Isolation, Identification And Anti-Tumor Ability Of Tetrastigma Hemsleyanum Root Tuber Flavonoids

Tetrastigma hemsleyanum Diels et Gilg（TH）belongs to Tetrastigma obtectum（Wall.）Planch,Vitaceae,and is now one of new developed“Zhebawei”medicinal materials.Due to the long-term folk eating habits,it has developed into a potential functional food.TH has been reported with capacities of anti-tumor,immunoregulation,liver protection,anti-virus and so on,among which its anti-tumor capacity has attracted widespread attention.Multiple studies have proved that TH could inhibit the proliferation of various cancer cells in vitro,whereas there exist few reports focused on it in vivo anti-tumor effects,as well as the key components and underlying mechanisms responsible for TH’s anti-tumor effects.Moreover,the application of TH in anti-tumor prevention and medication also needs further study urgently.Based on these backgrounds,the main findings are listed as follow:1.Water and ethanol were selected as solvents to extract flavonoids of TH root tuber,HPLC and UPLC-MS/MS analysis were adopted to identify the purified water-extracted（WTF）and ethanol-extracted TH flavone（ETF）.The four main components of WTF and ETF are quercetin-3-O-rutinoside and quercetin-3-O-glucoside,kaempferol-3-O-rutinoside and kaempferol-3-O-glucoside.The anti-proliferation abilities on different cancer cell lines（MDA,HeLa and A549）of WTF and ETF were compared.Results indicated that WTF inhibited cellular proliferation at lower dose,while strongest killing effects of WTF were observed treated with A549 cell line.2.An A549 tumor-bearing murine model was built to explore the anti-tumor capacity of WTF（called THTF）in vivo.THTF could inhibit A549 tumor growth via down-regulating proliferation-related proteins level and up-regulating the expression of apoptosis-related proteins,and unremarkable side effects were observed,implying THTF as a safe and effective agent to treat A549 tumor in vivo.The underlying mechanism of THTF’s anti-tumor effects were illustrated by i TRAQ-based proteome analysis.Based on the statistical analysis of differential expressed proteins,it was speculated and verified that THTF regulated the calcium signal pathway,altered the expression of cytoskeleton proteins,and thereby achieved tumor cell apoptosis.3.The anti-tumor effects and mechanisms of THTF’s four main components were compared and screened.Results revealed that kaempferol-3-O-rutinoside（KAE）could more significantly suppress cell proliferation,regulate cellular calcium levels and block cytoskeletal function,suggesting KAE as a crucial factor for the anti-tumor capacity of THTF.Furthermore,KAE could destroy cell calcium homeostasis,disrupt mitochondrial function,facilitate cytoskeleton collapse,thereby restraining cellular migration and proliferation,achieving anti-tumor effects.4.Inspired by KAE’s anti-tumor mechanism,M@CaCO₃@KAE nanoparticles were successfully synthesized and prepared with excellent uniform and dispersion,and their release regulations were clearly revealed.M@CaCO₃@KAE nanoparticles can effectively achieve calcium overload of A549 cells in vitro,destroy mitochondrial structure and function,facilitate cellular cytoskeleton collapse,inhibit cell migration,achieving synergistic cell apoptosis.The anti-tumor capacity and biosafety of cancer cell membrane functionalized M@CaCO₃@KAE nanoparticles were evaluated.M@CaCO₃@KAE nanoparticles can effectively target tumors,trigger calcium overload,achieving the tumor growth inhibition in vivo,and unremarkable toxicity of M@CaCO₃@KAE nanoparticles has been observed.M@CaCO₃@KAE nanoparticles sufficiently overcome the KAE’s disadvantages including low solubility and poor stability,as well as the difficulty of targeted delivery and supply of KAE and a large amount of calcium ions in vivo.In this way,safe,targeted,efficient and synergistic calcium overload cancer therapy can be achieved.In conclusion,this study has clarified the major functional components and anti-tumor mechanism of THTF,and constructed a nano-system to achieve anti-tumor effects efficiently both in vitro and in vivo.This study provides new ideas to overcome natural products’insufficiency such as low solubility,poor stability,rapid degradation,fast metabolism and poor bioavailability in vivo,and offers innovate options and strategies for food-borne functional components’applications in cancer therapy.