Hepatoprotective Effect of Lepidium
Sativum Linn (Cruciferae) Total Alkaloid Fraction
against CCl4 Induced Hepatotoxicity on
Rats
A. Shukla, P. Bigoniya*
Radharaman College of Pharmacy,
Ratibad, Bhopal-02, Madhya Pradesh, India
ABSTRACT:
The effect of
total alkaloid fraction extracted from the seeds of Lepidium
sativum Linn (Cruciferae) on body weight,
relative liver weight, biochemical parameters, in vitro bromosulphalein
(BSP) uptake and histology of liver in rats intoxicated with carbon tetra
chloride (CCl4) were evaluated in this study. The L. sativum alkaloidal fraction
(LSAF) was given in dose of 50, 150 and 250 mg/kg for fourteen days. LSAF showed dose
dependent protection against body weight loss. Silymarin and LSAF treatment showed hepatoprotection
by reducing the liver weight of CCl4 intoxicated rats from 4.342 ± 0.035 to 3.986 ± 0.025 gm/100 gm body weight
respectively. LSAF at 250 mg/kg dose significantly (p<0.01-0.001) reduced
the serum total and direct bilirubin, SGOT, SGPT,
ALP, cholesterol and triglyceride compared to CCl4 treated group.
Serum protein and albumin level was extreme significantly (p<0.01-0.001)
normalized at 250 mg/kg dose. LSAF at dose of 150 and 250 mg/kg showed 61.22
and 75.02% hepatoprotection in BSP uptake. LSAF treatment following CCl4 intoxication showed mild focal coagulative and centrolobular necrosis, with altered hepatic parenchyma
compared to severe hepatic damage caused by CCl4 in liver section.
KEYWORDS: Lepidium sativum, imidazole
alkaloid, hepatoprotective, bromosulphalein
INTRODUCTION
Lepidium sativum Linn (Cruciferae)
commonly known as Asaliyo, is an erect, glabrous
annual herb cultivated as a salad plant throughout India, Europe and United
States. It is an important medicinal plant since the Vedic era. The seeds are
bitter, thermogenic, depurative, rubefacient,
galactagogue, emmenagogue,
tonic, aphrodisiac, ophthalmic and diuretic. They are useful as poultices for
sprains and in leprosy, skin diseases, dysentery, diarrhoea,
splenomegal, asthma. The leaves are mild stimulant
and diuretic, serviceable in scorbutic diseases. The root is employed in skin
diseases1.
Literature search reveals that the plant has
antihypertensive effect2. The seeds have hypoglycemic activity and
used in the treatment of bronchial asthma3, 4. Patel et al. (2009)5
reported the presence of flavonoids, coumarins, sulphur glycosides, triterpenes, sterols and various imidazole
of alkaloids in L. sativum
seeds. The present investigation was taken to screen the hepatoprotective
effect of L. sativum
total alkaloid. Since no work has done on the alkaloid of the plant.
Collection and identification of plant material:
The seeds of L. sativum were purchased from local market of Bhopal,
Madhya Pradesh, India.
The seeds were identified with the help of
available literature and authenticated by Dr. H. B., Singh, Scientist, NISCAIR,
New Delhi, India. A voucher specimen was deposited in
the herbarium of department (L. sativum; No. NISCAIR/RHMD/Consult/-2009-10/1232/36).
Extraction of total
alkaloid:
Ground seeds were defatted with n-hexane in a soxhlet
extractor for 16 hr and subsequently extracted with methanol for 8 hr. The
resulting extract was evaporated to dryness, resuspended
in water, acidified with conc. hydrochloric acid and extracted three times with
ethyl acetate. The remaining aqueous layer was basified with conc. ammonia
and extracted again three times with ethyl acetate to separate total alkaloid
fraction. The ethyl acetate layer was pooled and concentrated under vaccum. The obtained alkaloidal
fraction from L. sativum
was designated as (LSAF).
Materials:
Silymarin was gift sample from Ranbaxy Laboratories, Dewas (M.P.). bromosulphthalein
was purchased from Himedia. All the other chemicals
were of analytical grade.
Test
animals:
Laboratory
bred Wistar albino rats of either sexes weighing
between 140-200 g were maintained under standard laboratory conditions at
25±2ºC, relative humidity 50±15% and photoperiod (12 h-dark and light).
Commercial pellet diet (Hindustan Lever, India) and water were provided ad-libitum. Animals were allowed to free access of water
and food during the experiment but no water and food were allowed before and
after one h of dosing. Ethical committee approval was obtained from
institutional animal ethical committee of Radharaman
College of Pharmacy, (Reg. no. 1169/ac/08/CPCSEA), Bhopal, before carrying out
the experiments.
Treatment
protocol:
Animals
were randomly divided into 6 groups with 5 rats in each. Group I, and III-VI
was treated with vehicle control, positive control (silymarin)
and different doses of LSAF continuously for 14 days. On 7th day two
hr after drug treatment all the animals including group II (negative CCl4 control)
were treated with CCl4 in liquid paraffin (1:1) in a dose of 1 ml/kg
b.wt. (p.o)
and afterwards on every alternate day for a week. Vehicle control group
animals were treated with normal saline (0.2 ml/100 gm, i.p).
Standard drug silymarin was prepared freshly in 1%
gum-accacia in normal saline. LSAF was dissolve in 1%
tween 80 as per the required quantity. All the LSAF
treatments were given intraperitoneal, silymarin and CCl4 by orogastric
intubation. Treatment plan was as following:
Group
I Vehicle
control group (0.2 ml/100 gm, i.p): 1% tween 80 for fourteen days
Group
II CCl4 control
group (1 ml/kg b.wt.): on seventh day onward every
alternate days for one week.
Group
III Silymarin (20 mg/kg b.wt.)
+ CCl4 (1 ml/kg b.wt.)
Group
IV LSAF (50
mg/kg b.wt.) + CCl4 (1 ml/kg b.wt.)
Group
V LSAF (150
mg/kg b.wt.) + CCl4 (1 ml/kg b.wt.)
Group VI LSAF (250 mg/kg b.wt.) + CCl4 (1 ml/kg b.wt.)
Assessment
of hepatoprotective activity:
Body
weights of all the animals were recorded on 1st day, 7th
day and on 14th day before sacrifice. On the 14th day, 2
hr after drug treatment animals of all groups were anaesthetized by light ether
anesthesia and blood was withdrawn by intracardiac
puncture. Blood was allowed to coagulate for 30 min at room temperature and
serum was separated by centrifugation at 3000 rpm for 15 min (Remi Centrifuge, Model RM 12 C). The serum was used to
estimate total and direct bilirubin6, SGPT, SGOT7, ALP8,
protein9, albumin10, cholesterol11 and
triglycerides12. The liver was harvested, washed in normal saline,
blotted in filter paper and weighed. Each liver was cut into three slices of 60
mg weight and used for bromosulphthalein (BSP) uptake
test following method described by Rajan and Subrahmanyam, (1965). Percent hepatoprotection
was calculated using the equation13:
H
= l - [T – V / C - V] ´
100
Where T is
mean value of group treated with test drug and C is mean value of group treated
with CCl4 alone and V is the mean value of control animals.
Rest of
the liver part was preserved in 10% neutral formalin for histopathological
assessment of liver damage. Preparation of permanent tissue slides and staining
(Hematoxylene and Eosin) was based on method of Nanji et al.
(2002)14.
Statistical
analysis:
The
results were expressed in term of Mean ± SEM. Experimental data of various
physical and biochemical parameters were analyzed using one way ANOVA followed
by Turkey-Kramer multiple comparisons using In Stat graph pad version 3.0.
p<0.05 were considered statistically significant.
RESULTS:
Vehicle
control group showed 21.42% and 32.14% increase in body weight respectively on
7th and 14th day. CCl4 treated group showed
7.35% decrease in body weight with reduced food consumption, silymarin showed 6.66% increase where as LSAF showed dose
dependent protection against body weight loss on 14th day. Silymarin and LSAF treatment showed hepatoprotection
by reducing the liver weight of CCl4 intoxicated rats from 4.342 ±
0.035 to 3.986 ± 0.025 gm/100 gm body weight respectively (Table 1).
CCl4
caused significant hepatic damage on rats as observed from elevated serum level
of total bilirubin, direct bilirubin,
SGOT, SGPT, ALP and cholesterol. Serum protein, albumin and triglycerides
levels were considerably reduced (Table 2). LSAF at 250 mg/kg dose
significantly (p<0.01-0.001) reduced the serum total and direct bilirubin, SGOT, SGPT, ALP, cholesterol and triglyceride
compared to CCl4 treated group.
Serum protein and albumin level was extremely significantly
(p<0.01-0.001) normalized at 250 mg/kg dose of LSAF.
LSAF
treated liver showed increased BSP uptake rate dose dependently after 30 min of
in vitro incubation. The percentage hepatoprotection
were 61.22 and 75.02% for LSAF at dose of 150 and 250 mg/kg body
weight respectively, where as standard drug silymarin
showed 83.57% protection as shown in Table 3.
Table 1: Effects of Lepidium sativum
total alkaloidal treatment on change in body weight
and relative liver weight
|
Treatment (mg/kg) |
Body weight |
Liver weight in gm/100 gm body
weight |
||||
|
Average body weight in gm (Mean
±SEM) |
% Change |
|||||
|
0 Day |
7th Day |
14th Day |
7th Day |
14th Day |
||
|
Vehicle control (0.2 ml/100 gm,
i.p) |
140.0 ±
18.61 |
169.9 ±
19.76 |
184.9 ±
21.34 |
21.42 |
32.14 |
3.782 ±
0.021 |
|
CCl4 (1 ml/kg, p.o) |
147.5 ±
16.45 |
160.8 ±
19.56 |
136.6 ±
16.29 |
9.03 |
− 7.35 |
4.342 ±
0.035 |
|
Silymarin (20, p.o)
|
145.0 ±
18.65 |
164.3 ±
20.41 |
154.6 ±
18.85 |
13.33 |
6.66 |
3.570 ±
0.091*** |
|
LSAF (50, i.p)
|
157.5 ±
20.76 |
173.2 ±
22.72 |
148.5 ±
20.51 |
10.0 |
− 5.71 |
4.323 ±
0.078ns |
|
LSAF (150, i.p)
|
168.7 ±
18.98 |
183.6 ±
23.89 |
170.8 ±
19.78 |
8.86 |
1.26 |
4.031 ±
0.030** |
|
LSAF (250, i.p)
|
190.0 ±
22.54 |
205.5 ±
24.62 |
193.5 ±
20.49 |
7.56 |
2.89 |
3.986 ±
0.025** |
The values are expressed as mean ±
SEM, n = 5 in each group. ***p<0.001 and **p<0.01when compared with CCl4
control.
Table 2: Effects of Lepidium sativum
total alkaloidal treatment on serum biochemical
parameter of CCl 4 intoxicated rats
|
Treatment (mg/kg) |
Serum biochemical parameters (M
± SEM) |
||||||||
|
Total bilirubin
(mg/dl) |
Direct bilirubin (mg/dl) |
SGOT (IU/L) |
SGPT (IU/L) |
ALP (IU/L) |
Protein (gm/dl) |
Albumin (gm/dl) |
Cholesterol (mg/dl) |
Triglycerides (mg/dl) |
|
|
Vehicle (i.p) |
0.73±
0.05 |
0.21 ±
0.02 |
44.25 ±
6.92 |
20.23 ±
2.68 |
253.14 ±
21.51 |
7.68 ±
0.89 |
4.33 ±
0.23 |
50.85 ±
6.21 |
61.32 ±
1.73 |
|
CCl4 (1, p.o) |
6.91 ±
0.47 |
4.56 ±
0.99 |
264.57 ±
21.32 |
120.37 ±
18.59 |
374.27 ±
19.87 |
4.35 ±
0.43 |
3.15 ±
0.26 |
68.12 ±
5.55 |
37.61 ±
4.89 |
|
Silymarin (20, p.o)
|
0.82 ±
0.03** |
0.46 ±
0.08*** |
70.07 ±
9.84*** |
29.27 ±
5.20*** |
206.57 ±
17.71*** |
7.42 ±
1.14*** |
4.05 ±
0.41* |
52.90 ±
8.22* |
43.15 ±
4.26ns |
|
LSAF (50, i.p)
|
1.10 ±
0.03** |
0.42 ±
0.03*** |
235.01 ±
26.73ns |
141.14 ±
17.36ns |
279.98 ±
22.44*** |
4.78 ±
0.49ns |
3.20 ±
0.37ns |
71.46 ±
6.76ns |
3.44 ±
4.44ns |
|
LSAF (150, i.p)
|
0.94 ±
0.07** |
0.26 ±
0.04*** |
107.78 ±
17.64*** |
72.55 ±
9.43*** |
248.59 ±
20.48*** |
5.21 ±
0.65ns |
3.81 ±
0.40ns |
58.72 ±
7.19ns |
54.20 ±
4.22*** |
|
LSAF (250, i.p)
|
0.88 ±
0.06** |
0.23 ±
0.03*** |
99.06 ±
22.16*** |
24.20 ±
2.06*** |
236.04 ±
18.27*** |
6.85 ±
0.99*** |
4.23 ±
0.52** |
37.74 ±
3.66*** |
64.75 ±
6.38*** |
The values are expressed as mean ±
SEM, n = 5 in each group. ***p<0.001, **p<0.01 and *p<0.05 when
compared with CCl4 control. SGOT = serum glutamate oxaloacetate transaminase, SGPT =
serum glutamate pyruvate transaminase
and ALP = alkaline phosphatase.
Table 3: Effects of Lepidium sativum
total alkaloidal treatment on in-vitro bromosulphalein uptake of CCl4 intoxicated rat
liver slices
|
Treatment (mg/kg) |
%
hepatoprotection |
||
|
After
10 min |
After
20 min |
After
30 min |
|
|
Vehicle control (0.2 ml/100 gm,
i.p) |
- |
- |
- |
|
CCl4 (1 ml/kg, p.o) |
- |
- |
- |
|
Silymarin (20, p.o)
|
55.65 |
67.75 |
83.57 |
|
LSAF (50, i.p)
|
25.30 |
32.05 |
49.43 |
|
LSAF (150, i.p)
|
42.72 |
59.46 |
61.22 |
|
LSAF (250, i.p)
|
25.86 |
45.40 |
75.02 |
n = 5 in each group. % hepatoprotection = 1 − [T – V / C − V] × 100.
Where, T = mean value of group treated with test drug, C = mean value of group
treated with CCl4 and V = mean value of group treated with vehicle.
Section of liver tissues
of CCl4 intoxicated
rat showed extensive diffuse vacuolar degeneration
engorged with blood and microvesicular fatty changes
in hepatocytes. Silymarin showed protection of liver tissue with minimal fatty
changes and focal necrosis. LSAF treatment
following
CCl4 intoxication showed
mild focal coagulative and centrolobular
necrosis, and slightly altered hepatic parenchyma (Fig. 3a ).
DISCUSSION:
Phytocontituents like alkaloids, flavanoid,
saponin and triterpenoid
are known to possess hepatoprotective activity. The
curative properties of herbs are due to presence of complex phytoconstituents
of varied composition in one or more parts of these herbs. Presence of
alkaloids, glycosides and saponin has been reported
in the plant L. sativum. Imidazoles
have been identified as anthelmintic, antifilarial agent, anti-inflammatory, antiviral,
anti-cancer, anti-bacterial, anti-fungal, anti
tubercular, lipo-oxygenase inhibitor and
anti-depressant. Methanolic extract of L. sativum seed has been reprted
to protect the liver from CCl4 insult15.
This study
compiles the effect of enriched imidazole alkaloid
fraction from L. sativum on hepatocellular damage caused on chronic exposure of CCl4.
Acute hepatic injury may be cytotoxic, cholestatic or of a mixed hepatocellular
type16. CCl4 induced hepatotoxicity
has chosen as the experimental model, since the changes associated with the CCl4
induced liver damage are similar to that of viral hepatitis oxidizes in hepatocyte activates accumulated CCl4 by chemolytic breakage of C-Cl bond
in hepatic endoplasmic reticulum via an enzyme system of electron transport
from reduced nicotinamide adenine dinucleotide
phosphate to oxygen. CCl4 is activated by cytochrome
CYP2E1, CYP2B1, or CYP2B2
and possibly CYP3A to form highly reactive and toxic metabolite tri chloromethyl radical.
Quantitative
estimation of SGOT and SGPT like marker enzymes gives an indication of the
extent and type of hepatocellular damage as they are
synthesized in the liver and increased in blood when hepatocytes
are damaged. SGPT is thought to be one of the indices of the degree of cell
membrane damage while SGOT is an indicator for mitochondrial damage since
mitochondria contains 80% of the enzyme17. The increased serum concentation of the liver marker enzymes SGPT, SGOT and ALP
in CCl4 treated rats indicated extensive damage to hepatic
architecture.
LSAF
showed extremely significant protection by reducing the rate of body weight
loss at 150 and 250 mg/kg doses. The results of our study demonstrated that
LSAF at the different doses caused significant reduction in the levels of SGPT,
SGOT and ALP elevated by CCl4. Depending on the type of cell and the
membrane involved, lipoperoxidation due to CCl4
results in hemolysis, which increases the serum bilirubin level18 which was evident in the study finding
also. LSAF showed an effective reversal in the elevated serum bilirubin level, signifying its potential application in
the acute condition of jaundice. Reduction of total serum protein concentration
observed in the CCl4 treated rats may be associated with the
decrease in the number of active hepatocytes which in
turn may result into decreased hepatic capacity to synthesize protein and
consequently decrease in the liver weight. LSAF given along with CCl4
showed significant increase in total serum protein indicating the hepatoprotective activity and also accounting for the
increase in the liver weight most probably promoting the hepatic cell
regeneration. CCl4 induced rise in serum levels of cholesterol has
been attributed to the damage in structural integrity of the liver leading to
release into circulation after cellular damages19
Decrease
in serum concentrations of triglycerides is probably due to the accumulation of
triglyceride in the hepatic cells blocking secretion into plasma, the basic
mechanism underlining the fatty liver induced in rat by CCl4 20.
Several phytoconstituents have the ability to induce microsomal enzymes either by accelerating the excretion of
CCl4 or by inhibition of lipid peroxidation
induced by CCl4 21. A possible mechanism of the alkaloid
fraction may be interference with cytochrome P450,
resulting in the hindrance of the formation of hepatotoxic
free radicals, thereby protecting the integrity of the membrane22.
Imidazoles are an important class of heterocycles
including many substances of both biological and chemical interest. They are
part of a large number of highly significant biomolecules
such as the essential amino acids and related compounds, biotin and the imidazole alkaloids. Methanol and aqueous extracts from
dehydrated hypocotyls of L. meyenii or maca containing imidazole
alkaloids (lepidiline A and B) exhibited cytoprotective effect against t-butyl hydroperoxide
intoxicated hepatocytes23. Raj et al. (2010) reported in
vitro and in vivo hepatoprotective effects of the
total alkaloid fraction of Hygrophila auriculata leaves24. Hepatoprotective
activity of Fumaria officinalis
arial parts containing alkaloid against CCl4-induced
liver damage in rats was reported by Sharma et al., (2012)25.
ACKNOWLEDGEMENT:
The authors are thankful to AICTE, New Delhi, for
providing financial support under Research Promotion Scheme (Sanction No.
8023/RID/RPS-55/2010-11) to carry out the project.
REFERENCES
1. Kirtikar KR and Basu BD. Indian
medicinal plants. Vol. 1. Popular Prakashan, India. 2004.
2. Maghrani et al. Antihypertensive
effect of Lepidium sativum
L. in spontaneously hypertensive rats. Journal of Ethnopharmacology. 22:100
(1-2); 2005: 193-197.
3. Eddouks et al. Study of hypoglycaemic
activity of Lepidium sativum
L. aqueous extract in normal and diabetic rat. Journal of Ethnopharmacology. 97; 2005: 391-395.
4. Archana PN, Mehta AA. A study on clinical efficacy of Lepidium sativum
seeds in treatment of bronchial asthma. Iranian Journal of
Pharmacology and Therapeutics. 5 (1); 2006: 55-59.
5. Patel,
et al. Evaluation of diuretic activity of aqueous and methanol extracts
of Lepidium sativum
garden cress (Cruciferae) in rats. Tropical
Journal of Pharmaceutical Research. 8; 2009: 215-219.
6. Jendrassik L and Gorf, P. Verainfachte photometrische Methoden zur Bestimmung
des Blutbilirrubins. Biochemische
Zeitschrift. 297 (1-2); 1938: 81-89.
7. Reitman S and
Frankel S. A colorimetric method for determination of serum glutamic oxaloacetic and glutamic-pyruvic transaminases.
American Journal of Clinical Pathology. 28;
1957: 56-63.
8. Kind PRN and King EJ..
Estimation of plasma phosphates by determination of
hydrolysed phenol with antipyrine. Journal
of Clinical Pathology.7; 1954: 322-330.
9. Lowry et al. Protein measurement with folin phenol reagent. Journal
of Biological Chemistry. 193; 1951: 265-275.
10. Doumas et al.
Albumin standards and the measurement of serum albumin with bromcresol
green. Clinica Chimica Acta;
International Journal of Clinical Chemistry. 31(1); 1971: 87-96.
11. Siedel et al. Reagent for the enzymatic
determination of serum total cholesterol with improved lipolytic
efficiency. Advances in Clinical Chemistry. 29 (6); 1983. 1075-1080.
12. Fossati P and Prencipe L. Serum
triglycerides determined colorimetrically with an
enzyme that produces hydrogen peroxide. Advances in
Clinical Chemistry. 28 (10); 1982: 2077-2080.
13. Rajan R and Subrahmanyam K.
Uptake of sodium phenol tetrabromophthalein (bromsulphalein) by rat liver slices under different
conditions. Indian Journal of Experimental Biology.
24; 1965:100-103.
14. Nanji et al. Increased severity of alcoholic liver
injury in female rats: Role of oxidative stress, endotoxin
and chemotoxin. American Journal
of Physiology-Gastrointestinal and Liver Physiology. 281; 2002:
G1348-G1356.
15. Shukla et al. Phytochemical
and Pharmacological Profile of Lepidium sativum Linn. “Bioactive Phytochemicals:
Perspective for Modern Medicine, Vol 1, M/S
Daya Publishing House: India. 2012;
437-455.
16. Zimmerman
HJ. Hepatotoxicity. Dis Mon 39 (10);
1993: 675–787.
17. Dabba MH and Abdel- Rahman MS. Hepatoprotective activity of thymoquinone
in isolated rat hepatocytes. Toxicology
Letters. 95(1); 1998: 23-29.
18. Recknagael R. Carbon tetrachloride hepatotoxicity.
Pharmacological
Reviews. 19; 1967:145-196.
19. Sallie R et al. Drugs and the liver. Part I. Testing
liver function. Biopharmaceutics and Drug
Disposition 12; 1991: 251-259.
20. Lombardi
B. Considerations on pathogenesis of fatty liver. Laboratory
Investigation. 15; 1966:1-20.
21. Mehta et al. Hepatoprotective activity of Trianthema portulacastrum. Indian
Drugs. 36; 1999: 241-244.
22. Tran
et al. Triterpene saponins
from Vietnamese ginseng (Panax
vietnamensis ) and their hepatocyteprotective
activity. Journal Natural Product. 64;
2001: 456-461.
23. Valentova et al. The in vitro biological activity of
Lepidium meyenii
extracts. Cell Biology and Toxicology. 22 (2);
2006: 91-99.
24. Raj
et al. In vitro and in vivo hepatoprotective
effects of the total alkaloid fraction of Hygrophila
auriculata leaves. Indian
Journal of Pharmacology. 42(2); 2010: 99–104.
25. Sharma
et al. Hepatoprotective Activity of Fumaria officinalis
against CCl4-induced Liver Damage in Rats. Pharmacologia. 3(1);
2012: 9-14.
Received on 28.01.2013
Modified on 06.02.2013
Accepted on 09.02.2013
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