A systematic approach to resolve the complexity of Danggui Buxue Tang, an angelica herbal decoction composed of astragali radix and angelica sinensis radix

Danggui Buxue Tang (DBT; 當歸補血湯) is a Chinese medicinal formula, which has been used for hundreds of years in China. Interestingly, the herbal composition of DBT is distinct in different ancient medical books, and three of them are still used today: (i) DBT₁₁₅₅, named after the recorded year composed of four herbs: Angelica Sinensis Radix (ASR), Astragali Radix (AR), Jujuba Fructus and Zingiberis Rhizoma Recens in a weight ratio of 12: 10 : 5 : 4; (ii) DBT₁₂₄₇ composed only two herbs, AR and ASR in a 5 : 1 ratio; (iii) DBT₁₆₈₇ composes of ASR, AR and Rehmanniae Radix Praeparata in a 2 : 1 : 1 ratio. Although all of them are recorded to have the function of nourishing “Qi” and enriching the “Blood”, DBT₁₂₄₇ is the one being used commonly. However, the systemic comparison among these three formulae has not been done. Here, we chose these three DBT as an example to explain the traditional Chinese medicine (TCM) theory by using chemical and biological approaches. The herbal extracts of three different DBT were standardized using rapid resolution liquid chromatography coupled with an electrospray ionization triple quadrupole tandem mass spectrometry. Eight marker chemicals, including AR-derived astragaloside III, astragaloside IV, calycosin, formononetin, ononin, calycosin-7-O-β-D-glucoside and ASR-derived ferulic acid and ligustilide were used to standardize DBT extracts. The highest amounts of AR-derived astragaloside III, astragaloside IV, calycosin, formononetin and ASR-derived ferulic acid were found in DBT₁₂₄₇. Upon the standardization of DBT₁₂₄₇ extract, the dynamic chemical change during the boiling process was studied. According to the traditional record, DBT₁₂₄₇ should be boiled with moderate heating. Under the moderate heating, the AR-derived ononin and calycosin-7-O-β-D-glucoside could be induced to hydrolyze in forming formononetin and calycosin. Both calycosin and formononetin had better biological functions and absorption rate. The current results supported the traditional practice of preparing DBT₁₂₄₇ by using moderate heating. The “Blood” enrichment effect of three DBT was evaluated on a myelosuppression anemic animal model, which was induced by an injection of cyclophosphamide. Daily intra-gastric administration of chemically standardized DBT for 14 days showed different effects: (i) the numbers of red blood cell and white blood cell, as well as the levels of hemoglobin and hematocrit, were increased; (ii) the serum level of IL-2 was increased; (iii) the levels of anti-oxidant enzymes were regulated; and (iv) the myelo-suppression was alleviated in the histopathology of bone marrow. These results suggested that three DBT not only had effect on hematological parameters but also affected the immune system and bone marrow. A series of biological analyses were applied to compare the biological efficacy of DBT. DBT₁₁₅₅ and DBT₁₂₄₇ were usually used to treat women’s disease, e.g. menopausal disorders, while DBT₁₆₈₇ was also used in men for the treatment of infertility. Here, DBT₁₂₄₇ showed the best effect on “erythropoetic system” by inducing the mRNA level of erythropoietin and increasing the hypoxia-responsive element-mediated transcriptional activity. The estrogenic activity of DBT₁₂₄₇ was robust in the phosphorylation of estrogen receptor α at S118 and of Erk1/2 in cultured MCF-7 cells. One the other hand, DBT₁₆₈₇ showed the best effect on circulation system by inhibiting platelet aggregation and increase nitric oxide production. Our study here well illustrated how different herbs can be selected and combined with flexibility to suit the specific syndromes under the guidance of the treatment principle. In addition, the roles of different Angelica roots (from China and Korea) in DBT₁₂₄₇ were elucidated. DBT₁₂₄₇ prepared with the Angelica roots from China showed high amount of marker chemicals, which also possessed better biological properties. Similar results were observed for DBT₁₂₄₇ prepared with AR as compared with that prepared with Hedysari Radix, the herb that is commonly used as adulterate of AR. These results recommend following the traditional formulation of TCM formula before fully realizing the rationale of traditional TCM theory. Our results here strongly supported the following TCM theory: (i) the traditional preparation of DBT₁₂₄₇ using moderate heating was proved to be reasonable, which should be complied with today; (ii) DBT₁₂₄₇ was the most famous and widely used Angelica decoction for the treatment of menopausal disease, however, special attention still should be paid in the clinic based on the syndrome differentiation; and (iii) DBT₁₂₄₇ should be prepared with AR and ASR from China, this special formulation should be followed to ensure the quality and effectiveness of TCM usage. By using chemical and biological assessment, a systematic approach for resolving the complexity of DBT was developed here: these methodologies could be applied in other TCM formulae.