Synergistic properties and compatibility of different ingredients are the basic principle of t̠raditional C̲hinese m̲edicine (TCM). But the exact efficacy of individual chemicals within a TCM decoction is not known, which is a major stumbling block to the internationalization of TCM. D̲anggui B̲uxue T̠ang (DBT) is a classical Chinese herbal decoction containing only two herbs, A̲stragali R̲adix (AR) and A̲ngelicae S̲inensis R̲adix (ASR) at the weight ratio of 5:1, which serves as dietary supplement for easing women menopause syndromes. However, the signaling mechanism and the active compounds contributed in DBT pharmacological functions remain unclear. Therefore, a specific chemical knock-out method, performed by semi-preparative HPLC, was coupled with transcriptomics, as to reveal the...[ Read more ]

Synergistic properties and compatibility of different ingredients are the basic principle of t̠raditional C̲hinese m̲edicine (TCM). But the exact efficacy of individual chemicals within a TCM decoction is not known, which is a major stumbling block to the internationalization of TCM. D̲anggui B̲uxue T̠ang (DBT) is a classical Chinese herbal decoction containing only two herbs, A̲stragali R̲adix (AR) and A̲ngelicae S̲inensis R̲adix (ASR) at the weight ratio of 5:1, which serves as dietary supplement for easing women menopause syndromes. However, the signaling mechanism and the active compounds contributed in DBT pharmacological functions remain unclear. Therefore, a specific chemical knock-out method, performed by semi-preparative HPLC, was coupled with transcriptomics, as to reveal the synergistic functions of individual chemicals within DBT.

Calycosin, a major flavonoid in AR, shares similar structure with estrogen, which is phytoestrogen in regulating estrogenic-like functions. Calycosin is hypothesized to be a critical compound within DBT. Ferulic acid, one of the most abundant bioactive chemicals found in ASR, is one of promising natural chemicals within DBT herbal decoction to decrease r̠eactive o̲xygen s̠pecies (ROS) formation. Both calycosin and ferulic acid were selected to be the targets for performing specific chemical knock-out from herbal mixtures. Calycosin-depleted DBT (DBT_∆cal) and ferulic acid-depleted DBT (DBT_∆fa) were generated. The authenticity of DBT_∆cal and DBT_∆fa were evaluated by HPLC, LC-MS/MS and ¹H-NMR. In addition, the purity of collected fractions were analyzed by LC-MS/MS and ¹H-NMR.

Several bioassays were performed for elucidating the role ferulic acid and calycosin within the herbal extracts. The estrogenic activity of DBT_∆cal was reduced in the phosphorylations of estrogen receptor and Erk1/2 in cultured MCF-7 cells. Authentic DBT showed better effect on "erythropoietic system" by stimulating mRNA level of erythropoietin and hypoxia-inducible factor on cultured Hep3B cells, as compared to DBT_∆cal. DBT_∆cal exhibited declined cardiovascular functions in terms of inducing nitric oxide production in cultured HUVECs. Our study well illustrated calycosin played an important role in enhancing estrogenic functions of DBT.

Similarly, the anti-oxidative properties of DBT and DBT_∆fa were systemically compared in rat cardiomyoblast H9C2 cells. Application of DST in cultures stimulated the transcriptional activity of anti-oxidant response element and increased the phosphorylation of Akt. The aforementioned anti-oxidative properties of DBT_∆fa in H9C2 cells were significantly reduced, as compared to DBT. Thus, our findings well illustrated the different chemical play their unique functions within the herbal extracts.

Osteoporosis is one of the clinical symptoms of menopause. However, the detailed signaling mechanisms were still remained unclear. Therefore, transcriptomics were employed to explore the osteogenic properties of chemical-modified DBT on cultured osteoblast. The total RNA from herbal decoction-treated osteoblasts was isolated for transcriptome analysis by RNA-seq. The gene expression of osteogenic differentiation markers, including Alpl, Runx2, Sparc, Sp7 and Spp1, were significantly increased under the treatment of DBT and DBT_∆fa. Pathway analysis by KEGG revealed that authentic DBT and DBT_∆fa were able to activate MAPK/Erk and Wnt/β-catenin pathway, both of these pathways were essential cascades for osteoblast formation, as compared to the untreated control. However, DBT_∆cal was not able to trigger Wnt/β-catenin signaling.

From the RNA-seq and biological results, it strongly supported: (i) the chemical depletion method was feasible and convenient; (ii) calycosin and ferulic acid exhibited their unique functions in DBT, which acted as linkers in orchestrating multi-components of DBT to achieve the maximal functions; and (iii) transcriptomics was a power technique to illustrate the functions of DBT in mitigating post-menopausal osteoporosis. Therefore, a systematic approach for resolving the complexity of DBT was well illustrated here, and these methodology could be applied in other TCM formulae for mechanism analysis