Effect
of Hexane Extract of Coleus forskohlii on Chemically Induced Liver Damage
Jyothi
Y.1*, Kiran Kumar2, Reddy Shashidhar3 and Dey
Tathagata1
1East Point College
of Pharmacy, Virgonagar Post, Bidarahalli, Bangalore-560 049,
2Aurobindo Pharma
Ltd, Jadcherla, Mahabub Nagar, Andra Pradesh.
3Syngene
International Ltd, Biocon Park, Bommasandra IV Phase, Bangalore- 560 099
ABSTRACT:
The hexane extract of Coleus forskohlii (CFHE)
was evaluated for its effect on liver injury induced by carbon tetrachloride,
paracetamol or thioacetamide. The CFHE was given in two different doses
(27.5mg/kg p.o. and 55mg/kg p.o.). Silymarin, a known hepatoprotective agent
was used as standard. The lower dose of CFHE (27.5mg/kg p.o.) significantly
reduced the elevated levels of serum marker enzymes and prevented the increase
in liver weight in all three models of liver injury, while the higher dose
showed mild hepatoprotective activity. The hepatoprotective effect of lower
dose of CFHE was supported by changes in histopathology. It was concluded that
hexane extract of CFHE in lower doses possess hepatoprotective activity.
KEYWORDS: Coleus forskohlii, hepatoprotective, carbon
tetrachloride, paracetamol, thioacetamide
INTRODUCTION
Liver diseases such as jaundice, cirrhosis
and fatty liver are very common worldwide. There are many factors for the
development of these diseases, one of the important factors being the use of
drugs. Coleus forskohlii (Labitae)
is a member of the mint family and grows in subtropical areas in India,
Burma, and Thialand. This plant is
known to possess a variety of activities such as anti-glaucomatic1,
anti-platelet2, a potential antimetastatic agent3,
bronchospasmolytic4, cadiotonic and hypotensive activity5.
Coleus forskohlii, forskolin, was
discovered in 1974 and has been the subject of many laboratory studies. The
compound has a vast array of effects on the body, working primarily on an
enzymatic level, raising the level of cyclic AMP (adenosine 3.5 -
monophosphate) a substance that activates all sorts of other cellular enzymes.
Many of the research papers tested this effect on cAMP as a starting point for
in-depth study of the pharmacological profile of forskolin. These studies,
which were not designed to examine the clinical effectiveness of forskolin,
nonetheless, revealed properties of forskolin promising to be of clinical use,
such as cardiovascular dilatation, bronchodilation, and reduction of
intra-ocular pressure. However, until now, there have not been convincing
preclinical studies conducted to support its use for any other indication.
The present study was undertaken to evaluate
the effect of hexane extract of Coleus forskohlii for hepatoprotective activity.
Experimental animals:
Albino Wister rats weighing 175-250 gm of either sex
were used. Institutional Animal Ethics Committee approved the experimental
protocol
and animals were maintained under standard conditions
in animal house approved by Committee for the Purpose of Control and
Supervision on Experiments on Animals (CPCSEA).
Acute toxicity study:
The acute oral toxicity study was performed according
to the OPPTS (Office of prevention, pesticide and toxic substance) Up and Down
procedure6.
Carbon tetrachloride (CCl4) induced acute
hepatitis7: The animals were divided into five groups consisting of
six animals. The animals were then subjected to either one of the following
treatments for 9 days.
Group 1: Tween 80 (2% p.o)
Group 2: Distilled water for 9 days + CCl4 (0.5ml/kg)
on ninth day
Group 3: Silymarin (100mg/kg/day, p.o) for 9 days + CCl4 (0.5ml/kg) on ninth day
Group 4: CFHE (27.5mg/kg/day, p.o) for 9 days + CCl4 (0.5ml/kg) on ninth day
Group 5: CFHE (55mg/kg/day, p.o.) for 9 days + CCl4 (0.5ml/kg) on ninth day
The CCl4 was administered after dilution
with liquid paraffin the ratio of 1:1. Food was withdrawn 12 hr before carbon
tetrachloride administration to enhance liver damage in animals of groups 2, 3,
4 and 5. The animals were sacrificed 24 hr after the administration of CCl4. Blood samples were collected and serum was
used for assay of marker enzymes such as aspartate aminotransferase (AST),
alanine aminotransferase (ALT), alkaline phosphatase (ALP) and serum bilirubin.
The liver was isolated and washed with normal saline, blotted with filter paper
and weighed immediately. The liver was then subjected to histopathological
examination.
Paracetamol (PCM) induced liver toxicity8:
The same procedure as mentioned above was followed except that the liver injury
was produced using PCM (2 g/kg, p.o)
diluted with sucrose solution (40%w/v). PCM was administered in 3 divided doses
on day 9 and animals were sacrificed 48 hr after administration of PCM.
Thioacetamide (TAA) induced liver necrosis9: The same procedure was followed.
Damage was induced by using TAA (100mg/kg s.c), which was prepared in
distilled water (2% solution).
The statistical significance was assessed using one way
analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test.
The values are expressed as mean + SEM and p<0.05 was considered
significant.
Preliminary
phytochemical investigation:
Acute oral toxicity study:
Lower dose of CFHE (27.5
mg/kg p.o) and silymarin (100 mg/kg p.o) significantly reduced the levels of
serum marker enzymes, ALT, AST ALP and bilirubin (p<0.001). Higher dose of CFHE (55 mg/Kg p.o) did not show any significant effect when compared to CCl4
treated group. Administration of CCl4 had produced a non-significant
increase in liver weight. Silymarin and low dose of CFHE (27.5mg/Kg p.o) showed a significant reduction in
the liver weight (p<0.05) when compared with CCl4 treated
group whereas high dose of CFHE (55mg/Kg p.o)
did not affect the liver weight (Table 1). Liver sections from CCl4
treated animals showed hydropic degeneration, inflammation and steatosis in
periportal region. The inflammation was more in the sinusoids with congestion.
In animals treated with either silymarin (100 mg/kg p.o) or CFHE
(27.5 mg/kg p.o), the inflammation, steatosis and congestion was
reduced. Liver sections from animals treated with higher dose (55 mg/kg p.o)
did not demonstrate any difference when compared to CCl4 control (Fig
1)
Table 1: Effect of
Silymarin and CFHE on Serum ALT, AST, ALP, Bilirubin level and liver weight in
CCl4 induced acute hepatitis in rats.
|
Treatment |
Dose (
p.o.) |
ALT (U/L) |
AST (U/L) |
ALP (U/L) |
Serum Bilirubin (mg/dl) |
Liver weight (g/100gm b.w) |
|
Vehicle control |
- |
49.73± 2.03 |
119.91 ± 4.73 |
424.86 ± 13.36 |
0.351 ± 0.020 |
2.83 ± 0.103 |
|
CCl4 control |
- |
320.40 ± 17.04a |
617.83 ± 15.42a |
755.26 ± 22.78a |
0.868 ± 0.038a |
3.93 ± 0.197a |
|
CCl4 + Silymarin |
100mg/kg |
137.15 ± 6.59*** |
436.63 ± 18.90*** |
575.46 ± 18.55*** |
0.530 ± 0.014*** |
2.94 ± 0.276* |
|
CCl4 + CFHE |
27.5mg/kg |
238.65 ± 15.25*** |
422.23 ± 15.22*** |
613.16 ± 13.51*** |
0.681 ± 0.022*** |
2.87 ± 0.277** |
|
CCl4 + CFHE |
55mg/kg |
329.96 ± 8.96ns |
504.61 ± 26.07ns |
651.90 ± 25.58* |
0.951 ± 0.030ns |
3.45 ± 0.075ns |
Forty eight hours after
administration of PCM, the serum levels of ALT, AST, ALP and bilirubin were
markedly increased. Pretreatment with CFHE (27.5mg/kg p.o) and silymarin reduced the levels of biochemical markers levels
significantly when compared to PCM treated control (p<0.001). The liver
weight of animals treated with either silymarin or lower dose of CFHE was
significantly less when compared to paracetamol control. Pretreatment with CFHE
(55mg/kg p.o) did not show
significant effect when compared with the PCM control (Table 2). Histological
examination revealed that paracetamol produced severe congestion of blood
vessels, mild hydropic degeneration, pyknosis of nucleus and occasional
necrosis. Liver sections from silymarin treated animals showed mild pyknosis of
the hepatocytes while those from BSHE lower dose showed mild hydropic
degeneration and no pyknosis and no congestion when compared to paracetamol
treated control. Sections obtained from animals treated with higher dose of
CFHE were similar to paracetamol treated control animals (Fig 2)
Thioacetamide induced liver necrosis:
A significant difference in
serum biochemical markers was observed between normal and thioacetamide treated
group (p<0.001). Pretreatment of animals with CFHE (27.5mg/Kg) and
silymarin significantly reduced the levels of AST, ALT, ALP and bilirubin (p<0.001). TAA induced acute toxicity
had increased the weight of liver significantly (p<0.01). When
compared with TAA treated group, only low dose of CFHE (27.5mg/Kg p.o) and silymarin had prevented the
increase in liver weight, while the high dose of CFHE (55mg/Kg p.o) did not produce significant change
in liver weight (Table 3). Thioacetamide administration produced perilobular
necrosis, inflammation and congestion with cytoplasmic vacuolations in liver
cells. Silymarin treatment reduced the inflammation and necrosis and CFHE lower
dose also reduced inflammation and there was no sign of necrosis when compared
to thioacetamide treated control. Higher dose of CFHE did not show any striking
difference in histopathology when compared to thioacetamide treated control
(Fig 3)
The hexane extract of Coleus forskohlii showed
hepatoprotective activity in lower doses (27.5 mg/kg p.o). The effect
produced was comparable to that produced by silymarin (100 mg/Kg p.o), a well-known hepatoprotective
agent. 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, trichloro methyl radical10.
Paracetamol induces liver damage by depletion of glutathione (GSH)11
and thioacetamide interferes with the movement of RNA from the nucleus to the
cytoplasm, which causes membrane injury12.
Table 2: Effect of Silymarin and CFHE on Serum ALT, AST,
ALP, Bilirubin level and liver weight in paracetamol induced liver damage in
rats.
|
Treatment |
Dose (
p.o.) |
ALT (U/L) |
AST (U/L) |
ALP (U/L) |
Serum Bilirubin (mg/dl) |
Liver weight (g/100gm b.w) |
|
Vehicle control |
- |
49.73± 2.03 |
119.91 ± 4.73 |
424.86 ± 13.36 |
0.351 ± 0.020 |
2.83 ± 0.103 |
|
PCM control |
|
284.43 ± 7.27a |
399.28 ± 19.73a |
946.21 ± 20.60a |
1.060 ± 0.121a |
4.30 ± 0.240a |
|
PCM + Silymarin |
100mg/kg |
136.15 ± 8.48*** |
268.53 ± 10.90*** |
580.51 ± 23.96*** |
0.585 ± 0.061*** |
2.88 ± 0.212*** |
|
PCM + CFHE |
27.5mg/kg |
203.10 ± 7.77*** |
286.35 ± 15.84*** |
561.11 ± 24.88*** |
0.683 ± 0.060*** |
3.39 ± 0.153* |
|
PCM + CFHE |
55mg/kg |
306.86 ± 18.97ns |
319.26 ± 16.61* |
878.63 ± 15.48ns |
0.951 ± 0.038ns |
4.00 ± 0.078ns |
Table 3: Effect of Silymarin and CFHE on
Serum ALT, AST, ALP, Bilirubin level and liver weight in thioacetamide (TAA)
induced acute hepatic necrosis in rats.
|
Treatment |
Dose (
p.o.) |
ALT (U/L) |
AST (U/L) |
ALP (U/L) |
Serum Bilirubin (mg/dl) |
Liver weight (g/100gm b.w) |
|
Vehicle control |
- |
49.73± 2.03 |
119.91 ± 4.73 |
424.86 ± 13.36 |
0.351 ± 0.020 |
2.83 ± 0.103 |
|
TAA control |
- |
393.33 ± 14.80a |
440.30 ± 8.24a |
785.93 ± 16.16a |
0.866 ± 0.041a |
4.01 ± 0.127a |
|
TAA + Silymarin |
100mg/kg |
169.66 ± 8.29*** |
321.95 ± 7.58*** |
625.81 ± 10.59*** |
0.363 ± 0.021*** |
3.07 ± 0.081*** |
|
TAA + CFHE |
27.5mg/kg |
321.01 ± 9.08*** |
320.25 ± 14.03*** |
664.84 ± 16.44*** |
0.506 ± 0.022*** |
3.11 ± 0.263*** |
|
TAA + CFHE |
55mg/kg |
382.50 ± 11.31ns |
392.50 ± 12.20* |
762.65 ± 12.72ns |
0.923 ± 0.066ns |
4.01 ± 0.124ns |
The lower dose of
CFHE (27.5 mg/kg p.o) and silymarin (100 mg/kg p.o) significantly
reduced the elevated levels of serum marker enzymes in animals treated with
either of the above hepatotoxicants. The hepatoprotective effect was supported
by histological changes produced by these drugs compared to hepatotoxin treated
control. It is suggested that cAMP is part of an endogenous mechanism for
down-regulating the inflammatory response and preventing the beneficial effects
of acute inflammation from progressing to chronic inflammation and its
associated tissue destruction13. There are reports to indicate that
a role for cAMP in a particular cell function can be inferred from the use of
agents that activate adenylate cyclase receptor-coupled activation or direct
activation with agents such as cholera toxin14 or forskolin15.
It is difficult to explain with the present data, the precise mechanism by
which Coleus is showing hepatoprotective action. However, it is
speculated that the hexane extract of the plant may activate adenylate cyclase
enzyme and hence activate cAMP in the hepatocytes and this may be responsible
for its hepatoprotective action.
The CFHE did not show dose dependent hepatoprotective
action. The higher dose of the extract was less effective than the lower dose.
In our study, we did not observe any aggravation of the hepatic damage induced
by known hepatotoxicants when CFHE was administered in higher dose (55 mg/kg p.o).
On the contrary, a partial hepatoprotective action was observed in all three
models as evidenced by a reduction in ALP in CCl4 induced liver
damage and a reduction in AST in paracetamol induced and thioacetamide induced
liver damage. However, the histological examination of the liver tissues did
not support the hepatoprotective effect. From the above results, it can be
suggested that CFHE at a dose of 55 mg/kg does not produce any hepatic damage.
To conclude, hexane extract of Coleus forskohlii possess good hepatoprotective effect in lower
doses and shows mild hepatoprotection in higher doses.
1.
Caprioli J and Sears M. Forskolin lowers intraocular
pressure in rabbits, monkeys and man. Lancet 1983; 1: 958-960.
2.
Wong S, Mok W and Phaneuf, S. Forskolin inhibits
platelet-activating factor binding to platelet receptors independently of
adenylyl cyclase activation. Eur J Pharmacol 1993; 245: 55-61.
3.
Agarwal KC and Parks RE Jr. Forskolin. Int J Cancer.
1983; 32: 801-804.
4.
Bauer K, Dietersdorfer F and Sertl K. Pharmacodynamic
effects of inhaled dry powder formulations of fenoterol and colforsin in asthma.
Clin Pharmacol Ther 1993; 43: 76-83.
5. Kramer W, Thormann
J, Kindler M and Schlepper M. Effects of forskolin on left ventricular function
in dilated cardiomyopathy. Arzneim Forsch 1987; 37: 364-7.
6. Health Effect Test
Guidelines, Acute Oral Toxicity, [Computer program] OPPTS 870, 1100 United
States Office of Prevention, Pesticides and Toxic Substances Environmental
Protection Agency (7101). [Available from: URL: http://www.epa.gov/opptsfrs/home/guidelin.htm.
5/6/2004].
7. Matsuda H, Samukawa
K and Kubo M. Anti-hepatic activity of Ginsenoside Ro. Planta Med 57 (1991) 523.
8. Asha VV and
Pushpangadan P. Preliminary evaluations of the antihepatotoxic activity of Phyllanthus
kozhikodianus, P maderaspatensis and Solanum indicum. Fitoterapia
LXIX 2 (1998) 135.
9. Ahmad A, Pillai K
K, Ahmed S J, Balani D K, Najmi A K, Marwah R and Hameed A, Evaluation of the
hepatoprotective potencial of Jingrine pretreatment on thioacetamide
induced liver damage in rats. Indian J Pharmacol 31 (1999) 416.
10. Shenoy KA, Somayaji
SN and Bairy KL. Hepatoprotective effect of Ginkgo biloba in carbon
tetrachloride induced hepatic injury in rats. Indian. J. Pharmacol 2001;
33: 260-266.
11. Udem SC, Madubunyy
I, Okoye JOA and Anika SM. Anti-hepatotoxic effects of the ethanolic extracts
of Combretum dolichopetalum root bark and Morinda lucida leaf.
Fitoterapia 1997; LXVIII, 1:21-25.
12. Kumar G, Sharmila
BG, Vanitha P, Sundararjan M and Rajesekara PM. Hepatoprotective acitivity of Trianthema
portulacastrum L. against paracetamol and thioacetamide intoxication in
albino rats. J Ethnopharmacol 2004; 92: 37-40.
13. Moore AR and
Walloughby. The role of cAMP in controlling inflammation.Clin Exp.Immunol 1995;
101:387-389.
14.
Pierce NF, Greenough WB and Carpenter CC. Jr. Vibrio
cholerae enterotoxin and its mode of action. Bacteriol Rev 1971; 35:1-13.
15.
Seamon KB and Daly JW. Forskolin: its biological and
chemical properties. Adv Cyclic Nucleotide Protein Phosphorylation Res 1986;
20:1-150.
Received on 04.08.2010
Accepted on 09.09.2010
© A&V Publication all right reserved
Research J. Pharmacology and
Pharmacodynamics. 2(6): Nov. –Dec. 2010, 380-383