethanolic Extracts (SEE) of Rivea hypocrateriformis (RH) which recorded highest polyphenolic contents was subjected to fractionation by column chromatography. All fractions were tested for in vitro antioxidant activity. Out of these fractions, SEE2 possessed significant antioxidant activity. This fraction was further tested for in vivo antioxidant and hepatotoxicity in rat liver using carbon tetrachloride (CCl4), showed significant activity. Further, this claim was confirmed by pretreatment of mice with SEE2 shortened the duration of pentobarbitone induced necrosis. The HPLC analysis was performed for same fraction evidenced the presence of gallic acid and lupeol along with other polyphenol when compared with markers.
August 14, 2011; Accepted: October 23, 2011;
Published: December 10, 2011
Presense of antioxidants in foods or body delays oxidation of oxidizable substrate.
Antioxidants may help the body to protect itself against various types of oxidative
damage caused by reactive oxygen species, which are linked to a variety of diorders
including cancer, diabetes, shock, arthritis, and acceleration of the ageing
process (Shahidi, 1997). Several groups of polyphenols
(anthocyanins, tannins, flavanones, isoflavones, resveratrol and ellagic acid)
are currently used as antioxidants in the industry as nutraceuticals or functional
foods (Espin et al., 2007). RH is climbing shrub
known for a large number of biological activities such as antidiabetic, antiimplantation,
in treatment of burning and piles, antidepressant, anticancer and analgesic
properties (Dhawan et al., 1980; Shivalingappa
et al., 1999). Chemically it known to contains amino acid and sugar
(Dhore et al., 2001). In literature survey, no
evidence was reported for biological and bio-chemical investigation of RH. In
the present study an attempt has been made for evaluation of antioxidant and
hepatoprotective activities along with phytochemical investigations.
MATERIALS AND METHODS
Plant material: Aerial parts of R. hypocrateriformis were collected in the month of August- September from Amravati District, Maharashtra and authenticated by Prof. Dr. Bhowagaokar, VIHS, Amravati, Maharashtra, India. A voucher specimen (AMT-36) has been preserved for future reference.
Extraction and fractionation: The dried powder material (10 kg) was
extracted successively extracted with petroleum ether (SPE), chloroform (SCE),
ethanol (SEE) and water (SAE) as per the increasing order of their polarity.
All solvents were evaporated to dryness under pressure using rotary flash evaporator
to obtain crude extracts. The Successive Ethanol Extract (SEE) was selected
for further fractionation based on polyphenolic content. SEE was subjected to
column chromatography over silica gel (60-120 mesh) using varying proportion
of chloroform: methanol (90:10, 75:25, 50:50, 0:100 v/v) as eluent. All fractions
were collected and concentrated to dryness on a rotary flash evaporator.
These fractions were screened for in vitro antioxidant activity.
Animals: Wistar albino rats (150-250 g) and Swiss albino mice (25-35 g) each of either sex, obtained from the Institutes animal house were used. Rats were housed under standard laboratory conditions and were fed commercial rat feed (Lipton India Ltd., Mumbai, India) and boiled water, ad libitum. All animal experiments were carried out according to institutional animal ethical committee (Approval letter no. GCPA/ IAEC/ 2011/ 1245).
Total phenolic content (TP): The TP content was determined by Folin-Ciocalteau
colorimetric method of Singleton et al. (1999).
The TP content was calculated from calibration curve of gallic acid and expressed
as gallic acid equivalents.
In vitro antioxidant capacity
DPPH and ABTS radical scavenging assay: ABTS and DPPHÿ•+
quenching ability was measured according to Kalaskar and
Surana (2011). The antiradical activity was expressed as IC50
Nitro blue tetrazolium (NBT) reduction assay: Effect of scavenging superoxide
radical was determined by the nitroblue tetrazolium reduction method of Fu
et al. (2010). The absorbance of samples was measured at 560 nm against
Lipid peroxidation inhibition activity by ferric thiocyanate method (FTC):
The FTC method was adapted from Fu et al. (2010).
All measurements were made in triplicate and averaged. The inhibition rate was
calculated using the equation.
where, Ac is absorbance of control; As is absorbance of sample.
CCl4 induced hepatotoxicity: CCl4 induced hepatotoxicity
in rats carried out according to Kalaskar and Surana (2011).
All studies were carried out in double dose, 50 mg and 100 mg kg-1
body wt. Samples of organ, livers and kidneys from all the animals were dissected
at the end of experiment, washed and used for histological studies and biochemical
Pentobarbitone-induced sleeping time studies: Pentobarbitone induced
sleep studies carried out according to Kalaskar and Surana
(2011). All groups of animals were given pentobarbitone (PBT, 40 mg kg-1,
IP), 2 h after CCl4/vehicle treatment. The time between loss of righting
reflex and its recovery was recorded.
Histopathological studies and Biochemical determinations: After blood draining, liver samples were excised from the control and treated groups of animals and washed with normal saline separately. All samples were fixed in 10% buffered formalin for 48 h. Each sample was stained with haematoxylin-eosin (H and E) for photomicroscopic observations of the liver histological architecture and homogenates of liver and kidney were used for determination of SOD, CAT, MDA, and GSH.
HPLC analysis of SSE2: HPLC analysis confirmed the presence of gallic
acid and lupeol in the SEE2 fraction using gallic acid and lupeol (Sigma-aldrich
Chemie, Steinheim, Germany) as standard markers. The separation of the components
was performed on Phenomenix C18 column (250x4.6 mm I.D., 5 μm particle
size). The separation of gallic acid was achieved using methanol + water (70:30
v/v) as mobile phase, flow rate was adjusted to 0.7 mL min-1 and
dectection was performed at 280 nm. While, for lupeol acetonitrile: water (90:10
v/v) was used as mobile phase, flow rate 1.0 mL min-1 and detected
at 230 nm.
Statistical analysis: Results were expressed as Mean±SEM. Data were analyzed using one-way Analysis of Variance (ANOVA) followed by Dunnetts test. Value of p<0.05 was considered to be statistically significant.
RESULTS AND DISCUSSION
Total phenolic contents: Phenolic compounds are considered to be the
major contributors to the antioxidant capacity of plants. Some of diverse biological
activities of plant may also be related to their antioxidant activity (Chung
et al., 1998). The phenolic content of RH was determined. The SEE
had highest concentration of phenolics 55.16% amongst the fractions 32.12, 39.21,
25.33% for SPE, SCE and SAE, respectively. The SEE further subjected to column
In vitro antioxidant activity: Antioxidant activity of different
fractions of SEE, SEE1, SEE2, SEE3 and SEE4 were evaluated by free radical scavenging
by ABTS and DPPH, superoxide scavenging activity and lipid peroxidation. The
concentration of each fraction required to inhibit each radical by 50% (IC50)
is shown in Table 1. The result revealed that the SEE2 fraction
exhibits the most robust radical-scavenging activity amongst all fractions.
Inhibition of DPPH and ABTS radical indicates its direct role in trapping free
radicals by donating hydrogen atom or electron. The superoxide anion radical
(•O¯2) is the most common free radical generated
in vivo. Suppression of •O¯2 in presense
of SEE was also observed. Also SEE inhibited lipid peroxidation may be due to
termination of the radical chain reaction (Zhou et al.,
Hepatoprotective and in vivo antioxidant activities: Based on our in vitro antioxidant assays, SEE2 was chosen as the most potent fraction which was shortlisted for evaluation in vivo antioxidant and hepatoprotective potential.
|| IC50 value of various fractions of SEE
|Values are the Mean±SEM, n = 3
|| Effects SEE2 on rat serum parameters after CCl4
|Values are the Mean±SEM, n = 6. SGOT: Serum glutamate
oxaloacetate transaminase, SGPT: Serum glutamate pyruvate transaminase,
ALP: alkaline phosphatase, DB: direct bilirubin, TB: total bilirubin, TP:
Total protein, PBT: Pentobarbitone (a) p≤0.01 when compared with control.
(b) p≤0.01 when compared with toxicant. (c) p≤0.05 when compared with
toxicant. (d) p≤0.05 when compared with toxicant.
|| Effects of SEE2 on liver and kidney SOD, CAT, MDA and GSH
in CCl4-intoxicated rats
|Values are mean±SD, Statistical significance is indicated
by asterisks and double asterisks. SOD, superoxide dismutase; CAT, catalase;
MDA, malondialdehyde; GSH, glutathione. Group I: Control, Group II: CCl4,
Group III: silymarin 100 (mg kg-1), Group IV: SEE2 50 (mg kg-1),
Group V: SEE2 100 (mg kg-1). (a) p≤0.01 when compared with
control. (b) p≤0.01 when compared with toxicant. (c) p≤0.05 when compared
with toxicant. (d) p≤0.05 when compared with toxicant
The CCl4-induced hepatotoxicity model extensively used for the evaluation
of hepatoprotective and in vivo antioxidant effects. CCl4
is accumulated in hepatic parenchyma cells and metabolized to the CCl•3
(Recknagel, 1983). CCl3•
radical reacts very rapidly with oxygen to yield a highly reactive CCl3OO•.
These radicals react with proteins and lipids. They remove hydrogen atoms from
unsaturated lipids thus initiating lipid peroxidation which causes loss of integrity
of cell membranes and damage to hepatic tissue (Zhou et
al., 2010). Concentration of enzymes viz., SGPT, SGOT, ALP, TP, TB and
DB are considerably high in the cytoplasm. When liver cells are injured, these
enzymes leak into the blood stream and causing significant rise in blood levels
of these enzymes. The extent of liver damage is in proportion with the elevated
serum levels of these enzymes. Alkaline phosphatase is the prototype of these
enzymes that reflects the pathological alteration in biliary flow (Kalaskar
and Surana, 2011). CCl4 induced elevation of enzymatic activity
in the serum is in line with high level of serum bilirubin content. The SEE2
induced suppression of the increased ALP activity with the concurrent depletion
of raised bilirubin, suggest the ability of SEE2 to stabilize biliary dysfunction
in rat liver. In this study pre-treatment of rats with SEE2 prior to CCl4
administration caused a significant change in the values of SGOT, SGPT, ALP,
TP, TB and SB) in a dose dependent manner. However, SEE2 (100 mg kg-1)
showed highest hepatoprotective activity (Table 2) almost
comparable to the standard, Silymarin (100 mg kg-1) treated group
which was also supported by histological studies. The histological architecture
of CCl4 treated liver section showed marked massive fatty changes,
necrosis, ballooning degeneration and the loss of cellular boundaries. However,
necrosis was not observed in any groups treated with standard and SEE2, indicates
that sufficient hepatotoxicity does not seem to be developed so as to cause
necrosis (Fig. 1). Alternatively, reduction in the prolongation
of pentobarbitone induced sleep in CCl4 poisoned rats is further
indicative of the hepatoprotective potential of the SEE2. It has been established
that since the barbiturates are metabolized almost exclusively in the liver,
the sleeping time after a given dose is a measure of hepatic metabolism.
Further levels of MDA, CAT, GSH and SOD reflect the extent of peroxidation
damage. The decrease of key components of the antioxidant defence system, CAT,
SOD and GSH may result in a lot of deleterious effects due to the accumulation
of superoxide radicals and hydrogen peroxide (Nanjan et
al., 2007). The fact that SEE2 treatment reduced elevated MDA and increased
levels of SOD, CAT, and GSH (Table 3), indicated its role
in prevention of lipids peroxidation.
The phytochemical analysis of SEE has shown high phenolic content. In the present
study, the presence of gallic acid and lupeol in SEE2 was phytochemically confirmed
|| Histopathological architecture in the liver of control and
experimental rats treated with CCl4 (H and E X100). a-Normal
control, b-CCl4 Control, c-Silymarin treated, d-SEE2 treated
(50 mg kg-1), e- SEE2 treated (100 mg kg-1)
|| HPLC chromatogram of (a) authentic standards (b) Compound
identified in SEE2, GA-Gallic Acid, LP-Lupeol
Perhaps gallic acid, lupeol and other related phenolic compounds present in
SEE2 may be responsible for its observed antioxidant and hepatoprotective activity.
The antioxidant and free radical scavenging property of gallic acid (Kawashima
et al., 1996) and lupeol (Preetha et al.,
2006) was previously reported. These results suggest that prevention of
superoxide radical generation (Yamashita et al.,
2002) and binding of the gallate compounds to lipid membrane (Shahrzad
et al., 2001) was the principal determining factor of antioxidant
Thus, it can be assumed that the possible mechanism of the hepatoprotective and anti-oxidant activities of SEE2 is due to the presence of polyphenolic constituents. It may be further concluded that the SEE2 is the most potent amongst all fractions, which may be due to presence of gallic acid, lupeol and other phenolics. In future further studies on identification of other phytoconstituents along with their biological evaluation should be necessarily carried out.
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