ISSN 0974-3618
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0974-360X (Online)
RESEARCH ARTICLE
Evaluation of Antibacterial action and
Hepatoprotective efficiency of Solanum
nigrum leaves extract on acetaminophen induced hepatotoxicity.
R. Bhavani1, G. Geetha1, J. Santhoshkumar2
and S Rajeshkumar1*
1PG and Research Department of
Biochemistry, Adhiparasakthi College of
Arts and Science, Kalavai – 632506, Vellore District, TN, India
2 Departmant of
Biochemistry, Periyar University, Salem - 636011, TN, India
*Corresponding Author E-mail: ssrajeshkumar@hotmail.com,
j3ssrajesh@gmail.com
ABSTRACT:
Solanum nigrum leaf is one of the most important
greens in throughout the world. Plants are playing a vital role in
hepatoprotective activity. In this present investigation we used Solanum nigrum for the antimicrobial
activity and hepatoprotective activity against acetaminophen induced liver
toxicity in male albino rats. The important chemical groups present in the
aqueous extract canbe determined using Fourier transform infrared spectroscopy
(FT-IR). Zone of inhibition in bacterial spread plate showing a antimicrobial
property of the plant extract. The aqueous extract of Solanum nigrum significantly decreases the serum enzyme alanine
amino transferase (ALT), asparate amino transferase (AST), triglycerides (TGL),
total cholesterol (TC) and significantly increased the total protein level.
Silymarin (100 mg/kg), a known hepatoprotective drug used for comparison
exhibited significant activity. The morphology of liver cells clearly
indicating the treatment of Solanum nigrum against acetaminophen induced liver
damage near to normal cells.
INTRODUCTION:
The
liver is the largest and special organ inside the body because it has the
wonderful capability to create new liver tissues from health liver tissues and
an important organ that we can survive only one or two days if it shuts down.
The liver play an vital role in many functions such as storage of nutrients,
carbohydrate metabolism, breakdown of erythrocytes, bile Secretion, synthesis
of plasma proteins, synthesis of cholesterol and etc.
Figure 1: Solanum
nigrum fresh plant
Solanum
nigrum is such
as amazing medicinal plant belonging to the family Solanaceae. It is
traditionally used as medicine for various health implications (fig 1). The
aqueous juice extract extracts of this leaves are very famous for its antiulcer
activity [1] used for the treatment of pain, inflammation fever [2,3] and
enteric diseases. [4]. The
phytochemicals such as glucoalkaloids, glycol proteins, polysaccharides and
polyphenolic compounds such as catechin, gallicacid, protocatechuic acid,
caffeic acid, rutin, and naringenin are major compounds (fig 2) of S. nigrum extract responsible for
pharmacological applications [5].
Scientific
classification
Kingdom:
Plantae
Order:
Solanales
Family:
Solanales
Genus:
Solanum
Species:
nigrum
Figure 2: Polyphenols of Solanum nigrum (Ravi et al., 2009) [5]
Table
1: Pharmacological applications of S. nigrum
|
S.
No
|
Applications
|
Part
Used / Phytochemcilas / Nature of extract
|
References
|
|
1
|
Hypoglycemic
or Antidiabetic activity
|
Aqueous
and Hydro-alcoholic extract of leaf, fruit and stem
Ethanolic
crude extact
|
(Akubugwo
et al., 2008). [6]
(Ali
et al., 2010) [7]
|
|
2
|
Antiulcerogenic Actvity on aspirin induced
ulceration
|
methanolic extract of Solanum nigrum berries
|
(Jainu
and Devi 2004) [1]
|
|
3
|
Immunostimulant
activity
for preventing fish diseases
|
Ethanol,
Methanol, chloroform, acetone and tolune extracts of S. nigrum
|
(Hanifa
2011) [8]
|
|
4
|
Protective
effect on lead acetate induced toxicity in brains of albino mice
|
Aqueous
leaf extract
|
(Chinthana
and Ananthi 2012) [9]
|
|
5
|
Antioxidant
and free radical scavenging activity
|
Methanolic extract of berries
Ethanolic extract of the dried fruit
|
(Jainu M and Devi 2004) [1]
( Rawani et al., 2010) [10]
|
|
6
|
Cytotoxic
activity
|
Ethanolic extract of the dried fruit
|
( Rawani et al., 2010) [10]
|
|
7
|
Cardioprotective activity
|
Methanolic extract of berries
|
(Balaji et al., 2011) [11]
|
|
8
|
Antidiarrhoeal activity on
castor oil induce diarrhoeain
|
ethanolic extract of the dried fruit
|
(Bhatia et al., 2011) [12]
|
|
9
|
Antimicrobial
activity Escherichia coli, Staphylococcus aureus, Enterobacter aerogenes and
Pseudomonas aeruginosa
|
methanol and water extracts of
leaves
|
(Kavishankar
et al., 2011) [13]
|
|
10
|
gram
negative bacteria namely Xanthomonas campestris (plant pathogen) and Aeromonas
hydrophila (animal pathogen)
|
methanol
and aqueous extracts of leaves
|
(Britto
AJD et al., 2011) [14]
|
|
11
|
Escherichia
coli, Citrobacter, Shigella flexenari, Staphylococcus aureus, Pseudomonas
aeruginosa and Yersinia aldovae
Fungal: Saccharomycescereviciae, Aspergillus
parasiticus, Trichophyton rubrum, Macrophomina, Fusarium solani and Candida
albicans
|
Methanolic
extracts of leaves and seeds of black and red
Varieties
|
(Sridhar
TM et al., 2011) [15]
|
|
12
|
Penicillium
notatum, Aspergillus niger, Fuserium oxisporium and Trichoderma
viridae
|
Ethanol
methanol and ethylacetate extracts of Solanum nigrum leaf, seed and
root
|
Sridhar
et al., 2011 [15]
|
|
13
|
S.
aureus and B.
sublitis
|
ethanolic
extract of the dried fruit
|
(Kaushik
et al., 2009) [16]
|
|
14
|
Anti-HCV
activity
|
Methanol and chloroform extracts of
seeds
|
(Javed et al., 2011)
[17]
|
|
15
|
Analgesic
activity
|
ethanolic extract of the dried fruit
|
(Bhatia et al., 2011) [12]
|
|
16
|
Carbon
tetrachloride induced hepatoprotective activity
|
aqueous and methanolic extracts
|
(Elhag et
al., 2011) [18]
|
With these points under consideration, the present study was carried
out to isolate the aqueous extracts of leaf of Solanum nigrum and to study their phytochemical constitution by
FT-IR studies, to investigate the antimicrobial activity against E.coli,
B. subtilis,
K. pneumoniae, B. cereus and
S.
aureus, assess the hepatoprotective activity of the
aqueous extracts of leaf of Solanum
nigrum and by studying the parameters such as changes in the activity of
AST, ALT, and the level of bilirubin, triglycerides, total cholesterol, and
protein. Finally the histological changes in normal and experimental rats were
compared.
MATERIALS AND METHODS:
Collection of
plant
The plant Solanum nigrum leaves were collected from Walaja, Vellore district,
Tamil nadu.
Preparation of
plant extract
Fresh leaves were collected and dried
under shade. The dried leaves were powdered by mixer grinder. 10 g of Solanum nigrum powdered was taken and
added 100 ml of distilled water in a beaker and boiled for 5 to 10 minutes and
are filtered through filter paper whatmann no 1. The extracts were allowed to
store and are used for experimental animals.
Chemicals
Acetaminophen and bacterial
media was purchased from M/s. Himedia Ltd., Mumbai. The rest of the chemicals
and biochemicals utilized were obtained from local firms and were of analytical
grade.
Antibacterial
Assay
Antibacterial activity of aqueous leaves
extracts of solanum nigrum was tested against Gram negative and Gram positive
strains such as E.
coli, B. subtilis, K. pneumoniae, B. cereus and S. aureus. By agar well diffusion method, significant activity of the
leaves extract was observed against the tested bacterial strain.
Animals
Adult male Wistar albino rats maintained at the college weighing
150-180 g were used for the hepatoprotective studies. The laboratory animal protocol used for this
study was approved by the Institutional Animals Ethics Committee.
Hepatoprotective
studies of S. nigrum aqueous
extract
Experimental
Design
The rats were randomly divided into five
groups of 6 animals each.
|
Group I
(Control)
|
:
|
Control rats,
received orally distilled water.
|
|
Group II
(Induced)
|
:
|
Induced rats,
orally received paracetamol (2 g/kg body weight) dissolved in water for 7
days.
|
|
Group III
(Paracetamol + Silymarin)
|
:
|
Standard rats,
orally received paracetamol (2 g/kg body weight), followed by silymarin (100
mg/kg body weight) dissolved in water for 7 days.
|
|
Group IV
(Paracetamol + ASN)
|
:
|
Treated rats,
orally received paracetamol (2 g/kg body weight), followed by aqueous extract
of Solanum nigrumleaf (300 mg/kg
body weight) dissolved in water for 7 days.
|
|
Group V
(Paracetamol + ASN)
|
:
|
Treated rats,
orally received paracetamol (2 g/kg body weight), followed by aqueous extract
of Solanum nigrum leaf (600 mg/kg body weight) dissolved in water
for 7 days.
|
Collection
of blood
Animals of all the groups were sacrificed by
cervical decapitation on the 8th day. Blood samples of each group
were collected separately into sterilized dry centrifuge tubes, and allowed to
coagulate for 30 min at 37 ºC. The clear
serum obtained after centrifugation was used for the estimation of serum
alanine amino transferase, serum aspartate amino transferase, alkaline
phosphatase, lactate dehydrogenase, serum bilirubin, serum protein, cholesterol
and triglycerides.
Biochemical
analysis
Assay of AST and ALT was performed according to the method of Reitmann and Frankel. Alkaline phosphatase was assayed by
the method of King and Armstrong. The Serum total bilirubin was
estimated according to the method of Malloy and Evelyn. The serum total protein
was estimated as per the method of Lowry. Serum total cholesterol was determined in serum by the method of
Parekh and Jung. Serum triglycerides were estimated by the method of Foster and
Dunn.
Histopathology
A portion of liver tissue in each group
was fixed in 10% formalin (formalin diluted to 10% with normal saline) and
processed for histopathology. After paraffin embedding, and block marking,
serial section of 5 μm thicknesses were made, stained with haematoxylin
and eosin and examined under microscope.
Statistical
Analysis
The statistical significance was assessed
using one-way analysis of variance (ANOVA) using SPSS 16 software. The values
are expressed as Mean ± SD and P < 0.05 was considered significant.
Figure 3: FT-IR
spectrum of Solanum nigrum plant
leaves extract
RESULTS AND DISCUSSION:
FT-IR Analysis
The figure 3 shows the FT-IR spectrum of
aqueous extract of S. nigrum. The
peaks of 3282.13, 2918.33 and
1613.47 corresponds to the functional
groups of C-H Stretch of alkynes, C≡C Stretch 0f alkynes and c-c=c
symmetric stretch respectively.
Antibacterial
Assay
Antibacterial activity (figure-4) of aqueous
leaves extracts of Solanum nigrum was
tested against Gram negative and Gram positive strains such as E. coli, B.
subtilis, K. pneumoniae, B. cereus and
S. aureus
shown in Table 2 using agar well diffusion
method, significant activity of the leaves extract was observed against the
tested bacterial strain. Among the various strains E. coli shows
the maximum zone of inhibition and minimum zone of inhibition was noted against
Staphyllococcus aureus. The increased
concentration of plant extract may responsible for high zone of inhibition [19].
Table :2 Antibacterial activity of Solanum nigrum aqueous
leaves extract
|
S.
No
|
Bacterial strain
|
Zone of Inhibition
(mM)
|
|
30 µl
|
60 µl
|
90 µl
|
Standard drug
|
|
1
|
E. coli
|
12 ± 02
|
14 ± 03
|
17 ± 08
|
15 ± 55
|
|
2
|
B. subtilis
|
8 ± 15
|
10 ± 12
|
11 ± 12
|
13 ± 21
|
|
3
|
K. pneumoniae
|
7 ± 21
|
8 ± 18
|
9 ± 17
|
12 ± 45
|
|
4
|
B. cereus
|
8 ± 0.8
|
10 ± 11
|
11 ± 58
|
10 ± 11
|
|
5
|
S. aureus
|
6 ± 12
|
7 ± 25
|
8 ± 32
|
9 ± 09
|
Figure 4:
Antibacterial activity of S. nigrum
Hepatoprotective activity of Solanum nigrum
Acetaminophen
is the drug and most commonly used as a liver damaging agent in animal studies.
The continues intake of acetaminophen will affect the metabolic functions
of liver may due to increased secretion
of enzymes like Aspartame transaminase, alanine trasaminase and alkaline
phosphatases [20] apart from it will increase the levels of Cholesterol,
Tryglycerides and Total protein level during induction.
Table
3 and figure-5 shows the effect of aqueous extract S. nigrum (300 mg/kg and 600 mg/kg) on serum biochemical markers in
paracetamol induced liver toxoxcity. Hepatic damage causes elevated level of
liver enzymes such as SGOT (serum glutamic oxaloacetic transaminase) and SGPT
(serum glutamic pyruvic transaminase). Treatment with S. nigrum at 600 mg/kg revealed comparable activity with reference
standard silymarin (25mg/kg). aqueous extract S. nigrum decreased the liver markers SGPT (102.33±4.23) and SGOT
(45.83 ± 1.94)
Table 3.Changes in
the activity of AST and ALT in Experimental Animals
|
Parameters
|
Group I
|
Group II
|
Group III
|
Group IV
|
Group V
|
Level of
Significance (p)
|
|
AST (IU/L)
|
100.67± 4.33
|
200.50±10.7
|
115.67±4.51
|
108.00±4.91
|
102.33±4.23
|
< 0.05
|
|
ALT (IU/ L)
|
44.83 ± 1.80
|
80.67 ± 4.82
|
56.33 ± 1.76
|
46.33 ± 2.21
|
45.83 ± 1.94
|
< 0.05
|
Group I – Control; Group II – Acetaminophen
induced; Group III – Acetaminophen+ Silymarin; Group IV – Acetaminophen + Aqueous
extract S. nigrum (300 mg/kg); Group V – Acetaminophen + Aqueous extract S.
nigrum (600 mg/kg) the observations are given as Mean ± SD of 6
rats of each group. Groups IV and V were compared with Group I.
Figure 5: Changes
in the activity of AST and ALT
Changes in the
Level of Serum Total Bilirubin
Table 5 and Fig. 6 present
the changes in the level of serum total bilirubin. Here, paracetamol-induced
liver damage was characterized by increased level of bilirubin and they return
to normal level after 7 days of treatment with aqueous extracts from S. nigrum leaf.
Figure 6: Changes
in the Activity of Bilirubin
Changes in the
Level of Serum Total Protein
Table 5 and Fig. 7 present
the changes in the level of serum total protein. Here, paracetamol-induced
liver damage was characterized by decreased level of total protein and they
return to normal level after 7 days of treatment with aqueous extracts from S. nigrum leaf.
Table 5.Changes in the levels of serum
Bilirubin and Total Protein in Control and Experimental Rats
|
Parameters
|
Group I
|
Group II
|
Group III
|
Group IV
|
Group V
|
Level of
Significance (p)
|
|
Bilirubin
(mg/dL)
|
0.19 ± 0.06
|
0.12 ± 0.15
|
0.15 ± 0.06
|
0.17 ± 0.02
|
0.18 ± 0.03
|
< 0.05
|
|
Total Protein
(g/dL)
|
7.49 ± 0.29
|
7.19 ± 0.23
|
7.33 ± 0.28
|
7.31 ± 0.33
|
7.41 ± 0.38
|
< 0.05
|
Figure 7: Changes in the activity of Total Protein
Changes in the
Level of Serum Total Cholesterol and Triglycerides
Table 6 and Fig. 8 present
the changes in the level of serum cholesterol and serum triglycerides. Here,
paracetamol-induced liver damage was characterized by increased level of total
cholesterol and triglycerides and they return to normal level after 7 days of
treatment with aqueous extracts from S. nigrum leaf.
Table 6: Changes in the level of total cholesterol
and triglycerides in control and experimental rats.
|
Parameters
|
Group I
|
Group II
|
Group III
|
Group IV
|
Group V
|
Level of
Significance (p)
|
|
Cholesterol
(mg/dL)
|
121.67 ± 5.62
|
349.50±15.6
|
115.00± 4.23
|
155.00±5.24
|
139.87±5.52
|
< 0.05
|
|
Triglycerides
(mg/dL)
|
108.50 ± 5.14
|
219.83±10.20
|
112.33± 5.40
|
135.00±6.02
|
123.17±5.62
|
< 0.05
|
Figure 8: Changes in the
Actvity TGL and cholesterol
Histopathology analysis
The
changes in the hepatocytes during different conditions are shown in the figure 9.
The normal cells and induced cells are having good difference are clearly shown
in the figures. And the treatment with standard drug and Solanum nigrum low and high dose treatments are look like same
structures explains the medicinal properties of the plnat extract.
(a) (b)
Figure9: Histaopathological changes of liver Normal,
Induced, Treatment Standard and Treatment with Solanum nigrum (a) 300 mg/kg
body and (b) 600 mg/kg body
CONCLUSION:
On the basis of results obtained, it can be concluded that
the aqueous extract of Solanum nigrum
leaves seems to possess hepatoprotective activity in male albino rats. The observed protective activity of Aqueous S. nigrum may be due to the identified
phytochemical compounds that are present in extracts. This finding justifies
the use of this plant in traditional medicine in treatment of microbial
infections, liver disease and supports the use of S. nigrum in the treatment of acetaminophen-induced hepatotoxicity.
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International Journal of Pharmacognosy 2(4): 178-185. DOI:
http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.2(4).178-8