The isolation of gallic acid from traditional medicine garidi-5 / Монголын Анагаах Ухаан

ABSTRACT

BackgroundThe preparations of multi-component have been the subject of chemical study for a long time. Therefore, when compounding the preparations of multi-component in traditional medicine, their taste is cautiously relied on, as the power of the one medicine should not be subdued with the power of another. Additionally the properties of the components and their regulating effects on the body systems are also considered. Our research group has been carrying out tests for raw materials,which are contained in multi-component preparations. However, it is a necessity to conduct phytochemical study on multi-component preparations in order to isolate pure biological active compounds and to identify their structure as well as to quantify its amount by modern techniques of analysis.GoalThe aim of the present study was to isolate pure biological active substances from Mongolian traditional medicine Garidi-5 and to elucidate their structures, which was used in Mongolian traditional medicine for the treatment of inflammation and as a pain relieving remedy.Objectives:1. To isolate pure substances from Garidi-5 and carry out tests to identify and determine their structure2. To quantify the amount of biological active substances.Materials and MethodsMongolian traditional medicine Garidi-5 has been selected as a biological natural product for the study. Garidi-5 is a traditional Mongolian medicine consisting of 5 medicinal herbs, namely Terminalia chebula Retz., Aconitum Kusnezoffii Reichb., Acorus calamus L., Saussurea lappa L., and musk of Moschus moschiferus and manufactured in the Drug factory of Traditional Medical Science Technology and Production Corporation of Mongolia. In this research, in order to determine the total content of phenolic compound was used the Folin–Ciocalteu method, which based on performing dark blue color complex compound. Isolated substance identification was determined by the TLC, UV and IR spectrophotometric methods. Inaddition it was checked melting point of the isolated substance. Determination of Gallic acid30g Garidi-5 was macerated in 60ml 80% methanol at room temperature for 24 h. After extraction, the extract was concentrated and vacuum evaporated. Different solvents from hexane, chloroform, ethyl acetate and n-butanol were used for theexperiment. All the extracts collected, evaporated and chromatographed on Silica gel column. Future purification of active fractions on Silica gel with methanol yielded the compound G1 which was further characterized as Gallic acid. Total phenolic content was determined spectrophotometrically according to the Folin–Ciocalteu’s method with slight modification. Gallic acid was used as a standard phenolic compound. Briefly, 1 ml of extract solution contains 1 mg extracts, in a volumetric flask diluted with distilled water (46 ml). One ml of Folin-Ciocalteu reagent was added and the content of the flask mixed thoroughly. After 3 min, 3 ml of Na2CO3 (2%) was added and then the mixture was allowed to stand for 2 h with intermittent shaking. The absorbance was measured at 760 nm in a spectrophotometer. All measurements were performed in triplicate.ResultsIn this research, TLC method on silica gel plates was used in order to identify the biological active pure substances from Garidi-5. Preliminary TLC experiments indicated the presence of Gallic acid in Garidi-5, which was isolated by column chromatography by comparing with reference standard substance (Gallic acid). Gallic acid was determined in the solvent system benzole-ethyl acetateformic acid- acetone (5520.5) in isolated substance (G1). It showed blue color, Rf =0.65, on TLC plate. [1] For the characterization of two samples it was carried out IR analysis for each. In the IR spectra of G1 and standard substance can be recognized by the following absorption frequency regions 700-900 cm-1 for Car-H; 1000-1300 cm-1 for vibration of bonds in various oxygen containing groups, 1350-1470 cm-1 for vibrations of –CH, -CH2 and –CH3 groups; 1500-1630 cm-1 for skeletal vibrations of aromatic rings, >C=O bonds; 2800-2950 cm-1 for stretching vibrations of –CH, -CH2 and -CH3 groups in saturated aliphatic structures; and 3030-3350 cm-1 for stretching associated vibrations of -OH groups in aromatic rings and aliphatic structures. As a result it was revealed that both IR spectra of G1 and standard substances were similar. [3]Further for the characterization of two samples it was carried out UV analysis of each. In the UV spectra of G1 and standard substance can be recognized by the following absorption frequency regions 260-280nm for benzole groups; 200-225nm for carbonic acids; 400-770nm >C=O bonds, which reveal the presence of Gallic acid. In addition, melting point of isolated substance G1 was analyzed and detected at 2410C, which was similar to the standard substance’s melting point. [4]Moreover, Mongolian traditional medicine Garidi-5 contains 24% of the biological active substance (total phenolic compounds). [2]Conclusions:As a result of current study on Mongolian medicine Garidi-5, it was isolated one essential substance from ethyl acetate fraction. The phytochemical analysis reveals the presence of Gallic acid in Garidi- 5, which was determined by thin layer chromatography, UV and IR spectrophotometric methods. Mongolian traditional medicine Garidi-5 contains 24% of the biological active substance. Thus, the isolation of Gallic acid from multi-component preparations and identification of its structure was first phytochemical study conducted in our laboratory.