Membrane Stabilization assay for Anti-inflammatory activity yields misleading results for samples containing traces of Methanol
Govind Navale1, Dipak D. Patil3, Aashutosh A. Patil2, Ketan B. Patil2, Narendra B. Patil2*
1Department of Bioanalytical, Sitec Labs Pvt Ltd.,Mahape (Navi Mumbai)
2Deprtment of Pharmacology, Ahinsa Institute of Pharmacy, Dhule-Road, Dondaicha.
3Department of Quality Assurance, H. R.Patel Institute of Pharmaceutical Education and Research, Shirpur.
There is an increased in the reports on erythrocyte haemolysis assay being used as a simple and rapid tool for screening of drugs and phytochemicals for anti-inflammatory activity. However, in case of hydroalchoholic extracts, there is a possibility of the trace amounts of methanol retained even after drying. These traces are not acconted in the assay units. The present study was designated to substantiate the effects of low concentrations of methanol on the heat induced haemolysis. It was observed that the presence of methanol at concentrations as low as 0.1 ml/ml exert significant membrane stabilizing activity. Thus, this assay may give misleading results if the test drug solution contains even slighest amount of methanol. Therefore, Present study suggested that the heat-induced erythrocyte haemolysis assay is not suitable for testing membrane stabilizing and anti-inflammmatory activity of the test samples containing methanol.
The in-vitro erythrocyte haemolyis assay is generally used for screening anti-inflammatory activity of drugs.1 Majority of the anti-inflammatory drugs stabilize the plasma membrane of mammalian erythrocyte and thereby inhibit the heat-induced and the hypotonicity-induced haemolysis.5
The plasma Mammalian red blood cells do not contains nuclei or internal membranes, So they are used as a source from which pure plasma membranes can be easily isolated for biochemical analysis.
The erythrocyte plasma membrane resemblances to the lyosomal membrane and hence the stabilizing effect drugs on erythrocyte membrane may correlate with its lyosomal membrane stabilizing effect.4
The lyosomal membrane stabilization leads to the inhibition of release of the inflammatory mediators and consequent inhibition of the process of inflammation.6 In the membrane stabilization assay, the erythrocytes are challenged with different haemolytic stimuli like heat, osmotic shock and free radicals.7 The heat induced and Hypotonicity induced haemolysis of erythrocytes is extensively used as a rapid, simple, economic and sensitive tool in determining the anti-inflammatory property of drugs. Due to this simplicity and economy, the researchers prefer to use this model in the preliminary screening of hydro-alcoholic extracts of medicinl plants. While standardizing this model in our laboratory, we came across the facts that methanol have significant membrane stabilizing activity. there is dearth of reports on the evaluation of lower concentrations of methanol on the eryrhrocyte membrane stabilization.5 in present work ,we investigated that membrane stablilzing effects on methanol against the heat-induced breakdown of human erythrocytes.
MATERIALS AND METHODS:
Methanol was procured from sigma aldrich. Ibuprofen was purchased from Glumex Pharmaceuticals Ltd., India and all the solutions used in this study were of analytical grade.
Phosphate buffer saline Solutions:
Weigh 8gm of NaCl, 0.2 KCl, 1.44 gm of Na2HPO4,0.24 gm KH2PO4 all weighing ingredients dissolved in up to 1 litre of distilled water and adjust the pH 7.4 with HCl and sterilised by autoclaving.
To prepare the erthyrocyte suspension.15 ml of Whole blood sample were collected through Vein Puncture from a healthy human volunteer. Each sample was mixed with equal amount of sterile phosphate buffered saline solution (PBS) containing 100 IU/ml of Heparin. These samples were centrifuged at 2500 rpm for 7 minutes and the erythrocyte pellets was washed thrice with heparinized PBS and finally suspended in PBS to contain 40% v/v of RBS in PBS.
We evaluated the effects of methanol in concentrations ranging from 100ml/ml to0.001ml/ml in the final assay medium.
Procedure for Heat-induced Haemolysis:
We evaluated the effects of methanol such as 100 ml/ml, 10 ml/ml, 1 ml/ml, 0.1 ml/ml, 0.01 ml/ml, 0.001ml/ml were prepared in PBS. The experiments were carried out in duplicates. In eppendrof tubes, equal amounts of each of PBS, stock erythrocyte suspension and methanol dilutions were taken. Negative controls in which methanol dilutions were replaced with PBS were also maintained. Positives control contained Ibuprofen at concentration of 50mg/ml of assay medium.
The eppendrof tubes were heated in a waterbath at 50°C for 18 minutes. After heating ,the reaction mixtures were centrifuged at 4500 rpm for 3 minutes and the absorbance of supernatant was measured using microplate reader (Powerwave XS, Biotek) at 540 nm.
RESULTS AND DISCUSSION:
Heating the assay units at 50°C for 18 minutes. After heating, the figure 1 depicts the haemolysis in the negative control samples as 100% and the relative percentage of haemolysis in the samples treated with various concentrartions of methanol. The samples treated with Ibuprofen were protected from heat induced haemolysis of erythrocytes.
The data were analysed by the one-way ANOVA. P < 0.01 was considered as statically significant. The assay units containing methanol were also protected from the haemolysis and this protective effects was statically significant even at methanol content assay medium as low asb 0.01 ml/ml. Our results indicate that even trace amount of methanol significantly protects the erythrocytes from heat induced haemolysis erythrocyte membrane stabilization model is widely used model and it is very simple, easy to carry out. It includes incubation of erythrocytes with drug and then heating is used to on duce haemolysis.
Membrane stabilization involves the process in which the integrity of the erythrocytes membrane and lyosomal membrane is maintained by anti-inflammatory drugs by stabilizing the membrane. The stabilizing effect of the drugs on erythrocyte membrane may be due to a stabilizing effect of the drugs on certain proteins in the membrane.8
Fig.No.1 Methanol protects the human erythrocytes againest the heat induced Haemolysis.
Our interest in this topic was developed as many studies have reported the membrane stabilizing activity of methanolic extract of various plant. For the sake of our interest we designed this study using the plain methanol at various concentrations. Because methanol are widely used as solvent for extraction of active phyto-constituents from plants as well as it is used in preparation of various homeopathic remedies.
Hence this assay may yield misleading results particularly for the hydroalcoholic extracts which may contains traces of methanol even after sufficient drying and reconstitution. It is suggested that this assay should be used with caution for the extracts and drug solutions which are suspected to contain methanol.
CONFLICT OF INTEREST:
No conflict of interest.
Authors are thankful to Sitec labs Pvt.Ltd., Mahape (Navi-Mumbai) and Principal of Ahinsa Institute of Pharmacy, Dondaicha.
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Received on 11.05.2019 Accepted on 10.06.2019
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Asian J. Pharm. Res. 2019; 9(3):169-171.