In our present study, composition of marker compounds in ELP aqueous extracts was determined by LC-MS. We showed that salidroside was the most abundant, followed by icariin, isopsoralen, and psoralen. It may be due to the fact that salidroside is highly soluble in water, while icariin, psoralen, and isopsoralen are water-insoluble in nature.
Comparing two antiresorptive drugs we used in the experiments, we found that alendronate was more effective than raloxifene in reducing overall total BMD loss. As shown in total BMD analysis, alendronate started to have the significant difference from OVX group from week 2, whereas raloxifene started from week 4. These findings were in line with those reported previously that alendronate has a higher efficacy than raloxifene in reducing the risk for osteoporotic fracture [20
]. This finding also echoed with the results of bone microarchitecture analysis, which illustrated that the efficacy of alendronate to increase trabecular bone formation of osteopenic bone was better than that of raloxifene. It could also support a current clinical review which reported that onset of efficacy for nonvertebral fracture was reduced by 12 months by alendronate but was reduced by 36 months by raloxifene [21
]. Significant reduction on hip fracture rate by alendronate, but not raloxifene, was also reported by Hopkins et al. [22
]. However, for the combination studies, we found that ELP extract could work synergistically with raloxifene in increasing BMD of osteopenic bone. These findings were further substantiated by bone microarchitecture analysis and revealed that ELP had a synergistic effect with raloxifene, but not alendronate, in increasing the new bone deposition on trabecular surface. This observation will be further confirmed by histological analysis.
Interestingly, although ELP extract itself did not cause significant osteo-protective effect on material (BMD) and architectural (microarchitecture) properties of femur, it improved femoral cortical bone strength significantly as shown in the biomechanical test. This unmatched observation might be due to the fact that biomechanical properties of bone depend not only on material and architectural factors, but also the geometric parameters of cortical bone on mid-shaft of long bone [23
]. Fracture load was correlated better with BSI (CSA) (a bone strength index including the cortical BMD and the cross-sectional area) of the bone than cortical BMD or total BMD alone in mid-shaft femur and humerus of goats. ELP might affect the geometry of the long bone cortex. Having not measured the geometric parameters was a limitation of this study.
Previously, it has already been demonstrated that ELP extract possessed beneficial effects in promoting bone health in aged ovariectomized rats [10
], tail-suspended rats [9
], and also in postmenopausal osteopenic women [11
]. For the purpose of health supplement and/or pharmaceutical products development, the information related to the herb-drug interactions between ELP and standard antiresorptive drugs is necessary, since some of the osteopenic individuals may consume them simultaneously. In the present study, we have demonstrated that ELP extract itself was able to significantly reduce total BMD loss in lumbar spine and increase biomechanical strength of the bone and serum osteocalcin levels. Provided that ELP and antiresorptive drugs act on different molecular targets in bone metabolism, we postulate that synergistic effects could be generated when individuals take both together, so that the dosage of these antiresorptive drugs can be reduced. In this study, we found that ELP could selectively enhance the therapeutic effects of raloxifene, but not alendronate. In addition, ELP could enhance the effects of raloxifene even at 1/10 of optimal dosage.
To elaborate on the selectivity of ELP actions, we need to look into the characteristics of the animal models and also the fundamental working mechanisms of alendronate and raloxifene. An estrogen deficiency caused by ovariectomy results in (i) inhibition of mature osteoblasts and promotes faster osteoblastic apoptosis; and (ii) stimulation of osteoclast formation and bone resorption; eventually causes a net effect of reduced bone mass [24
]. The antiresorptive mechanism of alendronate and raloxifene is not the same. Alendronate is an inorganic pyrophosphate, which preferentially inhibits osteoclast-mediated bone resorption without affecting bone formation [26
]. Raloxifene is an oral selective estrogen receptor modulator (SERM) that has estrogenic actions on bone and anti-estrogenic actions on the uterus and breast. It plays an analogous role to estrogens on bone tissue, and its osteoblastic action has recently been shown [27
]. It was reported that raloxifene required osteoblastic cells to achieve its anti-osteoclastic action [28
]. Previously, we found that ELP not only suppressed the osteoclast formation, but also enhanced the bone formation by increasing the osteogenesis of mesenchymal stem cells [9
] and the proliferation of osteoblast as well (unpublished data). The additional osteoblastic actions from ELP may favor the anti-osteoclastic activities of raloxifene in inhibiting bone resorption, as supported by the decrease in DPD level (). It may offer a possible explanation as to why the presence of ELP always synergistically enhances the osteoprotective effects of raloxifene against estrogen deficient bone loss in all studied parameters. This suggestion will be verified using different other known anabolic agents, such as strontium ranelate or parathyroid hormone, in combination with raloxifene on the ovariectomized rats. Further experiments are also required to understand the basic underlying mechanisms behind the synergistic actions of ELP on raloxifene using monocytes/macrophages and osteoblast coculture system [28