Evaluation
of Anti-Oxidant and Hepatoprotective activity of Desmostachya bipinnata
Leaf Extracts by Various Hepatotoxin Induced Albino
Rat Models
D. Benito Johnson*, Neethu.
P. Charles, Banshongdor H Mawlieh,
Timai Passah,
V. Venkatanarayanan
Department of Pharmacology, RVS college of
Pharmaceutical Sciences, Sulur, Coimbatore
*Corresponding Author E-mail: drbenitorvs@gmail.com
ABSTRACT:
Liver diseases are the major
medical problems faced by the people all over the world. About 20,000 deaths occur every year due to
liver disorders Liver diseases are mainly caused by toxic chemicals, excessive
intake of alcohol, infections and autoimmune disorders. Hepatotoxicity
mainly implies chemical-driven liver damage. Certain drugs when taken in
overdose and sometimes even when administered within therapeutic ranges may
injure many organs. Some chemical agents including those that are used in
laboratories (CCl4, paracetamol) and
industries (Lead, arsenic) and natural chemicals (microcystine,
aflatoxins) and herbal remedies (Cascara sagrada, Ephedra) can also
cause hepatotoxicity Chemicals which cause liver
injury are collectively known as hepatotoxins.
So the aim of this research
is to evaluate anti- oxidant and hepatoprotective
activity using Desmostachya bipinnata leaf
extract because very less
pharmacological studies have been carried out on the leaf of Desmostachya bipinnata
by emphasizing on the antioxidant and hepatoprotective
activity in Swiss albino rat model.
The whole plant materials
are useful for the symptoms of different diseases. Desmostachya
bippinata is indicated by several medicinal
treatments for jaundice and other gastro intestinal disorder, Plant is a common
weed plant mainly cultivated Northern parts of India.
KEYWORDS: Hepatoprotective, Anti oxidant, Desmostachya bipinnata.
INTRODUCTION:
The liver is one of largest gland in the body and
after the dermis1. The liver weights about three and a half pounds
(1.6 kg). It constitutes about 2.5% of an adult’s body weight2. It
is present in the upper part of the abdomen that aids in digestion and removes
the waste products and worn-out cells from the blood. Liver is connected to two
large blood vessels which include hepatic artery and portal vein2.
30 percentage blood was pumped by the heart for one minute for body’s chemical
factorial organ called liver. Liver cleanses blood and processes nutritional
molecule that are distributed to the tissues.
Liver accept nutritional red blood by portal circulation from lungs
which is filled with essential oxygen to be supplied to heart. Part of the body that receives more blood
than liver is brain. It is situated in the upper part of the abdominal cavity,
inferior to the diaphragm occupying the greater part of the right hypochondriac
region, part of the epigastric region and extending
into the left hypochondriac region. Its upper and anterior surfaces are mooth and curved to fit the under surface of the diaphragm
and its posterior surface is irregular in outline3. The different
types of cells propagate from the liver lobes are parenchymal
and non-parenchymal type of cells. Majority (about
80%) of the liver mass is filled by parenchymal type
of cells commonly known as hepatocytes. the other
type non-parenchymal type cells having forty
percentage of the total counts of the liver cells but it have 6.5% of its total
volume2. It also release about two and one-half ml of the bile in
its own ducts which is delivered by a gallbladder via congested tube called the
cystic duct for storage of these bile. Liver is regulated for this gland that
control as to whether these incoming substances was useful for body or whether
they are needless. Liver is an extremely important organ and exhibits multiple
functions1. Liver detoxifies
for blood cells by proper fixation of bile solution via chemical modification
to form less toxic substances, example alteration of ammonia to urea. Many chemical substances are inactivated by
liver through modification of chemical structure. Liver convert glucose to
glycogen as a storage form of energy and it produces glucose from disaccharides
and polysaccharides such as sugars, starches and protein molecules2
HEPATOTOXICITY
Liver diseases are the major medical problems faced
by the people all over the world3. About 20,000 deaths occur every
year due to liver disorders. In Africa and in Asia, the main causes of liver
diseases are viruses and parasitic infections, whereas in Europe and in North
America, a major cause is alcohol abuse3. Liver diseases are mainly
caused by toxic chemicals, excessive intake of alcohol, infections and
autoimmune disorders2. Hepatotoxicity due
to drug appears to be a common contributing factor. Liver is expected not only to carryout
physiological functions but also to protect against the hazardous of harmful
drugs and chemicals3. Drug induced chemical injury is responsible
for 5% of all hospital admissions and 50% of all acute liver failures. More
than 75% of cases of immunological reaction of drugs leading to liver
transplantation or death.
Hepatotoxicity mainly implies chemical-driven liver damage. Certain drugs when taken in
overdose and sometimes even when administered within therapeutic ranges may
injure many organs. Some chemical agents including those that are used in
laboratories (CCl4, paracetamol) and
industries (Lead, arsenic) and natural chemicals (microcystine,
aflatoxins) and herbal remedies (Cascara sagrada, ephedra) can also
cause hepatotoxicity. Chemicals which cause liver
injury are collectively known as hepatotoxins5.
MATERIALS AND METHODS:
Chemical reagents:
Nitro blue tetrazolium
(NBT) , Riboflavin , NaCN/EDA, Phosphate buffer, DPPH, Methanol5
SUPEROXIDE RADICAL SCAVENGING ACTIVITY
Procedure:
The reaction mixture contained EDTA (0.1 M), 0.3mM NaCN, Riboflavin (0.12mM), NBT (1.5 n moles), Phosphate
buffer (67mM, pH 7.8) and various concentrations of the seed oil extract in a
final volume of 3ml. The tubes were illuminated under incandescent lamp for
15min. The optical density at 560 nm was measured before and after
illumination. The inhibition of superoxide radical generation was determined by
comparing the absorbance values of the control with that of seed oil extract
and fraction-IV. Vitamin C was used as positive control. The concentration of
fraction-IV required to scavenge 50% superoxide anion (IC50 value)
was then calculated5.
DPPH RADICAL REDUCING ACTIVITY
Procedure:
Freshly prepared DPPH (187 µl) was taken in
different test tubes protected from sunlight. To this solution added different
concentrations (0, 25, 50, 75, 100, 150, 200µg/ml) of seed oil extract and
fraction-IV. The volume was made up to 1ml with methanol. Keep the tubes in
dark and after 20 min absorbance was measured at 515nm. Methanol was used as
blank and vitamin C was used as positive control. The concentration of test
materials to scavenge 50% DPPH radical (IC50 value) was calculated
from the graph plotted with % inhibition against Concentration5.
EVALUVATION OF HEPATOPROTECTIVE ACTIVITY
PARACETAMOL INDUCED LIVER
TOXICITY:
Methodology:
The paracetamol
suspension was freshly prepared at a dose of 2 gm/kg of paracetamol
in 1ml of distilled water11. Animals (except animals in control
group) received a single dose of paracetamol 2gm /kg
through oral route for 7 day. Animals were allowed to develop hepatotoxicity, which was identified by biomarkers. Hepatotoxicity induced animals were selected for 28 days
treatment7.
CCl4 INDUCED HEPATOTOXICITY:
Methodology:
The CCl4 solution was
freshly prepared at a dose of 2ml/kg of CCl4 diluted with liquid
paraffin (1:1). Animals (except animals in control group) received a single
dose of CCl4 2ml /kg through intra peritoneal route at 30 minutes
after administration standared and extracts6.
Hepatotoxicity induced animals were selected for 7
days treatment.
ETHANOL INDUCED LIVER TOXICITY:
Methodology:
The 75% Ethanol solution was
prepared at a dose of 4gm/kg. Animals (except animals in control group)
received a single dose of Ethanol 4gm /kg once a day through p.o route 1hr after
administration of standard and extracts for 30 days10. Animals were
allowed to develop hepatotoxicity. At termination
day, animals were anesthetized and blood collected. Hepatotoxicity
was identified by biomarkers9.
Histopathology Study:
·
Rats from all the treatment
groups and control groups were euthanized on the day 7. After gross
observation, liver was collected and fixed in 10% Neutral Buffer Formalin.8
·
Trimming: Tissues were trimmed
from all the lobes of liver.
·
Processing: Processing is done
with the help of Automated Tissue Processor (ATP) (Leica
ASP 300) for 16 hours.
·
Embedding: Processed tissues
were embedded in paraffin with the help of paraffin embedding station (Leica EG 1150 H).
·
Sectioning: Initially blocks
were trimmed at 25 microns and then sectioned at 4 microns with the help of
semi automatic Microtome (Leica RM 2245).
·
Staining: Slides were stained
by HandE stain at Multistainer
(Leica ST 5020).
·
All the H and E stained slides
were observed for pathological findings.
RESULT AND DISSCUSSION:
1.
IN VITRO ANTIOXIDANT
ACTIVITIES:
Effect of Desmostachya
bipinnata leaf extract on superoxide radical
scavenging activity:
Superoxide generated in the photo reduction of
riboflavin was effectively inhibited by the addition of varying concentrations
(0-10 mL/ml) of leaf juice. The concentration of the
DPLE needed to scavenge 50% superoxide anion (IC50) was found to be
5.8 mg/ml (figure 1) Vitamin C which was used as a positive control had an IC50
value of 4.5 mg/ml.
Figure
1: Superoxide radical reducing activity of DBLE and vitamin C.
Study of in vitro DPPH Radical Scavenging
Activity of DBLE
1. DPPH RADICAL REDUCING ACTIVITY :
The
DPPH radical was effectively scavenged by seed oil extract and Fraction- IV. A
dose dependent reduction of was observed within the range of concentrations
(0-100mg/ml) of reaction system (Fig.2). The IC50 value of DBLE was
found to be 71mg/ml. Vitamin C which was used as the positive control exhibited
an IC50 value of 21.6 mg/ml.
Figure
2: DPPH radical reducing activity of DBLE and vitamin C.
The yield of the whole plant extract shows
the amount of phyto constituents soluble in the
particular solvent used. Maximum percentage yield represents the more phyto constituents present in the extract. The percentage
yield of the ethanol and aqueous extracts were more, hence those extracts were
chosen for the pharmacological evaluation.
Hepatoprotective activity
Biochemical Parameters of Paracetamol Induced Model
TABLE 1: Showing values of biochemical parameters
in paracetamol (2gm/kg) induced model.
Treatment |
Dose mg/kg |
SGOT (IU/L) |
SGPT (IU/L) |
ALP (IU/L) |
Total
Protein (gm/dl) |
Total bilirubin (mg/dl) |
Control |
Vehicle |
72.46±0.24 |
29.82±0.17 |
31.02±0.00 |
8.54±0.07 |
0.8±0.02 |
Toxic control |
2 gm/kg |
125.05±0.2 |
126.18±0.16# |
56.45±0.22 |
4.11±0.08# |
3.21±0.04# |
Silymarine |
100 |
109.8±0.21 |
89.10±0.00 |
48.7±0.21 |
6.75±0.06 |
1.86±0.21 |
Ethanol extract |
200 |
107.4±0.2 |
88.5±0.22 |
47.6±0.16 |
6.92±0.01 |
1.75±0.06 |
Aqueous extract |
200 |
110.7±0.16 |
77.5±0.21 |
48.12±0.16 |
7.24±0.07 |
1.50±0.09 |
Figure 3: Comparison of
biochemical parameters against different groups in paracetamol
induced model .
HISTOPATHOLOGY:
Figure 4: Paracetamol Induced Model
Biochemical Parameters of CCl4 Induced
Model:
TABLE 2: Showing values of
biochemical parameters in CCl4 (2ml/kg) induced model
|
SGOT IU/L |
SGPT IU/L |
ALP IU/L |
Total Protein Mg/dl |
Total Bilirubin Mg/dl |
Control |
97.55 |
55 |
102.3 |
0.27 |
8.17 |
Toxic control |
197.31 |
146 |
139.2 |
3.27 |
4.78 |
Silymarine |
104.22 |
58 |
127.02 |
1.21 |
6.19 |
Ethanol extract |
105.24 |
59.01 |
124.49 |
1.22 |
7.2 |
Aqueous extract |
99.36 |
56.08 |
119.11 |
0.88 |
7.8 |
Values expressed as Mean ±SEM; Number of animals in
each group =6.*P˂0.001
Figure 5: Biochemical
parameters in CCl4 (2ml/kg) induced model
HISTOPATHOLOGY
Figure
6: Histopathology of CCl4 Induced Model
Biochemical
Parameters of Ethanol Induced Model
Table 3: Showing values of biochemical parameters in
Ethanol (4gm/kg) induced model
Treatment |
Dose mg/kg |
SGOT(IU/L) |
SGPT(IU/L) |
ALP(IU/L) |
Total Bilirubin
(mg/dl) |
Total Protein (gm/dl) |
Normal Control |
Vehicle |
76.4±3.38 |
27.81±1.26 |
103.77±2.79 |
0.63±0.04 |
8.94± 0.21 |
Toxic Control |
4g m/kg |
147.82±5.5 |
119.88±1.89 |
159.49±1.08 |
2.25±0.08 |
3.27±0.09 |
Silymarine (100mg/kg) |
100mg/kg |
109.23±2.16 |
45.05±2.55 |
115.91±3.25 |
1.24±0.15 |
6.00±0.18 |
Ethanol extract |
200mg/kg |
115.13±1.05 |
47.42±0.70 |
114.24±1.16 |
1.65±0.06 |
5.51±0.22 |
Aqueous extract |
200mg/kg |
104.22±2.03 |
39.26±1.89 |
104.66±1.60 |
1.40±0.12 |
6.90±0.13 |
Values
expressed as Mean ±SEM; Number of animals in each group =6; *P˂0.0001
Figure 7:
Comparison of biochemical parameters against different groups in Ethanol
induced model.
HISTOPATHOLOGY
Figure 8: Ethanol Induced
Model
CONCLUSION:
The
preliminary phytochemical screening of whole plant
extracts indicate in presence of flavanoid, alkaloid,
tannins, terpenoids and glycoside.
The
antioxidant studies particularly showed that BALE have slight antioxidant
potential but that not inferior than standard vitamin C .
Hepatoprotective study results shows that the levels of SGOT, SGPT,
ALP and Total Bilirubin were significantly
improvement may accounts hepatoprotective activity
All these
observation imply that the DBLE could be regarded as a favorable antioxidant
and hepatoprotective agents.
REFERENCES:
1. Gerald J Tortora
Bryan Derrickson, Principles of anatomy and
physiology, 13th edition, page 990-995.
2. Kmiec′Z (2001) .‟Cooperation of
liver cells in health and disease”. Adv Anat Embryol Cell Biol. 161:3-13, 1-151.
3. Anne Waugh, Allison Grant,
Ross and Wilson, Anatomy and Physiology in health and illness, 9th
edition, page307-309.
4. http://diagramreview.com/wp-content/uploads/simple-Human-Liver-Diagram.jpg
5. WebMD, Image collection: Human
Anatomy.
6. Friedman Scott E, Grendell James h, McQuaid,
Kenneth R. Current diagnosis and treatment in gastroenterology. New York: Lang
medical book/mcgraw-hill.2008; Pp664-679.
7. Essential Pathology, 3rd
edition, Harsh Mohan, 2007, page 361.
8. Miller-Keane Encyclopedia and
Dictionary of Medicine, Nursing and Allied Health, Seventh Edition 2003 by
Saunders, an imprint of Elsevier.
9. Wang CS; Chang Ting-Tsung; Yao Wei-Jen; Wang Shan-Tair;
Chou Pesus (2012). ‟Impact of increasing alanine amino transferase levels
within normal range on incident diabetes”. J Formos
Med Assoc. 3(4):201-8.
10. Ghouri N; Preiss David; Sattar Naveed (2010). ‟Liver enzymes, nonalcoholic fatty
liver disease and incident cardiovascular disease: a narrative review and
clinical perspective of prospective data”. Hepatology
52(3): 1156-61.
Received on 02.05.2016 Modified on 19.06.2016
Accepted on 29.06.2016
©A&V Publications All right reserved
Res. J. Pharmacognosy and Phytochem.
2016; 8(3):109-115.
DOI: 10.5958/0975-4385.2016.00020.0