Study On The Bioactive Chemical Constituents And Their Antioxidantive And Anti-proliferative Activities Of Crataegus Pinnatifida And Malus Doumeri Fruits

Fruits are promising potential source of bioactive phytochemicals,which exhibit diverse biological activities.Moreover,various epidemiological studies have demonstrated that increased consumption fruits are important for good health and disease prevention,especially in the prevention of chronic diseases.The fruit of Crataegus pinnatifida is a traditional Chinese medicinal herb and raw material of food industry,containing a variety of natural bioactive compounds.Thereby,C.pinnatifida is a potent source of raw material of functional foods.Malus doumeri(Bois)Chevalier,is one of well-known medical plant in south China.M.doumeri fruit is edible and also used as raw-food material in food industry.In the present work,the chemical composition,and antioxidant and anti-proliferative activities of the extracts from the fruits of C.pinnatifida and M.doumeri,including 3 varieties of C.pinnatifida,and 4 varieties of M.doumeri,were studied.Additionally,the major constituents responsible for the anti-proliferative activity of C.pinnatifida and M.doumeri fruits were obtained according to the results of anti-proliferative activities of different fractions isolated by Semi-preparative HPLC,respectively.Moreover,the possible mechanisms of anti-proliferative effect were analyzed as well.C.pinnatifida is a member of family Rosaceae native to Northern China.The phytochemical profiles and antioxidant properties of three varieties of C.pinnatifida were studied in the present work.The phenolics and flavonoids compounds of C.pinnatifida were mainly present in the free form.Among the three varieties,Shanlihong(SLH)variety exhibited elevated levels of total phenolics(58.43 mg GAE/g fruit,DW)and flavonoid contents(68.29 mg CE/g fruit,DW),including the free and bound form.A large amount of proanthocyanidins and triterpene acids were found in the free extract of C.pinnatifida fruits.And procyanidin B2 and ursolic acid were the most abundant proanthocyanidins and triterpene acids,respectively.Additionally,the free extract of SLH variety showed the highest oxygen radical absorbance capacity(ORAC),peroxyl radical scavenging capacity(PSC),and cellular antioxidant activity(CAA)values of 901.1 μmol TE/g DW,390.2 μmol VCE/g DW,and 789.9 and 1185 μmol of QE/100 g DW,respectively,compared to the other two varieties.Malus doumeri(Bois)Chevalier,belongs to genus Malus of Rosaceae family,also called ‘Daguoshanzha’ in Chinese.The present work aimed to evaluate the phytochemicals profiles,antioxidant properties of four varieties of M.doumeri fruits including two wild and two cultivars.As similar as C.pinnatifida,the phenolics and flavonoids compounds of C.pinnatifida were mainly present in the free form.Heyuanxiaoguo(HYXG)variety contained higher level of total phenolics(101.9 mg GAE/g fruit,DW),whereas Xinyishanzha(XYSZ)variety exhibited the highest total flavonoids(85.35 mg CE/g fruit,DW).Chlorogenic acid,phloretin-2′-xyloglucoside,and ursolic acid were the predominant compounds in the free extracts of M.doumeri fruits,their contents were highest in Limushan(LMS,1850 mg/100 g fruit,DW),Xinyishanzha(2212 mg/100 g fruit,DW),and Heyuanxiaoguo variety(506.3 mg/100 g fruit,DW),respectively.The free extracts of M.doumeri fruits showed strong antioxidant activities,i.e.higher PSC values was found in LMS variety,and the higher ORAC value and the significantly higher CAA values(1097 and 1832 μmol of QE/100 g DW)were observed in XYSZ variety.Among the 14 compounds identified,chlorogenic acid and phloretin glycosides accounted for 55.95% of the extracellular antioxidant capacity,while quercetin,luteolin,chlorogenic acid,and ursolic acid contributed significantly to the intracellular antioxidant activity.Furthermore,the intracellular antioxidant property of the XYSZ extracts was related to the activity of scavenging intracellular free radical,protection of endogenous antioxidant enzymes activities and inhibition of reactive oxygen species production.Fruits of M.doumeri were found to be good sources of phenolic compounds ranged from 70.7 to 101.9 mg GAE/g fruit,DW,much higher than that of C.pinnatifida fruits(48.68~58.43 mg GAE/g fruit,DW).As similar to M.doumeri,compounds like chlorogenic acid,hyperoside,isoquercitrin,epicatechin,ursolic acid,oleanolic acid,corosolic acid,and maslinic acid were found in C.pinnatifida fruits.These compounds may be responsible for the similar pharmacological activities of the fruits of M.doumeri and C.pinnatifida as recorded by Traditional Chinese Medicine.The ORAC values of free extract of C.pinnatifida were significantly higher than that of M.doumeri,and SLH variety showed significantly greater PSC values than M.doumeri fruits as well,while the CAA values of XYSZ and HYDG were significantly higher than that of C.pinnatifida fruits.The cytotoxicities and antiproliferative effects of free extracts from the fruits of C.pinnatifida and M.doumeri were evaluated.SLH and XYSZ varieties possessed significant antiproliferative effects against Hep G2,MCF-7,MDA-MB-231 and Siha human cancer cells lines,respectively.Triterpenoids-enriched fraction(S9 from SLH extract,X10 from XYSZ extract)prepared by Semi-preparative HPLC demonstrated remarkably antiproliferative activities for all the tested cancer cell lines,compared to other fractions.These results suggested that triterpenoids-enriched fractions were the major bioactive constituents mainly contributed to the anti-proliferative activities of C.pinnatifida and M.doumeri fruits.Western blotting analysis revealed that S9 fraction and X10 fraction significantly induced p21 up-regulation in MDA-MB-231 and Siha cells,followed by PCNA,CDK4,and Cyclin D1 down-regulation.Additionally,flow cytometer assays indicated that S9 fraction and X10 fraction significantly induced MDA-MB-231 and Siha cells apoptosis.Mitochondrial death and surface death receptor pathways were involved in these apoptosis as significantly induced caspase-9,caspase-8 and caspase-3 activation.The cytotoxicities and antiproliferative effects of major triterpene acids present in the fruits of C.pinnatifida and M.doumeri were evaluated.The results showed that ursolic acid was the dominant compounds responsible for the promising antiproliferative activities of C.pinnatifida and M.doumeri fruits.To further confirm the possible mechanisms of anti-proliferative effect of ursolic acid against MDA-MB-231 cancer cells at non-cytotoxic concentrations.Western blotting analysis revealed that ursolic acid significantly induced p21 up-regulation in MDAMB-231 cells,followed by PCNA,CDK4,and Cyclin D1 down-regulation,in time-and dosedependent manner.Additionally,flow cytometer assays indicated that ursolic acid exerted a dose-and time-dependent apoptosis in MDA-MB-231 cells.Mitochondrial death was involved in ursolic acid induced apoptosis as significantly increase the ratio of Bax/Bcl-2,induced caspase-9,and caspase-3 activation.Ursolic acid activated the phosphorylation of p53,p38 MAPK,c-Jun N-terminal kinase(JNK),and apoptosis signal-regulating kinase 1(ASK1),increased the expression of c-Jun protein,and suppressed the level of TNF receptor-associated factor 2(TRAF2).Accordingly,the anti-proliferative effect of ursolic acid on MDA-MB-231 cells might be through the activation of p38 MAPK and JNK pathways and activated p53,ultimately leading to cell cycle regulation and apoptosis induction.