Pharmaceutical composition for the prevention or treatment of inflammatory diseases or immune diseases containing ramalin
a technology of ramalin and composition, applied in the field of pharmaceutical composition, can solve the problems of inflammatory response, neuronal cell death, host cell or tissue damage, etc., and achieve the effect of inhibiting the excessive production of nitric oxid
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example 1
Isolation of Ramalin
[0056]672 g of a freeze-dried and ground Ramalina terebrata sample was extracted three times with a mixture of methanol and water (5 L, 80:20 v / v) and freeze-dried, thereby obtaining 83 g of a crude extract. The crude extract was dissolved in 1 L of distilled water, and then extracted with 1 L of n-hexane and chloroform (CHCl3) to obtain 12.7 g of a n-hexane extract, 9.1 g of a chloroform extract (CHCl3) and 61.0 g of a water-soluble extract. The water-soluble extract showed highly activity (IC50=9 μg / ml) against DPPH free radicals. A portion (5 g) of the water-soluble extract was then subjected to automated mild pressure liquid chromatography (MPLC) using a stepwise gradient solvent system of 0%, 20%, 40%, 60%, 80% and 100% methanol in water. The fraction dissolved in 0% methanol-water mixture showed high activity (IC50=8 μg / ml) against DPPH free radicals, and a portion (100 mg) of this extract was subjected to semi-preparative reverse phase HPLC using a C18ODS ...
example 2
Effect of Ramalin Treatment on the Production Of Nitric Oxide in Macrophages Treated with LPS
[0060]In order to examine the effect of pre-treatment with Ramalin on the LPS-induced production of NO, verification was performed using the mouse macrophage line RAQ 264.7 (ATCC, Rockville, Md., US). The cells were cultured in RPMI 1640 medium (GIBCO, Grand Island, N.Y., US) supplemented with 2 mM L-glutamine, 100 IU / ml penicillin, 100 μl / ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS: Carlsband, US). The cell culture was performed under the conditions of 37° C., 5% CO2 and wet air, and the medium was replaced twice a week.
[0061]The macrophages were cultured in a 96-well tissue culture dish at a concentration of 1×105 cells / well, and treated with Ramalin for 2 hours in order to examine cytotoxicity. The cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay which measures the mitochondrial activity of viable cells. The r...
example 3
Effect of Ramalin Treatment on the Production of iNOS in Macrophages Treated with LPS
[0066]In order to examine the effect of pretreatment with Ramalin on the LPS-induced production of iNOS, the production of mRNA was analyzed by RT-PCR, and the production of protein was analyzed by Western blot. This is because a decrease in the production of NO is attributable to the inhibition of production of iNOS. For RT-PCR, cells were treated with LPS and Ramalin as described in Example 2, and then the total RNA of the cultured cells was collected using Trizol (Invitrogen, US). Then, the total RNA was amplified by Superscript one step RT-PCR using a PLATINUM Taq kit (Invitrogen, US). The PCR product was loaded onto 1.2% agarose gel which was then stained with EtBr. As a control, GAPDH was used. The PCR amplification was performed using the following primer sequences: for iNOS-F, 5′-AGA CTG GAT TTG GCT GGT CCC TCC-3′(SEQ ID NO: 1); for iNOS-R, 5′-AGA ACT GAG GGT ACA TGC TGG AGC-3′ (SEQ ID NO: 2...
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