Astragalus L. (Leguminosae) is a large genus with over 2,000 species worldwide and more than 250 sections in angiosperm family Fabaceae (subfamily Papilionoideae). Both listed as the botanical sources of RA in Chinese Pharmacopoeia (2005) 3, Astragalus membranaceus (Fisch.) Bunge and Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P.K. Hsiao 45 are the most commonly used RA. The morphological appearances and chemical properties of RA and its adulterants show a remarkable resemblance 678. The DNA sequences of 5S rRNA spacer, ITS and 18S rRNA coding region were determined and compared among ten Astragalus taxa 678. With neighbor-joining and maximum parsimony analyses, phylogenetic trees were mapped according to their sequence diversity. A. membranaceus and A. membranaceus var.mongholicus have the highest sequence homology. The common substitute of RA in some parts of China is the roots of Hedysarum polybotrys which has very different genetic makeup from that of the Astragalus species 9 (Figure 1).

HPLC and spectrophotometry were used to determine the levels of isoflavonoids, astragalosides, polysaccharides, amino acids and trace elements, which are the main active constituents in different Astragalus species and RA collected in different seasons and of various ages. The results indicated that RA of three years of age from Shanxi, China (Figure 2a) contained the highest amounts of isoflavonoids, saponins and polysaccharides 610.

According to the Chinese Pharmacopoeia (2005) 3, RAS is the root of Angelica sinensis (Oliv.) Diels (family Umbellaceae); however, Angelica acutiloba (Sieb. et Zucc.) Kitag. and Angelica gigas Nakai, mainly found in Japan and Korea respectively, are also sold as RAS in the markets of South East Asia 11121314 (Figure 1). Studies have shown that the three commonly used Angelica roots vary in their chemical composition, pharmacological properties and efficacy 911. The 5S-rRNA spacer domains of the three species of Angelica were amplified and their nucleotide sequences were determined. The sequence of A. sinensis is 72.87% and 73.58% identical to those of A. acutiloba and A. gigas respectively, while A. acutiloba and A. gigas are 93.57% identical in their sequences 9. The phylogenetic tree clearly reveals that the three Angelica species are divided into two clusters: A. sinensis is in one cluster and A. acutiloba and A. gigas are in another.

The main chemical constituents of Angelica roots are ferulic acid, Z-ligustilide, angelicide, brefeldin A, butylidenephthalide, butyphthalide, succinic acid, nicotinic acid, uracil and adenine 9151617. The levels of ferulic acid and Z-ligustilide are often used as chemical markers for the quality control of Angelica roots 16. In A. sinensis roots from Gansu, China, the levels of ferulic acid and Z-ligustilide are about ten-fold higher than those of the roots of A. acutiloba (from Japan) and A. gigas (from Korea) 917 (Figure 2b). Su Jing (659 AD) in Tang Bencao and Li Shizhen (1596 AD) in Bencao Gangmu recorded that Angelica roots of two years of age produced in Gansu were the authentic source. RAS from Gansu contains about two-fold higher amounts of Z-ligustilide and ferulic acid than those RAS from Yunnan, Shanxi or Sichuan, China 9 (Figure 2b). To ensure the best quality of DBT decoction, we suggest that standardized RA from Shanxi and standardized RAS from Gansu should be used in all DBT preparations (Figure 2c).

Li Dongyuan (1247 AD) documented that RA and RAS combined at a ratio of 5:1 demonstrated the best efficacy. In a previous study 18, DBT was prepared by boiling the herbal mixture under various conditions and the results indicated that the 5:1 ratio indeed provided the maximum levels of active constituents of DBT. Furthermore, the levels of active constituents and biological activities of DBT extracts were investigated with preparations of RA and RAS at ratios of 1:1, 2:1, 3:1, 4:1, 5:1, 7:1 and 10:1.

Used as chemical markers, the main active constituents in DBT include RA-derived astragaloside IV, calycosin and formononetin, RAS-derived ferulic acid and ligustilide, and total saponins, total flavonoids and total polysaccharides 19. The detected levels of the chemical markers varied significantly among the seven preparations (Figure 3). The level of astragaloside IV of the 5:1 ratio preparation was the highest, 2-fold higher than the 10:1 ratio preparation which recorded the lowest level 19. The 5:1 ratio preparation also contained the highest level of calycosin, formononetin, and ferulic acid. As regards the levels of total saponins, total flavonoids and total polysaccharides, the 5:1 ratio DBT preparation recorded the highest levels (Figure 3) 19.

There are several possibilities for higher levels of active chemical constituents in DBT preparations. Firstly, compounds such as saponins (over 2% in total dry weight) 1017 in RA may help increase the solubility of other compounds extracted from RAS. For example, astragaloside increases the solubility of RAS-derived ferulic acid and ligustilide. Secondly, ferulic acid and ligustilide are readily oxidized under heat, which means they can be degraded when boiled 9. However, when RAS is boiled together with RA, compounds derived from RA may prevent this oxidization process, thereby producing a higher yield of ferulic acid and ligustilide in DBT preparations. Thirdly, the stability of those active constituents may be improved by having a cocktail of different chemicals. Further research is required for better understanding of this complexity.

According to TCM theories, DBT replenishes qi and nourishes xue (the blood). DBT is therefore used for treating menopausal symptoms 2. Due to a deficiency of ovarian hormones, especially estrogen, women in menopause often suffer from hot flashes, sweating, anxiety, mood swings and an increased risk for other health problems, such as reduction of bone mineral density and cardiovascular diseases 20. Apart from a lack of estrogen, the immune system is also involved in the menopausal symptoms. Steroid hormones may modulate the immune response 21 and immune reactions may also regulate the ovarian function 22. Various bioactivities related to menopausal symptoms, such as osteotropic effect, estrogenic effect, anti-platelet aggregation effect and immuno-modulatory effect have been used to evaluate the functional roles of DBT.

DBT extract was applied to a cultured human MG-63 osteosarcoma cell. Bone cell proliferation and differentiation were measured by 3-(4, 5-dimethylthioazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and alkaline phosphatase (ALP) assay. DBT induced both the proliferation and differentiation of osteoblast MG-63 cells in a dose-dependent manner. In both assays, DBT showed stronger effects than RA or RAS alone, In the MTT assay, the 5:1 ratio DBT extract stimulated MG-63 cell proliferation, which was 10–20% higher than the extracts of other ratios (Figure 4). For bone cell differentiation, the 5:1 ratio DBT preparation induced ALP activity to the highest level among all ratios and showed the strongest osteotropic effect 19.

The estrogenic effects of DBT were tested by a cellular reporter system of transcriptional activation of estrogen receptor/promoter. A promoter/reporter construct (pERE-Luc) corresponding to the responsive elements of estrogen receptor was stably transfected into MCF-7 cells. The DBT extracts of various ratios were applied onto the cultures for 2 days. Two parameters, namely cell number and promoter activity (luciferase activity), were determined. While DBT was not able to alter the proliferation of MCF-7 cells, the estrogen-driven promoter activity was markedly induced by DBT (Figure 4); the 5:1 ratio DBT showed the strongest effect in inducing the promoter activity than RA, RAS alone or the extracts of other ratios 19.

In anti-platelet aggregation assay, the activity of DBT in preventing ADP-induced platelet aggregation was determined. The ratios 5:1 and 7:1 DBT extracts demonstrated higher levels of activity in preventing platelet aggregation than either RA, RAS alone or the extracts of other ratios (Figure 4) 19.

In a study of immuno-modulatory effects, DBT preparations of various ratios were applied to cultured T-lymphocytes and macrophages. In cultured T-lymphocytes, DBT induced markedly cell proliferation, interleukin-2 secretion and the phosphorylation of extracellular signal-regulated kinase (ERK1/2). In addition, the phagocytosis of cultured macrophages was elevated by DBT treatment. The immuno-modulatory effects of 5:1 ratio DBT were the strongest 23 (Table 1).

In addition to the in vitro assays, the 5:1 ratio of RA and RAS in DBT was further tested and verified by animal studies. In DBT-administrated mice, the 5:1 ratio preparation was the most effective decoction in triggering immune responses 2425.

The pharmacological studies in animals also suggest that DBT has the ability to promote hematopoiesis, to stimulate blood circulation, to prevent osteoporosis and to counter oxidative stress 192627. Moreover, DBT is known to enhance myocardial mitochondria and glutathione status in red blood cells, thereby increasing their resistance to injury induced by oxidative stress 28. In rats, DBT protected against myocardial ischemia-reperfusion injury in a dose-dependent manner 28. A more potent cardio-protection was demonstrated in DBT-treated rats than in rats treated with either extracts of RA, RAS alone, or a mixture of RA and RAS (not boiled together). When the mice were administered orally with DBT, the serum collected from abdominal aorta was added to an in vitro cultivating system of mouse hematopoietic progenitor cells. The decoction-contained serum showed promoting actions to CFU-GM and CFU-E. Once again, the action of the 5:1 ratio DBT was 97.81% stronger than that of the 1:1 ratio extract 2930 (Table 2).

Fifty-four chemical peaks were detected in DBT extracts by an HPLC analysis (Figure 5a) and a total of over 100 DBT extracts from various preparations were analyzed 27. Among these 54 peaks, the markers for RA-derived astragaloside IV, calycosin and formononetin, and for RAS-derived ferulic acid and ligustilide were identified. In analysis of correlation, the identified 54 peak areas together with the contents of total saponins, total flavonoids and total polysaccharides were considered as independent variables. The results of the four bioactivities, namely proliferation and differentiation of MG-63 cells, estrogenic property in MCF-7 cells and anti-platelet aggregation activity, were considered as dependent variables. By analyzing the correlation of these two kinds of variables, coefficients of correlation between the HPLC data of the 57 chemicals and the bioassay data of the DBT extracts were obtained. The values of the coefficients indicate possible relationship of these chemical peaks with bioactivities, where positive values suggest positive effects of chemicals on bioactivities and negative values suggest negative effects.

In the assay of MG-63 cell proliferation, astragaloside IV, formononetin, total saponins and total flavonoids are correlated with the bioactivities (Figure 5b). In the assay of MG-63 cell differentiation, formononetin, total saponins and total flavonoids are correlated with the bioactivities. In the analysis of estrogen promoter in MCF-7 cells, ferulic acid are correlated with the bioactivities. Calycosin and total polysaccharides were two very important factors in the assay of anti-platelet aggregation. On the other hand, the amount of ligustilide showed negative effects in all bioassays (Figure 5b). Other components of DBT, such as those corresponding to peaks 5 to 15, have high correlation coefficients with the bioactivities, but are yet to be identified.

The estrogenic effects of DBT were investigated by determining the levels of phosphorylation of estrogen receptor α (ERα) and extracellular signal-regulated kinase 1/2 (ERK1/2) in cultured MCF-7 cells. In contrast to estrogen, DBT triggered the phosphorylation of ERα and ERK1/2 at both S118 and S167 in a time-dependent manner. Although the activity of the estrogen-responsive element in pERE-Luc stably expressing MCF-7 cells was activated by extracts of either RA or RAS alone, or by a mixture of RA and RAS, the phosphorylation of ERα at S167 and of ERK1/2 were only found in DBT-treated cultures. Interestingly, the specific estrogenic effects of DBT were not only shown in the MCF-7 cells 31.

In cultured T-lymphocytes, the phosphorylation of the ERK 1 (about 42 kDa) and ERK 2 (about 44 kDa) was increased by DBT 30. The induction was transient. An approximately eight-fold increase of ERK phosphorylation was detected 20 minutes after DBT was applied, whereas the phosphorylation was undetectable in the cultures treated with extracts of either RA or RAS alone 31. Moreover, the phosphorylation of ERK in T-lymphocytes could not be activated by a simple mixture of extracts of RA and RAS. This result suggests that boiling RA and RAS together is essential for DBT to exert estrogenic effects.

For decades, scientists mainly isolated pure chemicals from herbal extracts and then screened for biological activities and possible targets. This strategy does not garantee to isolate and/or identify active chemicals from well-known medicinal plants because a single chemical compound may not fully account for the overall effects of herbal extracts. Recent advances in genomics and proteomics have enriched our tool sets to reveal the complex nature of TCM decoctions. An experiment on DBT-regulated genes was carried out in our laboratory using gene chip (i.e. microarray) technology. Cultured MG-63 cells were treated with 1 mg/ml of RA, RAS or DBT for 24 hours. The isolated mRNAs were analyzed using microarray, which is a quantitative method to investigate the change of mRNA expression profiles between the control and treatment groups. A total of 8064 genes were screened. Significant changes in gene expression were found after the treatment of DBT, RA or RAS (Table 3). A total of 883 genes were either up or down regulated by DBT treatment of which 403 genes were DBT-specific; 660 genes were regulated by RA treatment of which 172 genes were RA-specific; 1,062 genes were regulated by RAS treatment of which 473 genes were RAS-specific. In addition, 279 genes were commonly regulated by the extracts of DBT, RA or RAS. The genomic analysis demonstrated not only the activation effect of DBT in stimulating the proliferation and differentiation of the cultured osteoblasts but also a set of candidates of biomarkers that are specifically activated by DBT. These DBT-specific changes in gene expression may be useful in developing biomarkers for quality control of DBT. After identification of these DBT-specific genes and their roles, it will be easier to elucidate the action mechanism of DBT.