Regulation and analysis of Simiao Yong'an Decoction fermentation by Bacillus subtilis on the diversity of intestinal microbiota in Sprague-Dawley rats.

Background and Aim: Simiao Yong'an decoction (SYD) is a classic traditional Chinese medicine (TCM) prescription that has the effects of clearing heat, detoxifying, promoting blood circulation, and relieving pain. In this study, we investigated the effect of SYD on the diversity of intestinal microbiota after fermentation by Bacillus subtilis. Materials and Methods: SYD was fermented using B. subtilis. Female Sprague-Dawley rats were randomly divided into the following four groups with six rats in each group: Negative sample group (NS), water exaction non-fermentation group (WE), B. subtilis group (BS), and fermentation liquid group (FL). All rats were orally administered for 14 days. High-throughput Illumina sequencing was used to analyze 16S rRNA expression in rat fecal samples. Results: A total of 2782 operational taxonomical units (OTUs) were identified in this study, and 634 OTUs were shared among all samples. Bacteroidetes (28.17%-53.20%) and Firmicutes (48.35%-67.83%) were the most abundant phyla identified among the four groups. The abundance of Escherichia and Alistipes was lower in the FL group than in the NS group, whereas the abundance of Bifidobacteria and Lactobacillus was increased in the FL group (p < 0.05). The abundance of Bifidobacterium was significantly upregulated in the FL group compared with the WE and BS groups (p < 0.05). Conclusion: After fermentation, SYD had a significantly better effect than SYD or B. subtilis. SYD significantly promoted the growth of intestinal probiotics, inhibited the growth of pathogenic bacteria, and maintained the balance of intestinal microbiota in SD rats. This study provides new insights into the development and use of SYD.

[Comprehensive identification of metabolites and metabolic characteristics of luteolin and kaempferol in Simiao Yong'an Decoction in rats by UHPLC-LTQ-Orbitrap MS/MS].

Simiao Yong'an Decoction is a classic prescription for treating gangrene. Modern medical evidence has proven that Si-miao Yong'an Decoction has therapeutic effects on atherosclerosis(AS), vascular occlusion angeitides, and hypertension, while its pharmacodynamic mechanism remains unclear. The evidence of network pharmacology, molecular docking, literature review, and our previous study suggests that luteolin and kaempferol are two major flavonoids in Simiao Yong'an Decoction and can inhibit macrophage inflammation and exert anti-AS effects. However, due to lack of the metabolism studies in vivo, little is known about the metabolic characteristics of luteolin and kaempferol. This study employed ultra-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS/MS) and relevant software to identify the metabolites and metabolic pathways of luteolin and kaempferol in rat plasma, urine, and feces, after oral administration of luteolin and kaempferol, respectively. After the administration of luteolin, 10, 11, and 3 metabolites of luteolin were detected in the plasma, urine, and feces, respectively. After the administration of kaempferol, 9, 3, and 1 metabolites of kaempferol were detected in the plasma, urine, and feces, respectively. The metabolic pathways mainly involved methylation, glucuronidation, and sulfation. This study enriches the knowledge about the pharmacological mechanism of luteolin and kaempferol and supplies a reference for revealing the metabolic process of other flavonoids in Simiao Yong'an Decoction, which is of great significance for elucidating the pharmacological effects and effective substances of this decoction in vivo.

Systematic investigation of the material basis, multiple mechanisms and quality control of Simiao Yong'an decoction combined with antibiotic in the treatment of sepsis.

BACKGROUND: Sepsis is one of the major threats to human health with high mortality. Simiao Yong'an decoction (SMYAD) has the efficacy of anti-inflammation, improving coagulation and microcirculation, which is applicable for the clinical assistance treatment of sepsis. Yet, its material basis and relevant mechanisms are still vague. PURPOSE: Explore the quality markers (Q-markers), biomarkers and potential mechanisms of SMYAD combined with imipenem/cilastatin sodium for anti-sepsis. METHODS: Linear-Trap-LC/MSn was employed to profile the compounds in the extract and medicated serum of SMYAD. Then, the components and targets obtained from databases were applied to network pharmacology. Q-markers' range was narrowed via the affinity of three times docking and determined as per its screening criteria. Also, the content of them was detected by HPLC. Next, cecal ligation and puncture (CLP) model was reproduced to observe the effect of SMYAD united antibiotic by survival rate, histopathology score, ELISA, western blot and qPCR. Finally, metabolomics based upon GC-MS was exerted to discover the differential endogenous metabolites, metabolic pathway and joint pathway of SMYAD combined with antibiotic for sepsis. RESULTS: The 25 serum migrant ingredients derived from 113 chemical compounds of SMYAD were identified for the first time, and 6 components were determined as the Q-markers of SMYAD. The enrichment analysis indicated that the potential mechanism was mainly associated with the IL-17 signaling pathway, complement-coagulation cascades signaling pathway and VEGF signaling pathway. Then, SMYAD united antibiotic declined the mortality of septic rats, restored cytokine levels, ameliorated histopathological lesions and decreased the mRNA and protein expression of target proteins in a dose-dependent way. Furthermore, 8 differential metabolites were regarded as latent biomarkers related to the antiseptic effect of SMYAD united antibiotic, which were mainly involved in the Citrate cycle (TCA cycle) metabolic pathway. CONCLUSIONS: Different skeletons of compounds, including iridoids, phenylpropanoids, organic acids, triterpenes and others, were the main compositions of SMYAD. Among them, 6 components were determined as the Q-markers, which provided a basis for the construction of quality standards for this ancient classic formula. The combination therapy of SMYAD and antibiotic obviously ameliorated inflammatory reaction, coagulation dysfunction and microcirculation abnormalities for sepsis by inhibiting IL-17 signaling pathway, complement-coagulation cascades signaling pathway and VEGF signaling pathway.

Simiao Yong'an decoction ameliorates murine collagen-induced arthritis by modulating neutrophil activities: An in vitro and in vivo study.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis (RA) is a common systemic autoimmune disease with high morbidity and disability rate. Currently, there is no effective allopathic treatment for RA, and most of the drugs provoke many adverse effects. Simiao Yong'an decoction (SMYAD) is a traditional Chinese prescription for the treatment of sore and gangrene caused by hot poison. With the development of pharmacology and clinical research, SMYAD has remarkable anti-inflammatory properties and has been used for RA treatments for years. AIM OF THE STUDY: This study aimed to investigate the anti-arthritic effect of SMYAD and further explore the immunopharmacological mechanisms. MATERIALS AND METHODS: Arthritis was induced in DBA/1 mice by two-time immunizations. Collagen-induced rheumatoid arthritis (CIA) mice were divided into 4 groups: control, model, methotrexate (MTX), and SMYAD group (n = 6). The administration groups were given MTX (0.5 mg/kg/3 d) and SMYAD (4.5 g/kg/d) by gavage from day 14. The arthritis index (AI) score was evaluated every 3 days after the second immunization. Hematoxylin and eosin (H&E) staining, Safranin-O fast green staining, Trap staining, and Micro-CT were used to measure the histopathology injuries and bone destruction of joints. Granulocyte changes in the spleen, bone marrow, and period blood were analyzed by flow cytometry. The expression of inflammatory cytokines and chemokines in joints were detected by qRT-PCR. SMYAD-containing serum was obtained from SD rats gavaged with SMYAD. Neutrophils were isolated from peripheral blood and bone marrow for the in vitro experiments of transwell cell assay, apoptosis assay, reactive oxygen species (ROS) generation and neutrophil extracellular traps (NETs) formation. RESULTS: SMYAD significantly relieved arthritis severity in CIA mice. The AI score was significantly decreased in the SMYAD group compared with the model group. Additionally, SMYAD alleviated inflammatory infiltration, cartilage damage, osteoclast formation, and bone damage in the ankle joints. In the flow cytometry assay, SMYAD significantly reduced granulocytes number in the spleen and bone marrow, while increased in peripheral blood. Furthermore, compared with the CIA group, SMYAD suppressed the mRNA levels of inflammatory factors including TNF-α, IL-1ß, IL-6 and chemokines CXCL1, CXCL2, and IL-8 in the inflamed joints. In the in vitro studies, 20% SMYAD-containing serum effectively inhibited the migration of neutrophils, promoted neutrophils apoptosis, reduced ROS production and NETs formation. CONCLUSION: Collectively, our results demonstrated that SMYAD effectively restrained arthritis in CIA mice by modulating neutrophil activities.

Comprehensive identification of metabolites and metabolic characteristics of luteolin and kaempferol in Simiao Yong'an Decoction in rats by UHPLC-LTQ-Orbitrap MS/MS / 中国中药杂志

Simiao Yong'an Decoction is a classic prescription for treating gangrene. Modern medical evidence has proven that Si-miao Yong'an Decoction has therapeutic effects on atherosclerosis(AS), vascular occlusion angeitides, and hypertension, while its pharmacodynamic mechanism remains unclear. The evidence of network pharmacology, molecular docking, literature review, and our previous study suggests that luteolin and kaempferol are two major flavonoids in Simiao Yong'an Decoction and can inhibit macrophage inflammation and exert anti-AS effects. However, due to lack of the metabolism studies in vivo, little is known about the metabolic characteristics of luteolin and kaempferol. This study employed ultra-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS/MS) and relevant software to identify the metabolites and metabolic pathways of luteolin and kaempferol in rat plasma, urine, and feces, after oral administration of luteolin and kaempferol, respectively. After the administration of luteolin, 10, 11, and 3 metabolites of luteolin were detected in the plasma, urine, and feces, respectively. After the administration of kaempferol, 9, 3, and 1 metabolites of kaempferol were detected in the plasma, urine, and feces, respectively. The metabolic pathways mainly involved methylation, glucuronidation, and sulfation. This study enriches the knowledge about the pharmacological mechanism of luteolin and kaempferol and supplies a reference for revealing the metabolic process of other flavonoids in Simiao Yong'an Decoction, which is of great significance for elucidating the pharmacological effects and effective substances of this decoction in vivo.

[Identification of chemical constituents in Simiao Yong'an Decoction based on UPLC-LTQ-Orbitrap-MS].

This study aims to identify the chemical constituents of Simiao Yong'an Decoction based on ultra-performance liquid chromatography coupled with linear quadrupole ion trap-orbitrap mass spectrometry(UPLC-LTQ-Orbitrap-MS). The elution was performed through a UPLC BEH C_(18) column(2.1 mm × 100 mm, 1.7 µm) with the mobile phase of water(containing 0.1% formic acid)-acetonitrile at a flow rate of 0.4 mL·min~(-1). LTQ-Orbitrap-MS with heat electrospray ion(HESI) source was employed to collect MS fragment information in the negative ion mode. A total of 72 compounds were identified based on reference substance comparison, fragmentation rules, accurate molecular weight, related reports and databases(MassBank and HMDB), including 30 iridoid glycosides, 9 organic acids, 15 flavonoids, 10 phenylpropanoids, 7 triterpenoids, and 1 saccharide. The method established in this study is comprehensive, rapid, and accurate, which can help summarize the fragmentation rules of constituents and provide reference for revealing the active constituents and pharmacodynamic mechanism of Simiao Yong'an Decoction.

Study on the Plasma Protein Binding Rate and Compatibility Regularity of the Constituents Migrating to Blood of Simiao Yong'an Decoction.

OBJECTIVE: To study the compatibility regularity of Simiao Yong'an decoction by determining the plasma protein binding rate with the constituents in Simiao Yong'an decoction and to preliminarily clarify the effects of the compatibility on the plasma protein binding rate of different components. METHODS: Based on the equilibrium dialysis method, high-performance liquid chromatography was used to determine the contents of six constituents, which were divided into a single group and combination groups, in Simiao Yong'an decoction in the internal and external dialysis solutions. The obtained plasma protein binding rate through calculations was an index to evaluate the binding of the above components to plasma protein in different conditions. RESULTS: Harpagide, harpagoside, sweroside and loganin showed low plasma protein binding rates, ferulic acid exhibited a moderate plasma protein binding rate, and glycyrrhizic acid showed a high plasma protein binding rate. The compatibility study showed that glycyrrhizic acid promoted the binding of ferulic acid to plasma protein. Glycyrrhizic acid and ferulic acid were the key compounds to promote the binding of harpagide to plasma protein. Glycyrrhizic acid, harpagide, harpagoside and loganin had a significant inhibitory effects on the binding of sweroside to plasma protein. The plasma protein binding capacities of harpagoside and loganin were reduced by the other five constituents. Glycyrrhizic acid had the strongest plasma protein binding effect, and the binding effect was not affected by other components. CONCLUSION: This study explores the effects of compound compatibility on effective components from the perspective of plasma protein binding by high-performance liquid chromatography combined with the equilibrium dialysis method, and lays a foundation for clarifying the compatibility rule of Simiao Yong'an decoction and also provides a new idea for the study of the compatibility of traditional Chinese medicine formulas.

[Exploration on origin of Simiao Yong'an Decoction].

Simiao Yong'an Decoction is composed of Lonicerae Japonicae Flos, Scrophulariae Radix, Angelicae Sinensis Radix, Glycyrrhizae Radix et Rhizome, which was chosen as one of the 100 classic prescriptions in Catalogue of Ancient Classics Prescription(the first batch). Through tracing to the source, It was found that the Simiao Yong'an Decoction(but not named) originated from the Shi Shi Mi Lu, and was later cited by books such as Ancient and Modern Book Integration-Full Record of Medical Department and New Edition of Useful Prescriptions. Literature shows that this prescription was not named until first reported in the Effect of Traditional Chinese Medicine on Arterial Embolism Gangrene in 1956 by a journalist LYU Min. This article recorded that SHIJIA Baoshan, a monk from Hebei Province, used self-named "Simiao Yong'an Decoction" to treat local arterial embolic gangrene. After comparison, there was two difference between ancient books and SHIJIA Baoshan's records. Firstly, according to ancient books, the composition and dosage of Simiao Yong'an Decoction is Lonicerae Japonicae Flos 90 g, Scrophulariae Radix 90 g, Angelicae Sinensis Radix 60 g, Glycyrrhizae Radix et Rhizome 30 g", and the ratio is 3∶3∶2∶1. By SHIJIA Baoshan's record, the composition and dosage are: Lonicerae Japonicae Flos 66 g, Scrophulariae Radix 132 g, Angelicae Sinensis Radix 99 g, Glycyrrhizae Radix et Rhizome 33 g, and the ratio changed to 2∶4∶3∶1. Secondly, ancient books show that patients can be healed after taking seven or ten days of the previous prescription, however, it would take 3 or 4 months, even 7 months in SHIJIA Baoshan's records. It can be considered that the previous prescription should be used at the beginning of gangrene, while the modified Simiao Yong'an Decoction by SHIJIA Baoshan is widely used in the middle and late stages of gangrene, even the critical condition, that is the reason for longer treatment and larger dosage. Nowadays, Simiao Yong'an Decoction is not limited to the treatment of gangrene and bulla in clinic. Relevant studies have confirmed that Simiao Yong'an Decoction has the effects such as anti-inflammatory, plaque stabilization, lipid-lowering, vascular protection, improvement of hemorheology, anticoagulation, inhibition of thrombosis and fibrinolysis, etc. In the follow-up, we should carry out the analysis of the compatibility of this four medicines, and redefine the scope of its clinical application under the theory of traditional Chinese medicine.

Exploration on origin of Simiao Yong'an Decoction / 中国中药杂志

Simiao Yong'an Decoction is composed of Lonicerae Japonicae Flos, Scrophulariae Radix, Angelicae Sinensis Radix, Glycyrrhizae Radix et Rhizome, which was chosen as one of the 100 classic prescriptions in Catalogue of Ancient Classics Prescription(the first batch). Through tracing to the source, It was found that the Simiao Yong'an Decoction(but not named) originated from the Shi Shi Mi Lu, and was later cited by books such as Ancient and Modern Book Integration-Full Record of Medical Department and New Edition of Useful Prescriptions. Literature shows that this prescription was not named until first reported in the Effect of Traditional Chinese Medicine on Arterial Embolism Gangrene in 1956 by a journalist LYU Min. This article recorded that SHIJIA Baoshan, a monk from Hebei Province, used self-named "Simiao Yong'an Decoction" to treat local arterial embolic gangrene. After comparison, there was two difference between ancient books and SHIJIA Baoshan's records. Firstly, according to ancient books, the composition and dosage of Simiao Yong'an Decoction is Lonicerae Japonicae Flos 90 g, Scrophulariae Radix 90 g, Angelicae Sinensis Radix 60 g, Glycyrrhizae Radix et Rhizome 30 g", and the ratio is 3∶3∶2∶1. By SHIJIA Baoshan's record, the composition and dosage are: Lonicerae Japonicae Flos 66 g, Scrophulariae Radix 132 g, Angelicae Sinensis Radix 99 g, Glycyrrhizae Radix et Rhizome 33 g, and the ratio changed to 2∶4∶3∶1. Secondly, ancient books show that patients can be healed after taking seven or ten days of the previous prescription, however, it would take 3 or 4 months, even 7 months in SHIJIA Baoshan's records. It can be considered that the previous prescription should be used at the beginning of gangrene, while the modified Simiao Yong'an Decoction by SHIJIA Baoshan is widely used in the middle and late stages of gangrene, even the critical condition, that is the reason for longer treatment and larger dosage. Nowadays, Simiao Yong'an Decoction is not limited to the treatment of gangrene and bulla in clinic. Relevant studies have confirmed that Simiao Yong'an Decoction has the effects such as anti-inflammatory, plaque stabilization, lipid-lowering, vascular protection, improvement of hemorheology, anticoagulation, inhibition of thrombosis and fibrinolysis, etc. In the follow-up, we should carry out the analysis of the compatibility of this four medicines, and redefine the scope of its clinical application under the theory of traditional Chinese medicine.