Chemical and biological assessment of Angelica herbal decoction: Comparison of different preparations during historical applications

Phytomedicine 19 (2012) 1042–1048

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Chemical and biological assessment of Angelica herbal decoction: Comparison of different preparations during historical applications Wendy Li Zhang a , Ken Yu-Zhong Zheng a , Kevin Yue Zhu a , Janis Ya-Xian Zhan a , Cathy Wen-Chuan Bi a , Jian-Ping Chen a , Crystal Ying-Qing Du a , Kui-Jun Zhao b , David Tai-Wai Lau a , Tina Ting-Xia Dong a , Karl Wah-Keung Tsim a,∗ a b

Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong Special Administrative Region Beijing Friendship Hospital, Affiliate of Capital University of Medical Sciences, 95 Yong An Road, Beijing 100050, China

a r t i c l e

i n f o

Keywords: Danggui Buxue Tang Astragali Radix Angelicae Sinensis Radix Herbal medicine Traditional Chinese medicine

a b s t r a c t The commonly used Angelica herbal decoction today is Danggui Buxue Tang (DBT), which is a dietary supplement in treating menopausal irregularity in women, i.e. to nourish “Qi” and to enrich “Blood”. According to historical record, many herbal decoctions were also named DBT, but the most popular formulation of DBT was written in Jin dynasty (1247 AD) of China, which contained Astragali Radix (AR) and Angelicae Sinensis Radix (ASR) with a weight ratio of 5:1. However, at least two other Angelica herbal decoctions recorded as DBT were prescribed in Song (1155 AD) and Qing dynasties (1687 AD). Although AR and ASR are still the major components in the DBT herbal decoctions, they are slightly varied in the herb composition. In order to reveal the efficiency of different Angelica herbal decoctions, the chemical and biological properties of three DBT herbal extracts were compared. Significantly, the highest amounts of AR-derived astragaloside III, astragaloside IV, calycosin and formononetin and ASRderived ferulic acid were found in DBT described in 1247 AD: this preparation showed stronger activities in osteogenic, estrogenic and erythropoetic effects than the other two DBT. The current results supported the difference of three DBT in chemical and biological properties, which could be a result of different herbal combinations. For the first time, this study supports the popularity of DBT described in 1247 AD. © 2012 Elsevier GmbH. All rights reserved.

Introduction Danggui Buxue Tang (DBT) is a very popular Chinese herbal decoction having a specific formulation prescribed for women in China as a remedy for menopausal symptoms, which improves their health by raising the “Qi” (vital energy) and nourishing the “Blood” (body circulation). According to historical usages in traditional Chinese medicine (TCM), at least three DBT formulae, namely DBT1155 , DBT1247 and DBT1687 , have been recorded. In “Chensuan Fuke Buji” written by Chen Suan of Song Dynasty (1155 AD), the first record of DBT (i.e. DBT1155 ) was revealed, which composed of four herbs: Angelicae Sinensis Radix (ASR), Astragali Radix (AR), Jujuba Fructus (JF) and Zingiberis Rhizoma Recens (ZRR) in a weight ratio of 12:10:5:4. DBT1247 was recorded in “Neiwaishang Bianhuo Lun” by Li Dongyuan in Jin dynasty (1247 AD), which composed only two herbs, ASR and AR in a 1:5 ratio. DBT1687 was recorded in “Bianzheng Lu” by Chen Shiduo in Qing dynasty (1687 AD), which composed of

Abbreviations: DBT, Danggui Buxue Tang; AR, Astragali Radix; ASR, Angelicae Sinensis Radix; TCM, traditional Chinese medicine. ∗ Corresponding author. Tel.: +852 2358 7332; fax: +852 2358 1559. E-mail address: [email protected] (K.W.-K. Tsim). 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.07.009

ASR, AR and Rehmanniae Radix Praeparata (RRP) in a 2:1:1 ratio. The three DBT formulations have one thing in common of having ASR and AR: both of them are considered as the key herbs in these decoctions. Clinically, the three herbal decoctions have very similar functions, i.e. prescribed to women suffering from menopausal symptoms. Under different scenarios, TCM practitioners could rearrange the herbal composition within DBT according to individual experience, as well as the syndrome differentiation and treatment variation of their patients. DBT1247 is frequently prescribed in herbal clinics today. In addition, DBT1247 is the most well studied herbal decoctions. DBT1247 was shown to possess the abilities to promote hematopoietic function, to stimulate cardiovascular circulation, to prevent osteoporosis, to increase anti-oxidation activity, to stimulate immune response and to increase insulin sensitivity (Dong et al. 2006; Gao et al. 2006, 2007a,b, 2008; Li et al. 2009; Zheng et al. 2011a). The optimized conditions for herbal extraction of DBT1247 have been also established (Song et al. 2004). The two herbs have to be boiled together as to achieve the maximum chemical and biological properties (Gao et al. 2007b; Choi et al. 2009). However, these analyses have not been done on DBT1155 and DBT1687 . Moreover, the rational of declining usage of DBT1155 and DBT1687 has not been revealed yet.

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In order to search the optimal formulation of DBT, the chemical and biological assessment of the three recorded DBT were performed and compared. Chemically, the amounts of AR-derived astragaloside III, astragaloside IV, calycosin, formononetin, ononin, calycosin-7-O-␤-d-glucoside and ASR-derived ferulic acid and ligustilide were determined in the herbal decoctions. In parallel, the biological properties of these herbal extracts in stimulating osteogenic, estrogenic and erythropoetic effects were determined in cell culture models, and the mechanisms of these DBT were investigated. Our results showed that the formulation of DBT1247 possessed the best chemical and biological properties: this included osteogenic, estrogenic and erythropoetic effects as compared to DBT1155 and DBT1687. Materials and methods Preparation of DBT Three-year-old Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) Hsiao roots (AR) from Shanxi Province (Ma et al. 2002) and two-year-old Angelica sinensis (Oliv.) Diels roots (ASR) from Minxian in Gansu Province (Zhao et al. 2003) were collected. Jujuba Fructus (JF, the fruit of Ziziphus jujube Mill.) and Zingiberis Rhizoma Recens (ZRR, the fresh rhizome of Zingiber officinale Rosc.) were collected in Heibei and Shangdong province, respectively. Rehmanniae Radix Praeparata (RRP, the processed root of Rehmannia glutinosa Libosch.) was purchased from Qingping herbal market in Guangzhou China. According to different formulations of DBT1155 , DBT1247 and DBT1687 , the appropriate amounts of crude herbs (grounded into powders) were weighed separately to form a combined weight of 30 g and then mixed by vortex. The mixture was boiled in 8 volume of water (v/w) for 2 h and extracted twice; this extraction followed the ancient recipe that had been shown to have the best extracting condition (Song et al. 2004; Dong et al. 2006; Zheng et al. 2010a). The extract was dried under vacuum and stored at −80 ◦ C. For the chemical analysis, an appropriate amount of each freeze drying powder was weighed into a 15-ml centrifugal tube, and 5 ml of water was added for sonication for 30 min. The supernatant was centrifuged at 16,000 × g at 4 ◦ C for 10 min before the rapid resolution liquid chromatography (RRLC) analysis. Chemical analysis by RRLC–QQQ-MS/MS Ferulic acid was from Sigma (St. Louis, MO); astragaloside III and astragaloside IV were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); calycosin, formononetin, ononin, calycosin-7-O-␤-d-glucoside and Z-ligustilide were kindly provided by Prof. Pengfei Tu, Medical College of Peking University. The purities of these marker chemicals, confirmed by high-performance liquid chromatography (HPLC), were higher than 98.0%. Analytical- and HPLC-grade reagents were from Merck (Darmstadt, Germany). An Agilent 1200 series system (Agilent, Waldbronn, Germany), equipped with a degasser, a binary pump, an auto-sampler, and a thermo-stated column compartment was used for the analysis. Chromatographic separations were carried out on an Agilent ZORBAX Eclipse XDB-C18 column (1.8 ␮m id, 50 mm × 4.6 mm). The mobile phase was composed of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B) using the following gradient program: 0–2 min, isocratic gradient 20.0–20.0% (A); 2–7 min, linear gradient 20.0–34.0% (A); 7–12 min, isocratic gradient 34.0–34.0% (A); 12–16 min, linear gradient 34.0–65.0% (A); 16–18 min, linear gradient 65.0–80.0% (A). A pre-equilibration period of 4 min was used between each run. The flow rate was 0.4 ml/min. The column

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temperature was at 25 ◦ C. The injection volume was 5 ␮l. For the calibration of eight markers, the standards were weighed and dissolved in methanol to give serial concentrations from 0.005 to 1 ␮g/ml, and two injections onto LC/MS/MS were performed for each dilution. The concentrations of these markers in the samples were calculated according to the regression parameters derived from the standard curve. For the MS/MS analysis, an Agilent QQQ-MS/MS (6410A) equipped with an ESI ion source was used for all analyses. The drying gas temperature was 325 ◦ C; drying gas flow: 10 l/min; nebulizer pressure: 35 psig; capillary voltage: 4000 V; delta electro multiplier voltage: 400 V. Two suitable transition pairs were chosen for acquisition in MRM mode for the 8 makers and two internal standards, ginsenoside Rg1 and polydatin. The fragmentor voltage and collision energy values were optimized to obtain the highest abundance. Agilent Mass Hunter workstation software version B.01.00 was used for data acquisition and processing. Cell proliferation and differentiation assay Human mammary epithelial carcinoma MCF-7 cells, human osteosarcoma MG-63 cells and human embryonic kidney fibroblast (HEK) 293T cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). MCF-7 cells and MG-63 cells were grown in modified Eagle’s medium (MEM), supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 0.1 mM nonessential amino acids, 1 mM pyruvate, 100 units/ml penicillin, and 100 units/ml streptomycin in a humidified CO2 (5%) incubator at 37 ◦ C. HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 units/ml streptomycin in a humidified CO2 (5%) incubator at 37 ◦ C. All culture reagents were purchased from Invitrogen Technologies (Carlsbad, CA). The cell proliferation was measured by 3-(4,5-dimethylthioazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Li et al. 2003). 17-␤-Estradiol (Sigma; 100 nM) and dexamethasone (Sigma, 50 nM) plus vitamin C (Sigma, 250 ␮M) served as positive controls for cultured MCF-7 and MG-63 cells, respectively. Osteogenic assay in MG-63 cells The proliferation of cultured MG-63 cells was performed by MTT assay. The differentiation of MG-63 cells was determined by the expression of alkaline phosphatase (ALP), an indicative bio-marker for later stage of osteoblast differentiation. ALP activity in MG-63 cells was measured by the hydrolysis of p-nitrophenyl phosphate (Sigma) as described previously (Lbbotson et al. 1986; Dong et al. 2006). Estrogenic assay in MCF-7 cells Three repeats of estrogen responsive elements (ERE: 5 -GGT CAC AGT GAC C-3 ) were synthesized, as described previously (Klinge 2011), and sub-cloned into a luciferase-reporter vector called pTALLuc (BD Biosciences Clontech, San Jose, CA) to form pERE-Luc. This reporter was stably transfected to MCF-7 cells to generate stable cells according to a reported protocol (Tung et al. 2004). To determine the estrogenic properties, different DBT extracts were applied onto the cultures for 48 h. Afterward, the medium was aspirated, and MCF-7 cells were washed by ice-cold PBS. The cell lysate was centrifuged at 16,000 × g and 4 ◦ C for 10 min, the supernatant was collected, and 50 ␮l of the supernatant was used to perform the luciferase assay (Tropix Inc., Bedford, MA); the activity was normalized by equal amount of protein. The phosphorylations of estrogen receptor (ER) at serine 118 (S118) are determined by Western blotting. Cultures were serum

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starved for 3 h before the drug applications. After drug treatments, the lysates were subjected to Western blot analysis to determine the phosphorylation of ER␣ at serine 118 as well as the total ER␣ as described previously (Zhu et al. 2007). Erythropoetic assay in HEK293T cells Six repeats of hypoxia responsive elements (HRE: 5 -TCG AGG CCC TAC GTG CTG TCT CAC ACA GCC TGT CTG ACG-3 ) were synthesized, concatemerized, and then cloned in tandem (head-to-tail orientation) into pBI-GL vectors (BD Biosciences Clontech) that had a downstream reporter of firefly luciferase gene (Zheng et al. 2010a). This vector was named as pHRE-Luc (Post and Van-Meir 2001). Cultured HEK293T cells were transiently transfected with pHRE-Luc by the calcium phosphate precipitation method (Xie et al. 2007). The transfection efficient was over 80%, as determined by another control plasmid of having a ␤-galactosidase, under a cytomergalovirus enhancer promoter. Other assays In the promoter activity assay, the amount of luciferase was performed by a commercial kit (Tropix Inc., Bedford, MA). In brief, cultures were lysed by a buffer containing 100 mM potassium phosphate buffer (pH 7.8), 0.2% Triton X-100, and 1 mM dithiothreitol. The luminescent reaction was quantified in a Tropix TR717 microplate luminometer, and the activity was expressed as absorbance (up to 560 nm) per mg of protein. The luciferase activity was normalized by the activity of ␤-galactosidase in the same amount of protein in each sample. Protein concentrations were measured routinely by Bradford’s method with a kit from Bio-Rad Laboratories (Hercules, CA). Statistical analysis Statistical tests were done by using one-way analysis of variance. Data were expressed as mean ± SD, where n = 4. Statistically significant changes were classified as significant (*) where p < 0.05, highly significant (**) where p < 0.01. Results Chemical analysis of different DBT preparations According to the historical usages, three DBT formulae, namely DBT1155 , DBT1247 and DBT1687 , have been recorded, and their herbal composition and clinical application were listed (Table 1). The herbal extracts were prepared and dried, and therefore the chemical compositions in these DBT were determined and standardized. Here, the AR-derived astragaloside III, astragaloside IV, calycosin, formononetin, ononin, calycosin-7-O-␤-d-glucoside and the ASRderived ferulic acid and ligustilide were determined as marker chemicals. The rationale to choose these chemicals for analyses was that they were reported to have known biological functions as

Fig. 1. Typical RRLC–QQQ-MS/MS chromatograms of marker chemicals in DBT. The chromatographic method was described in Materials and methods. The identification of calycosin-7-O-␤-d-glucoside (1), ferulic acid (2), ononin (3), calycosin (4), astragaloside IV (5), astragaloside III (6), formononetin (7), Z-ligustilide (8), and polydatin (internal standard, IS1), ginsenoside Rg1 (internal standard, IS2), were determined by a MS detector. Representative chromatograms are shown, n = 3.

described previously (Wagner et al. 1997; Sinclair 1998; Zheng et al. 2011a). The chemical structure of the markers (Supplementary Fig. 1) and their characteristics in the MS/MS (Supplementary Table 1) were revealed. In MS/MS analysis, a calibration curve was obtained from six different concentrations of marker chemicals. The squared values of all the correlation coefficients (r2 ) of these calibration curves were higher than 0.990 in MS/MS analysis. The limit of detection (LOD) and limit of quantification (LOQ) were determined at S/N of 3 and 10, respectively (Supplementary Table 2). The precision, repeatability and recovery were determined as described (Supplementary Table 3) (Zhu et al. 2010). The results showed that the RRLC–QQQ-MS/MS method was precise, accurate and sensitive enough for simultaneous, quantitative evaluation of marker chemicals in DBT formulae. The typical MS/MS chromatograms of the eight chemicals in the mixed standard, or in DBT, were shown in Fig. 1. The amounts of different marker chemicals were quantified according to their MS patterns. Table 2 shows the amounts of those chemicals within the three decoctions. DBT1247 contained the highest amounts of ARderived astragaloside III, astragaloside IV, calycosin, formononetin and ASR-derived ferulic acid: the difference of these marker concentrations could be at about 2-fold in comparison to the lowest groups. In contrast, the amounts of ononin and calycosin-7-O-␤d-glucoside were found to be higher in DBT1687 , and ligustilide was found to be the lowest in DBT1247 . In addition, the chemical quantitation could be served as a mean of standardized DBT for all biochemical assays as described below.

Table 1 Historical variation of different DBT formulae. Notationa

Record

Compositionb

Function

DBT1155

Chensu-an Fuke Bujie written in 1155 AD

ASR (12 g), AR (10 g), ZRR (4 g), JF (5 g)

DBT1247 DBT1687

Neiwaishang Bianhuo Lun written in 1247 AD Bianzhen Lu written in 1687 AD

ASR (6 g), AR (30 g) ASR (30 g), AR (15 g), RRP (15 g)

Deficient in Qi and Blood of woman, abnormality in menstruation and infertility Nourishing Qi, enriching Blood Deficient in Blood of man, yellow in facial color and infertility

a

The notation of DBT was corresponding to the year of recording. The herbal composition was calculated according to the historical record in g, where AR for the root of A. membranaceus var. mongholicus root; ASR for the root of A. sinensis; JF for the fruit of Z. jujube; ZRR for the fresh rhizome of Z. officinale; RRP for the processed root of R. glutinosa. b

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Table 2 Quantitative assessment of eight marker chemicals in three DBT formulae. Compound

Amounta DBT1155

Ligustilide Ferulic acid Formononetin Astragaloside III Astragaloside IV Calycosin Ononin Calycosin-7-O-␤-dglucoside

125.23 893.87 52.89 8.81 105.03 85.55 69.74

DBT1247 ± ± ± ± ± ± ±

12.29 47.88 2.97 0.60 12.45 2.56 7.09

119.97 1223.95 146.37 19.87 211.93 196.20 68.65

243.80 ± 5.08

DBT1687 ± ± ± ± ± ± ±

7.01 55.51* 3.12* 3.23** 16.56* 0.07* 6.94

229.77 ± 5.22

127.97 1021.01 94.86 6.54 155.92 134.95 89.14

± ± ± ± ± ± ±

8.37 26.79 4.74 1.42 18.78 4.85 0.06

322.87 ± 3.11

Values are expressed in ␮g/g dried extracts of DBT, mean ± SD, where n = 3, each with triplicate samples. * p < 0.05 as compared with two other DBT. ** p < 0.01 as compared with two other DBT. a

The osteogenic effect of three DBT The biological properties of DBT1155 , DBT1247 and DBT1687 were compared here, including the osteogenic, estrogenic and erythropoetic effects. MG-63 cell is a common cell line used in analyzing osteoblast differentiation (Lbbotson et al. 1986; Guo et al. 2011). Application of DBT extracts induced the proliferation of osteoblastic MG-63 cells in a dose-dependent manner: the cell induction was started to be obvious at 0.3 mg/ml of extracts, and the maximal response, in all cases, was achieved at ∼3 mg/ml (Fig. 2A). The sub-maximal concentration of DBT was revealed at about 1 mg/ml. Here, DBT1247 processed a significant stronger activity in stimulating the MG-63 cell proliferation, which was 10–15% higher than other two groups (Fig. 2A). The level of ALP, a differentiation marker of osteoblast, expressed by cultured MG-63 cells was increased by the applied herbal extracts. Again, DBT1247 induced the ALP expression higher than the other groups: the difference was more robust when the applied DBT was at sub-maximal dose of 1 mg/ml (Fig. 2B). The estrogenic effect of three DBT To investigate the estrogenic activity, MCF-7 cells stably transfected with pERE-Luc (Fig. 3A, upper panel) were employed. The extracts of different DBT were applied onto the cultures for 48 h. Two biological effects were subsequently determined, i.e. the promoter (ERE)-driven luciferase activity and cell viability. As shown in Fig. 3A, the expression of luciferase, carried in pERE-Luc, was markedly induced by DBT in a dose-dependent manner. DBT1247 possessed the best activity, ∼5-fold at sub-maximal concentration of 1 mg/ml, in stimulating the expression of luciferase, as compared to DBT1155 and DBT1687 . The difference was more robust under the low concentration of DBT, i.e. at 0.3 mg/ml: DBT1155 and DBT1687 at this low dose did not able to induce the promoter activity at all (Fig. 3A). A positive control, 17-␤-estradiol at 100 nM, caused ∼2-fold increase in the promoter activity. The application of DBT induced the phosphorylation of ER␣ at S118 (∼66 kDa) in a timedependent manner, and the total amount of ER␣ was unchanged (Fig. 3B). The degree of S118 phosphorylation, induced by DBT1247 , was stronger than the other two DBT. In term of cell proliferation, the three DBT did not show any significant effect (Fig. 3C).

Fig. 2. DBT increases the cell proliferation and enzymatic activity of alkaline phosphatase in cultured MG-63 cells. (A) The water extracts were applied onto cultured MG-63 cells for 48 h to determine the cell proliferation by MTT assay. (B) The water extracts of DBT were applied onto cultured MG-63 cells for 48 h to determine the enzymatic activity of alkaline phosphatase (ALP). Dexamethasone (DEX; 50 nM) together with vitamin C (VC; 250 ␮M) was used as a control in MG-63 cells. Dose–response curves of three DBT were performed. Values are expressed in percentage of increase as compared with control cultures (without herbal extract), and are in mean ± SD, where n = 4, each with triplicate samples. *p < 0.05; **p < 0.01.

construct (pHRE-Luc), containing six HREs derived from the promoter of EPO gene and tagged upstream of a luciferase gene (Post and Van-Meir 2001), was transfected into cultured fibroblasts. The pHRE-Luc expressing fibroblasts were validated in responding to hypoxia. The authentication of pHRE-Luc was confirmed by its activation in exposing to mineral oil or desferrioxamine (DFO), which was frequently used to mimic the effect of hypoxia (Zheng et al. 2011b). Under the hypoxia by oil layering or DFO, the expression of pHER-Luc was robustly induced (Fig. 4A). The three DBT showed significant induction on the pHER-Luc activity. After treating different DBT extracts in pHER-Luc-expressed cells for 48 h, the pHER-Luc activity was induced in a dose-dependent manner (Fig. 4B). The most potent DBT in the HRE activation was DBT1247 , and the maximal induction was over 2-fold at 1 mg/ml. DBT1155 had the lowest effect at 1.5-fold of the increase at 1 mg/ml (Fig. 4B). The difference of DBT1247 as compared to others was more obvious at the low concentration of the applied extract. Discussion

The erythropoetic effect of three DBT DBT1247 was shown to stimulate the production of erythropoietin (EPO), a specific hematopoietic growth factor, by activation of the hypoxia-mediated signaling cascade in cultured HEK293T cells. To reveal the transcriptional activity of HRE, a luciferase-reporter

According to traditional Chinese medicinal theory, the herbal decoction should be prepared in an unique methodology having specific combination of different herbs as a formula (named as Fu Fang). In a herbal formula, each decoction consists of four elements: Jun (king), Chen (minister), Zuo (assistant) and Shi (servant), which

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Fig. 4. Three DBT extracts stimulate the HRE-mediated transcriptional activity in cultured HEK293T cells. (A) Six repeats of hypoxia responsive elements (HRE: 5 TCG AGG CCC TAC GTG CTG TCT CAC ACA GCC TGT CTG ACG-3 ) were sub-cloned in an expression vector and was named as pHRE-Luc (upper panel). The pHRELuc-transfected HEK293T cells were treated with extracts (1 mg/ml) derived from DBT1155 , DBT1247 and DBT1687 for 48 h to determine the promoter-driven luciferase (pHRE-Luc) activity, the layer of mineral oil and DFO (50 ␮M) were used as positive controls. (B) Dose–response curves of three DBT were performed for assay as in (A), with treatment for 48 h. Values are expressed as the ratio to the basal reading where the control (untreated culture) equals to 1 and in mean ± SD, where n = 4, each with triplicate samples. *p < 0.05; **p < 0.01.

Fig. 3. DBT stimulates the activity of estrogen receptor but not cell proliferation in MCF-7 cells. (A) Three repeats of estrogen responsive elements (ERE: 5 -GGT CAC AGT GAC C-3 ) were sub-cloned into a luciferase-reporter vector called pERE-Luc (upper panel). This reporter was stably transfected to MCF-7 cells, and which were treated with DBT extracts at different dose for 48 h (lower panel). 17-␤-Estradiol (100 nM) was used as a positive control. (B) MCF-7 cells were serum-starved for 3 h before the addition of DBT extracts (1 mg/ml). The lysates were subjected to Western blot analysis to determine the phosphorylation of ER␣ at serine 118 as well as the total ER␣ (C). Cultures were treated as in (A) at 3 mg/ml (maximal dose) to determine the cell proliferation by MTT assay. Values are expressed in percentage of increase as compared with control cultures (without herbal extract), and are in mean ± SD, where n = 4, each with triplicate samples. *p < 0.05; **p < 0.01.

have to be worked harmoniously together in order to achieve a therapeutic purpose. In DBT, AR is considered as Jun, while ASR is Chen. Unfortunately, the action mechanisms, or the roles, of these elements in DBT have not been fully determined. In general, the compatibility amongst all the herbs within a decoction is an important principle of TCM theory, which ensures the full pharmacological properties of TCM decoctions. Under this compatibility principle, a herbal practitioner could develop or create new herbal formulae. Therefore, there are many herbal formulae that have the same name but with different herbal compositions, e.g. the existence of DBT1155 , DBT1247 and DBT1687 . There are several possible reasons to account for this confusion. First, Chinese herbal practitioners might revise the original old formula according to their own experience, the syndrome of their patients, the variation of their geographical location and the availability of herb sourcing; however, the name of formula was unchanged. Second, the loss of original historical recipe could be another reason for the emergence

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of different formulae, and thus different herbal formulae having the same name were arising. The principal herbs within DBT1155 , DBT1247 and DBT1687 are AR and ASR. AR is considered to be the best herb to treat the “Qi” (vital energy) deficiency. ASR is a herb for replenishing the “Blood” (body circulation), in particular ASR is commonly named as “the best herb for women”. The pairing of AR and ASR is commonly happened in ancient herbal formulae: this pairing enhances the pharmacological properties of the herbal decoction. According to traditional Chinese medicinal theory, “Qi” and “Blood” are closely related. “Blood” cannot be self-generated, and which requires the stimulation by “Qi” (Fan and Chen 2006). Thus, the AR-induced “Qi” could prelude the ASR-induced “Blood” functions, as to achieve maximal pharmacological activity. The compatibility of herbal medicine is fully recognized here by DBT of having AR and ASR. DBT1247 is the best amongst the tested three DBT in inducing the aforementioned pharmacological properties, and which contains higher amount of AR. The chemical and biological assessment of DBT1247 has been performed: the ratio of ASR and AR in 1:5 is the best in producing the herbal decoction (Song et al. 2004; Dong et al. 2006). In this 1:5 ratio, the amounts of AR-derived calycosin, formononetin, astragaloside IV and ASR-derived ferulic acid were found to be much higher as compared to other decoctions of AR and ASR having different herb ratios (Dong et al. 2006). In contrast, the ASR-derived ligustilide was found to be the lowest in this 1:5 ratio of DBT (Dong et al. 2006; Zheng et al. 2010b). In line to this result, DBT1247 contained higher amounts of calycosin, formononetin, astragaloside III, astragaloside IV and ferulic acid, as compared to that of DBT1155 and DBT1687 . Indeed, the higher concentration of these chemicals has been reported to be responsible for better DBT functions (Dong et al. 2006; Zheng et al. 2010a, 2011a). In parallel, these chemicals have been demonstrated to possess osteogenic, estrogenic and erythropoetic effects, e.g. calycosin and formononetin (Zheng et al. 2011a). For example, formononoetin, ononin, calycosin and calycosin-7-O-␤-d-glucoside could stimulate the expression of erythropoietin in cultured cells (Zheng et al. 2011a). These four flavonoids have been shown to induce estrogenic activities by activating the estrogen-responsive element in cultured MCF-7 cells, and calycosin has been shown to have better estrogenic effect than its glycoside calycosin-7-O-␤d-glucoside (Zhu et al. 2007). Lastly, calycosin could induce ALP activity in cultured osteoblasts (Guo et al. 2011). Higher amounts of those active ingredients revealed in DBT1247 could be due to: (i) higher amount of AR being used and (ii) the best ratio of AR and ASR in 1:5. On the other hand, DBT1247 contained slightly less ligustilide in the decoction, which was reported to be the negative regulator of DBT functions (Zheng et al. 2010b). Thus, our study fully supports why DBT1247 is the most widely used Angelica herbal decoction today. By using chemical and biological assessment, different historical DBT, i.e. DBT1155 , DBT1247 and DBT1687 , were compared. DBT1247 contains the highest amounts of AR-derived astragaloside III, astragaloside IV, calycosin and formononetin and ASR-derived ferulic acid, which also has stronger activities in osteogenic, estrogenic and erythropoetic effects. For the first time, the chemical and pharmacological approach was used here for the valuation of an ancient Chinese herbal formula, and the current results explain the reason of DBT1247 popularity, as compared to DBT1155 and DBT1687 .

Acknowledgments This research was supported by grants from Research Grants Council of Hong Kong (HKUST 6419/06M, N HKUST629/07, 662608, 661110), Croucher Foundation (CAS-CF07/08.SC03) to KWKT and DTWL.

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.phymed.2012.07.009.

References Choi, R.C.Y., Gao, Q.T., Cheung, A.W.H., Zhu, J.T.T., Lau, F.T.C., Li, J., Li, W.Z.M., Chu, G.K.Y., Duan, R., Cheung, J.K.H., Ding, A.W., Zhao, K.J., Dong, T.T.X., Tsim, K.W.K., 2009. A Chinese herbal decoction Danggui Buxue Tang, stimulates proliferation, differentiation and gene expression of cultured osteosarcoma cells: genomic approach to reveal specific gene activation. Evidence-Based Complementary and Alternative Medicine (Publish on-line). Dong, T.T.X., Zhao, K.J., Gao, Q.T., Ji, Z.N., Zhu, T.T., Li, J., Duan, R., Cheung, A.W.H., Tsim, K.W.K., 2006. Chemical and biological assessment of a Chinese herbal decoction containing Radix Astragali and Radix Angelicae sinesis: determination of drug ration in having optimized properties. Journal of Agricultural and Food Chemistry 54, 2767–2774. Fan, Y., Chen, X.Y., 2006. The research progress for the origin and the compatibility of Angelica decoction for tonifying blood. Chinese Journal of Experimental Traditional Medical Formulae 2, 61–65. Gao, Q.T., Cheung, J.K.H., Li, J., Chu, G.K.Y., Duan, R., Cheung, A.W.H., Zhao, K.J., Dong, T.T.X., Tsim, K.W.K., 2006. A Chinese herbal decoction Danggui Buxue Tang, prepared from Radix Astragali and Radix Angelicae Sinensis stimulates the immune responses. Planta Medica 72, 1227–1231. Gao, Q.T., Choi, R.C.Y., Cheung, A.W.H., Zhu, J.T.T., Li, J., Chu, G.K.Y., Duan, R., Cheung, J.K.H., Jiang, Z.Y., Dong, X.B., Zhao, K.J., Dong, T.T.X., Tsim, K.W.K., 2007a. Danggui Buxue Tang – a Chinese herbal decoction activates the phosphorylations of extracellular signal-regulated kinase and estrogen receptor alpha in cultured MCF-7 cells. FEBS Letters 581, 233–240. Gao, Q.T., Li, J., Cheung, J.K.H., Duan, J.A., Ding, A.W., Cheung, A.W.H., Zhao, K.J., Li, W.Z.M., Dong, T.T.X., Tsim, K.W.K., 2007b. Verification of the formulation and efficacy of Danggui Buxue Tang (a decoction of Radix Astragali and Radix Angelicae Sinensis): an exemplifying systematic approach to revealing the complexity of Chinese herbal medicine formulae. Chinese Medical 2, 12–22. Gao, Q.T., Cheung, J.K.H., Choi, R.C.Y., Cheung, A.W.H., Li, J., Jiang, Z.Y., Duan, R., Zhao, K.J., Ding, A.W., Dong, T.T.X., Tsim, K.W.K., 2008. A Chinese herbal decoction prepared from Radix Astragali and Radix Angelicae Sinensis induces the expression of erythropoietin in cultured Hep3B cells. Planta Medica 74, 392–395. Guo, A.J.Y., Choi, R.C.Y., Cheung, A.W.H., Chen, V.P., Xu, S.L., Dong, T.T.X., Chen, J.J., Tsim, K.W.K., 2011. Baicalin, a flavone, induces the differentiation of cultured osteoblasts an action via the Wnt/␤-catenin signaling pathway. Journal of Biological Chemistry 286, 27882–27893. Klinge, C.M., 2011. Estrogen receptor interaction with estrogen response elements. Nucleic Acids Research 29, 2905–2919. Lbbotson, K.J., Harrod, J., Gowen, M., D’Souza, S., Smith, D.D., Winkler, M.E., Derynck, R., Mundy, G.R., 1986. Human recombinant transforming growth factor alpha stimulates bone resorption and inhibits formation in vitro. Proceedings of the National Academy of Sciences of the United States of America 83, 2228–2232. Li, W.Z.M., Li, J., Bi, C.W.C., Cheung, A.W.H., Huang, W., Duan, R., Choi, R.C.Y., Chen, I.S.Y., Zhao, K.J., Dong, T.T.X., Duan, J.A., Tsim, K.W.K., 2009. Can Rhizoma Chuanxiong replace Radix Angelica Sinensis in the traditional Chinese herbal decoction Danggui Buxue Tang. Planta Medi 75, 602–606. Li, S.P., Zhao, K.J., Ji, Z.N., Song, Z.H., Dong, T.T.X., Lo, C.K., Cheung, J.K.H., Zhu, S.Q., Tsim, K.W.K., 2003. A polysaccharide isolated from Cordyceps sinensis, a traditional Chinese medicine, protects PC12 cells against hydrogen peroxide induced injury. Life Science 73, 2503–2513. Ma, X.Q., Shi, Q., Duan, J.A., Dong, T.T.X., Tsim, K.W.K., 2002. Chemical analysis of Radix Astragali (Huangqi) in China: a comparison with its adulterants and seasonal variations. Journal of Agricultural and Food Chemistry 50, 4861–4866. Post, D.E., Van-Meir, E.G., 2001. Generation of bidirectional hypoxia/HIF-responsive expression vectors to target gene expression to hypoxic cells. Gene Therapy 8, 1801–1807. Sinclair, S., 1998. Chinese herbs: a clinical review of Astragalus, Ligusticum, and Schizandrae. Alternative Medicine Review 3, 338–344. Song, Z.H., Ji, Z.H., Lo, C.K., Dong, T.T.X., Zhao, K.J., Li, O.T.W., Haines, C.J., Kung, S.D., Tsim, K.W.K., 2004. Chemical and biological assessment of a traditional Chinese herbal decoction prepared from Radix Astragali and Radix Angelicae Sinensis: orthogonal array design to optimize the extraction of chemical constituents. Planta Medica 70, 1222–1227. Tung, E.K.K., Choi, R.C.Y., Siow, N.L., Jiang, J.X.S., Ling, K.K.Y., Simon, J., Barnard, E.A., Tsim, K.W.K., 2004. P2Y2 receptor activation regulates the expression of acetylcholinesterase and acetylcholine receptor genes at vertebrate neuromuscular junctions. Molecular Pharmacology 66, 794–806. Wagner, H., Bauer, R., Xiao, P.G., Chen, J.M., Michler, G., 1997. Chinese drug monographs and analysis: Radix Astragali (Huangqi). In: Staudinger, A. (Ed.), Verlag für Ganzheitliche Medizin Dr. Erich Wühr GmbH. Kötzting/Bayer, Wald, Germany. Xie, H.Q., Choi, R.C.Y., Leung, K.W., Siow, N.L., Kong, L.W., Lau, F.T.C., Peng, H.B., Tsim, K.W.K., 2007. Regulation of a transcript encoding the proline-rich membrane anchor of globular muscle acetylcholinesterase: the suppressive roles of myogenesis and innervating nerves. Journal of Biological Chemistry 282, 11765–11775.

1048

W.L. Zhang et al. / Phytomedicine 19 (2012) 1042–1048

Zhao, K.J., Dong, T.T.X., Tu, P.F., Song, Z.H., Lo, C.K., Tsim, K.W.K., 2003. Molecular genetic and chemical assessment of Radix Angelica (Danggui) in China. Journal of Agricultural and Food Chemistry 51, 2576–2583. Zheng, K.Y.Z., Choi, R.C.Y., Xie, H.Q.H., Cheung, A.W.H., Guo, A.J.Y., Leung, K.W., Chen, V.P., Bi, C.W.C., Zhu, K.Y., Chan, G.K.L., Fu, Q., Lau, D.T.W., Dong, T.T.X., Zhao, K.J., Tsim, K.W.K., 2010a. The expression of erythropoietin triggered by Danggui Buxue Tang, a Chinese herbal decoction prepared from Radix Astragali and Radix Angelicae Sinensis, is mediated by the hypoxiainducible factor in cultured HEK293T cells. Journal of Ethnopharmacology 132, 259–267. Zheng, K.Y.Z., Choi, R.C.Y., Li, J., Xie, H.Q.H., Cheung, A.W.H., Duan, R., Guo, A.J.Y., Zhu, J.T.T., Chen, V.P., Bi, C.W.C., Zhu, Y., Lau, D.D.W., Dong, T.T.X., Lau, B.W.C., Tsim, K.W.K., 2010b. Ligustilide suppresses the biological properties of Danggui Buxue Tang: a Chinese herbal decoction composed of Radix Astragali and Radix Angelica Sinensis. Planta Medica 76, 439–443. Zheng, K.Y.Z., Choi, R.Y.C., Cheung, A.W.H., Guo, A.J.Y., Bi, C.W.C., Zhu, K.Y., Fu, Q., Du, Y.Q., Zhang, W.L., Zhan, J.Y.X., Duan, R., Lau, D.T.W., Dong, T.T.X., Tsim, K.W.K., 2011a. Flavonoids from Radix Astragali induce the expression of erythropoietin

in cultured cells: a signaling mediated via the accumulation of hypoxia-inducible factor-1␣. Journal of Agricultural and Food Chemistry 59, 1697–1704. Zheng, K.Y.Z., Guo, A.J.Y., Bi, C.W.C., Zhu, K.Y., Chan, G.K.L., Fu, Q., Xu, S.L., Zhan, J.Y.X., Lau, D.T.W., Dong, T.T.X., Choi, R.C.Y., Tsim, K.W.K., 2011b. The extract of Rhodiolae Crenulatae Radix et Rhizoma induces the accumulation of HIF-1␣ via blocking the degradation pathway in cultured kidney fibroblasts. Planta Medica 77, 894–899. Zhu, J.T.T., Choi, R.Y.C., Chu, G.K.Y., Cheung, A.W.H., Gao, Q.T., Li, J., Jiang, Z.Y., Dong, T.T.X., Tsim, K.W.K., 2007. Flavonoids possess neuroprotective effects on cultured pheochromocytoma PC12 cells: a comparison of different flavonoids in activating estrogenic effect and in preventing ␤-amyloid-induced cell death. Journal of Agricultural and Food Chemistry 55, 2438–2445. Zhu, K.Y., Fu, Q., Xie, H.Q., Xu, S.L., Cheung, A.W.H., Zheng, K.Y.Z., Luk, W.K.W., Choi, R.C.Y., Lau, D.T.W., Dong, T.T.X., Jiang, Z.Y., Chen, J.J., Tsim, K.W.K., 2010. Quality assessment of a formulated Chinese herbal decoction, Kaixinsan, by using rapid resolution liquid chromatography coupled with mass spectrometry: a chemical evaluation of different historical formulae. Journal of Separation Science 33, 3666–3674.