Yan-Qiu Liu1, Li-Bin Zhan2, Tiegang Liu3, Meng-Chun Cheng4, Xiao-Yan Liu4, Hong-Bin Xiao5. 1. College (Institute) of Integrative Medicine, Dalian Medical University, Dalian 116044, China; Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. 2. The Second Affiliated Hospital, Dalian Medical University, Dalian 116023, Liaoning Province, China; College (Institute) of Integrative Medicine, Dalian Medical University, Dalian 116044, China. 3. Beijing University of Chinese Medicine, Beijing 100029, China. 4. Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. 5. Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Electronic address: hbxiao@dicp.ac.cn.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Ecliptae herba, also known as "Mo-Han-Lian", has long been used in China to nourish Kidney and thereafter strengthen bones. Accumulating evidence indicates that extracts of Ecliptae herba have antiosteoporotic effect. However, the effective compounds and cellular mode of action are still unclear. To investigate the effect of ethyl acetate extract of Ecliptae herba (EAE) and its component wedelolactone on proliferation and differentiation of preosteoclastic RAW264.7 cells as well as proliferation of bone marrow stromal cells (BMSC). MATERIALS AND METHODS: RAW264.7 and BMSC were examined for proliferation by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. Tartrate-resistant acid phosphatase (TRAP) activity of RAW264.7 was measured by using p-nitrophenyl sodium phosphate (pNPP) assay after the cells were treated with 30ng/ml receptor activator for nuclear factor-κ B ligand (RANKL) plus various concentrations of EAE, wedelolactone or alendronate. The formation of multinucleated TRAP-positive RAW264.7 cells was observed by using a TRAP-staining kit. RESULTS: Treatment of RAW264.7 cells with EAE at high doses (20µg/ml and 40µg/ml) or wedelolactone at 10µg/ml resulted in a decrease in proliferation of RAW264.7 cells. Low doses of EAE (5, 10µg/ml) and wedelolactone (2.5µg/ml) inhibited RANKL-induced TRAP activity by 20.3%, 37.9%, and 48.3%. The inhibitory effect of wedelolactone is more potent than that of alendronate, an anti-resorptive drug. Morphological changes revealed that 5µg/ml EAE and 2.5µg/ml wedelolactone reduced the number of multinucleated osteoclast-like cells. At the high doses, EAE (20µg/ml) and wedelolactone (10µg/ml) inhibited the growth of BMSC. CONCLUSIONS: EAE and its component wedelolactone inhibited osteoclast RAW264.7 proliferation and differentiation at the low doses, but at the high doses, showed cytotoxic effect on BMSC. These results indicated that EAE and wedelolatone might be potential alternative therapy for osteoporosis.
ETHNOPHARMACOLOGICAL RELEVANCE: Ecliptae herba, also known as "Mo-Han-Lian", has long been used in China to nourish Kidney and thereafter strengthen bones. Accumulating evidence indicates that extracts of Ecliptae herba have antiosteoporotic effect. However, the effective compounds and cellular mode of action are still unclear. To investigate the effect of ethyl acetate extract of Ecliptae herba (EAE) and its component wedelolactone on proliferation and differentiation of preosteoclastic RAW264.7 cells as well as proliferation of bone marrow stromal cells (BMSC). MATERIALS AND METHODS: RAW264.7 and BMSC were examined for proliferation by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. Tartrate-resistant acid phosphatase (TRAP) activity of RAW264.7 was measured by using p-nitrophenyl sodium phosphate (pNPP) assay after the cells were treated with 30ng/ml receptor activator for nuclear factor-κ B ligand (RANKL) plus various concentrations of EAE, wedelolactone or alendronate. The formation of multinucleated TRAP-positive RAW264.7 cells was observed by using a TRAP-staining kit. RESULTS: Treatment of RAW264.7 cells with EAE at high doses (20µg/ml and 40µg/ml) or wedelolactone at 10µg/ml resulted in a decrease in proliferation of RAW264.7 cells. Low doses of EAE (5, 10µg/ml) and wedelolactone (2.5µg/ml) inhibited RANKL-induced TRAP activity by 20.3%, 37.9%, and 48.3%. The inhibitory effect of wedelolactone is more potent than that of alendronate, an anti-resorptive drug. Morphological changes revealed that 5µg/ml EAE and 2.5µg/ml wedelolactone reduced the number of multinucleated osteoclast-like cells. At the high doses, EAE (20µg/ml) and wedelolactone (10µg/ml) inhibited the growth of BMSC. CONCLUSIONS:EAE and its component wedelolactone inhibited osteoclast RAW264.7 proliferation and differentiation at the low doses, but at the high doses, showed cytotoxic effect on BMSC. These results indicated that EAE and wedelolatone might be potential alternative therapy for osteoporosis.