In Vitro Anti Inflammatory Activity on the Ethanolic bark extract of Perseaamericana M.

 

Zeena Fernandes*

Ullas D’ Souzaand Prasanna Shama Khandige, Department of Pharmacology, N.G.S.M Institute of Pharmaceutical Sciences, Nitte (Deemed to be university) Paneer, Derlakatte, Mangaluru-575018

 

ABSTRACT:

Thepresent study was designed to evaluate the in vitro anti-inflammatory effect of the ethanolic extract of Perseaamericana bark against the denaturation of protein. The anti-inflammatory property of the extract was evaluated by preparing the extract at different concentrations and incubating it with egg albumin under controlled experimental conditions. Absorbance of the extract at different concentrations was determined by using reference drug as diclofenac sodium.. The present findings exhibited a concentration dependent inhibition of protein (albumin) denaturation by the Perseaamericana bark.From the present study it can be concluded that Perseaamericana bark possessed marked in vitro anti-inflammatory effect against the denaturation of protein.

 

 

 

INTRODUCTION:

Nature can be called as a store house of remedies to cure ailments of mankind as it contributes us medicines from the plants and herbs to heal the diseases which are incurable, with few side effects.¹The natural products may have compounds that have little or no action by themselves but they can be made to produce potent drugs by modifying their chemical or biological activity to produce drugs that are of importance.²Inflammation is a normal protective response to tissue injury caused by physical trauma, noxious chemical or microbial agents. It is the body’s response to inactivate or destroy the invading organisms, to remove the irritants and set the stage for tissue repair

 

Perseaamericana is a medicinal tree or large bush which belongs to the Lauracea family known mostly for its edible fruit Avocado,which is commonly called as butter fruit.

 

 

Perseaamericana is native to the central and south America but is now widely cultivated in tropical climates throughout the world. The various morphological parts of the plant were reported to be effective against hepato-toxicity, cancer, hypertension, anti-inflammatory, in diarrhoea, dysentery, to lower blood cholesterol levels, analgesic and also in the management of childhood convulsions and epilepsy. Asno systematic studies were done on the anti-inflammatory activity of the ethanolic extract of the bark of Perseaamericana the present study was undertaken.

 

MATERIALS AND METHODS:

Plant material and preparation of Extract:

The plant bark was collected from Madikeri town, Kodagu District, Karnataka, India, and was authenticated by Dr. Noeline Pinto, Department of Botany, St Agnes College, Mangalore Karnataka, India. The fresh bark of Perseaamericana was sun dried and coarsely powdered and subjected for maceration. For the extraction, the powdered bark was macerated in ethanol and kept aside for 7 days with stirring occasionally. After 7 days, the total extract of the solvent was distilled off and concentrate was evaporated on a water bath to a syrupy consistency and was evaporated until dry. The extract was stored in a dessicator for further use. Preliminary phytochemical analysis was performed for testing the different chemical groups present in the ethanolic extract by standard procedure.³

 

Assessment of Invitro anti-inflammatory activity:

Inhibition of Albumin Denaturation⁴.

The extract at different concentrations was incubated with egg albumin in controlled experimental conditions and subjected to determination of absorbance to assess the anti -inflammatory property. Diclofenac sodium was used as the reference.The reaction mixture (5 ml) will be consisting of 0.2ml of egg albumin (got from fresh hen’s egg), phosphate buffer saline 2.8ml whose pH is 6.4 and 2ml ofethanolic extract of Perseaamericana of different concentrations so that we get a final concentration of 31.25, 62.5, 125, 250, 500, 1000 µg/ml. Distilled water was taken as control which was of same volume as the reaction mixture. Both these mixtures were incubated at (37±2) °c in a BOD incubator for 15 min and later heated for 5 min at a temperature of 70 °c. Once cooled, the absorbance was measured at 660nm (Shimadzu, UV 3600) by taking vehicle as the blank. The reference drug diclofenac sodium at concentrations 78.125, 156.25, 312.5, 625, 1250, 2500µg/ml was treated similarly and the absorbance was determined.

 

The turbidity obtained will be measured spectrophotometrically at 660 nm .The percentage inhibition of protein denaturation was calculated by the formula:

 

% inhibition = 100 x (Vt / Vc - 1)

V _c= absorbance of control. V_t= absorbance of test sample.

 

RESULTS:

Thein vitro anti-inflammatory studies on the effect of ethanolic extract of perseaamericana on the denaturation of egg albumin are summarised in Table 1. The experimental data obtained shows that there is a concentration dependent inhibition of protein denaturation by ethanolic extract of Perseaamericana all throughout the concentration range of 31.25-1000µg/mL. Diclofenac the standard drug which was used in the concentration of 78.25-2500µg/mL also showed  inhibition of protein denaturation in a concentration dependent manner (Table 2). But it was seen that the effect of the standard drug diclofenac sodium when compared to the ethanolic extract of Perseaamericana was found to be lesswhich was further confirmed by the comparison of the IC50 values.IC50 value of ethanolic extract of Perseaamericana was found to be 20 µg/mL whereas that of diclofenac was 140µg/mL

 

Table 1: Effect of ethanol extract of Perseaamericana on protein denaturation

	Concentration(µg/mL)	Absorbance	% Inhibition
1	Control	0.040	-
2	31.25	0.660	68.75
3	62.5	0.810	84
4	125	0.930	96.87
5	250	1.050	109.38
6	500	1.310	136.45
7	1000	1.390	144.79

 

Table 2:Effect of diclofenac sodium on protein denaturation

	Concentration	Absorbance	% Inhibition
1	Control	-	-
2	78.125	0.116	12.08
3	156.25	0.566	58.95
4	312.5	0.77	80.21
5	625	1.69	176.04
6	1250	1.76	183.33
7	2500	1.85	192.71

 

DISCUSSION:

There are certain problems in using animals in experimental pharmacological research, such as ethical issues and the lack of rationale for their use when other suitable methods are available or could be investigated. Hence, in the present study the protein denaturation bioassay was selected for in vitro assessment of anti- inflammatory property of ethanolic extract of Perseaamericana bark. One of the documented cause of inflammatory disease is denaturation of tissue proteins. Agents that can prevent protein denaturation therefore, would be worthwhile for anti-inflammatory drug development⁵. From the IC50 values ethanolic bark extract of Perseaamericana was found to be 20μg/mg and of the standard diclofenac sodium was 140 μg/mg. From these results it becomes evident that bark extract of Perseaamericana is more effective in lower concentration than diclofenac sodium and hence has anti- inflammatory property ^6,7. The major constituents of ethanol bark extract of Perseaamericanaare alkaloids, flavonoids, tannins andpolyphenols. Polyphenols are well known natural products known to possess several notable biological properties. In the present study, the in vitro anti-inflammatory activity of ethanol bark extract of Perseaamericanacan be attributed to its polyphenols content. The effect may be due to the synergistic effect rather than single constituent. It has been reported that one of the features of several non-steroidal anti-inflammatory drugs is their ability to stabilize (prevent denaturation) heat treated albumin at the physiological pH (pH: 6.2-6.5).

 

CONCLUSION:

Therefore, from the results of the present preliminary study it can be concluded that Perseaamericana possessed marked in vitro anti-inflammatory effect against the denaturation of protein.The active constituents and the exact mechanism for this anti-inflammatory action need to be ascertained. Further research work is neededto ascertain the mechanisms and constituents behind its anti-inflammatory actions.

 

ACKNOWLEDGEMENT:

The authors are thankful to Nitte (Deemed to be University) and N.G.S.M. Institute of Pharmaceutical Sciences for providing facilities for carrying out the work.

 

REFERENCES:

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Accepted on 13.10.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(12): 5517-5519.