[Researching on fingerprint of Inulacappa by HPLC].

OBJECTIVE: This study is to establish the fingerprint and find out the common chromatographic peaks of Inula cappa by HPLC. METHOD: The HPLC analysis was performed on an Agilent Eclipse Plus C18 column (2.1 mm x 150 mm, 1.8 µm) with 0.1% fomic acid aqueous solution-0.1% fomic acid acetonitrile solution as mobile phase at a flow rate of 0.3 · mL(-1) · min(-1); The detective wavelength is 325 nm; The column temperature is 45 °C. RESULT: The results indicated that 5 of 17 common peaks were identified . The similarity about 10 groups of Inulacappais is over 0.95. CONCLUSION: This method is able to be a scientific basis of quality assessment according to its convenient and reliable.

A HPLC-DAD-MS/MS Method for Simultaneous Determination of Six Active Ingredients of Salviae Miltiorrhizae and Ligustrazine Hydrochloride Injection in Rat Plasma and its Application to Pharmacokinetic Studies.

AIMS: This is a pharmacokinetic study of Salviae miltiorrhizae and ligustrazine hydrochloride injection. The study aimed to evaluate the mechanism of action, safety and rational clinical use of Salviae miltiorrhizae and ligustrazine hydrochloride injection. BACKGROUND: Salviae miltiorrhizae and ligustrazine hydrochloride injection is a compound preparation consisted of Salvia miltiorrhiza extract and ligustrazine hydrochloride for the treatment of cardiovascular and cerebrovascular diseases in China. OBJECTIVE: The study aimed to develop a rapid and sensitive high-performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC-DAD-MS/MS) method for simultaneous determination of six major active ingredients of Salviae miltiorrhizae and ligustrazine hydrochloride injection, namely danshensu, protocatechuic aldehyde, rosmarinic acid, lithospermic acid, salvianolic acid A, and ligustrazine hydrochloride, in rat plasma. METHODS: Plasma samples were precipitated with methanol, which was spiked with ascorbic acid and the supernatant was separated on a Waters Cortecs C18 column, by using a gradient mobile phase system of acetonitrile-water containing 0.05% formic acid (v/v). For internal standards, puerarin was selected for the five salvianolic acids, while isofraxidin was used for ligustrazine hydrochloride. Besides, electrospray ionization in negative mode and multiplereaction monitoring were used to identify and quantify the five salvianolic acids, whereas ligustrazine hydrochloride was quantified at 310 nm using the diode array detector. RESULTS: Noticeably, all calibration curves showed good linearity (R2>0.99) over the concentration range, with a lower limit of quantification between 0.00411 and 0.0369 µg/mL for salvianolic acids, and 1.74 µg/mL for ligustrazine hydrochloride. Next, the precision of the developed method was evaluated by intra- and inter-day assays, and the percentage of relative standard deviation was within 10%. Although the extraction efficiency of some salvianolic acids was not very satisfactory, the sensitivity of the analytical method met the analysis requirements of rat plasma samples. Moreover, the validated method was successfully applied to a pharmacokinetic study of Salviae miltiorrhizae and ligustrazine hydrochloride injection in the rat model. CONCLUSION: Linear pharmacokinetic characteristics were observed for the six active ingredients after intravenous infusion administration in rats within the dose range examined here. In summary, our study proposed a HPLC-DADMS/ MS method with the simultaneous determination of multiple ingredients, and demonstrated its applicability in pharmacokinetic studies.

Combination of Danshen and ligustrazine has dual anti-inflammatory effect on macrophages and endothelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia Miltiorrhiza Radix et Rhizoma (Danshen) and Chuanxiong Rhizoma (Chuanxiong) are both traditional Chinese medicines with vascular protective effects, and their combination is widely used in China to treat occlusive or ischemic diseases of the cerebrovascular or cardiovascular system. Although it is widely accepted that these diseases have high relevance to inflammation, little is known about the anti-inflammatory effect of Danshen, Chuanxiong, and their combination. AIM OF STUDY: We aimed to investigate the complex mode of action of Danshen, Chuanxiong, and their combination and the molecular mechanisms underlying their anti-inflammatory activity. Specifically, toll-like receptor (TLR1/2, 3, and 4)-triggered macrophages and endothelial cells, the two major cell players in atherosclerosis as well as in related cardiovascular and cerebrovascular injuries, were emphasized. METHODS: TLR1/2-, TLR3-, and TLR4-induced bone marrow macrophages (BMMs) and human umbilical vein endothelial cells (HUVECs) were treated with Danshen extract (S. miltiorrhiza extract, SME), ligustrazine (2, 3, 5, 6-tetramethylpyrazine, TMP), and their combination (S. miltiorrhiza and TMP injection, SLI), respectively. The proinflammatory cytokines interleukin 6 (IL-6), IL-12, and tumor necrosis factor alpha (TNF-α) were detected as the preliminary indicators of inflammation. In addition, RNA sequencing (RNA-seq)-based transcriptional profiling analyses were conducted for TLR2-activated BMMs to determine the molecular mode of action of SLI as well as the contribution of SME to SLI activity. RESULTS: SLI mitigated inflammation in both BMMs and HUVECs. Refer to the combination, SME had pronounced anti-inflammatory effect on BMMs but had only a slight effect on HUVECs. In contrast, TMP had considerable anti-inflammatory effect on HUVECs but not on BMMs. Bioinformatic analysis identified a broad spectrum of regulatory genes, in addition to IL-6 gene, and C-X-C motif chemokine ligand 10 (CXCL10) appeared to be another key molecule involved in the mechanism underlying SLI and SME effects. At the molecular level, SME was a major contributor of the anti-inflammatory activity of SLI. CONCLUSIONS: In TLR-activated inflammation, SLI exhibits a "multiple ingredient-multiple target" effect, with SME primarily affecting macrophages and TMP affecting HUVECs. Our study provides evidence for the clinical application of SLI in treating complex diseases involving inflammation-induced injury of both macrophages and epithelial cells. Further bioinformatics studies are required to reveal the entire molecular network involved in TMP, SME, and SLI activity.

Effect of Danshen on TLR2-triggered inflammation in macrophages.

BACKGROUND: Danshen (Salvia Miltiorrhiza Radix et Rhizoma) is a valued herbal plant widely used to treat cardiovascular diseases in Asian countries. In modern medicine, innate immunity-induced inflammation is considered a risk factor for cardiovascular diseases. However, little is known about the anti-inflammatory effects and molecular mechanism of Danshen. PURPOSE: To evaluate the molecular mechanisms of Danshen on Toll-like receptor (TLR) 2-triggered inflammation in macrophages and identify its bioactive components. METHODS: Pam3CSK4-stimulated bone marrow-derived macrophages (BMMs) were treated with Danshen water extract (DSE), and the levels of proinflammatory cytokines (interleukin (IL)-6, IL-12 and tumor necrosis factor (TNF)-α) were measured by both real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). RNA sequencing (RNA-seq)-based bioinformatics analyses were applied to reveal the novel molecular mechanisms of DSE, followed by western blotting for verification. Additionally, HPLC-UV analysis along with bioassays was performed to identify the bioactive ingredients of DSE. RESULTS: The results of RT-qPCR and ELISA showed that DSE significantly inhibited proinflammatory cytokine expression in a dose-dependent manner. Transcriptome analyses revealed that a wider panel of inflammatory cytokines responded to the regulatory effect of DSE, and that the TNF signaling pathway might have played a vital role. Western blotting data confirmed the involvement of extracellular signal-regulated protein kinases (ERK) and Jun N-terminal Kinase (JNK) related singling pathway. Among the seven components identified in DSE, Danshensu (DSS) and protocatechuic aldehyde (PA) were confirmed as bioactive ones with anti-inflammatory effects. CONCLUSION: DSE showed a promising effect against TLR2-triggered inflammation associated with the inhibition of the TNF cascade down-streamed mitogen-activated protein kinase (MAPK) signaling pathway, in which IL-6 acts as the key effective molecule, and ERK and JNK phosphorylation was inhibited. Notably, DSS and PA were considered bioactive components with anti-inflammatory bioactivity.

Evaluation of a multiple and global analytical indicator of batch consistency: traditional Chinese medicine injection as a case study.

This paper evaluates a multiple and global analytical indicator of batch consistency in traditional Chinese medicine injections (TCMIs) via a chemometrics tool, which is more comprehensive to appraise quality consistency of different batches of injections than the traditional method of fingerprint similarity. A commonly used TCMI, Salviae miltiorrhizae and ligustrazine hydrochloride injection (SLI), was employed as a model. With the aid of a chemometrics tool (principal component analysis, PCA), evaluation of multiple and global analytical indicators of batch consistency, which included saccharides, phenolic acids and inorganic salts (18 indicators in total), was carried out to appraise the quality consistency of 13 batches of injection provided by the Guizhou Baite Pharmaceutical Co., Ltd. (Guizhou, China). Compared with the traditional HPLC-UV fingerprint similarity evaluation, the method proposed in the paper can more comprehensively and correctly reflect the quality consistency of different batches of injections. In this paper, the multi-index evaluation result showed poor batch consistency, which was more consistent with the determination results, while the fingerprint similarity evaluation results still showed good batch consistency. The HPLC-UV fingerprint reflects only substances with UV absorption, but it is not able to reflect substances without UV absorption or weak UV absorption, which leads to inappropriate conclusions. Therefore, quality consistency of injections can be effectively appraised by evaluation of multiple and global analytical indicators, instead of HPLC-UV fingerprint only. For visualizing the batch consistency of the multiple and global analytical indicators, a heat map was used to represent the fluctuation. Furthermore, critical indicator identification was also applied to select several indicators that should be paid more attention during the process of quality control of injection. And the analysis result showed that Na+, fructose (Fru), glucose (Glc), manninotriose (Man), danshensu (DSS) and salvianolic acid B (SAB) are the indicators that should be given more attention when controlling the quality of injections, also called critical quality control indicators. The proposed method provides a reference for the quality control of TCMIs and has broad application potential.

Inhibition of nuclear factor kappa B as a mechanism of Danshensu during Toll-like receptor 2-triggered inflammation in macrophages.

Danshensu (DSS) is a water-soluble phenolic compound in Danshen (Salvia Miltiorrhiza Radix et Rhizoma). Although various pharmacological activities have been recognized, little is known regarding its anti-inflammatory effect and related molecular mode of action. In the current study, bone marrow-derived macrophages (BMMs) were activated by a Toll-like receptor 2 (TLR2) agonist Pam3CSK4 with or without DSS intervention. Production of pro-inflammatory cytokines interleukin-6 (IL-6) and interleukin-12 (IL-12) was detected by both enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR (RT-qPCR). Activation of signaling pathways involving nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) was assessed by Western blot. Additionally, RNA sequencing (RNA-seq) combined with bioinformatics analyses was applied to investigate the molecular mechanisms of DSS. Emphasis was placed on the construction of the protein-protein interaction (PPI) network and transcription factor (TF) enrichment analysis of data including co-regulated differentially expressed genes (DEGs) in the Pam3CSK4 vs. control and DSS vs. Pam3CSK4 groups. The RT-qPCR and ELISA results showed that DSS effectively inhibited the expressions of IL-6 and IL-12, indicating a significant anti-inflammatory effect. Western blot verified that DSS suppressed the phosphorylation of p65, which was in accordance with the results of the TF enrichment analysis. Additionally, the PPI network analysis showed several key molecules, including lactoferrin (Ltf), CC-chemokine receptor 7 (Ccr7), interferon-gamma (IFN-γ) and C-X-C motif chemokine ligand 9 (Cxcl9), to be regulatory genes that responded to DSS treatment. Overall, our study revealed that DSS has a pronounced anti-inflammatory effect involving TLR2 and macrophages through the NF-κB signaling pathway, which supports the novel application of DSS in the treatment of relevant diseases including atherosclerosis and ischemic or ischemic/perfusion injury of the heart and brain.