IN VIVO ANTIOXIDANT ACTIVITY OF PHYLLANTHUS EMBLICUS AGAINST CISPLATIN INDUCED OXIDATIVE STRESS IN MICE
Mamuna Naz, Uzma Saleem, Bashir Ahmad
Faculty of Pharmacy, The University of Lahore, Lahore-Pakistan.Assistant Professor, Faculty of Pharmaceutical Sciences, G. C. University, Faisalabad-Pakistan. Director / Professor, Riphah Institute of Pharmaceutical Sciences, Riphah International University, Lahore-Pakistan.
Keywords: In vivo antioxidant activity, hypolipidemic, hepato-renal curative
Abstract

Background: Plants are rich source of antioxidants. They can ameliorate the oxidative stress induced complications. Cisplatin is a cytotoxic drug which produced unwanted effects in the patients due to generation of free radicals inside the body. Phyllanthus emblicuspossessed in vitro antioxidant activity. Objective: The current study was aimed to explore in vivo antioxidant potential of Phyllanthus emblicus against the oxidative stress induced by cisplatin in mice. Method: oxidative stress was induced in micewith acute toxic dose (10 mg/kg) of cisplatin given i.p. Animals were divided into five groups (n = 5). Group I: negative control, group II: positive control group III and IV were given methanol extract ofPhyllanthus emblicus (250- & 500 mg/kg; orally respectively) and group V was standard group receiving orally vitamin C & E (200 mg/kg each) for 20 days. On 21st day, animals were sacrificed and oxidative stress biomarkers were quantified. Result: Phyllanthus emblicusextract showed vigorous in vivo antioxidant effect at 500mg/kg by increasing the SOD, CAT, and GSH (antioxidant enzymes) levels in heart, liver, kidney and brain homogentaes and MDA level decreased. Plant also displayed a cure against oxidative stress induced changes in renal, liver and lipid profile parameters. Conclusion: Phyllanthus emblicusraised antioxidant enzyme levels in mice. It manifested hypolipidemic, hepato-renal curative effects. Its adjuvant use with standard therapies may help to resolve unwanted effects.

Article Information

Identifiers and Pagination:
Year:2016
Volume:8
First Page:1
Last Page:6
Publisher Id:19204159.8:1.2016
Article History:
Received:October 06,2015
Accepted:November 13,2015
Collection year:2015
First Published:January 1, 2016

Introduction

Phyllanthus emblicus (common name: Amla) belongs to family Euphorbiaceae. Chemical constituents include ellagic acid, gallic acid, emblicanian A and emblicanian B [1].Number of researches unveiled its various pharmacological properties such as anti-tumor [2], antidiabetic [3], antimicrobial [4], gastroprotective [5], hypolipidemic [6], in vitro antioxidant activity[7, 8] antitussive [9] analgesic [10], anti- inflammatory [11], memory enhancing [12] and snake venom neutralizing effect[13]. Cisplatin is a cytotoxic drug which produced unwanted effects in the patients due to generation of free radicals (oxidative stress) inside the body.Oxidative stress is one of the causative agents in number of morbidities. Antioxidants help combat oxidative stress induced diseases. The current study was aimed to explore in vivo antioxidant activity of Phyllanthus emblicaagainst cisplatin induced oxidative stress in mice.

 

MATERIALS AND METHOD

Sample collection and preparation of extract

Fresh fruits of Phyllanthus emblica were collected fromsuburbs of Lahore-Pakistan. They were dried under shade and ground to fine powder. Extract was prepared in 70 % methanol by maceration and solvent was removed on rotary evaporator at 40oC.

Preparation of samples

Distilled water was used to prepare Phyllanthus emblicusand vitamin C solutions. Olive oil was used for vitamin E. All the samples were administered orally (p.o.).

Induction of oxidative stress

Acute toxic dose of cisplatin (10mg/kg) was given intraperitoneally (i.p). to induce oxidative stress.

Study Design

Twenty five mice of either sex weighing from 25 to 30 g were divided into five groups (n=5). Group I served as negative control, receiving chow and water ad libitum. Group IIwas positive control, received only cisplatin (10mg/kg). GroupIII and IV were given cisplatin (10mg/kg)via i.p. route three hours prior to administering plant extract at 250mg/kg and 500mg/kg dose levels respectively for 20 days. Group V was given two standard antioxidants i) vitamin C (200mg/kg p.o.) ii) vitamin E (200mg/kg p.o.) for 20 days.

Collection of blood sample

On 21st day animals were anaesthetized using chloroform and blood samples were collected by cardiac puncture. Serum was separated for analysis of renal function tests (RFT’s), liver function tests (LFT’s), lipid profile.

Preparation of tissue homogenates

Animals were sacrificed and organs i.e. liver, heart, kidney and brain were isolated and immediately stored in phosphate buffer (pH 7.4). Tissue homogenates were prepared by using ice cold phosphate buffer, it was then centrifuged for 10 minutes at 4000 rpm. Cellular debris was settled down and supernatant was separated and stored for quantification of antioxidant enzymes [glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) malondialdehyde (MDA)].

Quantitative analysis of oxidative stress biomarkers in tissues homogenates

GSH, CAT, SOD and MDA levels were quantified by adoptingMoron et al., 1979,Aebi, 1974, Kakkar et al.,1984, and Ohkawa et al. 1979methods respectively [14-17].

Determination of renal function tests, liver function tests and lipid profilefor quantitative analysis of oxidative stress biomarkers in serum

Renal function tests (BUNand serum creatinine), liver function tests (ALT, ALP, AST & total protein) and lipid profile (cholesterol, triglycerides, LDL & HDL) were determined by using randox UK kits..

Statistical analysis

The data were presented as mean ± SD. One way ANOVA followed by post hoc duncan test was applied using SPSS version 16.0. P <0.05 was considered significant.

RESULTS

Quantification of oxidative stress biomarkers in tissue homogenates

There was significant increase (P<0.05) inGSH, SOD, and CAT levels and decrease in MDA levels at 500 mg/kg dose of Phyllanthus emblicus in liver, brain, kidney and heart homogenates as compared to positive control group (Table 1-4). 

Table 1. Quantitative analysis of oxidative stress biomarkers in liver

Table. Quantitative analysis of oxidative stress biomarkers in brain

Table 3. Quantitative analysis of oxidative stress biomarkers in kidney

Quantitative analysis of liver function tests

Levels of alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) were increased in the positive control group (cisplatin 10mg/kg), while in the same group level of total protein (TP) was decreased. Treatment with Phyllanthus emblicus at 500mg/kg showed maximum decrease in the level of these enzymes and increase in total protein content indicating the hepato curative effect of the plant (Table 5).

Table 4. Quantitative analysis of oxidative stress biomarkers in heart

Table 5. Effect of various treatments on LFT’s

Quantitative analysis of renal function tests

Injection of cisplatin (10mglkg) increased the level of serum creatinine and BUN in positive control group, which was significantly decreased after various treatments. Maximum decrease in the BUN and creatinine levels was observed in the group of animals treated with Phyllanthus emblicus 500mg/kg (Table 6).

Table 6. Effect of various treatments on renal function tests

Quantitative analysis of lipid profile

There was increase in cholesterol, triglycerides, and LDL levels in positive control group while HDL value decreased as compare to negative control group.Phyllanthus emblicus (500mg/kg) showed maximum decrease in cholesterol, triglycerides, and LDLlevels whereas HDL increased. There was significant variation (P<0.05) in all lipid profile parameters of all the groups with respect to positive control

Table 7. Effect of various treatments on lipid profile

DISCUSSION

Results of current study showedsignificant in vivo antioxidant potential of Phyllanthus emblicus as it increased oxidative stress biomarkers in tissue homogenates. Biochemical tests on serum also revealed hepato-renal curative effect and hypolipidemic effect. From the results, it had been observed that Phyllanthus emblicus antioxidant activity was greater than the combined effect of vitamin E and C.It may be concluded from the findings that Phyllanthus emblicus extract at the dose of 500mg/kg could be an effective and novel approach for treating oxidative stress induced pathological conditions.

CONCLUSION

Phyllanthus emblicusraised antioxidant enzyme levels in mice. It manifested hypolipidemic, hepato-renal curative effects. Its adjuvant use with standard therapies may help to resolve unwanted effects.

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Prof. Dr. Cornelia M. Keck (Philipps-Universität Marburg)
Marburg, Germany

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Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

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