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