Int J Curr Pharm Res, Vol 9, Issue 5, 194-197Original Article


EVALUATION OF ETHANOLIC ROOT EXTRACT OF PARTHENIUM HYSTEROPHORUS LINN FOR ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITY

PANKAJ LOHUMI*, TIRATH KUMAR, LIPI NOGAI

Department of Pharmaceutical Sciences, Kumaun University, Bhimtal Campus, Nainital, Uttarakhand, India 263136
Email: Pankajlohani26@gmail.com

Received: 20 May 2017, Revised and Accepted: 22 Jul 2017

ABSTRACT

Objective: The work is aimed to draw out the health beneficial properties of a weed (Parthenium hysterophorus Linn). The present work is organized to evaluate the antioxidant and anti-inflammatory activity of the ethanolic root extract of Parthenium hysterophorus Linn.

Methods: In the present work the ethanolic extract was determined by using soxhlet apparatus. The antioxidant scavenging activity of this extract was determined by applying three different assay methods: (1) DPPH (1, 1-diphenyl-2-picryl hydrazyl) free radical method. (2) Nitric oxide scavenging assay and (3) Reducing power method. The anti-inflammatory activity was determined by in vivo method i.e. Carrageenan induced rat paw edema.

Results: DPPH radical scavenging activities of the standard antioxidant and extracts were found to be increased in dose dependent manner. The percentage inhibition increases from 4.19% to 97.09% within the concentration range of 10µg/ml to 160µg/ml. Parthenium hysterophorus Linn effectively reduced the generation of nitric oxide radicals from sodium nitroprusside solution in a concentration dependent manner and percentage inhibition increases from 3.53% to 55.21% within the concentration range 10µg/ml to 160µg/ml. All the concentrations of extract significantly showed higher absorbance than the absorbance of control reaction (0.9705) in reducing power assay. A Higher absorbance indicates high reducing power due to the formation of reduced intermediates. Parthenium hysterophorus Linn showed highly significant anti-inflammatory activity at a dose of 200 mg/kg and the lesser effect was observed at 100 mg/kg with the percentage change in paw volume at 0 min, 30 min, 60 min, 90 min and 120 min.

Conclusion: Thus, from above experimental observations, it can be stated that Parthenium is a natural antioxidant and bearing anti-inflammatory activity.

Keywords: Parthenium, Ethanolic extract, Antioxidant, Anti-inflammatory activity

© 2017 The Authors. Published Published Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijcpr.2017v9i5.22327

INTRODUCTION

Oxygen is an essential and basic component for all living organisms which helps in the process of oxidation and maintenance of biological system [1]. Production of energy by the reduction of dioxygen (O2) leads to the formation of reactive oxygen species. Free radicals are chemically unstable molecule and damage to cells, resulting imbalance between the generation of reactive oxygen species (ROS) and the antioxidant system collectively known as oxidative stress [2]. In the past decades, free radicals have aroused interest among the scientists. Their broad range of effects in biological systems has drawn the attention of many experimental works. Oxidative damage plays a significant pathological role in human diseases like arthritis, atherosclerosis, cancer etc [3]. Almost all organisms are well protected against free radical damage. There are many antioxidant defence systems in the body such as superoxide dismutase, catalase, α-tocopherol, ascorbic acid, glutathione, polyphenolic compounds, carotenoids etc. played an important role to maintain its integrity [4]. An antioxidant is a substance when present in low concentrations relative to the oxidizable substrate, significantly reduces oxidation process [5]. They scavenge the free radicals and prevent the tissue from damage. They can highly prevent the damage caused by free radicals by neutralizing the free radicals before they attack the cell. Flavonoids, ligans, phenolic acids and phenolic diterpenes are an example of phenolic compounds with the character of quenching oxygen-derived free radicals [6].

Inflammation is a protecting attempt by means of the organism to cast off the injurious stimuli and to initiate the healing manner. Although infection is resulting from a microorganism, inflammation is one of the response of an organism to the pathogen. However, for each pathogen, inflammation is a stereotyped response, and therefore it is considered as a mechanism of innate immunity, as compared to adaptive immunity [7]. Inflammation is a marker of the majority of diseases and number of people affected with inflammation, about 11% of adult men seen in urology clinics and 3% of children in globally balanitis. Chronic wounds counts 0.78% of population and the prevalence ranges from 0.18-0.32%. Globally the incidence of epiglottis was 15 per 1, 00,000 people [8].

Inflammation is a biochemical and cellular response that takes place in all vascularised tissue whose fitness and vitality or energy is threatened via either an internal or an external source. The important components of the inflammatory response can be observed within the blood, and mediators of inflammation function to increase the motion of plasma and infection fighting blood cells from the capillary bed into or across the injured tissue. The local inflammatory response is later observed via prominent systemic response called acute phase response {APR}. This response is marked by the induction of fever, anorexia, increased synthesis of hormones such as adrenocorticotropic hormone {ACTH}, increased leukocytosis and changed production of proteins in the liver. Proteins whose level changes during inflammation are known as acute phase protein {APP} [9].

Macrophages play principle roles within the immune and inflammatory responses involved in host protection. A number of different inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) reactive oxygen species (ROS), prostaglandin E2 (PGE2), nitric oxide (NO) are secreted by activated macrophages [10].

MATERIALS AND METHODS

Plant material

Congress grass (Parthenium hysterophorus) roots were collected from Haldwani, Uttarakhand, India and identified in the IIIM Jammu. The roots were cleaned and cut into small pieces and were subjected to dry at room temperature. The dried roots were grinded and powdered.

Preparation of extracts

The 30g of dried and powdered plant material was extracted with 300 ml of solvent ethanol by using soxhlet apparatus for 48h at a temperature not exceeding the boiling point of the solvents. The extract was filtered by using Whatman No. 1 paper. The extract was concentrated by using rotary evaporator then dried and was used for further investigations.

Antioxidant activity

DPPH radical scavenging activity assay

The stable 1, 1-diphenyl-2-picryl hydrazyl radical (DPPH) was used for determination of free radical-scavenging activities of the extracts, as per the method described by Hatano et al. A different concentration of each extract was added, at an equal volume, to an ethanolic solution of DPPH (0.1 mmol). After 30 minutes, at room temperature, the absorbance was recorded at 517 nm. The experiment was repeated for three times. Ascorbic acid was used as standard antioxidants. IC50 value represents the inhibitory concentration of the sample, which is required to scavenge 50% of DPPH free radicals [11].

Percentage inhibition was calculated as,

% scavenged DPPH radical = [(Abs control-Abs sample)]/(Abs control)] × 100

Where, Abs control is the absorbance of DPPH radical+methanol; Abs sample is the absorbance of DPPH radical+sample extract/ standard.

Reducing power method

Reductive ability of the extract was measured according to the method of Oyaizu (Oyaizu, 1986) [12]. Different concentrations (10-320µg/ml) of extract were mixed with 2.5 ml of sodium phosphate buffer (pH 6.6) and 2.5 ml of potassium ferricyanide (1%). The mixture was incubated at 50ͦC for 20 min. Trichloroacetic acid (2.5 ml of 10%) was added to it, the mixture was mixed and centrifuged at 650 rpm for 10 min. The upper layer (5 ml) was mixed with 5 ml of deionized water and 1 ml of ferric chloride (1%) and absorbance was measured at 700 nm. Control reaction contains all the reagents except test compound. Higher absorbance indicated higher reducing power.

Nitric oxide scavenging activity

Sodium nitroprusside (5 mmol) in phosphate buffer saline was mixed with different concentrations of ethanolic extract (10-320 µg/ml) dissolved in DMSO and incubated at 25 ͦC for 30 min. After 30 min, 1.5 ml incubated solution was removed and diluted with 1.5 ml of Griess reagent (1% suphanilamide, 2% orthophosphoric acid and 0.1% napthylethylene diamine dihydrocholride). The absorbance of the chromophore formed during diazotization of the nitride with sulphanilamide and subsequent coupling with napthyethylene diamine was measured at 546 nm along with a control [13]. The percentage inhibition of nitric oxide generated was measured by comparing the absorbance values of control and test samples using following formula.

Percent inhibition (%) = [((Acontrol-Asample)/Acontrol)]*100

Where, Acontrol is the absorbance of the control reaction (containing all reagents except test compound), and Asample is the absorbance of test compound. IC50 value (concentration of sample required to scavenge 50% of free radicals) were calculated from the regression equation, prepared from the concentration of the samples and percentage inhibition of free radical. Ascorbic acid was used as positive control and all tests were carried out in duplicate.

Carrageenan induced rat paw edema [14]

RESULTS AND DISCUSSION

DPPH free radical scavenging activity assay

DPPH radical scavenging activities of the standard antioxidants and extracts were found to be increased in dose dependent manner. The IC50 values of the standard antioxidant i.e. ascorbic acid was evaluated as 7.84 µg/ml. While the IC50 of the extract was calculated in various as 3.53 µg/ml. Therefore, it is concluded that Parthenium hysterophorus Linn is having excellent antioxidant activity against DPPH radical.

Fig. 1: DPPH scavenging activity of ethanol extract of Parthenium hysterophorus root with different concentration (µg/ml)

Reducing power method

The extract exhibited concentration dependent increase in absorbance. Absorbance indicated by all the concentrations of extract was significantly higher than the absorbance of control reaction (0.9705). Higher absorbance indicates high reducing power due to formation of reduced intermediates. Ascorbic acid has much high reducing ability than the ethanolic extract of Parthenium hysterophorus Linn.

Nitric oxide scavenging effect

The ethanolic extract of Parthenium hysterophorus Linn effectively reduced the generation of nitric oxide radicals from sodium nitroprusside solution in a concentration dependent manner. This showed significant nitric oxide scavenging activity of the extract (IC50 of ascorbic acid was found to be 7.84µg/ml.)

Carrageenan induced rat paw edema

The in vivo anti-inflammatory activity was performed by the carrageen an induced rat paw edema. Parthenium hysterophorus Linn showed significant anti-inflammatory activity at a dose of 200 mg/kg and lesser effect was observed at a dose of 100 mg/kg.

Fig. 2: Effect of different samples i.e. standard, control and test samples for reducing power assay

Fig. 3: Effect of different concentrations of ethanolic extract of Parthenium hysterophorus Linn on nitric oxide scavenging assay

Fig. 4: Effect of different samples (Control, Standard, T1 higher dose and T2 lower dose on Carrageenan induced rat paw edema)

Table 1: Variations in paw edema for different groups with respect to time

Group Dose Increase in paw edema
0 min 30 min 60 min 90 min 120 min
Control 10 ml/kg 0.2292±0.05966

0.3958±

0.0936*

0.5417±

0.0768***

0.7083±

0.0768***

0.8750±

0.0559
Standard 10 mg/kg 0.5833±0.1236

0.4167±

0.08333**

 0.1667±0.02635**

0.1458±

0.02083***

0.125±

0.00
Test T1 200 mg/kg 0.5833±0.08333

0.375±

0.0559**

 0.2708±0.05017***

0.1875±

0.02795***

0.1458±

0.02083
Test T2 100 mg/kg 0.5417±0.07683

0.375±

0.0559*

 0.2917±0.06972**

0.1875±

0.02795***

0.1458±

0.02083

All values are significant and are expressed in mean±SEM, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 indicates the level of statistical significance as compared to control.

In the present work, after cumulating the results, it could be considered that the congress grass root extract might be a potent source of antioxidant and possesses anti-inflammatory potential.

CONCLUSION

From the above experimental observation, it can be clearly stated that the Parthenium hysterophorus is a promising source of natural antioxidant and anti-inflammatory agent and surely provides an alternative towards synthetic antioxidant and opens a new aspect of research trend for Parthenium as a natural antioxidant and anti-inflammatory agent.

ACKNOWLEDGEMENT

I would like to express my heartfelt appreciation to all those who provided me the possibility to complete my report. A special gratitude to my guide, Dr. Tirath Kumar, research supervisor whose contribution in encouragement helped me out to coordinate my work throughout the year.

CONFLICT OF INTERESTS

Declare none

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