EVO Fusion

Evo Fusion - Danggui-Buxue-Tang (DBT) is a very popular Traditional Chinese Medicine prescription, which contains Angelica sinensis (Danggui) and Astragalus membranaceus (Huangqi) at a ratio of 1: 5. DBT is used widely in China for stimulating red blood cell production and enhancing cardiovascular function. They found that oral administration of DBT (3 or 6g/kg/ day for 4 weeks) could regulate blood lipid, decrease TC, TG, and LDL-C in diabetic atherosclerosis rats, which were induced by nitric oxide inhibition plus high-fat diet (Gao et al., 2007; Zhang et al., 2007).

Radix Astragali

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) (Zheng, 2005), Astragalus membranaceus (Fisch.) Bunge and Astragalus membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P.K. Hsiao (Sinclair, 1998; Hsiao, 1964) are the most commonly used RA. The morphological appearances and chemical properties of RA and its adulterants show a remarkable resemblance (Ma et al., 2000a,b; Dong et al., 2003). The DNA sequences of 5S rRNA spacer, ITS and 18S rRNA coding region were determined and compared among ten Astragalus taxa (Ma et al., 2000a,b; Dong et al., 2003). With neighbour-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 (Zhao et al., 2003).

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 contained the highest amounts of isoflavonoids, saponins and polysaccharides (Ma et al., 2000a; 2002).

Radix Angelicae Sinensis

According to the Chinese Pharmacopoeia (2005) (Zheng, 2005), 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 (Hu, 1989; Zhu, 1989; Zheng, 1997; Watanabe et al., 1998). Studies have shown that the three commonly used Angelica roots vary in their chemical composition, pharmacological properties and efficacy (Zhao et al., 2003; Hu, 1989). 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 (Zhao et al., 2003). 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 (Zhao et al., 2003; Wagner et al., 2001; Lao et al., 2004). The levels of ferulic acid and Z-ligustilide are often used as chemical markers for the quality control of Angelica roots (Wagner et al., 2001) . 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) (Zhao et al., 2003; Lao et al., 2004). 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 (Zhao et al., 2003). 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.

Functions of Danggui Buxue Tang, a Chinese Herbal Decoction Containing Astragali Radix and Angelicae Sinensis Radix, in Uterus and Liver are Both Oestrogen Receptor-Dependent and -Independent

Zierau O, et al. Evidence-Based Complementary and Alternative Medicine. Volume 2014 (2014), http://dx.doi.org/10.1155/2014/438531

Danggui Buxue Tang (DBT), an herbal decoction containing Astragali Radix (AR) and Angelicae Sinensis Radix (ASR), has been used in treating menopausal irregularity in women for more than 800 years in China.

Pharmacological results showed that DBT exhibited significant estrogenic properties in vitro, which therefore suggested that DBT could activate the nuclear oestrogen receptors. Here, we assessed the estrogenic properties of DBT in an ovariectomized in vivo rat model: DBT was applied to the ovariectomized rats for 3 days. The application of DBT did not alter the weight of uterus and liver, as well as the transcript expression of the proliferation markers including the oestrogen receptors α and β. However, DBT stimulated the transcript expression of the oestrogen responsive genes. In addition, the inductive role of DBT on the expression of members of the aryl hydrocarbon receptor family in uterus and liver of ovariectomized rats was confirmed. These responses of DBT however were clearly distinct from the response pattern detectable here for 17β-oestradiol.

Therefore, DBT exhibited weak, but significant, estrogenic properties in vivo; however, some of its activities were independent of the oestrogen receptor. Thus, DBT could be an exciting Chinese herbal decoction for an alternative treatment of hormone REPLACE ment therapy for women in menopause without subsequent estrogenic side effects.

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 hypoxia-inducible factor in cultured HEK293T cells

Zheng KYZ, Choi RCY, Xie HQH, et al. Journal of Ethnopharmacology. Vol 132, Issue 1, 28 October 2010, Pages 259–267 



Danggui Buxue Tang (DBT), a Chinese medicinal decoction that is being commonly used as hema"top"oietic medicine to treating woman menopausal irregularity, contains two herbs: Radix Astragali and Radix Angelicae Sinensis. Pharmacological results indicate that DBT can stimulate the production of erythropoietin (EPO), a specific hema"top"oietic growth factor, in cultured cells. It has been shown that the HIF pathway plays an essential role in directing DBT-induced EPO expression in kidney. These results provide one of the molecular mechanisms of this ancient herbal decoction for its hema"top"oietic function.

Aim of the study
In order to revealing the mechanism of DBT's hema"top"oietic function, this study investigated the activity of the DBT-induced EPO expression and the upstream regulatory cascade of EPO via hypoxia-induced signaling in cultured kidney fibroblasts (HEK293T).

Materials and methods: 
DBT-induced mRNA expressions were revealed by real-time PCR, while the change of protein expressions were analysed by Western blotting. For the analysis of hypoxia-dependent signaling, a luciferase reporter was used to report the transcriptional activity of hypoxia response element (HRE).

Results
: The plasmid containing HRE, being transfected into HEK293T, was highly responsive to the challenge of DBT application. To account for the transcriptional activation of HRE, DBT treatment was shown to increase the mRNA and protein expressions of hypoxia-inducible factor-1α (HIF-1α). In addition, the activation of Raf/MEK/ERK signaling pathway by DBT could also enhance the translation of HIF-1α, suggesting the dual actions of DBT in stimulating the EPO expression in kidney cells.

Conclusion
Our study indicates that HIF pathway plays an essential role in directing DBT-induced EPO expression in kidney. These results provide one of the molecular mechanisms of this ancient herbal decoction for its hema"top"oietic function.

Antifibrotic effect of the Chinese herbs, Astragalus mongholicus and Angelica sinensis, in a rat model of chronic puromycin aminonucleoside nephrosis

Hy, Li Jz, Yu L, Zhao Y, Ding W. Angelica sinensis, in a rat model of chronic puromycin aminonucleoside nephrosis” [Life Sciences 74 ISSUE 23, (2004) Pp.1645–58

Nephrotic syndrome has long been treated in China with two herbs, Astragalus mongholicus and Angelica sinensis, which may have antifibrotic effects. Methods: Rats with chronic puromycin-induced nephrosis were treated with Astragalus and Angelica 3 mL/d (n=7) or enalapril 10 mg/kg/d (n=7). Normal control rats (n=7) received saline rather than puromycin, and an untreated control group (n=7) received puromycin but no treatment. After 12 weeks, stained sections of the glomerulus and tubulointerstitium were evaluated for injury. Immunohistochemistry staining measured extracellular matrix components, transforming growth factor-β1 (TGFβ1), osteopontin, ED-1-positive cells, and α-actin. TGFâ1 mRNA was assessed by in situ hybridization. Renin, ACE activity, angiotensin, and aldosterone were measured by radioimmunoassay or colorimetry. In the untreated rats, chronic renal injury progressed to marked fibrosis at 12 weeks. Astragalus and Angelica significantly reduced deterioration of renal function and histologic damage. Expressions of type III and IV collagen, fibronectin, and laminin also decreased significantly. This anti-fibrotic effect was similar to that of enalapril. The herbs had no effect on the renin-angiotensin system but did reduce the number of ED-1-positive, and α-actin positive cells and expression of osteopontin compared to untreated controls.

The combination of Astragalus and Angelica has also been shown to slow the progression of renal fibrosis and deterioration of renal function with comparable effects of enalapril. These effects were not caused by blocking the intrarenal renin-angiotensin system, but associated with suppression of the overexpression of TGFâ1 and osteopontin, reduction of infiltrating macrophages, and lower activation of renal intrinsic cells.

Effects of Danggui Buxue Decoction on lipid peroxidation and MMP-2/9 activities of fibrotic liver in rats

Yuan Chen, Qian Chen, Jing Lu, Feng-hua Li, Yan-yan Tao and Cheng-hai Liu. Chinese Journal of Integrative Medicine. Volume 15, Number 6, December, 2009. DOI:10.1007/s11655-009-0435-y


Objective: to explore the mechanism of Danggui Buxue Decoction on the liver fibrosis related to hepatic lipid peroxidation and matrix metalloproteinases (MMP) −2/9 activities.

Methods: The liver fibrosis in 28 rats was induced by an injection of carbon tetrachloride (CCl4) and fed with high lipid and low protein diet for 6 weeks, the model rats were randomly divided into the model group and EVO Fusion (DBD) treated group, 14 in each group, and another 10 rats as the normal group were observed as well. Rats in the EVO Fusion (DBD) group were administered with EVO Fusion (DBD) at the dose of 6g/kg body weight for 6 weeks since CCl4 intoxication. The hepatic inflammation and fibrosis were examined with HE and Sirius red stain. The liver function including serum alanine aminotransamine (ALT), aspartate transamine (AST), albumin (Alb) and total bilirubin (TBIL), liver triglyceride (TG) and malondialdehyde (MDA) contents, superoxide dismutase (SOD) activity were assayed. Hepatic hydroxyproline (Hyp) content was detected with Jamall's method. The α-SMA expression was analysed by immunohistochemistry and the Western blot. Liver MMP-2 mRNA was analysed with Real-time PCR, and MMP-2/9 activities were measured with gelatine zymography and in situ zymography.

Results: Compared with the normal group, the levels of ALT, AST and TBIL, the content of Hyp, TG and MDA were remarkably increased, the Alb content and SOD activity were significantly decreased in the model group (P<0.05), and higher levels of MMP-2 mRNA and MMP-2/9 activities (P<0.01), the hepatic fatty degeneration and collagen accumulation and fibrosis at liver were observed. Compared with the model control, EVO Fusion (DBD) group showed slighter hepatic fatty degeneration and collagen deposition, and had lower levels of ALT, AST and TBIL activities, lower contents of MDA, TG and Hyp, but higher SOD level and Alb content (P<0.05), and EVO Fusion (DBD) also down-regulated MMP-2 mRNA expression and decreased MMP-2/9 activities in the fibrotic livers (P<0.01).

Conclusion: The action of Panaxea’s EVO Fusion (Danggui Buxue Decoction) against liver fibrosis is related to the prevention of lipid peroxidation and inhibition of MMP-2/9 activities in the fibrotic livers.

Verification of the formulation and efficacy of DBT (a decoction of Radix Astragali and Radix Angelicae Sinensis): an exemplifying systematic approach to revealing the complexity of Chinese herbal medicine formulae. 


Gao Q-t, Li J, Cheung KH, Duan J-n, Ding A-n, Cheung AWH, Zhao K-j, Li WZm, Dong TTx & Keung KW. Chinese Medicine. 2007; 2: 12. 2007 November 29. doi: 10.1186/1749-8546-2-12.

This article exemplifies a systematic approach to revealing the complexity of Chinese herbal medicine formulae through three levels of scientific research: standardization of herbs, verification of ancient formulae and mechanism studies. We used DBT as an example for this approach. Among thousands of traditional Chinese medicine herbal formulae, almost all of which consist of multiple herbs, DBT is one of the simplest. Containing only two herbs, namely Radix Astragali (RA) and Radix Angelicae Sinensis (RAS), DBT is traditionally used to treat ailments in women. The weight ratio of RA to RAS in DBT was prescribed to be 5:1 as early as in 1247 AD. In addition to advanced chemical analysis of herbal constituents, DNA genotyping techniques have been developed for reliable standardization of RA and RAS. Chemical evaluation shows that main active constituents in DBT, including astragaloside IV, calycosin, formononetin and ferulic acid, were most abundant after extraction at the RA to RAS ratio of 5:1, whereas other tested RA to RAS ratios only gave sub-optimal levels of the active constituents. Biological evaluation indicates that bioactivities of DBT, e.g. immuno-modulatory, osteotrophic and oestrogenic effects are also best exerted at the RA to RAS ratio of 5:1. Correlation analysis demonstrates statistically significant relationship between the tested chemical constituents and tested bioactivities. Up- and down-regulation of expression of some genes as potential biomarkers has been detected by using gene chip technology.

Standardization as the basis of modernization and internationalization of TCM is the key to ensure the safety and efficacy of TCM products. At present, lack of standardization in TCM products impedes the development of TCM. For instance, it is common that different herbs have the same name or a single herb has different names in the market. Some herbs cultivated in different regions or harvested in different seasons may vary considerably in their chemical and biological properties. Most of the TCM products do not have specific biomarkers. TCM is traditionally administered in the form of a decoction with a combination of different herbs. The complexity of biological effects of the interactions among different compounds within a decoction complicates experimental studies to reveal the action mechanisms.

Standardization of Radix Astragali and Radix Angelicae Sinensis

A reliably reproducible chemical composition of EVO Fusion is a prerequisite in delineating the biological effects of this Chinese medicine preparation. The quality of RA and RAS may be considerably influenced by weather, geographic location, soil conditions, and the methods of cultivation and processing. Some Chinese medicinal materials with excellent quality are only produced in certain regions of China, which are often referred to as ' the best growth region or 'Didao' . Therefore, how to authenticate and choose the best RA and RAS plays a critical role in ensuring the quality of EVO Fusion.

Biological evaluation

According to TCM theories, DBT replenishes qi and nourishes xue (the blood). DBT is therefore used for treating menopausal symptoms (Tsim, 2005). Due to a deficiency of ovarian hormones, especially oestrogen, 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 (Harlow, Signorello,. 2000). Apart from a lack of oestrogen, the immune system is also involved in the menopausal symptoms. Steroid hormones may modulate the immune response (Manyonda et al,. 1992) and immune reactions may also regulate the ovarian function (Bukovsky, Presl,. 1981). Various bioactivities related to menopausal symptoms, such as osteotrophic 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. For bone cell differentiation, the 5:1 ratio DBT preparation induced ALP activity to the highest level among all ratios and showed the strongest osteotrophic effect (Dong et al,. 2006).

The oestrogenic effects of DBT were tested by a cellular reporter system of transcriptional activation of oestrogen receptor/promoter. A promoter/reporter construct (pERE-Luc) corresponding to the responsive elements of oestrogen 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 oestrogen-driven promoter activity was markedly induced by DBT, the 5:1 ratio DBT showed the strongest effect in inducing the promoter activity than RA, RAS alone or the extracts of other ratios (Dong et al,. 2006).

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 (Dong et al,. 2006).

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 (Gao et al,. 2006).

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 (Wu et al,. 1989; Li et al,. 1992).

Findings

Model

Treatment

Reference

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 [24,25].

Cultured human MG-63 osteosarcoma cells

DBT of various ratios of RA and RAS, compared with β-oestradiol and negative control

Dong et al,. 2006

The 5:1 ratio DBT showed the strongest effect in inducing the oestrogen-driven promoter activity than RA, RAS alone or the extracts of other ratios.

Cultured MCF-7 cells

DBT of various ratios of RA and RAS, compared with β-oestradiol and negative control

Dong et al,. 2006

The ratios 5:1 and 7:1 DBT showed higher levels of activity in preventing platelet aggregation.

ADP induced-platelet aggregation in blood from adult New Zealand white rabbits

DBT of various ratios of RA and RAS, compared with ticlopidine and negative control

Dong et al,. 2006

DBT induced cell proliferation, interleukin-2 secretion and the phosphorylation of extracellular signal-regulated kinase (ERK1/2) in cultured T-lymphocytes. The 5:1 ratio DBT showed the strongest immuno-modulatory effects.

Cultured T-lymphocytes and macrophages

DBT of various ratios of RA and RAS, compared with PHA, PMA, Zymosan A and negative control

Gao et al,. 2006

The pharmacological studies in animals also suggest that DBT has the ability to promote haema"top"oiesis, to stimulate blood circulation, to prevent osteoporosis and to counter oxidative stress (Dong et al,. 2006; Wu et al,. 1999; Ning et al,. 2002). 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 (Mak et al,. 2006). In rats, DBT protected against myocardial ischemia-reperfusion injury in a dose-dependent manner (Mak et al,. 2006). 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 hema"top"oietic 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 (Zhang et al,. 1999a,b).

Findings

Model

Treatment

Reference

DBT had significantly higher RBC and Hb levels in both normal and anaemic mice than those in RA, RAS and control.

Kunming mice, male, RBC, Hb

Normal mice in 4 groups: RA, RAS, DBT and control; Anaemic mice in 4 groups: RA, RAS, DBT and control

(Wu et al,. 1989)

DBT was the most effective decoction in triggering immune responses.

Kunming mice, RBC, Hb, WBC, Plt, reticulocyte, nucleated cells of bone cavity, weight of pancreas and thymus

Mice in 5 groups: RA, RAS, DBT, RA+RAS (1:1) and control

(Li et al,. 1992)

DBT alleviated cardiac injury in ischemia reperfusion.

Wister rats (male), amplitudes of LVSP and ± dp/dtmax, arterial pressure, Na+-K+-ATP activity, level of MDA production, cAMP content

Rats in myocardial ischemia reperfusion injury; i.v.

(Wu et al,. 1999)

DBT increased the levels of RBC, WBC, and BMNC. Some DBT promoted the proliferation of BMNC and increased the level of CFU-Mix.

Kunming mice, ICR mice, Balb/c mice, RBC, WBC, reticulocytes and BMNC

Mice in 4 groups: normal, model, DBT without polysaccharides, DBT with polysaccharides

(Ning et al,. 2002)

DBT enhanced myocardial mitochondria and red blood cell glutathione status.

Rats, myocardial mitochondrial status, RBC glutathione status

Rats in 5 groups: RA, RAS, DBT, RA + RAS (not boiled together) and control; orally administered

(Mak et al,. 2006)

DBT inhibited growth of GM-CFU, while the decoction-containing serum promoted growth of GM-CFU.

Kunming mice, GM-CFU

DBT was administered orally; serum collected from abdominal aorta was added to an in vitro cultivating system of mouse hema"top"oietic progenitor cells.

Zhang et al,. 1999

The decoction-containing serum showed promoting actions to CFU-E. RA+RAS (5:1) was 97.81% stronger than RA+RAS (1:1).

Kunming mice, CFU-E

DBT was administered orally; serum collected from abdominal aorta was added to an in vitro cultivating system of mouse hema"top"oietic progenitor cells.

Zhang et al,. 1999

Specific oestrogenic and immuno-modulatory effects of DBT

The estrogenic effects of DBT were investigated by determining the levels of phosphorylation of oestrogen receptor α (ERα) and extracellular signal-regulated kinase 1/2 (ERK1/2) in cultured MCF-7 cells. In contrast to oestrogen, DBT triggered the phosphorylation of ERα and ERK1/2 at both S118 and S167 in a time-dependent manner. Although the activity of the oestrogen-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 EVO Fusion-treated cultures. Interestingly, the specific estrogenic effects of DBT were not only shown in the MCF-7 cells (Gao et al,. 2007).

In cultured T-lymphocytes, the phosphorylation of the ERK 1 (about 42 kDa) and ERK 2 (about 44 kDa) was increased by DBT (Zhang et al,. 1999b). 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 (Zhang et al,. 1999b). Moreover, a simple mixture of extracts of RA and RAS could not activate the phosphorylation of ERK in T-lymphocytes. This result suggests that boiling RA and RAS together is essential for DBT to exert oestrogenic effects.

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