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

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 ASR-derived 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.     

Danggui Buxue Tang,a Chinese herbal decoction containing Astragali Radix and Angelicae Sinensis Radix,improves mitochrondial bioenergetics in osteoblast

Osteoporosis is the process of bone loss, particular after menopause, when the production of estrogen in women is decreaing. Bioenergetic function is one of the critical roles in bone remodeling. Danggui Buxue Tang (DBT) is an herbal mixture containing Astragali Radix (AR) and Angelicae Sinensis Radix (ASR), and which is consumed for “Qi-invigorating”, i.e., stimulating energy metabolism, as a traditional Chinese medicine (TCM). However, the role of DBT in metabolism of osteoblast has not been examined. Here, we employed a metabolic flux to examine the mitochondrial functions of cultured osteoblast in the presence of herbal extracts, including DBT, ASR, AR, AR + ASR (single mixing of two herbal extracts), as well as DBT_∆cal (a DBT extract depeleting calycosin), to examine their roles in osteoblastic metabolism, e.g. glycolysis and energy kinetics. By revealing the rates of oxygen consumption and extracellular acidification of mitochrondia, the DBT-treated osteoblasts were markedly strengthened with increases of maximal respiration, spare capacity, glycolysis capacity and glycolysis reserve, in comparing to other herbal extracts. In addition, the bioenergetic metabolism was modulated by DBT via the signaling of cellular Ca²⁺ and reactive oxgen species (ROS). Furthermore, DBT affected the morphology of mitochondria, as well as mitochondrial dynamic. Here, we propose that DBT can be regarded as benefit herbal extract in improving osteoblastic metabolism for bone disorders via central energy metabolism and mitochondrial bioenergetics.     

Drosophila DBT Autophosphorylation of Its C-Terminal Domain Antagonized by SPAG and Involved in UV-Induced Apoptosis

Drosophila DBT and vertebrate CKIε/δ phosphorylate the period protein (PER) to produce circadian rhythms. While the C termini of these orthologs are not conserved in amino acid sequence, they inhibit activity and become autophosphorylated in the fly and vertebrate kinases. Here, sites of C-terminal autophosphorylation were identified by mass spectrometry and analysis of DBT truncations. Mutation of 6 serines and threonines in the C terminus (DBT^C/ala) prevented autophosphorylation-dependent DBT turnover and electrophoretic mobility shifts in S2 cells. Unlike the effect of autophosphorylation on CKIδ, DBT autophosphorylation in S2 cells did not reduce its in vitro activity. Moreover, overexpression of DBT^C/ala did not affect circadian behavior differently from wild-type DBT (DBT^WT), and neither exhibited daily electrophoretic mobility shifts, suggesting that DBT autophosphorylation is not required for clock function. While DBT^WT protected S2 cells and larvae from UV-induced apoptosis and was phosphorylated and degraded by the proteasome, DBT^C/ala did not protect and was not degraded. Finally, we show that the HSP-90 cochaperone spaghetti protein (SPAG) antagonizes DBT autophosphorylation in S2 cells. These results suggest that DBT autophosphorylation regulates cell death and suggest a potential mechanism by which the circadian clock might affect apoptosis.     

Enhanced Bone Tissue Regeneration by Porous Gelatin Composites Loaded with the Chinese Herbal Decoction Danggui Buxue Tang

Danggui Buxue Tang (DBT) is a traditional Chinese herbal decoction containing Radix Astragali and Radix Angelicae sinensis. Pharmacological results indicate that DBT can stimulate bone cell proliferation and differentiation. The aim of the study was to investigate the efficacy of adding DBT to bone substitutes on bone regeneration following bone injury. DBT was incorporated into porous composites (GGT) made from genipin-crosslinked gelatin and β-triclacium phosphates as bone substitutes (GGTDBT). The biological response of mouse calvarial bone to these composites was evaluated by in vivo imaging systems (IVIS), micro-computed tomography (micro-CT), and histology analysis. IVIS images revealed a stronger fluorescent signal in GGTDBT-treated defect than in GGT-treated defect at 8 weeks after implantation. Micro-CT analysis demonstrated that the level of repair from week 4 to 8 increased from 42.1% to 71.2% at the sites treated with GGTDBT, while that increased from 33.2% to 54.1% at GGT-treated sites. These findings suggest that the GGTDBT stimulates the innate regenerative capacity of bone, supporting their use in bone tissue regeneration.     

A Chinese herbal decoction, Danggui Buxue Tang, activates extracellular signal-regulated kinase in cultured T-lymphocytes

Danggui Buxue Tang (DBT) is prepared from Radix Astragali and Radix Angelicae Sinensis. This Chinese herbal decoction has been shown to stimulate the proliferation of T-lymphocytes; however, the action mechanism of this stimulation has not been revealed. In cultured T-lymphocytes, application of DBT markedly induced the cell proliferation, the release of interleukin-2, -6 and -10, as well as the phosphorylation of extracellular signal-regulated kinases (ERK). The pre-treatment of ERK inhibitor blocked the DBT-induced immune responses. In addition, the polysaccharide-enriched fraction of DBT showed marked responses on the cultured T-lymphocytes suggesting the important role of DBT polysaccharide in triggering such immune responses.     

Drosophila DBT lacking protein kinase activity produces long-period and arrhythmic circadian behavioral and molecular rhythms

A mutation (K38R) which specifically eliminates kinase activity was created in the Drosophila melanogaster ckI gene (doubletime [dbt]). In vitro, DBT protein carrying the K38R mutation (DBT^K/R) interacted with Period protein (PER) but lacked kinase activity. In cell culture and in flies, DBT^K/R antagonized the phosphorylation and degradation of PER, and it damped the oscillation of PER in vivo. Overexpression of short-period, long-period, or wild-type DBT in flies produced the same circadian periods produced by the corresponding alleles of the endogenous gene. These mutations therefore dictate an altered “set point” for period length that is not altered by overexpression. Overexpression of the DBT^K/R produced effects proportional to the titration of endogenous DBT, with long circadian periods at lower expression levels and arrhythmicity at higher levels. This first analysis of adult flies with a virtual lack of DBT activity demonstrates that DBT's kinase activity is necessary for normal circadian rhythms and that a general reduction of DBT kinase activity does not produce short periods.     

Efficient release of ferulic acid from sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis>) stems by chemical hydrolysis

An efficient method for release of ferulic acid from sweet potato stems was developed. Ferulic acid along with phenolic compounds were released from stems by acid and alkaline treatments. The base hydrolysis with 0.1 N NaOH yielded the highest quantity of total extracts (471.1 mg/g). The stems released more phenolic compounds when 0.0125∼0.025 N NaOH was employed. Where as ferulic acid release was maximal with 0.05 N H₂SO₄ (0.32 mg/g). Ferulic acid was separated from phenolics by column chromatography. Among the elution solvents, ethyl acetate fractions (80%) contained ferulic acid. Ethyl acetate eluants were further fractionated with n-hexane/ethyl acetate/formic acid (100/50/0.5, v/v/v). All fractions showed ferulic acid and phenolic compounds. Fraction V among them was ascribed to ferulic acid with an yield of 5.41 mg/g of dry sweet potato tissue.     

Fo Shou San,an Ancient Chinese Herbal Decoction,Protects Endothelial Function through Increasing Endothelial Nitric Oxide Synthase Activity

Fo Shou San (FSS) is an ancient herbal decoction comprised of Chuanxiong Rhizoma (CR; Chuanxiong) and Angelicae Sinensis Radix (ASR; Danggui) in a ratio of 2∶3. Previous studies indicate that FSS promotes blood circulation and dissipates blood stasis, thus which is being used widely to treat vascular diseases. Here, we aim to determine the cellular mechanism for the vascular benefit of FSS. The treatment of FSS reversed homocysteine-induced impairment of acetylcholine (ACh)-evoked endothelium-dependent relaxation in aortic rings, isolated from rats. Like radical oxygen species (ROS) scavenger tempol, FSS attenuated homocysteine-stimulated ROS generation in cultured human umbilical vein endothelial cells (HUVECs), and it also stimulated the production of nitric oxide (NO) as measured by fluorescence dye and biochemical assay. In addition, the phosphorylation levels of both Akt kinase and endothelial NO synthases (eNOS) were markedly increased by FSS treatment, which was abolished by an Akt inhibitor triciribine. Likewise, triciribine reversed FSS-induced NO production in HUVECs. Finally, FSS elevated intracellular Ca²⁺ levels in HUVECs, and the Ca²⁺ chelator BAPTA-AM inhibited the FSS-stimulated eNOS phosphorylation. The present results show that this ancient herbal decoction benefits endothelial function through increased activity of Akt kinase and eNOS; this effect is causally via a rise of intracellular Ca²⁺ and a reduction of ROS.     

The ferulic acid esterases of Chrysosporium lucknowense C1: purification, characterization and their potential application in biorefinery

Three ferulic acid esterases from the filamentous fungus Chrysosporium lucknowense C1 were purified and characterized. The enzymes were most active at neutral pH and temperatures up to 45 °C. All enzymes released ferulic acid and p-coumaric acid from a soluble corn fibre fraction. Ferulic acid esterases FaeA1 and FaeA2 could also release complex dehydrodiferulic acids and dehydrotriferulic acids from corn fibre oligomers, but released only 20% of all ferulic acid present in sugar beet pectin oligomers. Ferulic acid esterase FaeB2 released almost no complex ferulic acid oligomers from corn fibre oligomers, but 60% of all ferulic acid from sugar beet pectin oligomers. The ferulic acid esterases were classified based on both, sequence similarity and their activities toward synthetic substrates. The type A ferulic acid esterases FaeA1 and FaeA2 are the first members of the phylogenetic subfamily 5 to be biochemically characterized. Type B ferulic acid esterase FaeB2 is a member of subfamily 6.     

Danggui buxue tang--a Chinese herbal decoction activates the phosphorylations of extracellular signal-regulated kinase and estrogen receptor alpha in cultured MCF-7 cells

Danggui buxue tang (DBT), a Chinese herbal decoction used to treat ailments in women, contains radix Astragali (Huangqi; RA) and radix Angelicae Sinensis (Danggui; RAS). The weight ratio of RA to RAS used in DBT must be 5:1 as stipulated as early as AD 1247; however, DBT's mechanism of action has never been described. Here, the estrogenic effects of DBT were investigated by determining the phosphorylations of estrogen receptor alpha (ER alpha) and extracellular signal-regulated kinase 1/2 (Erk1/2) in cultured MCF-7 cells. The application of DBT triggered the phosphorylation of ER alpha and Erk1/2 in a time-dependent manner. In contrast to the effect of estrogen, DBT triggered ER alpha phosphorylation at both S118 and S167. This DBT-specific phosphorylation was not triggered by an extract of one of the individual herbs, or by mixing the extracts of RA and RAS. DBT-induced downstream signals are described here. These signals suggest the uniqueness of this Chinese herbal decoction that requires a well-defined formulation.     

Aquatic surface respiration,an adaptive response to hypoxia in the guppy,Poecilia reticulata (Pisces,Poeciliidae)

Synopsis Aquatic respiration at the air-water interface, herein termed aquatic surface respiration (ASR), is used by the guppy,Poecilia reticulata (Poeciliidae) to meet oxygen demand in hypoxid water. A specific position in which the head contacts the surface and the jaws open just beneath the surface is adopted. ASR is initiated at a P0₂ of about 50 torr and the percent time spent rises in a steep, linear fashion as PO₂ decreases. Below 4 torr more than 90% of the animal's time is spent in ASR. Males spend less time in ASR than do females. The percent time in ASR increases with increasing size of female guppies, but decreases with increasing size of male guppies. At low oxygen (18 torr) laboratory-born guppies derived from stocks likely to experience deoxygenation spend less time in ASR than do guppies derived from stocks less likely to experience deoxygenation. The percent time in ASR increases with temperature when PO₂ is held constant. Acclimation to low oxygen decreases the percent time in ASR.Guppies not permitted ASR die in 6–41 h at 14–17 torr and 10–15 min at 1–4 torr. Guppies performing ASR survive the duration of experiments at 13–35 torr (13 days), 14–17 torr (96 h), and 1–4 torr (9.5 h). Activity, courtship, and dive duration in response to a shadow stimulus are all reduced by low oxygen. ASR is an effective alternative to aerial respiration as an adaptation to hypoxic waters, but is probably energetically and temporally more costly.     

A Review on Potential Footprints of Ferulic Acid for Treatment of Neurological Disorders

Ferulic acid is being screened in preclinical settings to combat various neurological disorders. It is a naturally occurring dietary flavonoid commonly found in grains, fruits, and vegetables such as rice, wheat, oats, tomatoes, sweet corn etc., which exhibits protective effects against a number of neurological diseases such as epilepsy, depression, ischemia-reperfusion injury, Alzheimer’s disease, and Parkinson’s disease. Ferulic acid prevents and treats different neurological diseases pertaining to its potent anti-oxidative and anti-inflammatory effects, beside modulating unique neuro-signaling pathways. It stays in the bloodstream for longer periods than other dietary polyphenols and antioxidants and easily crosses blood brain barrier. The use of novel drug delivery systems such as solid-lipid nanoparticles (SLNs) or its salt forms (sodium ferulate, ethyl ferulate, and isopentyl ferulate) further enhance its bioavailability and cerebral penetration. Based on reported studies, ferulic acid appears to be a promising molecule for treatment of neurological disorders; however, more preclinical (in vitro and in vivo) mechanism-based studies should be planned and conceived followed by its testing in clinical settings.

     

In vitro and in vivo antithrombotic and cytotoxicity effects of ferulic acid

We discovered recently in vitro and in vivo antithrombotic and cytotoxicity effects of ferulic acid. The cytotoxicity assays showed that ferulic acid (～300 μg/mL) did not cause any significant toxicity on three cell lines, platelets, leukocytes, and erythrocytes. In vitro assays showed inhibitory effects of ferulic acid on thrombin (THR)‐ or collagen/epinephrine‐stimulated platelet activation by inhibiting platelet aggregation, and decreasing clot retraction activity. The in vitro effect of ferulic acid on THR‐stimulated platelet activation was proved by the decrease in the secretion of serotonin from the platelets. The anticoagulant effects of ferulic acid were confirmed by the prolongation of the intrinsic or/and extrinsic pathways and the delay of recalcification time in plasma coagulation. Ferulic acid had antithrombotic effect in acute thromboembolism model in vivo, and decreased the expression of α_IIbβ₃/FIB and phosphorylation of AKT in THR‐stimulated platelet activation in vivo, and their antithrombotic efficacies hold promise for therapeutic targeting in our ongoing studies.     

Evaluation of the Antioxidant Actions of Ferulic Acid and Catechins

We have evaluated the abilities of ferulic acid, (±) catechin, (+) catechin and (-) epicatechin to scavenge the reactive oxygen species hydroxyl radical (OH±), hypochlorous acid (HOCl) and peroxyl radicals (RO₂).

Ferulic acid tested at concentrations up to 5 mM inhibited the peroxidation of phospholipid liposomes. Both (±) and (+) catechin and (-) epicatechin were much more effective. All the compounds tested reacted with trichloromethyl peroxyl radical (CCl₃O₂) with rate constants > 1 × 10⁶M^?1s^?1.

A mixture of FeCl₃-EDTA, hydrogen peroxide (H₂O₂) and ascorbic acid at pH 7.4, has often been used to generate hydroxyl radicals (OH.) which are detected by their ability to cause damage to the sugar deoxyribose. Ferulic acid, (+) and (±) catechin and (-) epicatechin inhibited deoxyribose damage by reacting with OH. with rate constants of 4.5 × 10⁹M^?1s^?1, 3.65 × 10⁹M^?1s^?1, 2.36 × 10⁹M^?1s^?1 and 2.84 × 10⁹M^?1s^?1 respectively. (-) Epicatechin, ferulic acid and the (+) and (±) catechins exerted pro-oxidant action, accelerating damage to DNA in the presence of a bleomycin-iron complex. On a molar basis, ferulic acid was less effective in causing damage to DNA compared with the catechins.

A mixture of hypoxanthine and xanthine oxidase generates O₂ which reduces cytochrome c to ferrocytochrome c. (+) Catechin and (-) epicatechin inhibited the reduction of cytochrome c in a concentration dependent manner. Ferulic acid and (±) catechin had only weak effects.

All the compounds tested were able to scavenge hypochlorous acid at a rate sufficient to protect alpha-1-antiproteinase against inactivation. Our results show that catechins and ferulic acid possess antioxidant properties. This may become important given the current search for “natural” replacements for synthetic antioxidant food additives.     

The sulfur-fumigation reduces chemical composition and biological properties of Angelicae sinensis radix

Angelica Sinensis Radix (roots of Angelica sinensis; ASR) is a popular herbal supplement in China for promoting blood circulation. Today, sulfur-fumigation is commonly used to treat ASR as a means of pest control; however, the studies of sulfur-fumigation on the safety and efficacy of ASR are very limited. Here, we elucidated the destructive roles of sulfur-fumigation on ASR by chemical and biological assessments. After sulfur-fumigation, the chemicals in ASR were significantly lost. The biological activities of anti-platelet aggregation, induction of NO production and estrogenic properties were compared between the water extracts of non-fumigated and sulfur-fumigated ASR. In all cases, the sulfur-fumigation significantly reduced the biological properties of ASR. In addition, application of water extract deriving from sulfur-fumigated ASR showed toxicity to cultured MCF-7 cells. In order to ensure the safety and to achieve the best therapeutic effect, it is recommended that sulfur-fumigation is an unacceptable approach for processing herbal materials.     

Enzymatic release of ferulic acid fromIpomoea batatas L. (sweet potato) stem

Ferulic acid is a phenolic compound that serves as a major biosynthetic precursor of vanillin in higher plants. We investigated the ability of the 3 commercial enzymes—Ultraflo L, Viscozyme L, and α-Amylase—to induce the release ferulic acid from theIpomoea batatas L. (sweet potato) stem. The rate of release for ferulic acid was optimal when Ultraflo L (1.0%) was used compared with the other enzymes, whereas Viscozyme L was most effective for the release of vanillic acid and vanillin. Thus, these enzymes may be useful for the large-scale production of ferulic acid and other phenolic compounds from sweet potato stem.     

Ralstonia eutropha as a biocatalyst for desulfurization of dibenzothiophene

The potential of Ralstonia eutropha as a biocatalyst for desulfurization of dibenzothiophene (DBT) was studied in growing and resting cell conditions. The results of both conditions showed that sulfur was removed from DBT which accompanied by the formation of 2-hydroxybiphenyl (2-HBP). In growing cell experiments, glucose was used as an energy supplying substrate in initial concentrations of 55 mM (energy-limited) and 111 mM (energy-sufficient). The growing cell behaviors were quantitatively described using the logistic equation and maintenance concept. The results indicated that 2-HBP production was higher for the energy-sufficient cultures, while the values of the specific growth rate and the maintenance coefficient for these media were lower than those of the energy-limited cultures. Additionally, the kinetic studies showed that the half-saturation constant for the energy-limited cultures was 2 times higher than the energy-sufficient ones where the inhibition constant (0.08 mM) and the maximum specific DBT desulfurization rate (0.002 mmol g_cell ⁻¹ h⁻¹) were almost constant. By defining desulfurizing capacity (D _DBT) including both the biomass concentration and time to reach a particular percentage of DBT conversion, the best condition for desulfurizing cell was determined at 23% g_cell L⁻¹ h⁻¹ which corresponded with the resting cells that were harvested at the mid-exponential growth phase.

     

Review: Biocatalytic transformations of ferulic acid: An abundant aromatic natural product

In this review we examine the fascinating array of microbial and enzymatic transformations of ferulic acid. Ferulic acid is an extremely abundant, preformed phenolic aromatic chemical found widely in nature. Ferulic acid is viewed as a commodity scale, renewable chemical feedstock for biocatalytic conversion to other useful aromatic chemicals. Most attention is focused on bioconversions of ferulic acid itself. Topics covered include cinnamoyl side-chain cleavage; nonoxidative decarboxylation; mechanistic details of styrene formation; purification and characterization of ferulic acid decarboxylase; conversion of ferulic acid to vanillin;O-demethylation; and reduction reactions. Biotransformations of vinylgualacol are discussed, and selected biotransformations of vanillic acid including oxidative and nonoxidative decarboxylation are surveyed. Finally, enzymatic oxidative dimerization and polymerization reactions are reviewed.     

川芎晒干过程中阿魏酸和阿魏酸松柏酯含量的变化

为了解新鲜川芎采后干燥过程中阿魏酸和阿魏酸松柏酯含量的动态变化规律,采用高效液相色谱法测定了川芎晒干过程中总阿魏酸、游离阿魏酸和阿魏酸松柏酯的含量。结果显示,在整个晒干过程中(30 d),总阿魏酸、游离阿魏酸和阿魏酸松柏酯含量呈先升高后下降的变化趋势,晾晒第3 d时总阿魏酸含量最高(0.23%),因此在晾晒的第3 d利用快速干燥技术能较好地保留川芎药材中总阿魏酸含量,使其发挥更佳的药效。川芎药材中的阿魏酸松柏酯能水解产生阿魏酸,因此研究川芎干燥过程中的生理响应与含水量的关系对阿魏酸积累有重要意义。由于川芎在用药过程中是以总阿魏酸含量发挥药效的,所以以总阿魏酸含量作为川芎药材质量控制指标更加科学。     

Role of Vitamin D3 in Modulation of ΔNp63α Expression during UVB Induced Tumor Formation in SKH-1 Mice

ΔNp63α, a proto-oncogene, is up-regulated in non-melanoma skin cancers and directly regulates the expression of both Vitamin D receptor (VDR) and phosphatase and tensin homologue deleted on chromosome ten (PTEN). Since ΔNp63α has been shown to inhibit cell invasion via regulation of VDR, we wanted to determine whether dietary Vitamin D₃ protected against UVB induced tumor formation in SKH-1 mice, a model for squamous cell carcinoma development. We examined whether there was a correlation between dietary Vitamin D₃ and ΔNp63α, VDR or PTEN expression in vivo in SKH-1 mice chronically exposed to UVB radiation and fed chow containing increasing concentrations of dietary Vitamin D₃. Although we observed differential effects of the Vitamin D₃ diet on ΔNp63α and VDR expression in chronically irradiated normal mouse skin as well as UVB induced tumors, Vitamin D₃ had little effect on PTEN expression in vivo. While low-grade papillomas in mice exposed to UV and fed normal chow displayed increased levels of ΔNp63α, expression of both ΔNp63α and VDR was reduced in invasive tumors. Interestingly, in mice fed high Vitamin D₃ chow, elevated levels of ΔNp63α were observed in both local and invasive tumors but not in normal skin suggesting that oral supplementation with Vitamin D₃ may increase the proliferative potential of skin tumors by increasing ΔNp63α levels.