Isolation of triterpenoids and phytosterones from Achyranthes bidentata Bl. to treat breast cancer based on network pharmacology

Abstract Breast cancer (BC) is a cancer with a high incidence and mortality of female, threatening women's physical and mental health. Achyranthes bidentata Blume, a traditional Chinese medicine, has been popular in folk due to its effective pharmacology activities including anti-inflammatory and anti-tumour effects. In order to identify the active ingredients from the roots of A. bidentata for treating breast cancer, the separation guided by network pharmacology analysis was employed which led to the isolation of 7 triterpenoids and 10 phytosterones. According to the in vitro experiments, the isolated compounds showed significant inhibitory activities on MCF-7 cells as well as anti-inflammatory activities by attenuating the production of NO and TNF-α in LPS-induced macrophage cells. In conclusion, this study successfully predicted and confirmed the positive impact of triterpenoids and phytosterones on breast cancer, which provided a foundation for further in vivo studies. Graphical Abstract


Introduction
About 2.1 million newly breast cancer cases were diagnosed in 2018 worldwide, accounting for 24.2% of all newly diagnosed cancer cases and 15.0% of all cancerrelated death cases in women (Bray et al. 2018). Now, BC is the most commonly diagnosed cancer and the leading cause of cancer death of female. At present, surgical treatment is a common and effective method, but medical expulsive treatment is also of critical importance, among which traditional Chinese medicine (TCM) has attracted extensive attention for their moderate treatment effect and less side effect.
Achyranthes bidentata Blume is a famous TCM, widely distributed in Africa and Asia (Han and Niu 2007). Modern pharmacological studies showed that it could be able to manage various conditions, such as tumour, inflammation, osteoarthritis and aging (Gyu et al. 2012). It had been reported that A. bidentata extract could inhibit proliferation of BGC823 and K562 cells (Hu et al. 2005) and some phytosterones could overcome cancer multidrug resistance by regulating PI3K/Akt pathway, which is one of the main obstacles in the treatment of cancer (Martins et al. 2012). In addition, some phytosterones from A. bidentata exhibited anti-neuroinflammatory effects against LPSinduced NO production in RAW 264.7 macrophages (Liu et al. 2017). Total saponins from this plant could activate JAK/STAT3 signaling pathway through Dectin-1, which could stimulate the release of TNF-a and then exert anti-tumor effects (Chen et al. 2015).
Nowadays, network-based drug discovery is considered as a more cost-effective approach for drug development (Boezio et al. 2017). In this study, network pharmacology method was applied to investigate the bioactive constituents of A. bidentata for the treatment of BC and in vitro cytotoxic and anti-inflammatory assays were also investigated.

Compound-target network
Compound-target network was constructed to identify the relationships between 102 compounds (Supplementry material Table S1) and 13 predicted targets (Supplementry material Table S2). In Supplementry material Figure S1, if compounds (targets) engage in more interactions, the value of degree, an important topological parameter, will be larger and circles (hexagons) shown in the network will become bigger and darker. Supplementry material Figure S1b showed the classification of different components. Triterpenoids and phytosterols as the main constituents of A. bidentata also had good pharmacological effects to play a critical role in the treatment of BC, which should be further studied.
GO biological process was employed for all the predicted targets. The result showed that these targets were enriched to 26 biological process terms, including 375 genes that were highly related to BC (Supplementry material Figure S2). The KEGG pathways analysis of potential targets showed 18 pathways. Among them, 9 terms were intensively connected to the cancer related pathways (Supplementry material Figure S3).

The extraction and isolation of A. bidentata
Two main fractions, total phytosterones (ABP) and total triterpenoids (ABT) were extracted from the roots of A. bidentata. In order to correlate bioactivities with the chemical composition, 17 compounds (including 10 phytosterones and 7 triterpenoids) were also isolated. Their names and structures were listed in Supplementry material Table S3. All ADME properties of the isolated compounds were in acceptable range (Supplementry material Table S4).

In vitro experiment
As expected, in vitro experimental results were similar to that predicted by network pharmacology. The cytotoxic results from MTT assay were listed in the Supplementry material Table S5. All the isolated phytosterones had cytotoxicity effects on MCF-7 to varying degrees and most of triterpenoids also had good effects on BC. The antiinflammatory results (Supplementry material Table S6) showed that ABT could significantly inhibit the synthesis and release of NO and TNF-a in LPS-induced macrophage cells without obvious cytotoxicity at their effective concentrations. Besides, deglucose chikusetsu saponin IVa (30) showed the strongest inhibitory effect with IC 50 values of 11.20 ± 1.22 (NO) and 16.80 ± 1.54 mM (TNF-a), respectively. But ABP and the isolated phytosterones almost had no effects. Similar results have been reported, triterpenoid saponins showed strong anti-inflammatory effect in a dose-dependent manner, while the phytosterone showed no obvious anti-inflammatory effect (Yang et al. 2016).
The preliminary SARs showed that the glycosidation at C-3 of oleanane-type triterpenoids and the free carboxyl substitution at C-28 were crucial to the anti-tumour and anti-inflammatory effects of oleanane-type triterpenoids. The glycosidation on the C-3 could strengthen the effects as shown in deglucose chikusetsu saponin IVa (30). With the lengthening of sugar chain at C-3, zingibroside R1 (66) for example, the effects would be weakened. The glycosidation at C-28 will significantly lower down the activities, such as in 33, 34 and 42. Similar results were given by earlier studies on the cytotoxicity of oleanane-type triterpenoids (Wang et al. 2014).

Molecular docking
In order to further identify the interaction between potential targets and phytosterones. The binding models of the two compounds (6 and 22) against BC target were established. The most likely potential target might be PIK3CG, an important target associated with cancer. The results showed that compound 6 possesses the highest binding affinity (Docking score: À128.934), followed by compound 22 (Docking score: À107.362). As depicted in Supplementry material Figure S4-S5, H-bond interaction, p-alkyl interaction and alkyl interaction were involved between the active site residues and these two compounds.

Conclusions
It was the first time to search the active constituents of A. bidentata in BC treatment by the approach of the virtual screening with systematic pharmacology and biological assays. Ultimately, 10 phytosterones and 7 triterpenoids were isolated as the bioactive components based on the bioactivity-guided rapidly separation. Deglucose chikusetsusaponin IVa and oleanolic acid were the primary components displaying positive effects in vitro experiments. Meanwhile, achyranthesterone B was the most effective phytosterones that showed strongly inhibitory activities on MCF-7. They are potential leading compounds for later in vivo studies. The present studies could precisely provide a foundation for the further development of this plant to treat BC and provide a better bioactivity-guided separation method of active ingredients in the production of functional ingredients in pharmaceutical industry.