Experimental and molecular docking studies of estrogen-like and anti-osteoporosis activity of compounds in Fructus Psoraleae
Graphical abstract
Introduction
Estrogen is a sex hormone that plays a necessary role in a lot of biological processes, including that in maintaining gender identity and stable reproductive development in women (Oh et al., 2017). Abnormal estrogen levels can lead to high risk of breast cancer (Moore et al., 2016). Once a woman reaches menopause, endogenous estrogen levels drop significantly, triggering a series of menopause-related conditions, such as metabolic disturbance (Monteiro et al., 2014; Palmisano et al., 2017), reduced learning and memory ability (Korol and Pisani, 2015), osteoporosis (Rossini et al., 2013), insomnia (Ensrud et al., 2015), cardiovascular disease (Guivarch et al., 2018), etc. Many side effects of estrogen replacement therapy have been reported (Yusuf and Anand, 2002). Thus, research on estrogen replacement, including compounds with estrogen effects, has become a research hotspot.
Osteoporosis is a disease of the bone system which results from an imbalance between bone resorption and formation (Martin et al., 2014). There are two types in osteoporosis, namely primary osteoporosis and secondary osteoporosis. Primary osteoporosis, which accounts for 90% of osteoporosis cases, is divided into three classifications, namely postmenopausal osteoporosis (type I), senile osteoporosis (type II) and idiopathic osteoporosis (type III) (Manolagas and Jilka, 1995). Postmenopausal osteoporosis is the result of a high-bone-turnover state that increases bone formation, while senile osteoporosis is the result of a low-bone-turnover state that decreases bone formation (Tella and Gallagher, 2014). Research on osteoporosis has been ongoing for many years, and several therapies for the clinical treatment of osteoporosis have been developed, including hormone replacement therapy (HRT) (Gambacciani and Levancini, 2014), tumor necrosis factor superfamily member 11 (TNFSF11) inhibitors (Mo et al., 2019), selective estrogen receptor modulators (SERM) (Hadji et al., 2012), etc. However, long-term clinical studies of these therapies have shown they have serious adverse effects and thus new therapies to treat osteoporosis are needed.
The canonical Wnt/β-catenin signaling pathway governs a variety of biological processes (Yang et al., 2016), and has been implicated in various diseases. The key mediator of Wnt signaling, β-catenin, also has several functions (Voronkov and Krauss, 2013). The inhibition of Wnt/β-catenin signaling have been shown to have the function to suppress osteosarcoma cell survival and growth in vitro and in vivo (Ma et al., 2015; Chen et al., 2015; Dai et al., 2017). It has been reported that estradiol (E2) has some effects on osteoporosis through the estrogen receptor alpha (ERα), which is essential in the classical estrogen signaling pathway (Mieko et al., 2001). Mohammed et al. (2016) reported that there is a connection between ERα and the Wnt/β-catenin signaling pathway. Bhukhai et al. (2012) found that an estrogen receptor is required to inhibit β-catenin expression. Modder et al. (2012) found that ERα results in the suppression of Wnt/β-catenin signaling disruption in osteoblasts.
Fructus Psoraleae (FP) is the dry and mature fruit of the leguminous plant Cullen corylifolium (L.) Medik., which according to many traditional Chinese medicine (TCM) books, has a warming effect on the kidney to help yang, and on the spleen to stop diarrhea. Modern medicine studies have also demonstrated its beneficial effects in the clinical treatment of kidney yang deficiency-induced impotence, asthma and cold pain in waist and knee which were caused by kidney deficiency. TCM maintains that the lack of estrogen in middle-aged women can be explained by kidney deficiency, and thus the menopausal syndrome can be treated by supplementing the kidney (Fu et al., 2003). Accordingly, we hypothesized that the source of the significant kidney-enhancing effect of FP may be due to its strong estrogen-like activities. To investigate this hypothesis, we focused on evaluating the estrogen-like activities of FP and its components. Numerous research studies have been conducted on FP, for example, its extract was found to induce vasodilation (Kassahun et al., 2017), exhibit anticancer activity (Park et al., 2016) and have kidney-enhancing effect (Zhao et al., 2016). The psoralen compound in FP was found to have anticancer (Wang et al., 2018), anti-inflammatory, anti-bacterial and osteogenic effects (Li et al., 2018; Tang et al., 2011). Additionally, its isopsoralen compound was also found to have anticancer activity (Wang et al., 2017) and osteogenic effect (Li et al., 2019). It has also been certified that the psoralidin compound can inhibit proliferation of several cancer cells via different signaling pathways (Bronikowska et al., 2012; Jin et al., 2016a, 2016b). However, most research on the estrogenic effect of compounds in FP was focused on psoralen and isopsoralen (Yuan et al., 2016; Zhang et al., 2005; Lim et al., 2011), with only a few studies focusing on other compounds. This is a major reason we conducted this in-depth study on various types of compounds present in FP. At the same time, we also performed a structure-effect analysis guided by the experiment, and we anticipate it will be useful for later research.
Section snippets
Plant materials
The dry ripe fruits of Cullen corylifolium (L.) Medik. were bought from Yunnan, China. They were authenticated by Dr. Lihong Wu, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, China. The voucher specimen (PC-20190529-Yunnan) was stored in the Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine.
Preparation of extract
Powdered FP (50 g) was immersed in 500 ml of 75% ethanol at 90 °C for 2 h, and this procedure was repeated twice.
Activating estrogenic effect of 75% ethanol FP extract and 75% ethanol plus petroleum ether, ethyl acetate, n-butanol, water FP extracts
The cell proliferation analysis by CCK-8 assay showed that MCF-7 cells were able to proliferate when treated with the 75% ethanol FP extract, at four concentrations from 0.05 to 1 mg/ml (Fig. 1A). The luciferase reporter gene assay showed that the four concentrations of the 75% ethanol FP extract were able to activate both ER receptors, namely ERα and ERβ. The activation by the highest concentration can be attenuated by the specific ERα antagonist ICI182,780 (Fig. 1B and C). According to these
Discussion
The results of this study showed that the 75% ethanol extract of FP had activating estrogenic effect and the ethyl acetate extract was found to have the highest activity among all the extract fractions.
The results also revealed that the constituent compounds in the ethyl acetate extract belong to the categories of chalcones, dihydroflavones, isoflavones and pseudoestrogens. Therefore, we focused on further studying 13 compounds from FP belonging to these four categories.
Then, the experiments to
Conclusion
Our study provides the evidence to support our hypothesis that FP has compounds with high estrogen-like activity and the ethyl acetate extract fraction was found to have the highest estrogen-like activity among all the fractions. Additionally, we evaluated the estrogen-like and anti-osteoporosis effects of these13 compounds from FP. Moreover, molecular docking and structure-effect analysis was successfully performed to further validate the experimental results. We found some regulatory
Author contributions
Zijia Zhang and Xinyin Cai conceived and designed the experiments. Xinyin Cai, Jinglin Xiong and Meng Yang performed the experiments. Xinyin Cai analyzed the data. Zijia Zhang and Zhengtao Wang contributed reagents/materials/analysis tools. Xinyin Cai wrote the paper.
Funding
This work was supported by National Key R&D Program of China (2019YFC1711000), National Natural Science Foundation of China (NSFC) (81473322) and Natural Science Foundation of Shanghai (14ZR1441100).
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
The authors would like to thank Professor Lihong Wu (Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, China) for identifying the plant material.
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