Bioactivities And Bioavailability Of Hippopha? Rhamnoids L. Phenolics And Their Anticancer Mechanism Towards Human Breast Cancer Cells Both In Vitro And In Vivo

Breast cancer is one of the most common malignancies in women around the world,and the incidence is increasing year by year.Epidemiological and clinical studies have associated the consumption of larger amount of fruits and vegetables rich in phytochemicals with lower risks of different chronic diseases,such as cancer,cardiovascular diseases and diabetes.Diet control has become a safe and effective method with low side effects for preventing cancer.Hippopha?rhamnoides L.?sea buckthorn?is a shrub or small tree of the Hippopha?family.Its berry-like fruit is used as a traditional food-homologously Chinese medicine.In this study,the antioxidant and antiproliferative activities of phenolic phytochemicals in sea buckthorn fruit were systematically analyzed,and the cellular uptake and bioavailability of phenolic substances were evaluated.Cell model and tumor-bearing mice model were used to explore the synergistic inhibition effects of phenolic components on cancer cell proliferation and related modulation pathways.According to this study,we are aimed to provide a new theoretical basis for the prevention of breast cancer,and new ideas for the development of functional foods and anti-cancer drugs.Main results are listed as follows:?1?Phenolics in sea buckthorn:A large amount of free phenolics was found in four subspecies,ranged from 27.23±1.89 to 38.44±1.35 mg GAE/g DW,among which,Sinensis exhibited the highest,followed by Yunnanensis,Mongolica and Turkestanica.The content of flavonoids ranked the same order as phenolics,which also mainly existed in free form.The free phenolics and flavonoids contributed more than 98.5%to total phenolics and total flavonoids.Total flavonoids performed a contribution of 74.1 to 85.0%to total phenolics among subspecies.Isorhamnetin,quercetin,kaempferol and their glycosides were the dominant compounds in identified free phenolics,followed by phenolic acid and flavonols.?2?Bioactivities evaluation:ORAC,PSC,CAA and antiproliferative tests were taken to assess the antioxidative and antiproliferative activities of phenolics towards human live cancer cells HepG2,breast cancer cells MCF-7 and MDA-MB-231 as well as colon cancer cells Caco-2.Highest ORAC,PSC and CAA values under No PBS Wash protocol were found in phenolics of Sinensis subspecies,while the Yunnanensis owned the highest cellular antioxidant activities after model cells were washed by PBS.All the subspecies showed high antiproliferative activities against the four cell lines,of which subspecies Yunnanensis exhibited the strongest.When identified compounds were subjected to this evaluation,compound isorhamnetin presented highest antiproliferative activities with a lowest EC₅₀,followed by quercetin,kaempferol and their glucosides.?3?Cellular uptake and bioavailability:After simulated digestion,the total phenolics were decreased to 40%of chemical extracts,while more flavonols were released,leading to an enhancement of cellular antioxidative and antiproliferative activities.Using of Caco-2absorption model,the cellular uptake of isorhamnetin was improved 3.63 times by digestion,which was significant higher than pure isorhamnetin and its combination with quercetin and kaempferol.The bioaccessibility of isorhamnetin in pure,combination and digesta were0.40%,0.49%and 1.15%,respectively,which was extremely low and limited their bioactivities.Therefore,we developed an approach to improve the bioavailability of total flavonols by construction of self-microemulsion drug delivery system?SMEDDS?assisted with pseudo-ternary phase diagram and Design Expert optimization method.After loaded in SMEDDS?constituted of Cremophor EL,Transcutol HP and MCT at ratios of 60.38:26.36:13.26?with a droplet size around 65 nm,the relevant bioavailability of flavonols were improved 3.09-fold of free form.?4?Synergistic anti-proliferative effect and mechanism of flavonols in vitro:isorhamnetin and quercetin showed a significant synergestic antiproliferative activities against HepG2 when the inhibitory rate was 50%with CI value below 1.Highest synergestic effects were found in both MCF-7 and MDA-MB-231 cells treated with combination of isorhamnetin and kaempferol with inhibitory rate ranged from 20%to 60%,followed by combination of isorhamnetin,quercetin and kaempferol,while an antagonism effect was found between kaempferol and quercetin.Flow cytometer was used to detect the apoptosis and cell cycle of HepG2 cells after combination of isorhamnetin and quercetin treatment.It was shown that this combination could significantly increase cell apoptosis and arrest the cell cycle in G2 period.The expression of tumor-associated proteins in MDA-MB-231 cells modulated by isorhamnetin and kaempferol was evaluated by Western blot protocol.Result showed that both compounds could down-regulate the expression levels of Cyclin D1,PCNA,Bcl-2 and NF-?B,and up-regulate Bax expression,increase the phosphorylation level of p38MAPK in a dose-dependented manner.However,the expression levels of p-Akt were different.After 12hours‘treatment by isorhamnetin,the expression of p-Akt in cancer cells was significantly decreased in a dose-dependent manner,while which was in a U shape in kaempferol treated cells along with kaempferol concentrations increased from 40 to 80?M,the level of p-Akt was first suppressed and then up-regulated,indicating that the anticancer activities of kaempferol was not mainly via the inhibition of phosphorylation of Akt pathway.?5?Antitumor activities and mechanism of flavonols in combination and loaded form in vivo:a human breast cancer MDA-MB-231 cells xenografted tumor model was established in Balb/c nuld mice.After oral administration of isorhamnetin alone,kaempferol alone and their combination as well as isorhamnetin loaded in SMEDDS for 4 weeks,the tumor growth was inhibited in a range of 21.68%to 52.33%with a decrease of tumor weight ranged from 22.88%to 59.42%.Both parameters were decreased significantly when mice were administrated by durg combination or isorhamnetin loaded form.When subjected to the immunohistochemical detection,all the four administration methods could significantly decrease the integrated optical density?IOD?of PCNA,Bcl-2,Akt and p-Akt,while the positive expression of Bax,p38 and p53 was un-regulated.According to the RT-PCR measurement,the gene expression of Caspase-9 was significantly up-regulated.By contrast,drug treatment suppressed the PI3K,MMP-2 and MMP-9 levels in tumor tissue.Compared with the in vitro analysis,it is suggested that isorhamnetin and kaemferol might inhibit human breast cancer cell proliferation by modulating p38MAPK and PI3K/Akt signaling pathways and activating mitochondrial apoptosis reaction.More relevantly,both compounds could block tumor invasion and metastasis through down-regulation of MMPs.Combination and nano-scale delivery drug treatments could enhance this regulatory effect.