Studies On Chemical Constituents And Its Activity Of Rubus Pungens Var. Oldhamii

The plant Rubus pungens var. oldhamii belongs to the Rosaceae family, which includes many medicinal plants. In recent years, some researches were conducted on the extraction and isolation of the active pharmaceutical chemicals from rubus. It was found that the active constituents in rubus were abundant, including volatile oil, flavonoids, tannins, terpenoids, phenols, which own diverse bioactivities, such as antioxidant, antibacterial, anti-inflammatory and antitumor activities. Therefore, it has a high medicinal value and an important significance for human health caring. To our knowledge, the information regarding the bioactivities, main active compounds and exploitation of R. pungens var. oldhamii, which belongs to Genus Rubus, is limited. Therefore, the aim of the present study is to extract chemical constituents from R. pungens var. oldhamii and to determine the bioactivities. We epecially focused on the extraction and isolation of compounds functioned as antioxidant and antibacterial, which would provide a theoretical basis for the development of food preservatives, antioxidants from R. pungens var. oldhamii. The main results were summarized as following:The systematic pretests on chemical constituents of R. pungens var. oldhamii indicated that the chemicals in leaves of R. pungens var. oldhamii mainly contained sugar, phenols, flavonoids, terpenoids, tannins, and volatile oil. The results of activity assay found that the extracts of R. pungens var. oldhamii could inhibit the growth of some food spoilage bacteria. The60%ethanol extract had good antibacterial effects on Escherichia coli, Bacillus subtilis and Staphylococcus aureus, with the minimum inhibitory concentration (MIC) as6.25mg/mL,12.5mg/mL,12.5mg/mL, respectively. Meanwhile, the extracts of R. pungens var. oldhamii also demonstrated having high antioxidant capacity. In concentration of50mg/mL, the DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical-scavenging capacities of water and ethanol extracts were69.67%and83.02%, respctivelyWith the factors of extraction temperature, extraction time and ethanol concentration as the dependent variable and the antioxidant activity of extract as response value, the method of response surface methodology (RSM) combined with a Box-Behnken design (BBD), was applied to optimize the extraction process for antioxidant extracts of R. pungens var. oldhamii. The RSM results showed that, the factors of extraction time and ethanol concentration had significant influence on the response value. And the optimal extraction temperature, extraction time and ethanol concentration were80℃,85min,65%, respectively. Under these conditions, the predicted antioxidant activity of extract was214.64mg/g·DW. The experimental data were in good accord with the predicted values, which demonstrated that the BBD is an efficient approach to develop mathematical models for predicting antioxidant activity of extract.The extract was then purified with the diversified organic solvents. Through monitoring the antioxidant activity of various solvent extracts, the ethyl acetate (EA) was considered as the optimum solvent. The EA extract fraction was subjected to silica gel column chromatography to give167fractions. After the thin layer chromatography test, the167fractions were combined to F1-F19fracitons. The antioxidant compounds mainly existed in F3-F16fractions according to chromogenic of DPPH-methanol solution, among which, the fractions of F3,6and12-16had strong effects on DPPH free radical-scavenging capacity. Furthermore, the highest antioxidant activity was observed in F16fraction. And its50%inhibitory concentration (IC50values) was12.54μg/mL, which was comparable to that of antioxidants Vc (11.30μg/mL) and BHA (14.90μg/mL).The antioxidant components were further isolated and purified by column chromatography. The compound A was obtained in F6through a small silica gel column (1.0×25cm). The compound B was obstained in F16through a Sephadex LH-20column (24x700mm) and HPLC analysis. According the spectral data of 13C-NMR and1H-NMR, the compound A was identified as2-Hydroxybenzoic acid and the compound B was identified as Dibutyl phthalate. The salicylic acid has antioxidant activity, with IC50values on superoxide anion scavenging acitivity as420.56μg/mL, its antioxidant activity was higer than BHA.