The extract of Ziziphus jujuba fruit (jujube) induces expression of erythropoietin via hypoxia-inducible factor-1α in cultured Hep3B cells.

The fruit of Ziziphus jujuba Mill., known as jujube or Chinese date, is commonly consumed as health supplement or herbal medicine worldwide. To study the beneficial role of jujube in enhancing hematopoietic function, we investigated its roles on the expression of erythropoietin in cultured Hep3B human hepatocellular carcinoma cells. Application of chemically standardized jujube water extract stimulated erythropoietin expression in a dose-dependent manner, with the highest response by ~ 100 % of increase. A plasmid containing hypoxia response element, a critical regulator for erythropoietin transcription, was transfected into Hep3B cells. Application of jujube water extract onto the transfected cells induced the transcriptional activity of the hypoxia response element. To account for its transcriptional activation, the expression of hypoxia-inducible factor-1α was increased after treatment with jujube water extract: the increase was in both mRNA and protein levels. These results confirmed the hematopoietic function of jujube in the regulation of erythropoietin expression in liver cells.

Quality Control of Danggui Buxue Tang, a Traditional Chinese Medicine Decoction, by (1)H-NMR Metabolic Profiling.

Danggui Buxue Tang (DBT) is one of the simplest traditional Chinese medicine (TCM) decoctions, first described in China in 1247 AD. DBT is composed of 2 herbs, Astragali Radix (AR) and Angelica Sinensis Radix (ASR), boiled together in a 5 : 1 ratio. Clinically, DBT is prescribed to women as a remedy for menopausal symptoms. Here, H-NMR metabolic profiling was conducted for DBT and the water extracts of AR or ASR, to evaluate the potential of this chemical profiling method for quality control of the herbal decoction. Principal component analysis (PCA) showed that DBT could be readily distinguished from the water extracts of its constituent herbs by the metabolic profiles. More interestingly, the metabolic profile of DBT was not a simple sum of that of AR and ASR. Asparagine was found at significantly higher concentration in DBT than that in either AR or ASR extract, contributing mainly to the discrimination of DBT sample. In addition, we employed the same method to profile a commercial DBT powder, verifying its authenticity as compared to our prepared DBT. This study is the first to employ H-NMR metabolic profiling for the quality control of traditional Chinese medicine decoctions.

The standardized extract of Ziziphus jujuba fruit (jujube) regulates pro-inflammatory cytokine expression in cultured murine macrophages: suppression of lipopolysaccharide-stimulated NF-κB activity.

The fruit of Ziziphus jujuba Mill., known as jujube or Chinese date, is commonly consumed as a health supplement or herbal medicine worldwide. To study the beneficial role of jujube in regulating immune response, we investigated its roles on the expressions of pro-inflammatory cytokines in cultured macrophages. Application of chemically standardized jujube water extract for 24 h stimulated the transcriptional expression of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in cultured RAW 264.7 macrophages. In contrast, the pretreatment with jujube water extract suppressed the expression of IL-1ß and IL-6, but not for TNF-α in lipopolysaccharide (LPS)-stimulated macrophages. The IL-1ß and IL-6 cytokines in LPS-induced macrophages were suppressed by jujube water extract in both mRNA and protein levels. In parallel, the inhibition of jujube water extract on the transcriptional activity of nuclear factor-kappa B was revealed in LPS-induced macrophages. These results verified the bidirectional immune-modulatory roles of jujube by regulating the expressions of pro-inflammatory cytokines in macrophages.

Metabonomic analysis of water extracts from different angelica roots by ¹H-nuclear magnetic resonance spectroscopy.

Angelica Radix, the roots of the genus Angelica, has been used for more than 2,000 years as a traditional medicine in Eastern Asia. The Chinese Pharmacopoeia records more than 100 herbal formulae containing Angelica roots. There are two common sources of Angelica roots, Angelica sinensis from China and A. gigas from Korea. The two species of Angelica roots differ in their chemical compositions, pharmacological properties and clinical efficacy. ¹H-NMR metabolic profiling has recently emerged as a promising quality control method for food and herbal chemistry. We explored the use of ¹H-NMR metabolic profiling for the quality control of Angelica Radix. Unlike previous work, we performed the metabolic profiling on hot water extracts, so as to mimic the clinically relevant preparation method. Unsupervised principle component analyses of both the full spectral profile and a selection of targeted molecules revealed a clear differentiation of three types of Angelica roots. In addition, the levels of 13 common metabolites were measured. Statistically significant differences in the levels of glucose, fructose and threonine were found between different sources of Angelica. Ferulic acid, a marker commonly used to evaluate Angelica root, was detected in our samples, but the difference in ferulic acid levels between the samples was not statistically significant. Overall, we successfully applied ¹H-NMR metabolic profiling with water extraction to discriminate all three sources of Angelica roots, and obtained quantitative information of many common metabolites.

Ferulic acid enhances the chemical and biological properties of astragali radix: a stimulator for danggui buxue tang, an ancient Chinese herbal decoction.

Danggui buxue tang, an ancient formula composed of astragali radix and Angelicae sinensis radix, has been used for treating menopausal irregularity in women for more than 800 years in China. In danggui buxue tang, the complete functions of astragali radix require the assistance of Angelicae sinensis radix, and both herbs have to work harmoniously in order to achieve the maximal therapeutic purposes. In order to analyze the relationship of the two herbs, the role of ferulic acid, a major chemical within Angelicae sinensis radix, in chemical and biological properties of astragali radix was determined. Using ferulic acid in the extraction of astragali radix, the amounts of astragaloside IV, calycosin, and formononetin were increased in the final extract; however, the astragali radix polysaccharide showed a minor increase. The chemical-enriched astragali radix extract showed robust induction in osteogenic and estrogenic activities in cultured osteosarcoma MG-63 and breast MCF-7 cells. However, ferulic acid itself did not show such biological responses. The current results strongly suggest that Angelicae sinensis radix-derived ferulic acid is a positive regulator for danggui buxue tang, which enhanced the solubilities of active ingredients derived from astragali radix, and which therefore increased the biological efficacies of danggui buxue tang.

A standardized extract of the fruit of Ziziphus jujuba (Jujube) induces neuronal differentiation of cultured PC12 cells: a signaling mediated by protein kinase A.

The fruit of Ziziphus jujuba Mill., known as Chinese date or jujube, is consumed as a health supplement worldwide. To study the role of jujube in brain benefits, its effects on neuronal differentiation of PC12 cells were studied. Application of jujube water extract induced neurite outgrowth of PC12 cells, >25% of which were differentiated; this effect was similar to that of nerve growth factor. In parallel, the expressions of neurofilaments (NFs) in jujube-treated cultures showed a dose-dependent increase, with the highest inductions by ∼150% for NF68 and NF160 and by ∼100% for NF200. Application of H89, a protein kinase A inhibitor, attenuated jujube-induced neurite outgrowth of the cultures. Besides, using jujube extract induced the phosphorylation of cAMP responsive element binding protein on PC12 cells, which was blocked by H89. These results support the use of jujube as a food supplement for the prevention of neurodegenerative diseases in which neurotrophin deficiency is involved.

Chemical and biological assessment of Ziziphus jujuba fruits from China: different geographical sources and developmental stages.

Chinese date, the fruit of Ziziphus jujuba Mill., has thousands of years cultivation history, and about 700 cultivars of dates in China. Two types of dates are commonly found in the market: (i) fresh immature dates consumed as fruits, and (ii) dried mature dates used as Chinese medicines. Here, chemical and biological properties of these dates were revealed. Different sources of dates showed similar chemical profiles; however, the amounts of identified chemicals showed a great variation. The amount of nucleotides, flavonoids and polysaccharides in dates could be affected by its maturity and drying process. In parallel, the antioxidative functions of their extracts were compared. The date extracts protected PC12 cells against tBHP-induced cytotoxicity, and which also stimulated the transcriptional activity of antioxidant response element. The antioxidative effects were varied among different dates. The current results suggested the optimization of sources and specific usage of different maturity dates.

Optimizing combinations of flavonoids deriving from astragali radix in activating the regulatory element of erythropoietin by a feedback system control scheme.

Identifying potent drug combination from a herbal mixture is usually quite challenging, due to a large number of possible trials. Using an engineering approach of the feedback system control (FSC) scheme, we identified the potential best combinations of four flavonoids, including formononetin, ononin, calycosin, and calycosin-7-O- ß -D-glucoside deriving from Astragali Radix (AR; Huangqi), which provided the best biological action at minimal doses. Out of more than one thousand possible combinations, only tens of trials were required to optimize the flavonoid combinations that stimulated a maximal transcriptional activity of hypoxia response element (HRE), a critical regulator for erythropoietin (EPO) transcription, in cultured human embryonic kidney fibroblast (HEK293T). By using FSC scheme, 90% of the work and time can be saved, and the optimized flavonoid combinations increased the HRE mediated transcriptional activity by ~3-fold as compared with individual flavonoid, while the amount of flavonoids was reduced by ~10-fold. Our study suggests that the optimized combination of flavonoids may have strong effect in activating the regulatory element of erythropoietin at very low dosage, which may be used as new source of natural hematopoietic agent. The present work also indicates that the FSC scheme is able to serve as an efficient and model-free approach to optimize the drug combination of different ingredients within a herbal decoction.

Chemical and biological assessment of angelica roots from different cultivated regions in a chinese herbal decoction danggui buxue tang.

Roots of Angelica sinensis (Danggui) have been used in promoting blood circulation as herbal medicine for over 2000 years in China. Another species of Angelica roots called A. gigas is being used in Korea. To reveal the efficiency of different Angelica roots, the chemical and biological properties of Angelica roots from different cultivated regions were compared. Roots of A. sinensis contained higher levels of ferulic acid, Z-ligustilide, and senkyunolide A, while high amounts of butylphthalide and Z-butylenephthalide were found in A. gigas roots. The extracts deriving from A. gigas roots showed better effects in osteogenic and estrogenic properties than that of A. sinensis from China. However, this difference was markedly reduced when the Angelica roots were being prepared in a Chinese herbal decoction together with Astragali Radix as Danggui Buxue Tang. In contrast, the herbal decoction prepared from A. sinensis roots showed better responses in cell cultures. In addition, the extracts of A. gigas roots showed strong cell toxicity both as single herb and as Danggui Buxue Tang. This result revealed the distinct properties of Angelica roots from China and Korea suggesting the specific usage of herb in preparing a unique herbal decoction.

Importance of wine-treated Angelica Sinensis Radix in Si Wu Tang, a traditional herbal formula for treating women's ailments.

Si Wu Tang (Four Agents Decoction), a traditional Chinese decoction composed of Angelica Sinensis Radix, Chuanxiong Rhizoma, Paeoniae Radix Alba, and Rehmanniae Radix Praeparata in a ratio of 1 : 1 : 1 : 1, has been used to treat women's diseases for more than a thousand years. According to the original description of Si Wu Tang, Angelica Sinensis Radix should be treated with wine. However, the importance of this wine-treated Angelica Sinensis Radix in Si Wu Tang's function has not been identified. In this article, the chemical and biological properties of two decoctions processed in different ways (Si Wu Tang with crude Angelica Sinensis Radix and Si Wu Tang with wine-treated Angelica Sinensis Radix) were compared for examination. The herbal decoction Si Wu Tang prepared from wine-treated Angelica Sinensis Radix contained much different amounts of its active compounds. Compared with Si Wu Tang using crude Angelica Sinensis Radix, Si Wu Tang prepared from wine-treated Angelica Sinensis Radix had better biological responses. Therefore, these findings accentuate the functional importance of herbs treated with wine in the Chinese decoction.

Chemical fingerprinting and quantitative analysis of two common Gleditsia sinensis fruits using HPLC-DAD.

Gleditsiae Fructus Abnormalis and Gleditsiae Sinensis Fructus are obtained from different developmental stages of fruits from Gleditsia sinensis Lam. (Leguminosae). The possible interchangeable usage of the two fruits, however, has long been very controversial. Here, high performance liquid chromatography coupled with diode array detection was developed to explore their chemical fingerprinting profiles. Besides, the amounts of aglycones of saponin compounds, echinocystic acid and oleanolic acid in both fruits were quantified. The results indicated that there was no significant difference in the content of aglycones from the two types of fruits. However, their chromatographic fingerprints showed distinct characteristics. Therefore, the interchangeable application of these fruits has to be taken with a specific precaution.

Fo shou san, an ancient herbal decoction prepared from angelicae sinensis radix and chuanxiong rhizoma, induces erythropoietin expression: a signaling mediated by the reduced degradation of hypoxia-inducible factor in cultured liver cells.

Fo Shou San (FSS) is an ancient herbal decoction composed of Angelicae Sinensis Radix (ASR; Danggui) and Chuanxiong Rhizoma (CR; Chuanxiong) in a ratio of 3:2. FSS is mainly prescribed for patients having a deficiency of blood supply, and it indeed has been shown to stimulate the production of erythropoietin (EPO) in cultured cells. In order to reveal the mechanism of this FSS-induced EPO gene expression, the upstream regulatory cascade, via hypoxia-induced signaling, was revealed here in cultured hepatocellular carcinoma cell line Hep3B. The induction of EPO gene expression, triggered by FSS, was revealed in cultured hepatocytes by: (i) the increase of EPO mRNA; and (ii) the activation of the hypoxia response element (HRE), an upstream regulator of the EPO gene. The FSS-induced EPO gene expression was triggered by an increased expression of hypoxia-inducible factor-1 α (HIF-1 α) protein; however, the mRNA expression of HIF-1 α was not altered by the treatment of FSS. The increased HIF-1 α was a result of reduced protein degradation after the FSS treatment. The current results therefore provide one of the molecular mechanisms of this ancient herbal decoction for its hematopoietic function.

Chemical and biological assessment of Angelicae Sinensis Radix after processing with wine: an orthogonal array design to reveal the optimized conditions.

The roots of Angelica sinensis [Angelica Sinensis Radix (ASR)] have been used as a common health food supplement for women's care for thousands of years in China. According to Asian tradition, ASR could be processed with the treatment of wine, which subsequently promoted the biological functions of ASR. By chemical and biological assessments, an orthogonal array design was employed here to determine the roles of three variable parameters in the processing of ASR, including oven temperature, baking time, and flipping frequency. The results suggested that oven temperature and baking time were two significant factors, while flipping frequency was a subordinate factor. The optimized condition of processing with wine therefore was considered to be heating in an oven at 80 °C for 90 min with flipping twice per hour. Under the optimized processing conditions, the solubilities of ferulic acid and Z-ligustilide from ASR were markedly increased and decreased, respectively. In parallel, the biological functions of processed ASR were enhanced in both anti-platelet aggregation and estrogenic activation; these increased functions could be a result of the altered levels of ferulic acid and Z-ligustilide in wine-processed ASR. Thus, the chemical and biological assessment of the processed ASR was in full accordance with the Chinese old tradition.

Flavonoids from Radix Astragali induce the expression of erythropoietin in cultured cells: a signaling mediated via the accumulation of hypoxia-inducible factor-1α.

Radix Astragali (RA) is commonly used as a health food supplement to reinforce the body vital energy. Flavonoids, including formononetin, ononin, calycosin, and calycosin-7-O-ß-d-glucoside, are considered to be the major active ingredients within RA. Here, we provided different lines of evidence that the RA flavonoids stimulated the expression of erythropoietin (EPO), the central regulator of red blood cell mass, in cultured human embryonic kidney fibroblasts (HEK293T). A plasmid containing hypoxia response element (HRE), a critical regulator for EPO transcription, was tagged upstream of a firefly luciferase gene, namely, pHRE-Luc, which was being transfected into fibroblasts. The application of RA flavonoids onto the transfected cells induced the transcriptional activity of HRE. To account for the transcriptional activation after the treatment of flavonoids, the expression of hypoxia-inducible factor-1α (HIF-1α) was markedly increased: The increase was in both mRNA and protein levels. In addition, the degradation of HIF-1α was reduced under the effect of flavonoids. The regulation of HIF-1α therefore could account for the activation of EPO expression mediated by the RA flavonoids. The current results therefore reveal the function of this herb in enhancing hematopoietic functions.