Objective:To investigate the mechanisms of the active fractions from Hedyotidis Herba-Scutellariae Barbatae Herba (YDW11) in inducing apoptosis of triple negative breast cancer MDA-MB-231 cells. Method:Ultra high performance liquid chromatography (UPLC) was used to determine the chemical components in the active fractions

and methylthiazoletetrazolium(MTT) assay was used to evaluate the proliferation of cells in vitro. The colony formation assay was used to measure the changes of colony numbers. Flow cytometery combined with apoptosis detection kit was used to measure the ratio of apoptosis cells

and the changes in expression of proteins and signaling pathways were evaluated by Western blot. In addition

UPLC assay was carried out to identify the major constituents in YDW11. Result:The ethyl acetate fractions (YDW11

YDW12

YDW21) of the water extracts of Hedyotidis Herba-Scutellariae Barbatae Herba were prepared with three different ratios (1:1

1:2 and 2:1). YDW11 had four components:p-hydroxyacetophenone

scutellarin

luteolin and apigenin. By comparing the effect of these three fractions on the in vitro proliferation of normal mammary epithelial cells (10A1) and triple negative breast cancer cells (MDA-MB-231)

it was found that YDW11 at 25~50 mg·L-1 had no cytotoxicity to normal breast epithelial cells 10A1

but inhibited the in vitro proliferation of MDA-MB-231 (P<0.01)

and the inhibitory effect was stronger than that of YDW12 and YDW21.YDW11 inhibited the proliferation of three triple negative breast cancer cell lines (MDA-MB-231

HS578T

BT549) in a dose and time dependent manner (P<0.05

P<0.01)

but it had little effect on the proliferation of MCF-7 in vitro. YDW11 inhibited the colony formation of MDA-MB-231 cells in a dose-dependent manner and induced cells apoptosis (P<0.05

P<0.01). YDW11 increased the mRNA and protein expression of p21

and simultaneously inhibited the expression of proliferating cell nuclear antigen(PCNA) (P<0.05

P<0.01)

and reduced the phosphorylation level of p38

c-Jun N-terminal kinase (JNK) and extracellular regulated protein kinases (ERK) in mitogen-activated protein kinase(MAPK) signaling pathway (P<0.05). The phosphorylation expression level of VASP at both Ser157 and Ser239 sites were up-regulated

indicating that cyclic adenosine monophosphate(cAMP)and cyclic guanosine monophosphate(cGMP)signal pathways had been activated by YDW11 treatment (P<0.05

P<0.01). Conclusion:YDW11(25

50 mg·L-1) showed the strongest inhibition among fractions extracted from Hedyotidis Herba-Scutellariae Barbatae Herba in different ratios. YDW11 can especially inhibit the proliferation

colony formation and induce cells apoptosis of MDA-MB-231 cells in a dose and time dependent manner without cytotoxicity

elevated the expression of p21 at the mRNA and protein level

suppressed the expression of PCNA and the phosphorylation level of p38

JNK and ERK

and up-regulated the phosphorylation level of VASP at Ser157 and Ser239 sites. YDW11 induced the apoptosis of MDA-MB-231cells partly by activating cyclic adenosine monophosphate(cAMP)/cyclic guanosine monophosphate(cGMP)and inhibiting MAPK signal pathways.