Establishment Of Regeneration System Of V.negundo Var.cannabifolia And Effect Of Extract On Breast Cancer Cells

V.negundo var.cannabifolia is a plant of Vitex genus in Verbena family.It is rich in polyphenols,total flavonoids,terpenoids,etc.The bioactive substances have anti-oxidative,anti-inflammatory,anti-tumor and anti-aging pharmacological effects.The tissue culture is suitable for the production of high quality starting materials.However,the tissue culture technology suitable for standardized production of V.negundo var.cannabifolia is not mature yet.In this study,we developed effective direct and indirect regeneration systems using dormant buds as explants,and comprehensively evaluated the regenerated plants from the levels of genome,metabolism,physiology and cytotoxicity.The main research results are as follows:1.The high efficiency direct and indirect regeneration systems were established.Cluster buds could be rapidly induced by MS medium+1 mg/L 6-BA+0.2 mg/L NAA,and the proliferation coefficient was 20.3 Callus induction medium MS+1.5mg/L 2,4-D+1.5 mg/L NAA,the highest induction rate was 96.93%,and the callus was light yellow,loose and growing rapidly.The callus differentiation rate reached 81.10%on the medium of MS+1 mg/L6-BA+0.2 mg/L NAA+2.5 mg/L Cu SO₄·5H₂O.ISSR analysis of genetic stability showed that the fidelity was 98.44%and 95.58%,respectively.The callus-mediated regenerated plants produced 2.86%more variation than the directly induced regenerated plants.2.The antioxidant capacity of the in vitro propagated plants at the adult stage was equal to that of the wild plants（DPPH）,or even higher than that of the wild plants（FRAP）,and significantly higher than that of the seedlings.As for antioxidant enzymes,superoxide dismutase（SOD）,catalase（CAT）and ascorbate peroxidase（APX）activities of plants propagated in vitro at adult stage were the highest among the three tested materials.The antioxidant capacity of the plant was basically consistent with the contents of polyphenols,total flavonoids and enzyme activities,which indicated that these secondary metabolites played an important role in maintaining the antioxidant balance system of V.negundo var.cannabifolia.The inhibitory effect of regenerated seedlings on a-glucosidase was similar to that of wild seedlings.Among the extraction methods of acetone,ethyl acetate,ethanol and acid ethanol,acid ethanol was the most effective extraction method.The inhibitory rate of a-glucosidase of regenerated seedlings was 70.52%,slightly lower than that of wild seedlings（74.08%）.The IC₅₀of a-glucosidase determined by acid ethanol extract of wild seedling and regenerated seedling were 1.37 and 1.406μg/m L respectively.3.The plants propagated in vitro had similar cytotoxic effects to wild V.negundo var.cannabifolia at the seedling stage.V.negundo var.cannabifolia extracts was more cytotoxic than MCF7 in breast cancer cell line SUM159.The IC₅₀values of WGP,IVY and IVA in breast cancer cell line SUM159 were 37.06,88.14 and 36.67μg/m L,respectively,while the IC₅₀values in breast cancer cell line MCF7 were all higher than 143μg/m L.The extract of V.negundo var.cannabifolia did not show obvious cytotoxicity in MCF7 cell line.In the scratch experiment,the extracts of the two materials had obvious inhibitory effects on the proliferation and migration of breast cancer cell line SUM159,and the effect of the extracts on breast cancer cell line SUM159 was better than that on MCF7 cells.The inhibitory effect of regenerated vaccine on breast cancer was lower than that of wild vaccine.High concentration extract was better than low concentration extract.The results of flow cytometry showed that the apoptosis induced by vitexin in breast cancer cell line SUM159 was lower than that induced by V.negundo var.cannabifolia extracts.The apoptosis induced by V.negundo var.cannabifolia extracts was lower than that treated with paclitaxel.In breast cancer cell line MCF7,the apoptosis induced by vitexin was lower than that induced by extract of140μg/m L and 180μg/m L.4.The expression levels of apoptosis,migration and invasion related genes were measured after treatment in the experimental group.The results showed that the extracts of V.negundo var.cannabifolia mainly affected the expression of Caspase-9and NEXN-AS1 in breast cancer cell line SUM159.It may induce cell apoptosis by increasing the expression of Caspase-9 gene.It can also inhibit the migration and invasion of cancer cells by increasing the expression of NEXN-AS1 gene.In breast cancer cell line MCF7,the main genes changed were:Bcl-2,P21,RBBP6,KNL1,KIF20B,HMGN5.It may induce cell apoptosis through the mitochondrial apoptosis pathway by down-regulating the expression of Bcl-2 gene.Apoptosis can also be induced by up-regulation of P21 gene expression.Downregulation of the expressions of RBBP6,KNL1,KIF20B and HMGN5 genes inhibited the migration and invasion of cancer cells.This study showed that the effect of mature plants of in vitro regenerated seedlings was similar to that of wild seedlings.In conclusion,the regeneration system established in this study produced regenerated plants with high genetic fidelity.The cytotoxic activity of wild plants and regenerated plants against human breast cancer cell lines was found to be equivalent.This study will help to develop high-quality starting materials for V.negundo var.cannabifolia cultivation and lay the foundation for further research on the inhibition of breast cancer by V.negundo var.cannabifolia extract.