| Objective: The macroporous resin column chromatography was applied to separate the aqueous extract of Prunella vulgaris L.to obtain different eluted fractions.The antibreast cancer activities of different fractions were evaluated using breast cancer models in vitro and in vivo to determine the best effective anti-breast cancer fraction,which will be the candidate to be accessed its effect and mechanism on breast cancer.Methods:1.Separating the aqueous extract of Prunella vulgaris L.by D101 macroporous resin column chromatography to obtain different ethanol-water solvent gradient eluted fractions(100% water,20%,50% and 95% ethanol aqueous).The constituents of different eluted were preliminarily determined by LC and UPLC/MS.2.Evaluating the proliferation inhibitory activities of each eluted fraction on three breast cancer cell lines using the MTT assay.3.Detecting the effects of each eluted fraction on breast cancer by a combination of assays of Micro CT,tumor measurement and H-E staining in 4T1 tumor-bearing mice to confirm the best effective fraction along with a comprehensive consideration of the results of chapter 2.4.To further illustrate the anti-TNBC effect and its underlying molecular mechanisms of PV50,the MTT and colony formation assay were conducted to evaluate the effects of PV50 on 4T1 viability.To confirm the induced apoptosis activity of PV50,Hoechst 33342 staining was performed.Then,We quantified the effects of TFP on apoptosis with FCM after Annexin V/PI staining.We studied the effect of PV50 on cell cycle distribution by flow cytometry(FCM)analysis in 4T1.Wound-healing migration assays were performed to assess the effect of PV50 on 4T1 cell migration and invasion.5.Establishing a 4T1 tumor-bearing mice to test the anti-cancer effect of low-mediumhigh dose group of PV50 in vivo.The drugs were administered orally to mice continuously for 21 days.The body weight and tumor volume of the mice were recorded at intervals of 3 days.Micro CT,H-E staining,TUNEL and Immunohistochemical analysis assays were performed to evaluate the anti-breast effect of PV50 and its underlying mechanisms in vivo.Meanwhile,the mice were given a dose of 1000 mg/kg to carry out an acute toxicity test to examine the security of set doses.Results:1.The aqueous extract of Prunella vulgaris L.was eluted by macroporous resin column chromatography to obtain different fractions,which were PVW with a yield of 2.18%,PV20 with 4.55%,PV50 with 2.09%,and PV95 with the lowest yield of 1.09%.Spectral analysis revealed that the constituents of each fraction are almost different.2.In vitro,PV50 and PV95 showed significant proliferation inhibitory effects on breast cancer cell lines,especially for human-derived triple-negative breast cancer cell line MDA-MB-231 and mouse-derived triple-negative Breast cancer cell line 4T1,compared with other eluted fractions.3.In vivo,PV50 exhibited more inhibition of growth of established subcutaneous xenograft tumor than other eluted fractions.H-E staining assays showed that PV50 could effectively inhibit breast cancer metastasis to the lungs.4.Cell viability assay and colony formation assay showed that PV50 inhibited 4T1 proliferation in a dose-and time-dependent manner.Hoechst 33342 assay and Annexin V/PI staining assay showed that PV50 can induce apoptosis.PV50 can regulate the cell cycle and induce G0/G1 cell cycle arrest phase.Scratch experiments show that PV50 can inhibit cell migration.5.In vivo,PV50 significantly suppressed the growth of subcutaneous xenograft tumor without causing detectable side effects.In TUNLE assay,PV50 induced tumor cell apoptosis in dose-dependent manner.The expressions of cleaved caspase-3 was increased in the PV50 groups.The mice were orally administered with 1000 mg/kg of PV50 without any sign of toxicity.Conclusion: Prunella vulgaris L.can exert anti-breast cancer effect.PV50,which is eluted by macroporous resin column chromatography with 50% ethanol,exhibited more significant anti-breast cancer ability,can regulate cell cycle,inhibit cell proliferation,and induce apoptosis of 4T1.Therefore,it may provide a new idea for the development of Prunella vulgaris to exert anti-breast cancer effect and provide a new therapy for triplenegative breast cancer. |
