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Silkworm is an insect that feeds on mulberry leaves only, and silkworm powder has been reported to have antioxidant, hypoglycemic, and anticholesterol activities. In this study, we measured the content of 1-deoxynojirimycin (1-DNJ) in 24 different extracts from silkworm cocoons and also investigated the estrogenic activities of these extracts and their effects on the activation of intracellular signaling pathways in MCF-7 breast cancer cells. Among the 24 silkworm extracts, relatively high 1-DNJ content and estrogenic activity were shown by Extract 11 (30E3H20-1), which was prepared by a single extraction of silkworm powder with 20 times the volume of 30% ethanol against each powder weight for 3 h. The estrogen receptor activity of this extract was confirmed based on its promoting effects on the phosphorylation of the estrogen receptor α (ERα) and mRNA expression of the
ESR1
and
ESR2
genes. In addition, treatment with Extract 11 (30E3H20-1) increased the phosphorylation of AKT, p38, and JNK, which are downstream proteins of ERα. Based on our findings, a silkworm extract could be developed as a natural estrogen supplement in the future.