Hepatoprotective and
Antioxidant Potential of Calotropis gigantea in Cyclosporine–An Induced
Hepatotoxicity
Ajay Kshirsagar1*, Deepa Ingawale2,
Purnima Ashok3, Vrushali
Thorve2, Tanmay
Dodal2, Anurag Dodal2, Mahesh Kahane2
and Bharat Zope2
1Pad. Dr. D. Y. Patil Institute of Pharmaceutical
sciences and research, Pimpari, Pune-411 018, India.
2AISSMS College of
Pharmacy, Near RTO, Kennedy road, Pune-411 001, India.
3Department of
Pharmacology, K.L.E.S’s College of Pharmacy, Bangalore-560010, India.
ABSTRACT:
The ethanolic fraction of Calotropis
gigantea flowers (CGFE) was evaluated for its possible hepatoprotective
potential in Wistar rats. The CGFE (250 mg/kg and 500 mg/kg, bw p.o.) showed a
remarkable hepatoprotective activity against cyclosporine-A induced
hepatotoxicity as judged from the level of serum markers for liver damage.
Cyclosporine-A induced a significant rise in serum glutamate oxaloacetate transaminase (SGOT), serum
glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP) and lipid
profile levels. The cotreatment of animals with CGFE (250 mg/kg and 500 mg/kg,
p.o.) significantly decreased the elevated serum marker enzyme and lipid
profile levels near to normal. The activity of the CGFE was comparable to the
standard drug, silymarin (100 mg/kg, p.o.). Further histopathological studies
support the above finding.
KEYWORDS: Antioxidant,
Calotropis gigantea, Cyclosporine-A, Hepatotoxicity.
INTRODUCTION
The liver is a
vital organ of paramount importance involved in the maintenance of metabolic
functions and detoxification from exogenous and endogenous challenges like
xenobiotics, drugs, viral infections and chronic alcoholism. Drug induced liver
injury is an unresolved problem and often limits drug therapy in clinical
practice1. Indiscriminate uses of certain category of drugs such as
analgesics2, antimalarials3, anti-tubercular4,
antidepressants and/or immunosuppresants5 etc. are potential threats
to the integrity of liver. Quite often certain drugs even in therapeutic dose
may cause hepatic damage in susceptible individual. The spectrum of drug induced
liver injury ranges from asymptomatic increase in marker enzyme (markers of
hepatic damage) levels to fulminant hepatic failure6. Cyclosporine-A (Cs-A) is a cyclic undecapeptide of fungal
origin, which has been used successfully in organ transplantation and in the
treatment of autoimmune disorders during the past 15 years7.
It has been reported that impaired hepatic function occurs in 20 to 50 % of
Cs-A treated patients8.
Conventional
drugs used in the treatment of liver diseases are sometimes inadequate and can
have serious adverse effects9. It is therefore, necessary
to search for alternative drugs for the treatment of liver disease to replace
currently used drugs of doubtful efficacy
and safety. In spite of tremendous strides in drug discovery and modern medical
practices, there are hardly any drugs that stimulate liver functions or offer
protection to the liver against injurious substances or help in regeneration of
hepatic cells. The presently used agents like folic acid, multivitamins and few
polyherbal preparations provide only a supportive therapy and do not play an
effective role in providing hepatic protection10.
Calotropis
gigantea R. Br. belonging
to the family Asclepiadaceae (Synonym- Swallowort,
Maddar) commonly known as “Ruvi” having habitat wild throughout India, in drier
and warmer areas, upto an altitude of 1050 m11. Traditionally the C.
gigantea flowers were used to cure jaundice, inflammation, ulcer and asthma like
diseases12. The plant is used as a purgative, alexipharmic,
anthelmintic and it cures leprosy, leucoderma, ulcers, tumors, piles, diseases
of the spleen, liver and abdomen13.
The C. gigantea
flowers were previously reported for their analgesic, anti-inflammatory
properties14, 15. The aerial part of the C. gigantea has been
reported for its anti-diarrhoeal property in castor-oil induced diarrhoea16.
The C. gigantea roots reported for CNS stimulant and pregnancy interceptive
properties17, 18. Since, there is no systematic or scientific data
available on flowers for their hepatoprotective potential. The present study
was planned to evaluate antihepatotoxic potential of C. gigantea flowers using
various in-vivo and in-vitro models.
MATERIALS AND METHODS:
Plant
material and preparation of extract:
The
flowers of C. gigantea were procured and authenticated from Regional
Research Institute (RRI/BNG/SMP/Drug Authentication/2007-08 964), Bangalore,
India. The shade dried flowers of about 500 g were subjected for size reduction
to coarse powder. The powder was defatted with petroleum ether (60-80°C) and
then extracted with 90% ethanol using soxhlet apparatus. The CGFE was
concentrated under vacuum to get the residues
and stored at – 20oC in deep freezer till further use.
Animals:
Wistar albino rats
(200-250 gm) of either sex were used. They were maintained under standard
environmental conditions (temp 25 ± 2°C and relative humidity of 45 ± 10% and
12 h light: 12 h dark cycle). Animals were allowed to take specified amount of
standard laboratory feed (Amrut feed, Sangali) and water ad libitum. The experimental protocols were carried out as per the
CPCSEA guidelines for laboratory animal facility and approved by the
Institutional Animal Ethics Committee (IAEC).
Phytochemical
analysis of extract:
The preliminary
phytochemical analysis of CGFE was carried out by using different
pharmacognostic tests19.
In-vitro methods:
Nitric oxide scavenging
activity:
Nitric oxide
radical scavenging activity was determined according to the method reported by
Garrat20. In brief 2 ml of 10 mM sodium nitroprusside in 0.5 ml
phosphate buffer saline (pH 7.4) was mixed with 0.5 ml of CGFE at various
concentrations and the mixture incubated at 25oC for 150 min. From
the incubated mixture 0.5 ml was taken out and added into 1.0 ml sulfanilic
acid reagent (33% in 20% glacial acetic acid) and incubated at room temperature
for 5 min. Finally, 1 ml naphthylethylenediamine dihydrochloride (0.1% w/v) was
mixed and incubated at room temperature for 30 min. The absorbance at 540 nm
was measured with a spectrophotometer. The nitric oxide radicals scavenging
activity was calculated according to the following equation:
% Inhibition = (A0-A1)
/ A0 × 100)
Where, A0
was the absorbance of the control (blank, without CGFE) and A1 was
the absorbance in the presence of the CGFE.
Superoxide anion scavenging activity:
The scavenging
activity of the C. gigantea towards superoxide anion
radicals was measured by the method of Liu, Ooi, and Chang21. In these
experiments the superoxide anion was generated in 3 ml of Tris-HCl buffer
(100 mM, pH 7.4) containing 0.75 ml of NBT (300 μM) solution, 0.75
ml of NADH (936 μM) solution and 0.3 ml of different concentrations
of the CGFE. The reaction was initiated by adding 0.75 ml of PMS (120
μM) to the mixture. After 5 min of incubation at room temperature, the
absorbance at 560 nm was measured in spectrophotometer. The superoxide anion
scavenging activity was calculated according to the above equation of %
inhibition.
In- vivo method:
Cyclosporine-A induced hepatotoxicity in rats:
Wistar rats (200-250 gm) of either sex were distributed into five
groups comprising six animals in each. The animals of group I (normal control)
received only distilled water. The animals of group II (positive control)
received only Cs-A (25 mg/kg) for 21 days. In test group III and IV the animals
were treated with CGFE 250 and 500 mg/kg respectively and Cs-A (25 mg/kg) for
21 days. The animals of group V (standard) received silymarin (100 mg/kg) and
Cs-A (25 mg/kg) for 21 days. All the treatments were given orally by means of a
gastric tube5.
The
treatments were continued for 21 days. On the 22nd day all animals
were sacrificed under light ether anesthesia and blood collected without the
use of anti-coagulant for serum preparation. The blood samples were collected
by direct cardiac puncture and allowed to stand for 10 min before being
centrifuged at 2,000 rpm for 10 min and the serum was collected using rubber
micropipette. The levels of ALP was analyzed by the method of Wright et al.22,
SGPT and SGOT levels were analyzed according to Reitman and Frankel23.
The
levels of cholesterol (CH),
triglycerides (TG), low density lipoproteins (LDL), very
low density lipoproteins (VLDL) and high
density lipoproteins (HDL), were
determined with a span diagnostic kit by autoanalyser (300 TX, E. Merck-Micro
Labs, Mumbai)5.
Histopathological
examination of the liver:
The liver of the
sacrificed experimental animals were fixed in 10% formalin prior to routine processing in paraffin-embedded
blocks. Sections of 5μm thick were cut and stained using hematoxylin-eosin
(H and E) stain. Sections observed under microscope and photomicrographs were
taken for histopathological screening.
Statistical
analysis:
Data for the in-vitro antioxidant activity was
expressed as Mean ± SD from three separate observations and for
hepatoprotective activity expressed as
Mean ± S.E.M. The data
were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s
test and p<0.05 considered as
statistical significant.
RESULTS AND DISCUSSION:
The shade dried
flowers of C. gigantea was extracted
with 90% ethanol. The preliminary phytochemical screening of CGFE revealed the
presence of steroids, triterpenoids and flavonoids. Whereas glycosides,
saponins, carbohydrates, alkaloids and proteins were found to be absent.
The in-vitro antioxidant
activity of C. gigantea was
evaluated using nitric
oxide scavenging and superoxide anion scavenging methods. The CGFE
exhibited concentration dependent free radical scavenging activity when
compared with ascorbic acid as a reference standard (Fig. 1 and Fig 2). The above antioxidant effect may be due to
the presence of terpenoids (α-amyrin and β-amyrin) and flavonoids as
suggested by Francisco et al24. and Sen et al25..
Fig 1: Nitric Oxide scavenging activity of CGFE and vitamin-C.
Each value represents means ± SD (n=3).
Fig 2: Superoxide Anion scavenging activity of CGFE and vitamin-C.
Each value represents means ± SD (n=3).
Liver is the key organ responsible for multitude of
essential functions and plays an essential role in metabolism of foreign compounds
entering the body26. It has also been reported that Cs-A causes
oxidative phosphorylation that in turn leads to the enhanced generation of free
radicals. Hence, the role of oxidative stress has been strongly documented in
the pathogenesis of Cs-A induced hepatotoxicity27. In present study,
the animals treated with Cs-A showed a significant liver damage, as elicited by
the increased activities of serum enzymes28 and this rise has been
attributed to damage of structural integrity of liver. The effect of C. gigantea on different serum marker
enzymes is represented in Fig. 3; whereas the effects on lipid levels are
reported in Table I. The levels of various serum marker viz. SGOT, SGPT, ALP,
CH, TG, LDL, VLDL and HDL levels were markedly
elevated in positive control group as compared to normal control group
indicating severe hepatic damage. The CGFE at dose of 250 and 500 mg/kg of bw
exhibited significant decrease in SGOT, SGPT, ALP, CH, TG, LDL, VLDL and HDL
levels as compared with positive control group. The effect of CGFE was
comparable with that of standard drug silymarin. The significant reduction of
serum marker enzyme levels in CGFE and silymarin co-treated groups might be
possibly due to its effect of preserving the cellular membrane of the hepatocytes
from breakage by the reactive metabolites29.
In normal control group, histopathological studies of
rat liver tissue show normal hepatic cells (HC) with central vein (CV) and
sinusoidal spaces (SS) (Fig. 4a). In positive control group (group II) hepatotoxicity
was observed by severe necrosis (N), disappearance of hepatocytes in areas of
inflammation (IF) and increased sinusoidal spaces (Fig. 4b). Mild degree of
necrosis (N) with normalization of cells (HC), central vein (CV) and reduced
sinusoidal dilation was observed in Group III (Fig. 4c), whereas the Group IV
animals showed normalization of hepatocytes (HC) with some regenerating hepatic
cells, reduced sinusoidal dilation (SS) along with mild inflammogens (Fig. 4d).
Fig 3: Effect of CGFE on serum enzymes against Cs-A
induced hepatotoxicity in rats.
SGOT = Serum glutamate oxaloacetate transaminase, SGPT = Serum glutamate
pyruvate transaminase, ALP = alkaline phosphatase. The SGOT, SGPT and ALP are expressed as
IU/L. The results were expressed as Mean ± SEM (n=6). The data was analysed using ANOVA followed by Tukey’s- test. #p<0.05, ##p<0.01 and ###p<0.001.
Fig
4: Histopathology of Cs-A induced heaptotoxicity model in rat (H and E stains,
400X).
Where, Control (A), Cs-A group (B), CGFE 250 and 500
mg/kg (C and D), Silymarin 100 mg/kg (E)
The Group V animals treated with standard drug
silymarin showed the normal hepatocytes (HC) with central vein (Fig. 4e), hence
the histopathological investigation supports the fact that the co-treatment of
CGFE at 250 and 500 mg/kg bw can be useful for prevention of Cs-A induced
hepatotoxicity in rats.
Clinically, the
Cs-A induced hepatic adverse effects manifest by elevated serum bile acid
levels30, hyperbilirubinemia31 and sometimes an increase
in serum aminotransferases and alkaline phosphatase activities32.
There seem to be similarities between the hepatic adverse effects in rat and in
man. The underlying mechanisms leading to the hepatic side effects are not yet
fully understood. The role of oxidative stress in the pathogenesis of various
diseases has been reported33. Many xenobiotics and drugs cause their
deleterious effects by oxygen free radical production, leading to functional
and structural cellular changes34.
Above findings
support the fact that Cs-A induces liver toxicity by increased oxidative
stress. The CGFE showed significant in-vitro
antioxidant activity and hence has a preventive role against increased
oxidative phosphorylation due to Cs-A administration. The mechanism can be
attributed to the free radical scavenging and antioxidant potential. The above
findings were further substantiated by histopathological observations. The
hepatoprotective effect of the CGFE may be due to the presence of principle
phytoconstituents like β-sitosterols, triterpenoids (α-amyrin and
β-amyrin) as reported previously24, 25.
CONCLUSIONS:
Cs-A induces
liver toxicity in rats by increased oxidative stress. The co-treatment of CGFE
at 250 and 500 mg/kg bw in rats showed significant hepatoprotective potential.
Hence the CGFE can be useful in therapeutics for prevention of Cs-A induced
hepatotoxicity.
REFERENCES:
1.
Braunwald E, Fauci AS, Casper DL, Hauser SL, Longo DL
and Jameson JL. Harrison's Principles of Internal Medicine. The McGraw-Hill
Companies, Inc., USA. 2001.
2.
Bhanwra S, Singh J and Khosla P. Effect of Azadirachta
indica leaf aqueous extract on paracetamol-induced liver damage in rats. J
Physiol Pharmacol. 2000; 44: 64-8.
3.
Dass EE and Shah KK. Paracetamol and conventional
antimalarial drugs induced hepatotoxicity and its protection by methionine in
rats. Indian J Exp Biol. 2000; 38: 1138-42.
4.
Saraswathy SD, Suja V, Gurumurthy P and Shyamala devi
CS. Effect of Liv.100 against antitubercular drugs (Isoniazid, Rifampicin and
Pyrazinamide) induced hepatotoxicity in rats. Indian J Pharmacol. 1998; 30: 233-8.
5.
Josephine A, Nithya K, Amudha G, Coothan
KV, Sreenivasan PP and Palaninathan V. Role
of sulphated polysaccharides from Sargassum Wightii in Cyclosporine
A-induced oxidative liver injury in rats. BMC Pharmacol. 2008; 8: 40.
6.
Herfindal ET and Gourley DR. Text Book of
Therapeutics Drug and Disease Management. Williams and Wilkins, USA. 1996.
7.
Borel JF, Baumann G, Chapman I, Donatsch P, Fahr A, Mueller EA and Vigouret JM. In vivo Pharmacological
effects of cyclosporine and some analogues.
Adv Pharmacol. 1996; 35: 115–246.
8.
Deters M, Strubelt O and Younes M. Reevaluation of
cyclosporine induced hepatotoxicity in the isolated perfused rat liver.
Toxicol. 1997; 123: 197-206.
9.
Mohamed BA and Khater MR. Evaluation of the
protective potential of Ambrosia maritime extract on
acetaminophen-induced liver damage. J. Ethnopharmacol. 2001; 75: 169–74.
10.
Gulati RK, Agarwal S and Agrawal SS. Hepatoprotective
studies on Phyllanthus emblica Linn. and Quercetin. Indian J. Exp. Biol.
1995; 33: 261-8.
11.
Publications and Information Directorate, CSIR. The
Wealth of India, Dictionary of Indian Raw Materials and Industrial Products,
New Delhi.
12.
Kshirsagar AD and Ashok P.
Evaluation of Calotropis gigantea R.
Br. flowers extract on alcohol induced hepatotoxicity. J. Cell Tissue Res. 2008; 8: 1551-6.
13.
Kirtikar KR and Basu BD. Indian Medicinal Plants. Prabasi Press, Calcutta. 1980.
14.
Pathak K and Argal A. Analgesic activity of Calotropis gigantea flower. Fitoterpia.
2007; 78: 40–2.
15.
Kshirsagar AD, Patil PA, Ashok P and Hulkoti B.
Anti-inflammatory and anti-ulcer effects of Calotropis
gigantea R. Br. flowers in rodents. J. Nat. remed. 2008; 8: 183-90.
16.
Chitme HR, Chandra R and Kaushik S. Studies on
anti-diarrhoeal activity of Calotropis gigantea R. Br. in experimental animals. J. Pharm.
Pharmaceut. Sci. 2004; 7: 70-5.
17.
Argal A and Pathak AK. CNS activity of Calotropis
gigantea roots. J. Ethnopharmacol. 2006; 106: 142–5.
18.
Srivastavaa SR, Keshria G, Bhargavana B, Singh C and
Singha MM. Pregnancy interceptive activity of the roots of Calotropis gigantea Linn. in rats. Contracep. 2007; 75: 318– 22
.
19.
Khandelwal KR. Practical Pharmacognosy, Techniques Experiments. Nirali
prakashan, Pune. 2001.
20.
Garrat DC. The
quantitative analysis of drugs. Chapman and Hall, Japan. 1964.
21.
Liu F, Ooi VEC and Chang ST. Free radical scavenging
activity of mushroom polysaccharide extracts. Life Sci. 1997; 60: 763-71.
22.
Wright PJ, Leathwood PD and Plummer DT. Enzymes in
rats urine: Alkaline phosphatase. Enzymology. 1972; 42: 317-27.
23.
Reitman S and Frankel S. Colorimetric method for the
determination of serum glutamate oxalo-acetate and glutamae pyruvate
transaminase. Am.J. Clin. Path. 1957; 28: 56- 63.
24.
Francisco AO,
Mariana HC, Fernanda RCA, Roberto CPL, Regilane MS, Juliana LM, Gerly ACB,
Flavia AS and Vietla SR. Protective effect of alpha- and beta
amyrin, a triterpene mixture from Protium heptaphyllum (Aubl.) March.
trunk wood resin, against acetaminophen-induced liver injury in mice. J.
Ethnopharmacol. 2005; 98: 103-8.
25.
Sen S, Sahu PN and Mahato
SB. Flavonol glycosides from Calotropis gigantea.
Phytochem. 1992; 31: 2919-21.
26.
Abdel-Rahman MK and Abd
El-Megeid AA. Hepatoprotective Effect of Soapworts (Saponaria officinalis),
Pomegranate Peel (Punica granatum
L) and Cloves (Syzygium
aromaticum linn) on Mice with CCl4 Hepatic Intoxication. World J. Chem.
2006, 1: 41-6.
27.
Salducci MD, Chauvet-Monges AM, Berland Y, Dussol B,
Elsen R and Crevat A. The restoration
of ATP synthesis may explain the protective effect of calcium antagonist against cyclosporine A nephrotoxicity. Life Sciences. 1992; 50: 2053-8.
28.
Recknagel RO. Carbon
tetrachloride hepatotoxicity. Pharmacol.
Rev. 1967: 19: 145-208.
29.
Shaarawy SM, Tohamy AA, Elgendy SM, Abd Elmageed ZY,
Bahnasy A, Mohamed MS, Kandil E and Matrougui K. Protective Effects of Garlic
and Silymarin on NDEA-Induced Rats Hepatotoxicity. Int. J. Biol. Sci. 2009; 5:
549-57.
30.
Schade RR, Guglielmi A, Van Thiel DH, Thompson ME,
Warty V, Griffith B, Sanghvi A, Bahnson H
and Hardesty R. Cholestasis in Heart Transplant Recipients Treated
Cyclosporine. Transplant. Proc. 1983; 25: 2757–60.
31.
Roman ID, Monte MJ, Gonzalez-buitrago JM, Esteller A
and Jimenez R. Inhibition of hepatocytatory vesicular transport by cyclosporin
A in the rat: Relationship with Cholestasis and Hyperbilirubinemia. Hepatology.
1990; 12: 83–91.
32.
Mentha G and Houssin D. La Ciclosporine: Un
Immunosuppresseur Selectif en Hepatogie, Gastroenterol. Clin. Biol. 1986; 10: 641–7.
33.
Ames BN, Shigenaga MK and Hagen TM. Oxidants,
antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci.
U.S.A. 1993: 90; 7915–22.
34.
Kehrer JP. Free Radicals as Mediators of Tissue Injury
and Disease. Crit. Rev. Toxicol. 1993; 23: 21–48.