Spatholobi Caulis Research Articles

Predicting Pharmacokinetics of Active Constituents in Spatholobi caulis by Using Physiologically Based Pharmacokinetic Models

Background/Objectives: Spatholobi Caulis (SPC) is a medicinal plant that mainly grows in China and Southeast Asian countries and commonly used in clinics; the pharmacokinetic characteristics in humans need to be determined. This study was to establish the physiologically based pharmacokinetic (PBPK) models of multiple active constituents from SPC in rats, and predict the pharmacokinetic properties of rats with different dosages and extrapolated to humans. Methods: The parameters were collected based on our previous study and by prediction using ADMET Predictor software predict. The PBPK models for 3′-methoxydadizein (1), 8-O-methylretusin (2), daidzin (3), and isolariciresinol (4) administered orally to rats were established using GastroPlus software. These models were employed to simulate the pharmacokinetic properties in rats across various dosages, and subsequently extrapolated to humans. The calculated parameters including Cmax, Tmax, and AUC were compared with observed values. The accuracy of the PBPK models was assessed using fold-error (FE) values. Result: The FE values ranged from 1.03 to 1.52, meeting the PBPK model regulations where FE should be less than 2. The sensitivity analysis focusing on the absorption amount and AUC0→t of these four constituents in humans was also conducted. These results confirm the successful establishment of PBPK models of these four constituents from SPC in this study, and these models were applicable to predict pharmacokinetics across various doses and extrapolate across species. Conclusions: The PBPK models of four constituents can be used to predict the pharmacokinetic characteristics in humans after oral administration of SPC and provide useful data for safe and rational medication in clinical practice.

Self-healing hydrogels loaded with Spatholobi Caulis alleviate disc degeneration by promoting autophagy in nucelus pulposus

Intervertebral disc degeneration (IDD) is a common degenerative disease of the spine that has a significant impact on both society and human health. Many studies have confirmed that there is a close relationship between IDD and senescence and apoptosis, and autophagy can combat apoptosis and senescence. Spatholobi caulis (SC) is an herb that contains various active compounds that are effective in tissue repair and regeneration, but it has not been explored in field of IDD. In this study, it was first found that SC can boost autophagy and reduce the apoptosis and senescence of Nucleus pulposus cell (NPCs). However, our animal studies revealed limited absorption of SC. To improve the bioavailability and efficacy of SC, we developed a hydrogel incorporating quaternary ammonium chitosan (QCS) and oxidized starch (OST) as carriers for SC. The QCS-OST/SC hydrogel exhibits excellent compatibility with cells, can be easily injected, and can release SC durably. At the cellular level, the QCS-OST/SC hydrogel enhances cell viability, initiates autophagy and release of the extracellular matrix (ECM), and inhibits cellular senescence and apoptosis. The injection of the QCS-OST/SC hydrogel via microneedles (MNs) into discs had successfully diminished disc degeneration in rats, which shows that this hydrogel has broad potential in the treatment of IDD.

First Report in China of Leaf Blight on Spatholobus suberectus Dunn Caused by Diaporthe tulliensis.

Spatholobus suberectus Dunn is an important Chinese medicinal plant, which is the only officially recognized authentic source of the Chinese herbal medicine Spatholobi caulis in the China Pharmacopeia (Qin et al. 2024). In November 2023, a serious leaf disease was observed in the S. suberectus planting base in Nanning city (108.22E, 22.51N), Guangxi, China, with an incidence rate of 80%. Initially the symptoms appeared in the diseased leaves tips and was irregular in shape. The necrosis displayed discoloration of the gray color with a deep brown margin and this gradually expanded through into the lamina. To isolate the causal agent, 10 symptomatic leaves were collected and small segments (5×5 mm) of infected tissues were cut, and then they were surface sterilized (30s in 75% EtOH, 2 min in 2.5% NaOCl, and rinsed three times in sterilized distilled water), placed on potato dextrose agar (PDA) and incubated at 28℃ for 5 days. The hyphal tips were transferred to PDA to obtain pure cultures. 36 isolates were obtained, 23 of them had similar morphology, which were isolated from all the symptomatic leaves and were considered pathogens, and named YY-3-S. After 7 d, the colonies were white, fluffy, with white aerial mycelium. Sporulation was induced using pine needle medium (Su et al. 2012). After 30 days, the globose pycnidia developed, which were superficial to semi-immersed on the pine needle medium, and they appeared to be orange to black in color, with pale yellow conidial droplets. The alpha conidia spores were 3.53 to 6.85×1.75 to 3.70 μm (n = 50). They were unicellular, hyaline, aseptate and ellipsoidal to fusiform. These morphological features were consistent with Diaporthe sp (Sun et al. 2021). For further molecular identification, isolate YY-3-S underwent sequencing of the internal transcribed spacer (ITS), beta-tubulin (TUB2), and translation elongation factor 1-alpha (TEF1) sections (Huang et al. 2021). Obtained sequences of ITS, TUB2 and TEF1 (Genebank accessions nos. PP838571, PP865050 and PP865051) displayed a 99% similarity to Diaporthe tulliensis (Genebank accessions nos. OP163562, KR936132, OR662168, respectively). Phylogenetic analysis was based on a combination of ITS, TUB2 and TEF1 sequence data using MEGA 11 software to construct a phylogenetic tree with Neighbor-Joining. In the phylogenetic tree, the combined sequences attributed YY-3-S to D. tulliensis. The pathogenicity test was conducted on 1-year-old S. suberectus seedlings. We sprayed the mixed suspension of spores and mycelia on the unwounded leaves, while control seedlings received sterile water. 3 leaves per plant and 5 plants per treatment were selected for assessment. All seedings were placed in pots in a moist chamber at 28°C and 90% humidity. After 5 d, browning was observed on all the inoculated leaves tips. 14 d after inoculation, necrotic lesions expanded outward and their symptoms like those in the field, while control plants remained healthy. D. tulliensis was reisolated from the inoculated leaves, fulfilling the Koch's postulates. D. tulliensis has been reported to necrotic spots and leaf blight on Rambutan (Serrato-Diaz et al. 2023), leaf spot on bodhi tree (Li et al. 2022), stem canker on Jasmin (Hsu et al. 2023). To our knowledge, this is the first report of D. tulliensis causing leaf blight on S. suberectus in China. It is important to ensure timely management of the newly emerging disease.

Research of the mechanism of spatholobi caulis for immune-inflammatory of osteoarthritis based on clinical data mining,network pharmacology and cell experiment verification

BackgroundTraditional Chinese medicine is widely used in the treatment of osteoarthritis. This study explores the therapeutic effect of Traditional Chinese Medicine (TCM) on osteoarthritis (OA), as well as the mechanism by which Spatholobi Caulis (SPC) improves the immune-Inflammatory response of OA. MethodsThe clinical data mining techniques to evaluate the efficacy of Caulis spatholobi in treating OA, and elucidated its mechanism of action using network pharmacology, molecular docking, and cell experiments. The random walk model was applied to systematically evaluate the therapeutic efficacy of Chinese herbal medicine on OA. ResultsThe evaluation based on the clinical data mining showed that TCM is effective in improving ESR, CRP, C3, C4, and PLT. Data mining results showed significant OA treatment. Network analysis results showed that it may fight OA by targeting JUN, ESR1 and PPARG; molecular docking results showed good JUN, ESR1, PPARG and active component formononetin. Finally, in vitro experiments conducted the preliminary validation of formononetin for the core target of FLS. Cell proliferation was measured by CCK-8, and JUN, ESR1, PPARG protein, and mRNA expression was measured by quantitative real-time RCR at 48 h after drug intervention. ConclusionThe present study demonstrated that the SPC effectively relieved immune inflammation indicators in OA patients. Formononetin, the main active component of the chicken blood vine, acts to reduce the immune validation response in OA patients by reducing the expression of ESR1, JUN, and PPARG.

Effect of Spatholobi Caulis extract from ethyl acetate on immune killing function of NK cells

This study aims to investigate the regulatory effect of the Spatholobi Caulis extract from ethyl acetate(SEA) on natural killer(NK) cells under physiological conditions and elucidate the underlying mechanism. The C57BL/6 mice were randomized into NC and SEA groups, and NK-92 cells were respectively treated with 0, 25, 50, and 100 μg·mL~(-1) SEA. The body weight and immune organ index of the mice were compared between groups. The lactate dehydrogenase(LDH) assay was employed to examine the cytotoxicity of NK-92 cells treated with SEA and the killing activity of mouse NK cells against YAC-1 cells. The cell-counting kit-8(CCK-8) was used to examine the impact of SEA on the proliferation of NK-92 cells. Flow cytometry was employed to measure the number of NK cells in the peripheral blood as well as the expression levels of natural killer group 2 member A(NKG2A) and natural killer group 2 member D(NKG2D). The enzyme-linked immunosorbent assay(ELISA) was performed to determine the interferon(IFN)-γ secretion in the serum. Semi-quantitative PCR was conducted to determine the mRNA levels of NKG2A, NKG2D, and IFN-γ in spleen cells. Western blot was employed to investigate the involvement of phosphoinositide 3-kinase(PI3K)/extracellular regulated protein kinase 1(ERK1) signaling pathway. The results showed that SEA exhibited no adverse effects on the body, while significantly enhance the number of NK cells and augment the cytotoxicity of NK-92 cells against YAC-1 cells. Moreover, it suppressed the expression of NKG2A, enhanced the expression of NKG2D, promoted IFN-γ secretion, and upregulated the protein levels of PI3K and ERK. The findings suggest that SEA has the potential to enhance the immune recognition and effector function of NK cells by increasing the cell number, modulating the expression of functional receptors, and promoting IFN-γ secretion via the PI3K/ERK signaling pathway.

Ethyl-acetate extract of Spatholobi Caulis blocked the pro-metastatic support from the hemato-microenvironment of colon cancer by specific disruption of tumor-platelet adhesion

BackgroundWithin the pro-metastatic hemato-microenvironment, interaction between platelets and tumor cells provides essential support for tumor cells by inducing Epithelial-Mesenchymal Transition (EMT), which greatly increases the stemness of colon cancer cells. Pharmacologically, although platelet deactivation has proved to be benefit against metastasis, its wide application is severely restricted due to the bleeding risk. Spatholobi Caulis, a traditional Chinese herb with circulatory promotion and blood stasis removal activity, has been proved to be clinically effective in malignant medication, leaving its mechanistic relevance to tumor-platelet interaction largely unknown. MethodsFirstly, MC38-Luc cells were injected into tail-vein in C57BL/6 mice to establish hematogenous metastasis model and the anti-metastasis effects of SEA were evaluated by using a small-animal imaging system. Then, we evaluated the anti-tumor-platelet interaction efficacy of SEA using a tumor-specific induced platelet aggregation model. Platelet aggregation was specifically induced by tumor cells in vitro. Furthermore, to clarify the anti-metastatic effects of SEA is mainly attributed to its blockage on tumor-platelet interaction, after co-culture with tumor cells and platelets (with or without SEA), MC38-Luc cells were injected into the tail-vein and finally count the total of photons quantitatively. Besides, to clarify the blocking pattern of SEA within the tumor-platelet complex, the dependence of SEA on different fractions from activated platelets was tested. Lastly, molecular docking screening were performed to screen potential effective compounds and we used β-catenin blockers to verify the pathways involved in SEA blocking tumor-platelet interaction. ResultsOur study showed that SEA was effective in blocking tumor-platelet specific interaction: (1) Through CCK-8 and LDH assays, SEA showed no cytotoxic effects on tumor cells and platelets. On this basis, by the tail vein injection model, the photon counts in the SEA group was significantly lower than model group, indicating that SEA effectively reduced metastasis. (2) In the "tumor-platelet" co-culture model, SEA effectively inhibited the progression of EMT and cancer stemness signatures of MC38 cells in the model group. (3) In mechanism study, by using the specific inhibitors for galectin-3 (GB1107) andWNT (IWR) respectively, we proved that SEA inhibits the activation of the galectin-3-mediated β-catenin activation. ConclusionBy highlighting the pro-metastatic effects of galectin-3-mediated tumor-platelet adhesion, our study provided indicative evidence for Spatholobi Caulis as the representative candidate for anti-metastatic therapy.

Molecular mechanism of Spatholobi Caulis treatment for cholangiocarcinoma based on network pharmacology, molecular docking, and molecular dynamics simulation.

Cholangiocarcinoma (CCA) is a type of malignant tumor originating from the intrahepatic, periportal, or distal biliary system. The treatment means for CCA is limited, and its prognosis is poor. Spatholobi Caulis (SC) is reported to have effects on anti-inflammatory and anti-tumor, but its role in CCA is unclear. First, the potential molecular mechanism of SC for CCA treatment was explored based on network pharmacology, and the core targets were verified by molecular docking and molecular dynamics simulation. Then, we explored the inhibitory effect of SC on the malignant biological behavior of CCA in vitro and in vivo and also explored the related signaling pathways. The effect of combination therapy of SC and cisplatin (DDP) in CCA was also explored. Finally, we conducted a network pharmacological study and simple experimental verification on luteolin, one of the main components of SC. Network pharmacology analysis showed that the core targets of SC on CCA were AKT1, CASP3, MYC, TP53, and VEGFA. Molecular docking and molecular dynamics simulation indicated a good combination between the core target protein and the corresponding active ingredients. In vitro, SC inhibited proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of CCA cells. In vivo experiments, the results were consistent with in vitro experiments, and there was no significant hepatorenal toxicity of SC at our dosage. Based on KEGG enrichment analysis, we found PI3K/AKT signaling pathway might be the main signaling pathway of SC action on CCA by using AKT agonist SC79. To explore whether SC was related to the chemotherapy sensitivity of CCA, we found that SC combined with DDP could more effectively inhibit the progression of cholangiocarcinoma. Finally, we found luteolin may inhibit the proliferation and invasion of CCA cells. Our study demonstrates for the first time that SC inhibits the progression of CCA by suppressing EMT through the PI3K-AKT signaling pathway, and SC could enhance the effectiveness of cisplatin therapy for CCA.

Mechanism of herb pair containing Astragali Radix and Spatholobi Caulis in the treatment of myelosuppression based on network pharmacology and experimental investigation

Ethnopharmacological relevanceThe Astragali Radix and Spatholobi Caulis herb pair (ARSC) is one of the most commonly used herbal combinations for bone marrow suppression. According to traditional Chinese medicine, Astragali Radix strengthens the spleen and replenishes qi, while Spatholobi Caulis is a hematinic agent that promotes blood circulation and enrichment. The compatibility of the two helps the body to tonify the spleen and kidneys and compensate for visceral deficiencies. However, the multi-target mechanism of ARSC in bone marrow suppression has remained largely unknown. Aim of the studyThe aim of this study is to explore the key targets and signaling pathways of the traditional Chinese herbal pair ARSC for the treatment of bone marrow suppression. Materials and methodsThe active components of ARSC and targets for myelosuppression were screened using network databases. Cytoscape 3.8.0 was used to construct compound-target, compound-disease-target and protein-protein interaction (PPI) networks. Go-function and pathway enrichment analyses were performed to explore the potential mechanism. In vivo animal experiments were conducted to verify the molecular mechanisms. ResultsThe 36 active compounds were identified from the ARSC, and a total of 108 genes involved in myelosuppression were screened. VEGFA, IL6, TNF, JUN, STAT3, PTGS2, CASP3 and MMP9 genes were identified as potential drug targets in the PPI network analyzed by CytoHubba. Enrichment analysis indicated that ARSC may treat myelosuppression through various biological processes, such as apoptosis, TNF-α signaling pathway via NF-κB, PI3K/AKT/mTOR signaling pathway, IL6/JAK/STAT3 signaling pathway, P53 signaling pathway and G2/M checkpoint signaling pathway. The results of the experiment showed that the aqueous extract of ARSC significantly alleviated myelosuppression, reduced the apoptosis rate of bone marrow cells, upregulated the mRNA expression levels of TNF-α, IL-6 and VEGF, and promoted NF-κB phosphorylation in myelosuppressed mice. ConclusionsThis study identified the active components and relevant mechanisms of ARSC in the treatment of myelosuppression. Our findings predicted that ARSC could treat bone marrow suppression through multiple components, multiple targets and multiple pathways. Pharmacological experiments showed that ARSC alleviated fluorouracil-induced myelosuppression by reducing the apoptosis rate of bone marrow cells and regulating the TNF-α/NF-κB signaling pathway.

Unraveling the Antioxidant Capacity of Spatholobi caulis in Nonalcoholic Fatty Liver Disease: A Multiscale Network Approach Integrated with Experimental Validation.

Nonalcoholic fatty liver disease (NAFLD) is a global health problem that is closely associated with obesity and metabolic syndrome. Spatholobi caulis (SC) is a herbal medicine with potential hepatoprotective effects; however, its active compounds and underlying mechanisms have not been fully explored. In this study, we combined a multiscale network-level approach with experimental validation to investigate SC's antioxidant properties and their impact on NAFLD. Data collection and network construction were performed, and active compounds and key mechanisms were identified through multi-scale network analysis. Validation was conducted using in vitro steatotic hepatocyte models and in vivo high-fat diet-induced NAFLD models. Our findings revealed that SC treatment improved NAFLD by modulating multiple proteins and signaling pathways, including AMPK signaling pathways. Subsequent experiments showed that SC treatment reduced lipid accumulation and oxidative stress. We also validated SC's effects on AMPK and its crosstalk pathways, emphasizing their role in hepatoprotection. We predicted procyanidin B2 to be an active compound of SC and validated it using a lipogenesis in vitro model. Histological and biochemical analyses confirmed that SC ameliorated liver steatosis and inflammation in mice. This study presents SC's potential use in NAFLD treatment and introduces a novel approach for identifying and validating active compounds in herbal medicine.

Biological analysis of constituents in Spatholobi Caulis by UFLC-MS/MS: Enhanced quantification and application to permeability properties study in Caco-2 cell monolayer model

Major chemical constituents in medicinal materials are often used as the marker compounds of traditional Chinese medicine (TCM) for treating various diseases. For spatholobi caulis (SPC), it contains a variety of flavones, phenolic acid esters, and lignans which exert many pharmacological effects. However, the absorption and permeability properties of these constituents of SPC are still unclear and require further investigation. Different types and major compounds of SPC were chosen as representative constituents to study their absorption and transepithelial transport characteristics in the human intestinal epithelium-like Caco-2 cell monolayer model. 35 constituents of SPC were evaluated by using ultra fast liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UFLC-MS/MS) method, acetonitrile and water containing with 0.5 mM ammonium acetate were used as mobile phase, these analytes with good linear relationships (R2 was within 0.9967−0.9998), precision (CV values were less than 10.23 %, LLOQ was less than 13.69 %), accuracy (Mean of inter- and intra-day were within 85.02 %−111.61 % and 85.50−112.97 %, respectively) and stability (The mean was within 85.07 %−113.93 %), among which 16 analytes showed good permeability, 5 analytes were considered to be poorly permeable compounds, and the other 14 analytes were assigned for the moderately absorbed compounds in Caco-2 cell monolayer model. The further results showed that the absorption mechanism of 7 well absorbed compounds, 8-O-methylretusin (1), genistein (7), spasuberol B (16), naringenin (18), isoliquiritigenin (19), 4-hydroxy-3-methoxy cinnamic acid methyl ester (23) and (+)-epipinoresinol (31) in SPC was mainly passive diffusion, their bidirectional transport rate was correlated with the concentration and transport time. The chemical structures of these compounds could affect the permeability properties on the cell monolayer. This study demonstrated the utility of Caco-2 cell monolayer model for evaluating the absorption properties and initial mechanisms of compounds in SPC in vitro, and provided important basis for predicting oral bioavailability of SPC compounds.

Pharmacodynamic Material Basis and Potential Mechanism Study of Spatholobi Caulis in Reversing Osteoporosis.

To elucidate the mechanism of Spatholobi Caulis (SC) in treating osteoporosis (OP) integrated zebrafish model and bioinformatics. Skeleton staining coupled with image quantification was performed to evaluate the effects of SC on skeleton mineralization area (SSA) and total optical density (TOD). Zebrafish locomotor activity was monitored using the EthoVision XT. Bioactive compounds of SC and their corresponding protein targets were acquired from Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Potential therapeutic targets for OP were summarized through retrieving 5 databases, and then, the overlapping genes between SC and OP were acquired. The core genes were selected by CytoHubba. Subsequently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) functional analysis of the intersection target genes were carried out by R software. Finally, the molecular docking simulation was manipulated between the ingredients and the hub genes. Compared with the model group, SC significantly increased the SSA and TOD at 10 mg/mL and improved the locomotor activity in a dose-dependent manner (p < 0.001). 33 components of SC were associated with 72 OP-related genes including 10 core genes (MAPK1, VEGFA, MMP9, AKT1, AR, IL6, CALM3, TP53, EGFR, and CAT). Advanced Glycation End Product (AGE) Receptor for AGE (RAGE) signaling pathway was screened out as the principal pathway of SC in anti-OP. The bioactive components (Aloe-emodin, Emodin, Formononetin, Licochalcone A, Luteolin, and Lopac-I-3766) have excellent affinity to core genes (MAPK1, VEGFA, MMP9, AKT1, and IL6). SC had the hierarchical network characteristics of "multicomponents/multitargets/multifunctions/multipathways" in reversing OP, but AGE-RAGE signaling pathway may be the main regulatory mechanism.

Antioxidant Activity of Aqueous Extract of Spatholobi Caulis and Its Ameliorative Effect on CCl4 Induced Acute Liver Injury in Mice

The acute liver injury model of mice induced by carbon tetrachloride (CCl₄) was used to explore the protective effect of aqueous extract of Spatholobi Caulis on acute liver injury. Methods: The contents of total phenols and total flavonoids in the aqueous and ethanol extracts of Caulis spatholobi were determined, and the antioxidant activity was evaluated by DPPH, ABTS free radical scavenging rate and FRAP total reducing capacity. 30 C57BL/6J male mice were randomly divided into model group, bifendate group, the low-dose, medium-dose and high-dose groups of aqueous extract of Spatholobi Caulis (n = 6). The animal model of acute liver injury was established by intraperitoneal injection of CCl₄ after 7 days with corresponding drugs. The pathological changes of liver tissue were observed by H&E staining. Serum levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor α (TNF-α) were determined by ELISA. The indexes of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and antioxidant effects of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were determined. Results: Liver histomorphology observation showed that aqueous extract of Spatholobi Caulis could significantly improve liver cell injury and inflammatory cell infiltration, and significantly increased the activities of CAT, SOD and GSH-Px antioxidant enzymes in liver, decreased the contents of MDA and ALT and AST in serum, significantly down-regulated the levels of IL-1β, IL-6 and TNF-α in serum, and up-regulated the levels of IL-10 and other inflammatory factors. Conclusion: The aqueous extract of Spatholobi Caulis can improve acute liver injury induced by CCl₄ in mice through improving antioxidant and anti-inflammatory effects.

A Comprehensive Study on the Chemical Constituents and Pharmacokinetics of Erzhi Formula and Jiawei Erzhi Formula Based on Targeted and Untargeted LC-MS Analysis.

Erzhi formula (EZF) is a traditional Chinese medicine prescription, which has been widely used in the treatment of osteoporosis and premature ovarian failure. To enhance curative effects, the other two herbal medicines, including Spatholobi Caulis (SC) and Achyranthes bidentata Blume (ABB), were added into the original EZF formula to obtain two new Jiawei-EZF (JW-EZF) preparations. To clarify the effect of the compatibility of herbs for original formulas, the chemical constituents and bioactive compounds in vivo were detected. An efficient and sensitive targeted and untargeted UHPLC/ESI-Q-Orbitrap MS method, together with mass defect filter and precursor ion list, was established firstly for the profiling of different EZF formulas. Furthermore, eleven absorbed compounds (apigenin, luteoloside, luteolin, oleuropein, wedelolactone, acteoside, specnuezhenide, 11-methyloleoside, ecliptasaponin A, formononetin, and β-ecdysone) were simultaneously quantified in rat plasma. A total of 124, 162, and 177 compounds were identified or tentatively identified in EZF, JW-3-EZF (EZF+SC) and JW-4-EZF (EZF+SC+ABB), respectively. 110 compounds were found to be common constituents in the three formulas. Moreover, 66 prototypes were unambiguously identified in the rats' plasma after oral administration of the three formulas using the same strategy. 11 out of the 66 absorbed components were simultaneously quantitated in the pharmacokinetic (PK) study. Compared to the original EZF, the plasma AUC(0-24h) and AUC(0-∞) of apigenin, 11-methyloleoside, luteolin, luteoloside, wedelolactone, and acteoside were found to be significantly increased after oral administration of JW-3-EZF, and plasma AUC(0-24h) and AUC(0-∞) of apigenin, wedelolactone, and acteoside, were also found to be significantly increased after JW-4-EZF administration. The combined qualitative and quantitative methods were used to provide a potential approach to the characterization and quality control of the Traditional Chinese Medicine (TCM) and its preparations.

Chemical Profiling and Quantification of Potential Bioactive Components in Gandouling Pill by Ultra-High Performance Liquid Chromatography Coupled with Diode Array Detector/Quadruple-Qrbitrap Mass Spectrometry

Gandouling (GDL) Pill is a novel Traditional Chinese medicinal drug to treat Wilson's disease in clinics. It is composed of six separate herbal medicines, including Rhei Radix ET Rhizoma, Coptidis Rhizoma, Salviae Miltiorrhizae Radix ET Rhizoma, Spatholobi Caulis, Curcumae Rhizoma, and Curcumae Longae Rhizoma. In this study, a strategy was proposed to investigate the chemical constituents and to quantify the potential bioactive components in GDL Pill. Firstly, the mass fragmentation behaviors of representative compounds were investigated, and, in total, 69 compounds were characterized in GDL Pill using full scan/dd-MS2 scan mode by ultra-high-performance liquid chromatography (UPLC)/Q-Orbitrap mass spectrometry (MS). These compounds included 18 alkaloids, 18 ketones, 16 phenolic compounds, 11 organic acids, and 6 tanshinones. Seventeen of the compounds were unambiguously identified by comparison with reference standards. Secondly, the absorption components of GDL Pill in rat plasma were investigated by using target-Selected Ion Monitoring (t-SIM) scan mode built in Q-Orbitrap MS. A total of 18 components were detected, which were considered as potential bioactive components of GDL Pill. Thirdly, 10 major absorption components were simultaneously determined in six batches of samples by UPLC/diode array detector (DAD). The method was fully validated with respect to linearity, precision, repeatability, stability, and recovery. Alkaloids from Coptidis Rhizoma, such as coptisine (8), berberine (18), palmatine (19), were the most abundant bioactive compounds for GDL Pill that possess the potential be used as quality markers. The proposed strategy is practical and efficient for revealing the material basis of GDL Pill, and also provides a simple and accurate method for quality control.

Coordination of AMPK and YAP by Spatholobi Caulis and Procyanidin B2 Provides Antioxidant Effects In Vitro and In Vivo.

The liver is vulnerable to oxidative attacks from heavy metals, such as iron, as well as some drugs, including acetaminophen. It has been shown that enhanced oxidative stress in the liver leads to excessive ROS production and mitochondrial dysfunction, resulting in organ injury. The beneficial effects of Spatholobi Caulis (SC), a natural herbal medicine, include treating ischemic stroke, inhibiting tumor cell invasion, pro-angiogenic activities, and anti-inflammatory properties. Scientific studies on its effects against hepatotoxic reagents (e.g., iron and acetaminophen), as well as their underlying mechanisms, are insufficient. This study examined the antioxidant effects and mechanisms of SC in vitro and in vivo. In cells, the proinflammatory mediator, arachidonic acid (AA), plus iron, significantly induced an increase in ROS generation, the damage in mitochondrial membrane potential, and the resulting apoptosis, which were markedly blocked by SC. More importantly, SC affected the activation of AMP-activated protein kinase (AMPK)-related proteins, which were vital to regulating oxidative stress in cells. In addition, SC mediated the expression of Yes-associated protein (YAP)-related proteins. Among the active compounds in SC, the procyanidin B2, but not liquiritigenin, daidzein, and genistein, significantly inhibited the cytotoxicity induced by AA + iron, and activated the LKB1-AMPK pathway. In mice, the oral administration of SC alleviated the elevations of ALT and histological changes by the acetaminophen-induced liver injury. These results reveal the potential of SC and a key bioactive component, procyanidin B2, as antioxidant candidates for hepatoprotection.

Unveiling Potential Mechanisms of Spatholobi Caulis against Lung Metastasis of Malignant Tumor by Network Pharmacology and Molecular Docking.

Background Lung metastasis of malignant tumor signifies worse prognosis and immensely deteriorates patients' life quality. Spatholobi Caulis (SC) has been reported to reduce lung metastasis, but the mechanism remains elusive. Methods The active components and corresponding targets of SC were obtained from the Traditional Chinese Medicine Database and Analysis Platform (TCMSP) database and the SwissTargetPrediction database. The disease targets were acquired from DisGeNET and GeneCards databases. Venn map was composed to figure out intersection targets by using R. The PPI network was constructed through STRING and Cytoscape, and MCODE plug-in was used to sift hub targets. Gene Ontology (GO)-Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was carried out by utilizing clusterProfiler package (R3.6.1) with adjusted P value <0.05. Network of SC-active components-intersection targets-KEGG pathway was accomplished with Cytoscape. Molecular docking between hub targets and active components was performed, analyzed, and visualized by AutoDockTools, AutoDock Vina, PLIP Web tool, and PYMOL. Results 24 active components and 123 corresponding targets were screened, and the number of disease targets and intersection targets was 1074 and 47, respectively. RELA, JUN, MAPK1, MAPK14, STAT3, IL-4, ESR1, and TP53 were the 8 hub targets. GO analysis and KEGG analysis elucidated that SC could ameliorate lung metastasis mainly by intervening oxidative stress, AGE-RAGE signaling pathway, and microRNAs in cancer. All 8 hub targets were proven to combine successfully with active components of SC. Conclusion Inflammation is the core factor that integrates all these targets, biological process, and signaling pathways, which indicates that SC prevents or reduces lung metastasis mainly by dispelling inflammation.

The prescription rule analysis of the new theory on spleen dampness syndrome by academician TONG Xiaolin

ObjectiveTo analyze the basic characteristics, drug features, prescription rules, and drug-symptom relationships of patients in the splenic deficiency and impairment stage, by data mining of medical records under the New Theory on Spleen Dampness Syndrome (Pi Dan Xin Lun, 《脾瘅新论》). MethodsMedical records listed in the “New Theory on Spleen Dampness Syndrome – Understanding and Treatment of Metabolic Syndrome from the Perspective of Traditional Chinese Medicine”, and which were diagnosed with the spleen dampness syndrome at the splenic deficiency and impairment stage, during January 2004 and December 2016 were selected. These patients’ data, including basic information, clinical symptoms, laboratory examination results, traditional Chinese medicine (TCM) and western medicine diagnoses, treatment methods, prescriptions, etc., were collected. The collected data were subsequently compiled into a medical record database using the Epidata 3.1 data management software, followed by the use of Apriori algorithm provided in the SPSS Modeler 14.2 statistical software to investigate the association rules between drug-drug, drug-symptom, and drug-western medicine indices. Results(i) A total of 51 medical records were included, involving 17 types of syndromes. Among them, the top three with frequency ≥3 included “Phlegm and blood stasis, and thoracic obstruction” “Deficiency-weakness of the spleen Qi, and static blood blocking collaterals”, and “Deficiency-weakness of the spleen Qi, and static blood blocking collaterals”. Alternatively, of the 14 treatment methods, the top three treatments with frequency of ≥3 included “Activating Yang and eliminating turbidity, and removing phlegm and dredging channel blockage” “Strengthening the spleen and benefiting Qi, and eliminating phlegm to activate the channels”, and “Warming Yang and benefiting Qi, and expelling cold to remove obstructions”. Among the 15 prescriptions, the top three used with frequency ≥3 included Huangqi Guizhi Wuwu Tang (黄芪桂枝五物汤), Gualou Xiebai Banxia Tang (瓜蒌薤白半夏汤), and Ganjiang Huangqin Huanglian Renshen Tang (干姜黄芩黄连人参汤). Lastly, of the 83 drugs used for a total of 476 times, those with frequency ≥15 included Huanglian (Coptidis Rhizoma), Huangqi (Astragali Radix), Jiudahuang (Wine-processed Rhei Radix et Rhizoma), Jixueteng (Spatholobi Caulis), Shengjiang (Zingiberis Rhizoma Recens), Huangqin (Scutellariae Radix), and Guizhi (Cinnamomi Ramulus). (ii) For the drug-drug associations, under the criteria of support ≥15% and confidence = 100%, 7 s-order association rules, seven third-order rules, and six fourth-order roles were identified. The top-ranking rule of each was “Huangqin (Scutellariae Radix) → Huanglian (Coptidis Rhizoma)” “Ganjiang (Zingiberis Rhizoma) + Huangqin (Scutellariae Radix) → Huanglian (Coptidis Rhizoma)”, and “Baishao (Paeoniae Radix Alba) + Guizhi (Cinnamomi Ramulus) + Jixueteng (Spatholobi Caulis) → Huangqin (Scutellariae Radix)”, respectively. Alternatively, the drug-symptom associations were analyzed under the criteria of support ≥5% and confidence = 100%, which derived 8 s-order association rules, 31 third-order rules, and 30 fourth-order rules. The top-ranking association rule of each order was “Huangqi (Astragali Radix) → Limb edema” “Guizhi (Cinnamomi Ramulus) + Jixueteng (Spatholobi Caulis) → Limb numbness and pain”, and “Guizhi (Cinnamomi Ramulus) + Jixueteng (Spatholobi Caulis) + Huangqi (Astragali Radix) → Limb numbness and pain”, respectively. Similarly, the drug-western medicine index associations were investigated under the criteria of support ≥5% and confidence = 100%, and 5 s-order association rules, 16 third-order rules, and 16 fourth-order rules were identified. In this category, the top-ranking association rule of each order was “Qinpi (Fraxini Cortex) → Uric acid” “Huanglian (Coptidis Rhizoma) + Ganjiang (Zingiberis Rhizoma) → Glycated hemoglobin”, and “Huanglian (Coptidis Rhizoma) + Ganjiang (Zingiberis Rhizoma) + Huangqin (Scutellariae Radix) → Glycated hemoglobin”, respectively. ConclusionThrough association rule mining, this study objectively and quantitatively demonstrated the drug-drug, drug-symptom, and drug-physicochemical index associations of patients with the spleen dampness syndrome at the splenic deficiency and impairment stage treated by Academician TONG Xiaolin. The results indicated that treatment for these patients adopted the “state-target” syndrome differentiation method. The drug combination was characterized by “small prescriptions”, targeting both the patient’s symptoms and signs (syndrome target) and western medicine indices (treatment target). This study could provide references for future research on the academic thoughts and medical experience of Academician TONG Xiaolin.

Molecular mechanism of Spatholobi Caulis in treatment of ovarian cancer based on network pharmacology and experimental verification

The present study explored the main active ingredients and the underlying mechanism of Spatholobi Caulisin the treatment of ovarian cancer(OC) by network pharmacology, molecular docking, and in vitro cell experiments. The active ingredients and their predicted targets(AITs) were first acquired online with the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). Theoretical disease targets(DTs) were obtained through professional databases including GeneCards, OMIM, PharmGkb, TTD, and DrugBank. The common targets in the intersection of AITs and DTs were used for the construction of a &quot;drug-ingredient-disease-target&quot; network by Cytoscape 3.7.1. STRING database was used to construct a protein-protein interaction(PPI) network. R 4.0.5 was used for GO and KEGG functional enrichment analyses. Schr9 dinger Maestro was used to perform and optimize the molecular docking and virtual screening.Twenty-three active ingredients of Spatholobi Caulis were screened out, involving 75 OC targets and 178 signaling pathways.Network analysis revealed that Spatholobi Caulis presumedly exerted an anti-OC effect by acting on key protein targets such as GSK-3β, Bcl-2, and Bax. Molecular docking showed that GSK-3β possessed goodbinding activity to prunetin. In vitro cell experiments preliminarily verified the core targets and pathways of prunetin, the active ingredient of Spatholobi Caulis against human OC SKOV3 cells.CCK-8 assay was used to detect the cell proliferation, and flow cytometry was used to detect the effect of prunetin on apoptosis of human OC SKOV3 cells.The expression of prunetin targets and related regulatory proteins was detected by Western blot.In vitro cell experiments demonstrated that prunetindisplayed significant inhibitory effects on the proliferation of OC cells and could induce apoptosis of SKOV3 cells. Western blot showed that prunetin could induce SKOV3 cell apoptosis by inhibiting GSK-3β phosphorylation and regulating the expression of downstream Bcl-2 and Bax proteins. This study reveals the scientific nature of network pharmacology in the prediction and guidance of experimental design, confirming that prunetin can treat OC by blocking the GSK-3β/Bcl-2/Bax cell signal transduction pathway. The findings are expected to provide a basis for the investigation of the mechanism of Spatholobi Caulis in the treatment of OC.

Effect of Spatholobi Caulis Extract in Acute Liver Injury

This study aimed to examine the protective effect of the water extract from Spatholobi Caulis (SC) on rats with acute liver injury (ALI) induced by thioacetamide (TAA) and its underlying mechanism. ALI was induced by intraperitoneal injection with TAA for 3 days in a row and the rats were administered SC (100 or 200 mg/kg) 1 h 30 min prior to the TAA treatment. Myeloperoxidase (MPO) was measured in serum and malondialdehyde (MDA) was measured in serum and liver tissue, as markers related to oxidative stress. Further, inflammation-related proteins and apoptosis proteins were investigated using western blot analysis. SC treatment significantly reduced the levels of MPO and MDA. Further, SC treatment significantly reduces the mitogen-activated protein kinase (MAPK) signaling pathway, including p-JNK, p-ERK, and p-p38, and reduces the expression of pro-inflammatory signaling factors. Furthermore, it effectively decreased the expression of Bax, Cytochrome C, and Cleaved Caspase-3 in liver tissue. Taken together, SC treatment alleviate0d the severity of ALI via inhibition of inflammation through both the MAPK pathway and Matrix metalloproteinase (MMPs)-dependent apoptosis.

Simultaneous determination of twenty-five compounds with anti-inflammatory activity in Spatholobi Caulis by using an optimized UFLC-MS/MS method: An application to pharmacokinetic study

As a kind of commonly used Traditional Chinese Medicine in clinical, Spatholobi Caulis (SPC) contains a wide variety of bioactive compounds, including protocatechuate (1), nicotinic acid (2), p-hydroxybenzoic acid (3), salicylic acid (4), 6,9-dihydroxy megastigma-4,7-dien-3-one (5), 8,9-dihydroxy megastigma-4,6-dien-3-one (6), daidzin (7), genistin (8), isolariciresinol (9), ononin (10), 4′,8-dimethoxy-7-O-β-d-glucopyranosyl isoflavone (11), 3′-methoxydaidzein (12), odoratin (13), spasuberol A (14), (+)-pinoresinol (15), 4-hydroxy-3-methoxy cinnamic acid methyl ester (16), (+)-epipinoresinol (17), calycosin (18), 8-O-methylretusin (19), formononetin sodium (20), formononetin (21), biochanin A (22), butesuperin A (23), homovanillyl-4-oxo-nonanoate (24) and (6aR,11aR)-maackiain (25). The pharmacokinetic characteristics of these twenty-five compounds in rat plasma were quantitatively and simultaneously studied using a fast, sensitive and precise ultra fast liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UFLC-MS/MS) method after oral administration of aqueous extract of SPC to rats. The mobile phase consists of acetonitrile and 0.5 mM ammonium acetate in water, and these compounds were well separated at a gradient elution program with flow rate of 0.35 mL/min. Carbamazepine was employed as the internal standard (IS) and all samples were precipitated with MeOH-ACN (2:1, v/v). The analytical method has been proved to be good linearity (R2 ≥ 0.9957), precise, accurate, stable, recovery and matrix effect, which applicated becomingly to study the pharmacokinetic processes of these compounds in rat plasma. In addition, these twenty-five compounds exhibited anti-inflammatory activity on the inflammatory model of NO over production in RAW264.7 cells stimulated by lipopolysaccharide (LPS). Isoflavones, especially compounds 20-22 (The IC50 of which were 22.75 μM, 21.11 μM and 48.29 μM, respectively.) might be the important constituents for anti-inflammatory activity of SPC. This study provides reference values for the clinical application, in-depth study on new dosage forms and pharmacological activities of SPC.