| Rubus chingii Hu is a typical traditional Chinese medicine in China,which possesses a variety of biological activities and is of great prospect for research and development.The active ingredients of R.chingii are complex,which are not comprehensively investigated.Moreover,the study of functional components in R.chingii needs to be further in-depth discussion,which is to promote the development and utilization of R.chingii resource.The active ingredients of R.chingii were purified and identified in terms of modern chromatographic and spectroscopic techniques,which illustrated the main active components in R.chingii,including essential oil,bioactive compounds,polysaccharides,and so on.The possible molecular mechanisms of the anti-inflammatory effect of flavonoid glycoside(tiliroside)in LPS-stimulated macrophage RAW 264.7 cells were explored.The underlying mechanisms of proliferation inhibitions and apoptosis of human breast carcinoma cells(MCF-7)induced by triterpenoid(tormentic acid)were illustrated.The extraction,purification,physico-chemical properties,structural analysis and evaluation of anti-inflammatory activity of R.chingii polysaccharides were investigated.The comprehensive investigation of R.chingii could clarify the active ingredients responsible for the pharmaceutical activities of R.chingii and provide theories and methods for the development of relative products.(1)Three different methods,steam distillation extraction(SDE),Soxhlet extraction(SE)with ethanol,and SE with ether,were employed to extract the essential oils from R.chingii.The yields of essential oils were 0.15 %(SDE),2.12 %(SE-ethanol)and 1.98 %(SE-ether)(w/w),respectively.Chemical compositions of the three essential oils were identified by GC-MS.A total of 58 compounds were identified,including 9 saturated hydrocarbons,10 unsaturated hydrocarbons,9 alcohols,2 carbonyl compounds,11 esters,7 organic acids,8 oxides and epoxides and 2 others.The unsaturated fatty acids 9,12-Octadecadienoic acid(Z,Z)-and 9,12,15-Octadecatrienoic acid(Z,Z,Z)-showed the highest levels.Biological evaluation displayed that the essential oils extracted by SE-ether exhibited the highest anticomplementary activity,even higher than heparin(control).The essential oils extracted by SDE and SE-ethanol both showed certain cytotoxicity on A549 cell lines,and the former was higher than the latter.These results indicated the importance of selecting appropriate methods in practice.(2)In order to analyze its effective ingredients,polysaccharides,flavonoids,saponins and alkaloids were extracted from R.chingii.Biological evaluation showed that total flavonoids exhibited an obviously antioxidant effect and anti-proliferative activity against human lung adenocarcinoma A549 cells.Total flavonoids and saponins displayed a significant inhibitory effect on the complementary system,even stronger than the positive control.Given that flavonoids expressed higher multi-bioactivities than the other three ingredients,the extraction process of flavonoids was optimized as follows: extraction temperature 75 °C,ethanol concentration 30%,extraction time 2.5 h,and a liquid/solid ratio of 20 : 1.The practical mean value was 3.2 %,which was close to the theoretical value.The most abundant compound in flavonoids of R.chingii was isolated and identified as tiliroside.Tiliroside treatment suppressed cell proliferation and induced apoptosis in A549 cell lines.(3)R.chingii was extracted with 95% ethanol and then partitioned into four fractions: petroleum ether fraction,ethyl acetate fraction,n-butanol fraction,and water fraction.Results showed that the ethyl acetate fraction not only had good antioxidant activity and excellent anti-inflammatory activity,but also had significant cytotoxicity against human cancer cell lines(HepG-2,Bel-7402,A549 and MCF-7),which indicated that the main active ingredients were concentrated in the ethyl acetate fration after solvents grading extraction.Nine compounds were isolated from the ethyl acetate fraction by various column chromatography and their structures were identified in terms of nuclear magnetic resonance spectrum(NMR)and mass spectrum(MS),and they were identified as ent-16α,17-dihydroxy-kauran-19-oic acid,tormentic acid,oleanolic acid,quercitrin,hyperoside,tiliroside,β-daucosterol,astragalin and kaempferol 3-rutinoside,in which,ent-16α,17-dihydroxy-kauran-19-oic acid and tormentic acid were isolated and identified from R.chingii for the first time.(4)The flavonoid glycosides(quercitrin,hyperoside,tiliroside and astragalin)isolated from R.chingii displayed different degrees of anti-inflammatory activity,among which tiliroside showed stronger activity than others.Further analysis showed that the anti-inflammatory activity of tiliroside might be exerted through inhibiting the activation of ERK and JNK to decrease NO and pro-inflammatory cytokines(TNF-α,iNOS and IL-6)production.(5)Among all compounds isolated from ethyl acetate fraction,tormentic acid showed the better inhibitory effect of four kinds of tumor cells and one kind of normal cells determined by MTT experiments,especially on MCF-7 cells,which showed strong cells selectivity.Tormentic acid exhibited a dose-dependent inhibitory effect on MCF-7 cells,which was caused by apoptosis and G0/G1 phase arrest.Intracellular ROS levels induce mitochondrial dysfunction,G0/G1 arrest and the activation of the NF-κB and MAPKs pathways.Mitochondrial dysfunction results in the release of apoptogenic factors into cytosol,and then activates caspase-3 and caspase-9.In addition,treatment with TA induces the down-regulation of cyclin D1 and CDK4 levels,thereby finally contributing to apoptosis and G0/G1 phase arrest.(6)Polysaccharides of R.chingii Hu fruit and leaf were extracted to compare their antioxidant,anti-inflammatory,and anti-cancer activities against breast cancer cells MCF-7 and liver cancer cells Bel-7402.All tested bioactivities(antioxidant,anti-inflammatory capacities,and anti-cancer activities against MCF-7 and Bel-7402)of L-Ps were better than F-Ps.Given the results,the extraction process of L-Ps was optimized as follows: extraction temperature 88 °C,extraction time 3 h,and ratio of water to raw material 30 m L/g.Under these conditions,the experimental yield was 9.57%,which corresponded with the predicted value well.Additionally,polysaccharides from fruit and leaf were characterized and their contents of total sugars,proteins and uronic acid were compared.It was found that polysaccharides from fruit and leaf were similar in IR and UV absorption,but significantly different in contents of total sugars,protein and uronic acid.Their elution profiles of DEAE-Sepharose fast flow column were different too.The main peak of F-Ps with an average molecular weight 81 kDa was eluted using 0.3 mol/l NaCl,while the main peak of L-Ps with an average molecular weight 17 kDa was eluted with deionized water.In addition,F-Ps-3 was consisted of rhamnose,arabinose,xylose,glucose and galactose in an approximate molar ratio of 4.21:14.72:1.63:1:3.22,in which the content of arabinose was highest;while L-Ps-1 was composed of rhamnose,arabinose,xylose,glucose and galactose with the molar ratio of 2.47:4.75:4.12:1:2.48,in which the content of arabinose and xylose were higher.L-Ps showed better active capacities compared with F-Ps,therefore,the different characterizations of F-Ps and L-Ps resulted in their different bioactivities.L-Ps may be better sources for food additives and health products. |
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