Use of betel leaf extract to induce IFN-gamma production from human peripheral blood T cells and as a Th1 type immunomodulator

Abstract

This invention relates to use of betel leaf extract to induce IFNγ from human peripheral blood mononuclear cells and as a Th 1 type immuno modulator, wherein the said method comprises the steps of, preparing water extract of betel leaf, preparation of human peripheral blood mononuclear cells, incubation of hPBMC with betel leaf extract for a period of 18-48 hours, extraction of RNA for cytokine specific RT-PCR or for flow cytometry for the detection of intracellular cytokine protein, subjecting RNA for RT-PCR to obtain PCR products using IFNγ specific known primers, and enhancement of IFNγ as reflected by IFNγ specific band.

1 . (canceled)  
     
     
         2 . A method for inducing IFNγ produced from human peripheral blood mononuclear cells wherein the said process further comprises alternate steps of: 
 a) subjecting incubated cells for intracellular staining for IFNγ,    b) analysis of stained cells in flow cytometer, and    c) enhancement of IFNγ positive cells to at least seven fold.    
     
     
         3 - 10 . (canceled)  
     
     
         11 . A pharmaceutical composition useful as Th 1  type immunodulator, said composition comprising an effective amount of betel leaf aqueous extract or lyophilized betel leaf extract together with or associated with an additive.  
     
     
         12 . A composition as claimed in  claim 11  wherein, the lyophilized extract is obtained by freeze drying the aqueous extract by conventional methods.  
     
     
         13 . A composition as claimed in  claim 11  wherein, the additive is selected in such a manner that does not interfere with the activity of betel leaf extract.  
     
     
         14 . A composition as claimed in  claim 11  wherein, the additive is selected from nutrients such as proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste and/or pharmaceutically acceptable carriers, excipient, diluent or solvent.  
     
     
         15 . A composition as claimed in  claim 11  wherein, the aqueous extract, lyophilized product or the composition is administered orally or intramuscularly.  
     
     
         16 . A composition as claimed in  claim 11  wherein, the oral route is in the form of capsule, syrup, concentrate, powder or granules.  
     
     
         17 . A composition as claimed in  claim 11  wherein the ratio of betel leaf extract to the additive is in the range between 10-1:1-10.  
     
     
         18 . A composition as claimed in  claim 11  wherein, the betel leaf extract, lyophilized extract or the composition comprising the betel leaf extract is administered at a dosage level between 5 to 10 mg/kg of body weight at least once in a day for one month.  
     
     
         19 . A composition as claimed in  claim 11  wherein, the lyophilized betel leaf extract has the following properties: 
 i) the dried sample is a dark colored material soluble in water and dimethyl sulfoxide,    ii) thin layer chromatography of the active material shows five spots having R f  0.75, 0.64, 0.50, 0.40 and 0.33 in the solvent system of n-butanol, acetic acid and water in the ratio of 9:5:7 respectively, and    iii) the HPLC analysis of the active material using Intersil ODS-3 (4.6×250 mm) analytical column, solvent system methanol and water in the ratio of 4:1 and a flow rate of 1.0 ml/min., detection at 217 nm resolved the material into eleven peaks with the retention time of 2.69, 4.27, 5.95, 6.97, 7.49, 9.39, 11.20, 12.40, 15.53, 18.90 and 21.49 min.    
     
     
         20 . (canceled)  
     
     
         21 . A use of betel leaf extract or its lyophilized extract or a composition comprising effective amount of betel leaf extract for inducing IFNγ from human peripheral blood mononuclear cells or as a Th 1  type immunomodulator.  
     
     
         22 . Use as claimed in  claim 21  wherein, the said composition comprising betel leaf extract associated with or in combination with a pharmaceutically acceptable additive.  
     
     
         23 . Use as claimed in  claim 21  wherein, the additive is selected in such a manner it does not interfere with the activity of lyophilized betel leaf extract.  
     
     
         24 . Use as claimed in  claim 21  wherein, the additive is selected from nutrients such as proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, starch-gelatin paste and/or pharmaceutically acceptable carriers, excipient, diluent or solvent.  
     
     
         25 . Use as claimed in  claim 21  wherein, the aqueous extract, lyophilized product or the composition is administered orally or intramuscularly.  
     
     
         26 . Use as claimed in  claim 21  wherein, the oral route is in the form of capsule, syrup, concentrate, powder or granules.  
     
     
         27 . Use as claimed in  claim 21  wherein, the ratio of betel leaf extract to the additive is in the range between 10-1:1-10.  
     
     
         28 . Use as claimed in  claim 21  wherein, the betel leaf extract, lyophilized extract or the composition comprising the betel leaf extract is administered at a dosage level between 5 to 10 mg/kg of body weight at least once in a day for one month.