Screening of Anti-inflammatory Potential of Berberis coriaceae Leaves by HRBC Membrane Stabilization

Amit Roy^1*, Ram Kumar Sahu²

¹Columbia Institute of Pharmacy, Tekari, Raipur (C.G.), India

²Oriental College of Pharmacy, Patel Nagar, Bhopal (M.P.), India.

ABSTRACT:

The present study was undertaken to screen anti-inflammatory activity of alcohol and aqueous extracts of leaves from Berberis coriaceae against HRBC membrane stabilization. The prevention of hypotonicity induced HRBC membrane lysis was taken as a measure of the anti-inflammatory activity. Both the extracts showed a biphasic effect on the membrane stabilization. Their activities are comparable to that of the standard drug diclofenac sodium. However their activities decreased with time.

KEY-WORDS: Berberis coriaceae, HRBC, Diclofenac sodium

INTRODUCTION:

Inflammation is the complex biological response of vascular tissues to harmful stimuli including pathogens, irritants or damaged cells. It is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. Inflammation, however, if runs unchecked, led to onset of diseases such as rheumatoid arthritis and atherosclerosis. It is believed that current drugs available such as opoids and nonsteroidal anti-inflammatory drugs are not useful in all cases of inflammatory disorders, because of their side effects and potency. As a result, a search for other alternatives is necessary. Through medicinal plants, having a wide variety of chemicals, novel anti-inflammatory agents could be discovered. Research on the biological activities of plants during the past two centuries has yielded numerous compounds for the development of modern drugs^1-3. Berberis coriaceae (Berberidaceae) known in Hindi as Kashmal is an erect spiny shrub, ranging between 2 and 3 meters in height wood, hard and yellow; leaves, yellow to brown from outside and deep yellow from inside, removable in longitudinal strips by hand; spines (which, in fact, are modified leaves), three-branched and 1.5 cm long⁴. The objective of present study was to evaluate anti-inflammatory activity of leaves of Berberis coriaceae by HRBC membrane stabilization.

MATERIAL AND METHODS:

Plant material

The proposed study of Berberis coriaceae leaves were collected from the Sunder Nagar, Mandi, Himachal Pradesh, with the help of local tribal and field botanist. Care was taken to selected healthy plant and for normal leaves. The leaves were shade dried, reduced to coarse powder and stored in airtight container till further use.

Preparation of extract

The dried and powdered leaves (300 gm) were successively extracted on a Soxhlet apparatus, employing petroleum ether, ethanol and distilled water respectively. The extracts were further concentrated under reduced pressure with a rotary evaporator.

Anti-inflammatory activity

The HRBC membrane stabilization has been used as method to study the ant inflammatory activity. Blood was collected from healthy volunteers. The collected blood was mixed with equal volume of sterilized Also ever solution (25dextrose, 0.8% sodium citrate, 0.5% citric acid and 0.42% sodium chloride in water). The blood was centrifuged at 3000 rpm and packed cell were washed with isosaline (0.85%, pH 7.2) and a 10% (v/v) suspension was made isosaline.

The assay mixture contained the drug (concentration as mentioned in Table 1), 1 ml of phosphate buffer (0.15 M, Ph 7.4), 2 ml of hyposaline (0.36%) and 0.5 ml of HRBC suspension. Diclofenac was used as the reference drug. Instead of hyposaline 2 ml of distilled water was used in the control. All the assay mixture were incubated at 37°C for 30 min and centrifuged. The hemoglobin content in the supernatant solution was estimated using spectrophotometer at 560 nm. The percentage hemolysis produced in the presence of distilled water as 100%. The percentage of HRBC membrane stabilization or protection was calculated using the formula^5-7.

O.D. of drug treated sample

% Protection = 100 – -------------------------------------------X 100

O.D. of control

Statistical analysis

Results are expressed as Mean ± SEM.

RESULT AND DISCUSSION:

The lysosomal enzymes released during inflammation produced a variety of disorders. The extracellular activity of these enzymes is said to be related to acute or chronic inflammation. The Diclofenac drugs act either by inhibiting these lysosomal enzymes or by stabilizing the lysosomal membrane.

Table 1 Effect of concentration on the activity of ethanol and aqueous extracts of B. coriaceae

Concentration (µg/ml)	Activity (Prevention of lysis %)
Concentration (µg/ml)	Ethanolic Extract	Aqueous Extract	Diclofenac
10	49.25±0.12	44.63±1.52	-
50	60.32±1.03	55.71±1.21	54.23±1.46
100	39.47±1.35	40.26±0.86	-
200	32.58±0.36	35.62±1.42	-

Values are expressed as mean ± SEM (n=6)

Table 2 Variation of activity with time (drug concentration 10 µg/ml in all)

Concentration (µg/ml)	Activity (Prevention of lysis %)
Concentration (µg/ml)	Ethanolic Extract	Aqueous Extract	Diclofenac
10	58.52±0.52	50.63±1.15	71.32±0.75
20	48.63±1.32	44.86±1.47	65.42±0.69
50	38.68±1.12	39.46±0.89	57.51±1.01
100	34.52±0.65	35.58±1.34	49.22±0.88
200	31.45±1.45	30.43±1.11	45.38±1.18

Values are expressed as mean ± SEM (n=6)

Since HRBC membrane is similar to lysosomal membrane components, the prevention of hypotonicity induced HRBC membrane lysis is taken as a measure of anti-inflammatory activity of drugs. Both extracts of the leaves of B. coriaceae showed biphasic effects on HRBC membrane stabilization (table 1). They increasing activity at low concentration levels but decreasing activity with high concentrations. They have a critical concentration (50 µg/ml) at which their activities are maximum. The activities of both extracts are comparable to that of Diclofenac at concentration of 50 µg/ml the variation of activity with time was studied at 10 µg/ml concentration, the activities in general decreased with time (table 2). The results of anti-inflammatory revealed that both extracts displayed anti-inflammatory activity, while ethanolic extract has more significant as compared to aqueous extract.

CONCLUSION:

B. coriaceae plant was found to posses good membrane stabilizing property which is one of the preliminary steps involved in the screening of anti-inflammatory property. Hence it can be concluded that the plant may have the anti-inflammatory activity. But further in vivo studies have to be performed to confirm the claim. Since leaves of this plant are rich in multiple phytoconstituents, further studies have to conducted on the isolation, identification and characterization of the pharmacologically potent moiety responsible for the activity.

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Received on 10.11.2011

Modified on 05.12.2011

Accepted on 27.12.2011

Research J. Pharmacology and Pharmacodynamics. 4(1):Jan. - Feb., 2012, 5-6