ETHNOPHARMACOLOGICAL RELEVANCE: Er Zhi Wan (EZW), a Chinese medicinal preparation, has been used clinically for treating menopausal syndrome for its kidney-invigorating function, which contains simply two herbs, Ecliptae Herba (EH) and Ligustri Lucidi Fructus (LLF). Although this herbal extract has been used for many years, there is no scientific basis about its effectiveness on menopausal symptom. Here, we aimed to evaluate the estrogenic activities of EZW and to study the compatibilities of two herbs including different processed-LLF in single and mixed preparation of EZW. Moreover, the weight ratio of EH to LLF in EZW was determined according to their estrogenic activities. MATERIALS AND METHODS: The extractions of LLF, processed-LLF and EH were prepared separately by extracting the powders with water, 50% alcohol or 95% alcohol. Steamed-LLF and EH were extracted separately, or together, in preparing EZW extracts. A promoter-reporter construct (pERE-Luc) containing three repeats of estrogen responsive elements (ERE) was stably transfected into MCF-7 cells, and this stable breast cancer cell line was used to determine the estrogenic property. The cell proliferation was measured by MTT assay. RESULTS: The results showed that EZW could significantly induce the expression of luciferase driven by an estrogen responsive element in a pERE-Luc vector. The proliferation of MCF-7 cells was not altered by this herbal treatment. The best preparation of EZW was from: (i) LLF was firstly steamed over water and then dried to make steamed-LLF; and (ii) steamed-LLF and EH were extracted separately by 95% alcohol and then mixed together according to a weight ratio of 1:1. CONCLUSIONS: Under the optimized extracting method, EZW possessed robust effect in activating the estrogenic activity, but which did not alter the proliferation of cultured MCF-7 cells. Thus, EZW is an effective and safe estrogenic herbal extract.
ETHNOPHARMACOLOGICAL RELEVANCE: Er Zhi Wan (EZW), a Chinese medicinal preparation, has been used clinically for treating menopausal syndrome for its kidney-invigorating function, which contains simply two herbs, Ecliptae Herba (EH) and Ligustri Lucidi Fructus (LLF). Although this herbal extract has been used for many years, there is no scientific basis about its effectiveness on menopausal symptom. Here, we aimed to evaluate the estrogenic activities of EZW and to study the compatibilities of two herbs including different processed-LLF in single and mixed preparation of EZW. Moreover, the weight ratio of EH to LLF in EZW was determined according to their estrogenic activities. MATERIALS AND METHODS: The extractions of LLF, processed-LLF and EH were prepared separately by extracting the powders with water, 50% alcohol or 95% alcohol. Steamed-LLF and EH were extracted separately, or together, in preparing EZW extracts. A promoter-reporter construct (pERE-Luc) containing three repeats of estrogen responsive elements (ERE) was stably transfected into MCF-7 cells, and this stable breast cancer cell line was used to determine the estrogenic property. The cell proliferation was measured by MTT assay. RESULTS: The results showed that EZW could significantly induce the expression of luciferase driven by an estrogen responsive element in a pERE-Luc vector. The proliferation of MCF-7 cells was not altered by this herbal treatment. The best preparation of EZW was from: (i) LLF was firstly steamed over water and then dried to make steamed-LLF; and (ii) steamed-LLF and EH were extracted separately by 95% alcohol and then mixed together according to a weight ratio of 1:1. CONCLUSIONS: Under the optimized extracting method, EZW possessed robust effect in activating the estrogenic activity, but which did not alter the proliferation of cultured MCF-7 cells. Thus, EZW is an effective and safe estrogenic herbal extract.