Ashok Kumar BS, Saran G, Harshada R, Manoj B, Archana PG. Evaluation of anti-arthritic activity of vitex negundo by in vitro protein denaturation method. J Traditional Med, 2014; 1(1): 0000001.

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Journal of Traditional Medicine

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IntroductionVitex negundo belongs to the family Verbenaceae is a large aromatic shrub or a small tree of about 3M in height. It is claimed

to possess anti-arthritic, antileprotic, anti-inflammatory, anti-spasmodic and anti-asthmatic, astringent, anthelmintic; promotes the growth of hair; useful in disease of the eye, consumption, enlargement of the spleen, painful teething of children [1-3]. Vitex negundo Linn reported to possess laxative Activity [4], Anti-inflammatory activity, antihistaminic, membrane stabilizing and antioxidant activities [5].

Rheumatoid arthritis is a chronic inflammatory disease characterized by inflammation of the synovial membrane, pain and restricted joint movement [6]. arthritis is one of the commonest autoimmune diseases, is a chronic, progressive, systemic inflammatory disorder affecting the synovial joints and typically producing symmetrical arthritis that leads to joint destruction, which is responsible for the deformity and disability. The consequent morbidity and mortality has a substantial socio-economic impact [7].

The prevalence of arthritis is approximately in the West [8]. The prevalence of RA in India subcontinent is 1.5-2 percent of population. The epidemiological ratio of arthritis in female: male is 3:1 and the prevalence is 1% of the world population. Progression of the disease results in joint destruction deformity and significant disability [9].

The present study is aimed to investigate antiarthritic activity of Vitex negundo by in vitro protein denaturation method.

Materials and MethodsCollection of plant material and extraction

Leaves of Vitex negundo were collected from surroundings of Chickballapur, Karnataka and was authenticated by Prof. B.K.Venkatesh, Department of Botany, Government First grade College, Chickballapur, Karnataka. A voucher specimen (SKVCP 11) was deposited in college herbarium. Leaves were dried and made into coarse powder and the coarse powder was subjected to extraction

Evaluation of anti-arthritic activity of vitex negundo by in vitro protein denaturation method

Research Article Volume 1 • Issue 1 • 2014

AffiliationsaDepartment of Pharmacognosy, Sri K.V.College of Pharmacy, Chickballapur, Karnataka, IndiabDepartment of Pharmaceutical Chemistry, Sri K.V.College of Pharmacy, Chickballapur, Karnataka, India

*Corresponding author: Ashok Kumar BS, Department of Pharmacognosy, Sri K.V.College of Pharmacy, Chickballapur, Karnataka, India, E-mail: ashok4vani@gmail.com

AuthorsAshok Kumar BSa*, Saran Ga, Harshada Ra, Manoj Bb and Archana PGa

Article historyReceived - July 05, 2013

Accepted - January 20, 2014

Published - January 23, 2014

KeywordsVitex negundo; Diclofenac sodium; Antiarthritic

activity; Denaturation of protein

Copyright© Ashok Kumar BS et al. 2014

AbstractIntroduction: Vitex negundo (Verbenaceae), traditionally used as anti-arthritic and anti-inflammatory. The main aim of the study is to screen antiarthritic activity of aqueous extract of leaves of Vitex negundo by protein denaturation.

Methods: The extract at different concentrations was incubated with egg albumin and bovine albumin in controlled experimental conditions and subjected to determination of absorbance to assess the anti-inflammatory property. Diclofenac sodium was used as the reference drug.

Results: The present findings exhibited a concentration dependent inhibition of protein (albumin) denaturation by the Vitex negundo. The effect of diclofenac sodium was found to be less when compared with the Vitex negundo (1000 µg/ml).

Conclusion: From the present study it can be concluded that Vitex negundo showed marked in vitro antiarthritic effect against the denaturation of protein.

Ashok Kumar BS, Saran G, Harshada R, Manoj B, Archana PG. Evaluation of anti-arthritic activity of vitex negundo by in vitro protein denaturation method. J Traditional Med, 2014; 1(1): 0000001.

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with water by soxhlet apparatus and extract was concentrated to dryness in vacuum.

Preliminary phytochemical screening

The methanol extract of A.spinosus was screened for the presence of various phytoconstituents like steroids, alkaloids, glycosides, flavonoids, carbohydrates, proteins and phenolic compounds [10].

Evaluation of in vitro anti-arthritic activity

Protein denaturation by using Egg albumin [11]: The reaction mixture (5 ml) consisted of 0.2 ml of egg albumin (from fresh hen’s egg), 2.8 ml of phosphate buffered saline (PBS, pH 6.4) and 2 ml of varying concentrations of aqueous extract of Vitex negundo so that final concentrations become 100,200,400,800 and 1000 µg/ml. Similar volume of double-distilled water served as control. Then the mixtures were incubated at (37±2)°C in a Biochemical oxygen demand (BOD) incubator for 15 min and then heated at 70°C for 5 min. After cooling, their absorbance was measured at 660 nm. Diclofenac sodium (100 µg/ml) was used as reference drug. The percentage inhibition of protein denaturation was calculated by using the following formula:

Percentage inhibition = (Abs control – Abs test sample) X 100/ Abs control

Whereas Abs- Absorbance.

Protein denaturation by using Bovine albumin [12]: The reaction mixture was consisting of aqueous extract of Vitex negundo at different concentrations and 1 % of aqueous solution of bovine albumin. The samples were incubated at 37°C for 20 min and then heated at 57°C for 20 min. after cooling the samples. Absorbance of turbidity was measured at 660 nm. Diclofenac sodium (100 µg/ml) was used as reference drug.Percentage of inhibition of protein denaturation was calculated as follows:

Percentage inhibition = (Abs control – Abs test sample) X 100/ Abs control

Whereas Abs- Absorbance.

Results Preliminary phytochemical screening

On preliminary phytochemical analysis of aqueous extract of Vitex negundo showed the presence of flavonoids, saponins, alkaloids, carbohydrates, phenolic compounds and proteins.

In vitro anti-arthritic activity

The effect of aqueous extract of Vitex negundo was evaluated against denaturation of egg albumin and bovine albumin. The results are summarized in table 1 and 2. The presenting findings exhibited a concentration dependent inhibition of protein denaturation by Vitex negundo throughout the concentration range of 100 to 1000 µg/ml. diclofenac sodium at concentration 100 µg/ml showed less effect compared to Vitex negundo at concentration (1000 µg/ml).

DiscussionIn the present study the protein denaturation bioassay

was selected for in vitro assessment of antiarthritic activity of aqueous extract of leaves of Vitex negundo. Denaturation of tissue proteins is one of the well-documented causes of inflammatory and arthritic diseases. Production of auto antigens in certain rheumatic diseases may be due to in vivo denaturation of proteins. The mechanism of denaturation probably involves alteration in electrostatic, hydrogen, hydrophobic and disulphide bonding [13]. Agents that can prevent protein denaturation therefore, would be worthwhile for antiarthritic drug development. The increments in absorbance of test samples with respect to control indicated stabilization of protein i.e. inhibition of heat-induced protein (albumin) denaturation by Vitex negundo and reference drug diclofenac sodium [14]. Vitex negundo contains alkaloids, flavonoids, tannins and a phenolic acid are known to promote antiarthritic activity [15].

ConclusionFrom present study it can be concluded that Vitex negundo

possessed marked antiarthritic effect against the denaturation of protein.References

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Table 1: Effect of Vitex negundo on protein denaturation (Fresh egg albumin).

Treatment Concentration (µg/ml)

Percentage of inhibition (%)

Aqueous extract of Vitex negundo

100 36.11200 41.66400 57.40800 79.621000 88.40

Diclofenac sodium 100 80.24

Table 2: Effect of Vitex negundo on protein denaturation (Bovine serum albumin).

Treatment Concentration (µg/ml)

Percentage of inhibition (%)

Aqueous extract of Vitex negundo

100 20.22200 44.4400 58.6800 66.251000 70.88

Diclofenac sodium 100 72.04

Ashok Kumar BS, Saran G, Harshada R, Manoj B, Archana PG. Evaluation of anti-arthritic activity of vitex negundo by in vitro protein denaturation method. J Traditional Med, 2014; 1(1): 0000001.

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This is an Open Access article under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The authors are thankful to K.V. Naveen Kiran, chairman, Sri K.V.College of Pharmacy, for providing facilities to carry out the work.

Acknowledgements