| Polygonum bistorta L.(quan shen) belongs to family Polygonaceae and is one of the strongest traditional herb astringents that have been used around the world for centuries. Its roots and leaves are cooked and eaten in raw form. It has traditionally been used as a cure of diarrhea, cholera, scrofula, oral ulcer, dysentery with bloody stools, leucorrhoea, and venomous snakebites. Recent studies have shown its bioactive properties such as anticancer, antibacterial, antiviral, antimutagenic, antioxidant and anti-inflammatory activities. Chemical studies have shown that it possesses different classes of constituents such as phenolic compounds, flavonoids, steroids, triterpenoids, tannins and others.In the first research work, an optimized, simple, sensitive and reliable HPLC separation method for the rhizome of Polygonum bistorta L. was developed. The optimized extraction conditions including soaking, sonication for30minutes and heating at80℃for one hour were selected to achieve the maximum number of peaks with maximum peak height. Different chromatographic conditions, most importantly the mobile phase composition and pH were studied and compared in the Reversed Phase separation mode to acquire fingerprinting profiles.30distinct peaks were obtained and3phenolic acids (gallic acid, protocatechuic acid and chlorogenic acid) with anticancer bioactivity were identified by comparing their retention times with reference marker compounds in the optimized method. The repeatability was tested and%RSD values were calculated for retention times and peak areas and the resulting values were less than0.5%and1.2%, respectively in any case for these respective compounds. Resolution of the peaks of marker compounds with respect to the adjacent peaks (next to the respective compounds) were also calculated which were found to be not less than1.6in any case that ascertained a good separation for such a complex sample. The differences in the resolution values of peaks in three experiments were calculated as their%R.S.D which were not greater than3.02%in any case. The developed RP-HPLC method can be used for quality assessment and further identification, quantification and purification of the important bioactive constituents present in the rhizome of Polygonum bistorta L.The second study was designed to specifically identify and evaluate the phenolic content of Polygonum bistorta L. for cytotoxicity. For this purpose methanol-water (40:60v/v) extract was subjected to conventional preparative high pressure liquid chromatography and13fractions were obtained. Constituents of obtained fractions were separated and identified with the help of GC-MS and LC-DAD-ESI-MS. Phenolic compounds such as gallic acid, protocatechuic acid,p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, syringic acid, catechol,4-methyl catechol, syringol and pyrogallol were separated from different fractions. Fractions were evaluated for their cytotoxic activity on human hepatocellular carcinoma cell line (HCCLM3).11fractions showed good to strong cytotoxicity in a range of200μg/mL800μg/mL, whereas2fractions did not show any activity even at800μg/mL and no phenolic content was detected from them.50percent growth inhibition (GI50) values for five most active fractions were calculated and results were in a range of86.5(±3)μg/mL-126.8(±3) μg/mL.3out of these5most active fractions were found to contain phenolic content in them whereas all other fractions containing phenolic content did possess cytotoxic activity that strengthens the relationship between phenolic content and anticancer activity. Moreover, the results also showed a definite dose dependent relationship between the phenolic content and cytotoxic activity of Polygonum bistorta L. In addition to these phenolic constituents, different other constituents including the ones related to other bioactive properties were also separated and identified from these fractions and the raw sample of this traditional medicine.In third study, essential oils present in rhizome of Polygonum bistorta L. that were obtained from two different Asian regions (China and Pakistan) were extracted by hydrodistillation, analyzed and identified by GC-MS and confirmed by calculating their retention indices (RI).79compounds were obtained in different amounts and75were identified successfully. Main constituents found were furfural (6.31%), palmitic acid (5.21%),24(E)-ethylidenecycloartanone (5.19%), oleic acid (3.74%), linoleic acid (3.01%) and cosanes from sample of Chinese origin sample while oleic acid (8.87%), oleic acid methyl ester (8.63%), palmitic acid (6.58%), linoleic acid methyl ester (4.01%) and cosanes from Pakistani origin. These two essential oils were separately tested against3Gram-positive and3Gram-negative bacterial strains for antibacterial activity. The Lowest MIC value obtained was0.063mg/mL against G. oxydan and the highest was≥2mg/mL against A. pretiosum. Comparison of the results from samples of different origins showed that the difference was found, not only in their percentage composition but also in their antibacterial activities, which could possibly be due to difference in growing and cultivating conditions of two different regions. Moreover, to further study and support this conclusion (plants of different origins exhibit different chemical composition),20%methanol-water extract obtained from two different regions was run and separated through HPLC and results strongly supported this phenomenon. |
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