The Study Of Screening Anti-osteoporosis Constituents Of Maca (Lepidium Meyenii) And Mechanism

Osteoporosis is a bone metabolic disease, which causing disability to the elderly and enormous long-term care expenses. Along with the increased population of elderly persons, the demand for preventing osteoporosis products has grown rapidly. However, there are many problems in efficacy and safety of anti-osteoporosis drug. Thus, developing an effective and safe functional food will be an effective way for the people who had not yet been onset and mild symptoms.Maca(Lepidium meyennii Walp.) has many physiological functions such as improving menopause syndrome and preventing postmenopausal osteoporosis. But the efficacy material base of maca on preventing postmenopausal is unclear, severely hindering the exploitation and application of maca anti-osteoporosis products. In this paper, maca compound library was built, and potential active ingredients to estrogen receptors in the library were discovered by virtual screening, and then its action target were predict through reverse docking, further were evaluation on cell and animal models. The innovative results as follow:(1) Macamides were screened out as active constituent of maca in preventing osteoporosis by using virtual screening against estrogen receptor, N-benzyl-palmitamide was one of the best structures. Maca compound library was constructed by literature survey. By AutoDock Vina, the compounds were screened out for estrogen receptor (PDB:1 GWR), which free binding energy were between 7.7 and 2.1 kcal/mol. The binding energy of macamides were lowest, between 7.7 and 7.0 kcal/mol, which suggest macamides were most likely as maca anti-osteoporosis active substances. Furthermore, phenyl, amide bond and the carbon chain length is greater than 15 were key structures for macamides to bind with estrogen receptor. Among macamides, N-benzyl-palmitamide was one of the best structures.(2) Through reverse docking, there was found that N-benzyl-palmitamide could regulate bone metabolism by affectting retinoic acid signaling pathways related protein, such as cellular retinoic acid-binding protein, retinol binding protein, retinoic acid receptors and transthyretin, and vitamin D3 receptor and carbonic anhydrase 2. The targets in the body of N-benzyl-palmitamide were found by PharmMapper, including cellular retinoic acid-binding protein 2, transthyretin, amine oxidase [flavin-containing] B, retinol-binding protein 4, retinoic acid receptor beta, vitamin D3 receptor, alpha-tocopherol transfer protein, serum albumin, carbonic anhydrase 2, which relate to visual, nervous, immune, reproductive, skeletal and basal metabolism. Among them, retinoic acid binding protein, retinol-binding protein, transthyretin, vitamin D3 receptor and carbonic anhydrase 2 were closely associated with bone metabolism, which suggest that macamides were anti-osteoporosis active substances.(3) The effect of N-benzyl-palmitamide on promoting bone formation were confirmed on cell model. By MC3T3-E1 Subclone 14 cell, the effects of N-benzyl-palmitamide on osteoblast were researched. The result shown that N-benzyl-palmitamide could promote MC3T3-E1 cell proliferation, differentiation and mineralization, showed promoting bone formation, which might through increasing the expression levels of estrogen receptor and the key factor of osteogenesis, such as BMP-2, Cbfal, COL1 and ALP.(4) The effects of N-benzyl-palmitamide on anti-osteoporosis were further confirmed on animal model. By constructing retinoic acid induced osteoporosis rats model, the effects of N-benzyl-palmitamide on anti-osteoporosis were researched. The results shown that N-benzyl-palmitamide could improve bone mineral density of femur and spine, increasing bone ash and calcium content, improve bone morphological structure, showed anti-osteoporosis effect. N-benzyl-palmitamide might regulate bone metabolism by promoting the ALP activity, resisting decline in estrogen levels and increase in TRAP activity caused by retinoic acid.(5) The effects of maca alcohol extract containing N-benzyl-palmitamide and N-benzyl-palmitamide were compared. The result shown the effects of extract in promoting osteoblast proliferation, improve the density of femur and spine, and improve bone physical parameters and morphological structure were similar to N-benzyl-palmitamide alone, while in promoting ALP activity and improve bone ash and bone calcium content significantly superior to N-benzyl-palmitamide alone. N-benzyl-palmitamide in maca is a important anti-osteoporosis ingredients, but also contain other has certain anti-osteoporosis effect or synergy. In the experiment using doses, maca alcohol extract and N-benzyl-palmitamide were not exhibit obvious adverse reactions, such as uterine weight.In conclusion, N-benzyl-palmitamide were screened out as an active constituent of maca in preventing osteoporosis by using virtual screening against estrogen receptor, the effect of which were verified on osteoblasts and animal. N-benzyl-palmitamide might regulate bone metabolism by estrogen signaling pathways, BMP-2 signaling pathways and retinoic acid signaling pathways.