Effect of Boerhaavia diffusa against dimethylnitrosamine induced liver cirrhosis

Abstract

Boerhaavia diffusa (Nyctaginaceae) has been used for the chronic liver disease. The worldwide use of B. diffusa roots to treat liver disorders was validated when researchers demonstrated, in 1980 and 1991, that its root extract had antihepatotoxic properties1. Roots have been widely used for the treatment of dyspepsia, jaundice, enlargement of spleen, abdominal pain. In present study alcoholic and aqueous extract of whole plant of Boerhaavia diffusa given orally exhibited anticirrhosis activity against Dimethylnitrosamine induced liver cirrhosis in rat's model. The activity was assessed using Increases in life span (ILS), histopathological studies of liver, biochemical and hematological studies. The oral administration of EEBD & AEBD shows significant increase in the survival time (life span), a decrease in cirrhotic nodules. The biochemical and hematological parameters were also corrected by EEBD & AEBD in dimethylnitrosamine induced liver cirrhosis in rats. These observations are suggestive of the protective effect of EEBD & AEBD in dimethylnitrosamine induced cirrhosis in rats. However, out of these two extracts the anti-cirrhosis activity was maximally observed with the ethanol extract of Boerhaavia diffusa (EEBD) as compared to the aqueous extract of Boerhaavia diffusa (AEBD).

Full text

EFFECT OF BOERHAAVIA DIFFUSA AGAINST DIMETHYLNITROSAMINE INDUCED LIVER
CIRRHOSIS
Research Article
P.V. AJMIRE1*, DR. N. CHIDAMBARANATHAN1, D.R. DEWADE2, M.B. NARKHEDE3, A.E. WAGH3
1K.M. College of Pharmacy, Uthangudi, Madurai-625107, (TN) ,2 S.G.S.P.S. Institutes of Pharmacy, Kaulkhed, Akola-444004, ,3
Received: 23 July 2011, Revised and Accepted: 21 Nov 2011
I.B.S.S. College
of Pharmacy, Malkapur, Dt-Buldhana. (MS) India. Email: prashantajmire@gmail.com
ABSTRACT
Boerhaavia diffusa (Nyctaginaceae) has been used for the chronic liver disease. The worldwide use of B. diffusa roots to treat liver disorders was
validated when researchers demonstrated, in 1980 and 1991, that its root extract had antihepatotoxic properties1
Keywords: Boerhaavia diffusa; Dimethylnitrosamine; Anticirrhosis; Antihepatotoxic, EEBD, AEBD.
. Roots have been widely used for
the treatment of dyspepsia, jaundice, enlargement of spleen, abdominal pain. In present study alcoholic and aqueous extract of whole plant of
Boerhaavia diffusa given orally exhibited anticirrhosis activity against Dimethylnitrosamine induced liver cirrhosis in rat’s model. The activity was
assessed using Increases in life span (ILS), histopathological studies of liver, biochemical and hematological studies. The oral administration of
EEBD & AEBD shows significant increase in the survival time (life span), a decrease in cirrhotic nodules. The biochemical and hematological
parameters were also corrected by EEBD & AEBD in dimethylnitrosamine induced liver cirrhosis in rats. These observations are suggestive of the
protective effect of EEBD & AEBD in dimethylnitrosamine induced cirrhosis in rats. However, out of these two extracts the anti-cirrhosis activity
was maximally observed with the ethanol extract of Boerhaavia diffusa (EEBD) as compared to the aqueous extract of Boerhaavia diffusa (AEBD).
INTRODUCTION
The approach to new drugs through natural products has proved
to be the single most successful strategy for the discovery of new
drugs2
Boerhaavia diffusa, commonly known as “Punarnava” in Sanskrit,
is an herbaceous plant of the family Nyctaginaceae
. Many herbal remedies have been employed in various
medical systems for the treatment and management of different
liver diseases. However, most of the drugs showed limited
efficacies due to the development of various side effects. This
fostered our attempts to evaluate some plant products against
cirrhosis as they are less likely to cause serious side effects. Many
Indian spices and plants are quoted to be useful in different types
of liver diseases.
3,4. Boerhaavia
diffusa is indigenous to India; it is found throughout the warmer
parts of the country up to an altitude of 2000 m in the Himalayan
region. It grows well on wastelands and in fields after the rainy
season5,6. Different parts of the Boerhaavia diffusa plant have been
widely used by indigenous tribes in the traditional system of
medicine. In India, number of tribes uses the roots of this plant to
treat liver ailments. A decoction of whole plant is taken with milk
in early morning to cure jaundice and weakness by tribes of south
India. The tribal population in south Garhwal used the whole plant
in the treatment of liver enlargement7. The roots have been widely
used for the treatment of dyspepsia, jaundice, enlargement of
spleen, abdominal pain, abdominal tumours, and cancers8
A vast literature collection fails to produce a scientific evidence
to prove the anti-cirrhosis activity of Boerhaavia diffusa. Hence
this study was planned to evaluate the effect of Boerhaavia
diffusa against Dimethylnitrosamine (DMN) induced cirrhosis in
rats.
.
MATERIALS AND METHODS
The plant Boerhaavia diffusa was collected from Algarkovil Temple,
Madurai, Tamilnadu. This plant was authenticated by Department of
Botany, The American College, Madurai.
The Male wistar albino rats weighing 150-200gms were selected for
this study. [Approved by the institution animal ethical committee
(Reg.No.KMCP/09/3-27)]. The rat’s were housed in clean
polypropylene cages having 6 rat’s per cage and maintained under
temperature controlled room (27±2° C) with photoperiod of 12h
light and 12h dark cycle. The animals were fed with commercially
available food pellet diet and water ad libitum.
Preparation of Drug
The shade dried plant leaves of Boerhaavia diffusa was
powdered coarsely and about 200g of plant powder was
extracted (soxhlet) with 70% ethyl alcohol and aqueous for 72h.
The extract was dried in vacuum and resuspended in water
before use. The Phytochemical screening proves the presence of
flavonoids, alkaloids, steroids, triterpenoids, lipids,
carbohydrates, proteins, and glycoprotein’s9.
Effect against DMN induced cirrhosis
Animals were divided into five group’s viz. G1, G2, G3, G4 and G5 of
six each. For comparison, G1 designated as normal control group
was used which was neither injected with DMN nor treated with
EEBD and AEBD. To induce Liver cirrhosis, DMN dissolved in sterile
saline was intraperitoneally injected (10 μl/kg) to rats three times
per week for 3 week, and then on the fourth week, the rats were
subjected to three consecutive daily DMN injections and housed for
5 days without further treatment. Cirrhotic rats were randomly
distributed to four groups (n=6 per treatment group). As the group
G2 was reserved as cirrhosis control, it was not treated with EEBD
and AEBD. Group G3 served as the positive control, was treated with
50mg/Kg of Silymarin dissolved in 0.05% carboxy methylcellulose
by oral route10. Group G4 & G5 was treated with AEBD and EEBD
orally11
1. Derived parameter {Body weight & Life span (%)}
. The treatment was continued for 28 days. The mortality and
body weight were monitored during the 4 week of treatment.
Surviving animals were sacrificed on day 29 & following parameters
were estimated,
2. Hepatic morphology was assessed by light microscopy.
Determination of hematological parameters
Apart from above mentioned parameters, the effect of EEBD and
AEBD on hematological parameters was also studied in the rats of all
groups. Blood was collected from the all rat in the groups by
puncturing retro-orbital plexus and counted for RBC, WBC, Platelets
and Haemoglobin.
Blood chemistry
Alanine aminotransferase (ALT), aspartate aminotransferase
(AST), cholesterol, bilirubin, total proteins, and albumin in
plasma were analyzed using Spectrum, an automatic blood
chemistry analyzer.
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ISSN- 0975-1491 Vol 3, Suppl 5, 2011
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Statistical analysis
The results are expressed as mean ± SEM. The evaluation of the data
was done using one way ANOVA followed by Newman – Keul’s
multiple range tests. Difference below P<0.05 implied significance.
RESULT
Liver cirrhosis is a condition in which the liver slowly deteriorates and
malfunctions due to chronic injury12. Dimethylnitrosamine (DMN)
induced liver cirrhosis in rat is a well established, reproducible model
and has several similarities with human liver cirrhosis13
Anticirrhosis effects of Boerhaavia diffusa against DMN
.
Table 1 shows that large fraction of vehicle-treated cirrhotic rats
died within the first 3 wk. EEBD and AEBD treatment (orally for 4
wk) improved the survival rate of these rats to 75% and 65%
respectively on day 28 compared with 48% in vehicle-treated
cirrhotic animals.
However the average life span of standard drug SILYMOL treatment
was found to be 85%. Rats treated with EEBD and AEBD had
significantly greater body weight gain on day 28 than that of vehicle-
treated ones.
Table 1: Effect of B. Diffusa on the life span and body weight of cirrhosis induced rats.
Groups
% ILS Life span
Body Wt.(gms)
on 30th
Body Wt.(gms)
day on 58th day
Control
>>
60 days
216.08±2.7
219.78±1.02
Cirrhotic Control
48%
194.28±4.8
192.98±3.2
a**
Standard
a**
85%
194.95±3.84
213.06±1.24
a**
Treatment (AEBD)
b**
65%
197.35±1.7
206.48±1.63
a**
Treatment (EEBD)
b**
75%
199.38±5.04
209.16±1.47
a**
b**
All values are expressed as mean ± SEM for 6 animals in each group. **a – Values are significantly different from control (G1). **b – Values are
significantly different from cirrhotic control (G2
Table 2 shows that the level of serum albumin is regarded as an
important index of liver function. The decreased synthesis of
albumin in the liver accompanies edema and ascites formation. The
plasma albumin, which was significantly decreased to 48% of
control in cirrhotic rats, was restored by EEBD and AEBD to 92%
and to 80% respectively of healthy control animals.
In the present study, ALT and AST activities were elevated to
certain extents in cirrhotic rats. The plasma AST activity was
significantly decreased by 60-65% and 40% with EEBD and AEBD
treatment respectively.
). *P (<0.05). All values are found out by using one way ANOVA followed by Newman Keul’s multiple
range tests. EEBD- Ethanolic extract of Boerhaavia diffusa. AEBD- Aqueous extract of Boerhaavia diffusa.
The total bilirubin content was assessed as representative index for
the liver function14
Table 2 shows that treatments with DMN caused 40% to 48%
decreases in the total plasma protein and albumin contents, which
were restored to 80% to 90% by EEBD and in fewer amounts by
AEBD. There is rise in total serum cholesterol in Cholestasis,
probably due to retention of cholesterol which is normally
excreted in bile. In this study cholesterol level of cirrhotic group
raises two folds (165.43±5.66) relative to control group EEBD and
AEBD decrease the level of cholesterol by 60% at 40%
respectively.
. Table 2 shows DMN caused an 8-fold increase in
the bilirubin content, relative to control, whereas EEBD and AEBD
almost completely prevented an increase in the plasma total
bilirubin by DMN.
Table 3 shows RBC, Hb, platelets were decreased and WBC count
was significantly increased in the cirrhotic control group compared
to the normal control group. Treatment with EEBD and AEBD
significantly increased the RBC, Hb, platelets and significantly
decreased the WBC count to near standard level. All these results
suggest the effective nature of Boerhaavia diffusa.
Table 2: Effect of Boerhaavia diffusa. on serum Enzymes and lipid proteins
Groups
Total Proteins
(g %)
Albumin
(g /dl)
AST
(U/L)
ALT
(U/L)
Bilirubin
(mg %)
Cholesterol
(mg/dl)
Control
7.18 ±0.16
4.46 ±0.19
145.6 ±1.57
78.61±3.62
0.65 ±0.08
85.08 ±11.7
Cirrhotic Control
4.05 ±0.15
2.18±0.14
**
278.25 ±1.62
a**
179.9±4.04
a**
4.87 ±0.12
a**
165.43 ±5.66
a**
Standard
a**
6.21 ±0.17
3.60 ±0.10
b**
191.16 ±2.83
b**
103.4 ±2.72
b**
0.91±0.23
b**
118.68±0.85
b**
Treatment (AEBD)
b**
5.5 ±0.07
3.90 ±0.08
b**
218.6 ±1.73
b**
117.65±2.57
b**
2.78 ±0.25
b**
133.05±1.31
b**
Treatment (EEBD)
b**
5.85 ±0.22
4.25 ±0.06
**
199.10 ±3.76
b**
110.11±1.33
b**
1.74 ±0.11
b**
125.91±1.40
b**
b**
All values are expressed as mean ± SEM for 6 animals in each group. **a – Values are significantly different from control (G 1). **b – Values are
significantly different from cirrhotic control (G2
). *P (<0.05). All values are found out by using one way ANOVA followed by Newman Keul’s multiple
range tests. EEBD- Ethanolic extract of Boerhaavia diffusa. AEBD- Aqueous extract of Boerhaavia diffusa.
Table 3: Effect of Boerhaavia diffusa on Hematological Parameters
Groups
RBC
(millions/mm3
WBC
) (cells/mm3
Hemoglobin
) ( g/dl)
Platelets
(lakhs/mm3)
Control
Cirrhotic Control
Standard
Treatment (AEBD)
Treatment (EEBD)
8.10 ±0.10
5.05 ±0.14
7.16 ±0.12
**
6.56 ±0.12
b**
6.70 ±0.18
b**
11.80±0.18
**
14.66±0.26
12.36±0.07
a**
12.80±0.14
b**
12.51±0.10
b**
16.46 ±1.57
b**
10.15±0.14
14.65±0.31
a**
13.65±0.20
b**
14.13 ±0.10
b**
1.13±0.03
b**
0.93±0.03
0.99 ±0.01
a**
0.95±0.01
b**
0.97±0.01
b**
b**
All values are expressed as mean ± SEM for 6 animals in each group. **a – Values are significantly different from control (G1). **b – Values are
significantly different from cirrhotic control (G2). *P (<0.05). All values are found out by using one way ANOVA followed by Newman Keul’s multiple
range tests. EEBD- Ethanolic extract of Boerhaavia diffusa. AEBD- Aqueous extract of Boerhaavia diffusa.

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368
Histopathological analysis
Dimethylnitrosamine (DMN) exerts hepatotoxic and carcinogenic
effects in animals, and induces hepatic necrosis and subsequent
fibrosis probably through metabolic activation by cytochrome P450
2E1. EEBD and AEBD markedly reduced the number of cirrhotic
nodules and the staining intensities of nodular capsules15
To determine whether cirrhosis could be treated with
Boerhaavia diffusa, we histopathologically examined the
formation of cirrhotic nodules, extent of liver fibrosis,
intralobular hepatocytes degeneration, and portal inflammation
of surviving cirrhotic rats after 4 wk of vehicle or drug
treatment.
.
Fig. 1: (Normal control) It shows of liver parenchyma with central vein and radiating column of hepatocytes. Portal tracts appear normal.
No evidance of cirrhosis seen
Fig. 2: (Cirrhotic control) It shows Section shows liver parenchyma with foci showing fatty change. This indicates liver cirrhosis
occurrence. Masson’s trichrome staining revealed that extracellular matrix was heavily accumulated around and within thick multiple
fibrotic nodules, particularly in proximity to portal spaces in the liver of cirrhotic rats. DMN treatment caused thick multiple fibrotic
nodules
Fig. 3: Treatment of cirrhotic rats with 50 mg/kg of silymarin for 4 wk almost disappeared of liver fibrotic nodules. Section shows
parenchyma of liver with central vein and radiating column of hepatocytes also portal tracts appear normal as compared to cirrhotic
control group

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Fig. 4: It shows treatment of cirrhotic rats with AEBD for 4 wk notably decreased the intensities of liver fibrotic nodules
Fig. 5: It shows Liver fibrotic nodules completely disappeared after EEBD treatment. Only marginal fibrous bands were detected
In Table 4 anti-cirrhotic effects were further supported by decreases in Knodell score a general marker of LC and inflammation15
Table 4: Fibrosis and Knodell scores in the livers of rats
.
Groups Fibrosis Score Knodell Score
Control
0
0
Cirrhotic Control
4.8±0.41**
18.5±2.8**
a
Standard
a
2.7±0.58**
9.5±1.6**
b
b
Treatment (AEBD)
3.8±0.51**
14.1±1.8**
b
Treatment (EEBD)
b
3.1±0.67**
11.9±1.4**
b
b
All values are expressed as mean ± SEM for 6 animals in each group. **a – Values are significantly different from control (G1). **b – Values are
significantly different from cirrhotic control (G2
). *P (<0.05). All values are found out by using one way ANOVA followed by Newman Keul’s multiple
range tests. EEBD- Ethanolic extract of Boerhaavia diffusa. AEBD- Aqueous extract of Boerhaavia diffusa.
DISCUSSION
Plants have served as a good source of anti-cirrhosis agents, several
studies have been conducted on herbs under a multitude of
ethanobotanical grounds. A large number of plants possessing anti-
cirrhotic properties have been documented16
Laboratory cirrhotic rats produced by DMN administrations
simulate the clinical features of human LC such as mortality, ascites,
hepatic parenchymal cell destruction, formation of connective tissue,
and nodular regeneration, providing a preclinical model to evaluate
therapeutic efficacy of drug and underlying mechanism. In this study
treatment with Boerhaavia diffusa markedly reduced the number of
cirrhotic nodules and the staining intensities of nodular capsules.
Anticirrhotic effects were further supported by decreases in Knodell
score, a general marker of LC and inflammation
.
17
Treatment with Boerhaavia diffusa effectively increases the life span
of cirrhotic rats as well as it prevents the loss of body weight
compare to cirrhotic control group. The decreased synthesis of
albumin was restored in present study. Hence, Boerhaavia diffusa
improved liver function.
.
The plasma transaminase activity is increased with biliary
obstruction in cirrhotic patients18
We monitored the plasma total bilirubin content as a liver function
test. Our treatment prevented an increase in the total plasma
bilirubin level induced by DMN, which represented the protective
efficacy against DMN-induced liver injury.
. In the present study, the plasma
AST and ALT activity in treatment control group was decreased
significantly.
Also this treatment was active in restoring the total plasma proteins
and albumin contents in rats treated with DMN over a 4-week
period.
The reversal of Hb content, RBC, Platelets and WBC by the present
treatment towards the value of the normal group clearly indicate
that Boerhaavia diffusa possessed protective action on the
haemopiotic systems.
ACKNOWLEDGEMENT
We thank Prof.M.Nagarajan and Dr. A.J.M. Cristina for his
encouragement and support throughout this study.
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