Anticonstriction Effect of MCA in Rats by Danggui Buxue Decoction

Objective: Danggui Buxue decoction (DBD), consisting of Angelicae Sinensis Radix (ASR) and Astragali Radix (AR), is a famous prescription with the function of antivasoconstriction. This study intends to probe its mechanisms on the relaxation of the middle cerebral artery (MCA).Methods: Vascular tension of rat MCA was measured using a DMT620 M system. First, the identical series of concentrations of DBD, ASR, and AR were added into resting KCl and U46619 preconstricted MCA. According to the compatibility ratio, their dilatation effects were further investigated on KCl and U46619 preconstricted vessels. Third, four K+ channel blockers were employed to probe the vasodilator mechanism on KCl-contracted MCA. We finally examined the effects of DBD, ASR, and AR on the vascular tone of U46619-contracted MCA in the presence or absence of Ca2+.Results: Data suggested that DBD, ASR, and AR can relax on KCl and U46619 precontracted MCA with no effects on resting vessels. The vasodilator effect of ASR was greater than those of DBD and AR on KCl-contracted MCA. For U46619-contracted MCA, ASR showed a stronger vasodilator effect than DBD and AR at low concentrations, but DBD was stronger than ASR at high concentrations. Amazingly, the vasodilator effect of DBD was stronger than that of AR at all concentrations on two vasoconstrictors which evoked MCA. The vasodilator effect of ASR was superior to that of DBD at a compatibility ratio on KCl-contracted MCA at low concentrations, while being inferior to DBD at high concentrations. However, DBD exceeded AR in vasodilating MCA at all concentrations. For U46619-constricted MCA, DBD, ASR, and AR had almost identical vasodilation. The dilation of DBD and AR on KCl-contracted MCA was independent of K+ channel blockers. However, ASR may inhibit the K+ channel opening partially through synergistic interactions with Gli and BaCl2. DBD, ASR, and AR may be responsible for inhibiting [Ca2+]out, while ASR and AR can also inhibit [Ca2+]in.Conclusion: DBD can relax MCA with no effects on resting vessels. The mechanism may be related to ASR’s inhibition of KATP and Kir channels. Meanwhile, the inhibition of [Ca2+]out by DBD, ASR, and AR as well as the inhibition of [Ca2+]in by ASR and AR may contribute to dilate MCA.

Main Active Components and Cell Cycle Regulation Mechanism of Astragali Radix and Angelicae Sinensis Radix in the Treatment of Ox-LDL-Induced HUVECs Injury and Inhibition of Their Cell Cycle

To explore the main active components and effects of cell cycle regulation mechanism of Astragali radix (AR) and Angelicae sinensis radix (ASR) on oxidative damage in vascular endothelial cells, a model of oxidative damage in human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein (ox-LDL) treatment was developed. Based on the “knock-out/knock-in” model of the target component, cell viability, intracellular reactive oxygen species (ROS), and lactate dehydrogenase (LDH) leakage were assessed by Cell Counting Kit-8 assay, fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and colorimetric assay, respectively, to evaluate the protective effect of the active components of AR and ASR (astragaloside IV (AS IV), astragaloside I (AS I), formononetin (FRM), calycosin (CAL), calycosin-7-O-β-D glucoside (CLG), and ferulic acid (FRA)) against oxidative damage. The cell cycle and expression of genes encoding cyclins and cyclin-dependent kinases (CDKs) were observed using flow cytometry and quantitative real-time polymerase chain reaction. The results showed that the combination of active components (ACC) significantly inhibited the decrease in cell viability as well as the increase in ROS and LDH release in HUVECs induced by ox-LDL treatment. AS IV and FRM promoted the proliferation of HUVECs but the proliferation index was decreased in the AS I and FRA groups; this inhibitory effect was counteracted by the ACC. The ACC reduced and increased the proportion of positive cells in G1 and S phases, respectively, followed by the upregulation of cyclin A (CCNA), cyclin E (CCNE), and CDK2 mRNA expression and downregulation of cyclin B (CCNB), cyclin D1 (CCND1), CDK1, CDK4, and CDK6 mRNA expression, which significantly mitigated inhibition of HUVECs proliferation induced by ox-LDL treatment. Taken together, AS IV, AS I, FRM, CAL, CLG, and FRA were the primary pharmacodynamic substances of AR and ASR that alleviated oxidative injury in HUVECs. ACC mitigated the upregulation of intracellular ROS levels and LDH release induced by ox-LDL treatment, which promoted the cell cycle procession of HUVECs by regulating the expression of genes encoding cyclins and CDKs and thus preventing oxidative damage in HUVECs.

The Osteogenic Function of Danggui Buxue Tang, a Herbal Decoction Containing Astragali Radix and Angelicae Sinensis Radix, Is Optimized by Boiling the Two Herbs Together: Signaling Analyses Revealed by Systems Biology

The therapeutic efficacy of a herbal mixture, being multi-target, multi-function and multi-pathway, is the niche of traditional Chinese medicine (TCM). Systems biology can dissect the network of signaling mechanisms in a complex biological system. In preparing TCM decoctions, the boiling of herbs together in water is a common practice; however, the rationale of this specific preparation has not been fully revealed. An approach of mass-spectrometry-based multi-omics was employed to examine the profiles of the cellular pathways, so as to understand the pharmacological efficacy of Danggui Buxue Tang (DBT), a Chinese herbal mixture containing Astragali Radix and Angelicae Sinensis Radix, in cultured rat osteoblasts and mesenchymal stem cells. The results, generated from omics analyses, were compared from DBT-treated osteoblasts to those of treating the herbal extract by simple mixing of extracts from Astragali Radix and Angelicae Sinensis Radix, i.e., herbal mixture without boiling together. The signaling pathways responsible for energy metabolism and amino acid metabolism showed distinct activation, as triggered by DBT, in contrast to simple mixing of two herbal extracts. The result supports that boiling the herbs together is designed to maximize the osteoblastic function of DBT, such as in energy and lipid metabolism. This harmony of TCM formulation, by having interactive functions of two herbs during preparation, is being illustrated. The systems biology approach provides new and essential insights into the synergy of herbal preparation. Well-defined multiple targets and multiple pathways in different levels of omics are the key to modernizing TCM.

Integrated Omics Reveals the Orchestrating Role of Calycosin in Danggui Buxue Tang, a Herbal Formula Containing Angelicae Sinensis Radix and Astragali Radix, in Inducing Osteoblastic Differentiation and Proliferation

Systems biology unravels the black box of signaling pathway of cells; but which has not been extensively applied to reveal the mechanistic synergy of a herbal formula. The therapeutic efficacies of a herbal formula having multi-target, multi-function and multi-pathway are the niches of traditional Chinese medicine (TCM). Here, we reported an integrated omics approach, coupled with the knockout of an active compound, to measure the regulation of cellular signaling, as to reveal the landscape in cultured rat osteoblasts having synergistic pharmacological efficacy of Danggui Buxue Tang (DBT), a Chinese herbal formula containing Angelicae Sinensis Radix and Astragali Radix. The changes in signaling pathways responsible for energy metabolism, RNA metabolism and protein metabolism showed distinct features between DBT and calycosin-depleted DBT. Here, our results show that calycosin within DBT can orchestrate the osteoblastic functions and signaling pathways of the entire herbal formula. This finding reveals the harmony of herbal medicine in pharmacological functions, as well as the design of drug/herbal medicine formulation. The integration of systems biology can provide novel and essential insights into the synergistic property of a herbal formula, which is a key in modernizing TCM.

Simultaneous Determination of Eight Chemical Components in Angelicae Sinensis Radix and Its Herbal Products by QAMS

A HPLC method has been developed for simultaneously detecting chlorogenic acid, ferulic acid, senkyunolide I, senkyunolide H, coniferyl ferulate, senkyunolide A, ligustilide, and levistolide A in Angelicae Sinensis Radix through quantitative analysis of multicomponents by single-marker (QAMS) method with ferulic acid as internal standard substance. The relative analysis correction factors of each component in Angelicae Sinensis Radix have good reproducibility under different chromatography conditions. In addition, no significant difference of results was found between quantitative analysis of multicomponents by single-marker (QAMS) method and external standard method in determining content of these components of different Angelicae Sinensis Radix and its 12 kinds of preparations. As a result, the established QAMS method for Angelicae Sinensis Radix analysis with ferulic acid as internal standard substance is accurate and feasible, which could be used as an effective and economical method to control quality of Angelicae Sinensis Radix and its herbal products.

Establishment of Raman Fingerprints and Localization of Main Components of Radix Angelicas sinensis from Different Origins

In this paper, confocal micro-Raman spectrum (CMR) was used to investigate the distribution of main components (ferulic acid and nicotinic acid) in Radix Angelicae sinensis (RAS). The ferulic acid and nicotinic acid were scanned to obtain their Raman spectra of single component; the RAS from different origins were scanned to obtain the Raman spectrum of RAS, and the Raman spectrum was compared with single component. The Raman spectrum of a single component was introduced into the map review to localize ferulic acid and nicotinic acid in RAS. In the results, the RAS from different origins showed strong similarity, with obvious characteristic peaks at 120 cm–1 140 cm–1210 cm–1,480 cm–1 and 1050 cm–1. The characteristic peaks of ferulic acid are mainly concentrated in the spectrum range of 100-1100 cm1 with obvious characteristic peaks around 160 cm150 cm–1 and 1800 cm–1 while the peaks of nicotinic acid mainly concentrated in the spectrum range of 100-1100 cm–1, with obvious characteristic peaks around 120 cm–1, 140 cm–1,810 cm–1and 1050 cm–1 The red and the green color represent the locations of ferulic acid and nicotinic acid, respectively. In conclusion, CMR can be used to establish the Raman fingerprint library of RAS for a quick and accurate identify, and locate the known active components.

Rapid Geographical Origin Identification and Quality Assessment of Angelicae Sinensis Radix by FT-NIR Spectroscopy

Angelicae Sinensis Radix is a widely used traditional Chinese medicine and spice in China. The purpose of this study was to develop a methodology for geographical classification of Angelicae Sinensis Radix and determine the contents of ferulic acid and Z-ligustilide in the samples using near-infrared spectroscopy. A qualitative model was established to identify the geographical origin of Angelicae Sinensis Radix using Fourier transform near-infrared (FT-NIR) spectroscopy. Support vector machine (SVM) algorithms were used for the establishment of a qualitative model. The optimum SVM model had a recognition rate of 100% for the calibration set and 83.72% for the prediction set. In addition, a quantitative model was established to predict the content of ferulic acid and Z-ligustilide using FT-NIR. Partial least squares regression (PLSR) algorithms were used for the establishment of a quantitative model. Synergy interval-PLS (Si-PLS) was used to screen the characteristic spectral interval to obtain the best PLSR model. The coefficient of determination for calibration (R2C) for the best PLSR models established with the optimal spectral preprocessing method and selected important spectral regions for the quantitative determination of ferulic acid and Z-ligustilide was 0.9659 and 0.9611, respectively, while the coefficient of determination for prediction (R2P) was 0.9118 and 0.9206, respectively. The values of the ratio of prediction to deviation (RPD) of the two final optimized PLSR models were greater than 2. The results suggested that NIR spectroscopy combined with SVM and PLSR algorithms could be exploited in the discrimination of Angelicae Sinensis Radix from different geographical locations for quality assurance and monitoring. This study might serve as a reference for quality evaluation of agricultural, pharmaceutical, and food products.