Oxysophocarpine suppresses TRAF6 level to ameliorate oxidative stress and inflammatory factors secretion in mice with dextran sulphate sodium (DSS) induced-ulcerative colitis.

PURPOSE: Ulcerative colitis is an inflammation-related disease with a high recurrence risk. Oxysophocarpine (OSC) is a traditional Chinese medicine isolated from legumes and exerts vital functions on many human diseases. However, the OSC's role in ulcerative colitis has not been fully elucidated. This research aimed to investigate the OSC's impact on ulcerative colitis and its mechanisms. METHODS: A mouse model of ulcerative colitis was induced by dextran sulphate sodium (DSS). The effect of OSC on ulcerative colitis was examined using Disease Activity Index detection, hematoxylin-eosin (HE) staining, and enzyme-linked immunosorbent assay (ELISA). Meanwhile, the mechanism of OSC in ulcerative colitis was assessed by immunohistochemistry assay, Western blot, HE staining, and ELISA. RESULTS: For the OSC's function in ulcerative colitis, OSC increased the mice weight, decreased Disease Activity Index scores, and alleviated colitis cell infiltration and epithelial cell destruction in DSS-induced ulcerative colitis. Also, OSC mitigated oxidative stress (decreased PGE2, MPO levels, and increased SOD levels) and inflammation (decreased IL-6, TNF-α and IL-1ß levels) in DSS-induced ulcerative colitis. For the OSC's mechanism in ulcerative colitis, OSC inhibited the level of tumor necrosis factor receptor-associated Factor 6 (TRAF6) and the phosphorylation of nuclear factor-κB (NF-κB). TRAF6 overexpression abolished the effect of OSC on DSS-induced colon injury and its associated oxidative stress and inflammatory properties in ulcerative colitis. CONCLUSION: OSC decreased the TRAF6 level to reduce oxidative stress and inflammatory factors secretion in mice with DSS induced-ulcerative colitis.

Oxysophocarpine inhibits airway inflammation and mucus hypersecretion through JNK/AP-1 pathway in vivo and in vitro.

Asthma is a high-incidence disease in the world. Oxysophocarpine (OSC), a quinolizidine alkaloid displays various pharmacological functions including anti-inflammation, neuroprotective, anti-virus and antioxidant. Here, we established mice and cell asthmatic model to explore the effects of OSC for asthma treatment. Mice were sensitized and challenged with ovalbumin (OVA) and treated with OSC before challenge. Enzyme-linked immuno sorbent assay (ELISA), hematoxylin and eosin (H&E), periodic acid-schiff (PAS), tolonium chloride staining and immunohistochemical assay were performed. OSC treatment inhibited inflammatory cell infiltration and mucus secretion in the airway, reduced IgE level in mouse serum and decreased IL-4, IL-5 production in bronchoalveolar lavage fluid (BALF). OSC also reduced the spleen index to regulate immune function. Meanwhile, NCI-H292 cells were induced by lipopolysaccharide (LPS) to simulate airway epithelial injury. OSC pretreatment decreased the IL-6 and IL-8 cytokine levels, mucin 5 AC expression, and mucin 5 AC mRNA level in the cell model. Further, OSC suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1, Fos and Jun). These findings revealed that OSC alleviated bronchial asthma associated with JNK/AP-1 signaling pathway.

Oxysophocarpine suppresses FGFR1-overexpressed hepatocellular carcinoma growth and sensitizes the therapeutic effect of lenvatinib.

AIMS: Hepatocellular carcinoma (HCC) is an aggressive solid tumor with restricted therapeutics. Lenvatinib is the second approved frontline drug for advanced HCC, however lenvatinib-resistant cases have been reported in clinical. Overexpression of fibroblast growth factor receptor (FGFR1) has been found to be associated with advanced HCC. This study was aimed to investigate the relationship between FGFR1 overexpression and lenvatinib resistance, and explore the potential candidate that can sensitize lenvatinib against FGFR1-overexpressed HCC. MAIN METHODS: Development of FGFR1 overexpression was accomplished in Hep3B and HepG2 cell lines by pCDH-FGFR1 lentiviral vector. In vitro, cell proliferation, colony formation, cell migration and cell apoptosis assays were used to explore the effect of lenvatinib and Oxysophocarpine. In vivo, BALB/c nude mice were burdened with subcutaneous FGFR1-overexpressed Hep3B tumor to assess the therapeutic effect of lenvatinib and Oxysophocarpine. qRT-PCR and western blotting were further used to identify the underlying mechanism. KEY FINDINGS: Here, we revealed that overexpressed FGFR1 and its downstream AKT/mTOR and ERK signaling activation could induce lenvatinib resistance in HCC. In vivo and in vitro results showed Oxysophocarpine inhibited the proliferation and induced the apoptosis of FGFR1-overexpressed HCC cells. Oxysophocarpine could further sensitize FGFR1-overexpressed HCC cells to lenvatinib treatment. Mechanism studies revealed that Oxysophocarpine downregulated FGFR1 expression along with downstream AKT/mTOR and ERK signaling to sensitize lenvatinib against FGFR1-overexpressed HCC. SIGNIFICANCES: These data collectively provided evidence that FGFR1 overexpression could be a potential cause of lenvatinib resistance and Oxysophocarpine could be an ideal combined therapy with lenvatinib in HCC treatment.

Oxysophocarpine suppresses hepatocellular carcinoma growth and sensitizes the therapeutic blockade of anti-Lag-3 via reducing FGL1 expression.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with limited effective treatments and ranks as the second most lethal tumor. Immunotherapy has brought great hope for HCC treatment. Oxysophocarpine is a bioactive alkaloid which poses various pharmacological functions including neuroprotective, anti-virus, anti-convulsant, and anti-nociception. However, there is little systematic study of Oxysophocarpine against HCC and its underlying potential and mechanism combined with immunotherapy in HCC treatment remain poorly unknown. This study was aimed to investigate whether Oxysophocarpine can distinctly suppress HCC cells and sensitize the immunotherapy of CD8+ T cells against HCC. We used HepG2, Hepa1-6, and primary CD8+ T cells to perform in vitro assays and Hepa1-6 subcutaneous tumor to conduct in vivo assay. Oxysophocarpine inhibited the proliferation and increased the apoptosis of HepG2 and Hepa1-6 cells, meanwhile suppressed the migration of HepG2 and Hepa1-6 cells. Oxysophocarpine sensitized the Lag-3 immunotherapy effect of CD8+ T cells against HCC in vivo and in vitro by decreasing Fibrinogen-like protein 1 (FGL1) expression through downregulating IL-6-mediated JAK2/STAT3 signaling, whereas Oxysophocarpine treatment had a little effect of CD8+ T cells cytotoxicity function against HCC with PD-1, Tim-3, or TIGIT blockade. Our studies provided preclinical basis for clinical application of Oxysophocarpine.

Oxysophocarpine reduces oxidative stress and inflammation in tuberculosis-infected neutrophils and mouse lungs.

Tuberculosis (TB) is a chronic inflammatory infectious disease caused by Mycobacterium tuberculosis (Mtb), which induces irreversible pulmonary damage. Oxysophocarpine (OSC) is a natural alkaloid that exhibits multiple pharmacological activities, including anti-inflammation; however, the protective effects of OSC against TB and the mechanisms involved are unknown. Here, we established murine and cellular models of TB with C3HeB/FeJ mice and neutrophils infected with H37Rv to investigate the biological functions of OSC in TB. We found that OSC reduced the mortality, inhibited the pulmonary H37Rv growth, and alleviated the lung pathology injury in the Mtb-infected mice. OSC also repressed neutrophil recruitment to the lesions of the Mtb-infected mice as evidenced by a decrease in the number and percentage of neutrophils in the lungs. OSC hampered the production of proinflammatory cytokines and chemokines, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, macrophage inflammatory protein-2 (MIP-2), granulocyte colony stimulating factor (G-CSF), and keratinocyte chemoattractant (KC) in the lungs of Mtb-infected mice. The results of the in vitro experiments showed that OSC repressed the adhesion and F-actin polymerization of the Mtb-infected neutrophils by inhibiting the toll-like receptor 2/myeloid differentiation primary response gene 88/Src/extracellular signal-regulated kinase 1/2 signaling. Moreover, OSC abolished the Mtb-induced expression and release of TNF-α, IL-1ß, IL-6, MIP-2, G-CSF, and KC in neutrophils. Overall, these findings indicate that OSC can treat TB partly by lessening the neutrophilic recruitment and inflammation.

Oxysophocarpine protects airway epithelial cells against inflammation and apoptosis by inhibiting miR-155 expression.

Oxysophocarpine (OSC) has been documented for anti-inflammatory activity. However, the mechanisms of OSC in anti-inflammation are unclear. Aim: To investigate the protective effects of OSC on inflammation and apoptosis induced by lipopolysaccharide in NCI-H292 and human primary airway epithelial cells. Materials & methods: MTT and Annexin V-FITC/PI staining were used to detect cells viability. Inflammatory responses were determined by ELISA. The quantitative real-time PCR (qRT-PCR) and western blot were used to detect mRNA/miRNA and protein expressions respectively. Co-immunoprecipitation was investigated for protein interactions. Results & conclusion: miR-155 mimics significantly induced cell apoptosis, inflammatory responses and MAPK and NF-κB pathways. NDFIP1 was identified as the target of miR-155. OSC protected cells against apoptosis and inflammatory responses and compromised miR-155 activity by attenuating MAPK and NF-κB pathways.

Sophora alopecuroides L.: An ethnopharmacological, phytochemical, and pharmacological review.

ETHNOPHARMACOLOGICAL RELEVANCE: Sophora alopecuroides L., which is called Kudouzi in China, is a medicinal plant distributed in Western and Central Asia, especially in China, and has been used for decades to treat fever, bacterial infection, heart disease, rheumatism, and gastrointestinal diseases. AIM OF THE REVIEW: This review aims to provide up-to-date information on S. alopecuroides, including its botanical characterization, medicinal resources, traditional uses, phytochemistry, pharmacological research, and toxicology, in exploring future therapeutic and scientific potentials. MATERIALS AND METHODS: The information related to this article was systematically collected from the scientific literature databases including PubMed, Google Scholar, Web of Science, Science Direct, Springer, China National Knowledge Infrastructure, published books, PhD and MS dissertations, and other web sources, such as the official website of Flora of China and Yao Zhi website (https://db.yaozh.com/). RESULTS: A total of 128 compounds, such as alkaloids, flavonoids, steroids, and polysaccharides, were isolated from S. alopecuroides. Among these compounds, the effects of alkaloids, such as matrine and oxymatrine, were extensively studied and developed into new drugs. S. alopecuroides and its active components had a wide range of pharmacological activities, such as anticancer, antiviral, anti-inflammatory, antimicrobial, analgesic, and neuroprotective functions, as well as protective properties against pulmonary fibrosis and cardiac fibroblast proliferation. CONCLUSIONS: As an important traditional Chinese medicine, modern pharmacological studies have demonstrated that S. alopecuroides has prominent bioactivities, especially on gynecological inflammation and hepatitis B, and anticancer activities. These activities provide prospects for novel drug development for cancer and some chronic diseases. Nevertheless, the comprehensive evaluation, quality control, understanding of the multitarget network pharmacology, long-term in vivo toxicity, and clinical efficacy of S. alopecuroides require further detailed research.

Study on Vasodilatory Effect of Oxysophocarpine on Isolated Thoracic Aortic Rings of Rats and Its Mechanism / 中国药房

OBJECTIVE： To study the vasodilatory effect of oxysophocarpine （OSC） on isolated thoracic aortic rings of rats and its possible mechanism. METHODS： Thoracic aortic rings of rats were collected （called “vascular ring” for short）. Using K-H nutrient solution as blank control and the diastolic rate as index， the effects of different concentrations （0.2-1.0 mg/mL） of OSC on normal vascular rings in basal state， normal or endothelium-free vascular rings pre-contracted by norepinephrine （PE， 1×10－6 mol/L） were investigated. After pre-culturing normal thoracic aortic rings by nitric oxide synthase inhibitor L-nitro-arginine methyl ester（L-NAME）and cyclooxygenase inhibitor indomethacin（INDO），as well as pre-culturing endothelium-free vascular rings by potassium ion channel blocker BaCl2，tetraethylammonium（TEA）and 4-aminopyridine（4-AP）， the diastolic effects of OSC of different concentrations （0.2-1.0 mg/mL） on the above vascular rings were investigated by using the same method. RESULTS： Compared with blank control， there was no significant effects of different concentrations of OSC on the diastolic rate of normal vascular rings in basal state （P＞0.05）， but 0.4-1.0 mg/mL OSC could significantly improve the diastolic rate of normal or endothelium-free vascular rings pre-contracted by PE （P＜0.01）， in concentration-dependent manner. After preculturing with L-NAME， INDO， 4-AP and BaCl2， different concentrations of OSC had no significant effect on the diastolic rate of normal or endothelium-free vascular rings pre-contracted by PE （P＞0.05）. After pre-culturing with TEA and Gli， 0.4-1.0 mg/mL OSC could significantly reduce the diastolic rate of endothelium-free vas- cular rings pre-contracted by PE （P＜0.01）. CONCLUSIONS： OSC did not significantly dilate the thoracic aortic rings of rats in the basal state within the dose range （0.2-1.0 mg/mL）， but OSC of 0.4-1.0 mg/mL have significant diastolic effects on the normal or endothelium-free thoracic aortic rings of rats pre-contracted with PE. The mechanism of thoracic aortic rings dilation is endothelium-independent， which may be associated with receptor operational calcium channel，Ca2+-activated potassium channels and ATP-sensitive potassium channels.

Optimization of Extraction Process for Active Ingredients in Seeds of Sophora alopecuroides by Plackett-Burman Design and Response Surface Analysis / 中国药学杂志

OBJECTIVE: To optimize extraction process for active ingredients in seeds of Sophora alopecuroides, to provide a reference for scale production. METHODS: Active ingredients from Sophora alopecuroides were extracted by ethanol, with average yield of oxysophocarpine and oxymatrine as index, some factors affecting index were firstly evaluated by Plackett-Burman design, then taking oxysophocarpine and oxymatrine as indexes respectively, extraction conditions were optimized by Box-Behnken design, experimental data was fitted by multiple linear regression and binomial formula fitting, extraction process was optimized by response surface method, and prediction was carried out through comparing the observed and predicted value. RESULTS: Extracting times, crushing degree and solvent times had significant effects on yields of oxysophocarpine and oxymatrine; binomial equation fitted well with good predictability. optimum extraction technology of Sophora alopecuroides was as following:crushed through 65 mesh sieve, extracted 4 times with 12-fold the amount of 60% ethanol for 2 h each time; yield of oxysophocarpine and oxymatrine was 92.3%, 78.6% respectively, both deviations were small by comparing with the predicted value. CONCLUSION: This extraction process is reasonable and feasible by Plackett-Burman design and response surface analysis with good predictability. This study can provide experimental basis for further scale production of Sophora alopecuroides.

Oxysophocarpine Retards the Growth and Metastasis of Oral Squamous Cell Carcinoma by Targeting the Nrf2/HO-1 Axis.

BACKGROUND/AIMS: Nuclear factor erythroid 2-related factor 2 (Nrf2) is an oncogene in various types of cancers, including oral squamous cell carcinoma (OSCC). Oxysophocarpine (OSC) is a natural alkaloid that has multiple pharmacological activities. However, the biological functions and molecular mechanism underlying the effects of OSC on the growth and metastasis of OSCC are unclear. METHODS: Nrf2 levels were determined in OSCC tissues and non-cancerous specimens by quantitative real-time PCR, western blotting, and immunohistochemistry (IHC) assays. The effects of OSC on OSCC cell growth and metastasis were explored (1) using 5-ethynyl-20-deoxyuridine staining and Cell Counting Kit-8, colony formation, flow cytometry, wound-healing, Transwell, and tube formation assays in vitro; and (2) by establishing a xenograft nude mouse model in vivo. The molecular mechanisms underlying the effects of OSC on the growth and metastasis of OSCC were investigated in vitro by western blotting, caspase-3 activity, and enzyme-linked immunosorbent assays, and in vivo by western blotting and IHC assays. RESULTS: The expression levels of Nrf2 in OSCC tissues and in cell lines were much higher than in non-cancerous tissues and normal oral keratinocytes. The upregulation of Nrf2 was positively correlated with a high incidence of lymph node metastasis and advanced histological grade and TNM stage, but inversely associated with differentiation and survival of OSCC patients. OSC reduced the expression of Nrf2 and heme oxygenase 1 (HO-1) in OSCC cells. OSC also inhibited proliferation, migration, invasion, and pro-angiogenesis of OSCC cells. Moreover, OSC induced cell cycle arrest, enhanced apoptosis of OSCC cells in vitro, and decreased OSCC tumor growth in vivo. Mechanically, OSC reduced the aggressive behavior of OSCC cells by inactivation of the Nrf2/HO-1 signaling pathway. CONCLUSION: Our findings provide evidence that OSC inhibits the growth and metastasis of OSCC by targeting the Nrf2/ HO-1 axis, suggesting that OSC may be a potential therapeutic agent for OSCC.

Neuroprotective Effect of Oxysophocarpine by Modulation of MAPK Pathway in Rat Hippocampal Neurons Subject to Oxygen-Glucose Deprivation and Reperfusion.

Oxysophocarpine (OSC), an alkaloid isolated from Sophora flavescens Ait, has been traditionally used as a medicinal agent based on the observed pharmacological effects. In this study, the direct effect of OSC against neuronal injuries induced by oxygen and glucose deprivation (OGD) in neonatal rat primary-cultured hippocampal neurons and its mechanisms were investigated. Cultured hippocampal neurons, which were exposed to OGD for 2 h followed by a 24 h reoxygenation, were used as an in vitro model of ischemia and reperfusion. 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) assay were used to confirm neural damage and to further evaluate the protective effects of OSC. The concentration of intracellular-free calcium [Ca2+]i and mitochondrial membrane potential (MMP) were measured to determine the intracellular mechanisms and to further estimate the degree of neuronal damage. Changes in expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, p-ERK1/2, p-JNK1/2, and p-p38 MAPK were also observed in the in vitro model. It was shown that OSC (0.8, 2, or 5 µmol/L) significantly attenuated the increased absorbance of MTT, and the release of LDH manifests the neuronal damage by the OGD/R. Meanwhile, the pretreatment of the neurons during the reoxygenation period with OSC significantly increased MMP; it also inhibited [Ca2+]i the elevation in a dose-dependent manner. Furthermore, the pretreatment with OSC (0.8, 2, or 5 µmol/L) significantly down-regulated expressions of IL-1ß, TNF-α, p-ERK1/2, p-JNK1/2, and p-p38 MAPK in neonatal rat primary-cultured hippocampal neurons induced by OGD/R injury. In conclusion, OSC displays a protective effect on OGD-injured hippocampal neurons by attenuating expression of inflammatory factors via down-regulated the MAPK signaling pathway.

Discussion and consideration on safety of Sophorae Tonkinensis Radix et Rhizoma and its rational use / 中草药

Sophorae Tonkinensis Radix et Rhizoma is known as the important drug for treating sore throat and is widely used in clinic. Because of the coexisting of its efficacy and poison, Sophorae Tonkinensis Radix et Rhizoma often leads to a series of safety problems in the clinical application process. In this study, we attempt to analyze the ultimate cause of the toxicity of Sophorae Tonkinensis Radix et Rhizoma by reviewing nearly 20 years of literature, discuss the substance of its efficacy/toxicity from the point of view of chemical composition, and put forward exploratory thinking on its clinical rational application.

Oxysophocarpine inhibits lung injury induced by respiratory syncytial virus.

Oxysophocarpine (OSC) has various pharmacological effects. This study was designed to investigate whether OSC confers protection against respiratory syncytial virus (RSV) infection-induced lung injury. Here, we found that OSC inhibited RSV replication and increased the viability of RSV-infected lung epithelial A549 cells. OSC suppressed the RSV-increased production and release of pro-inflammatory cytokines and chemokines [tumor necrosis factor-α, interleukin-6 (IL-6), IL-8, regulated on activation in normal T-cell expressed and secreted, macrophage inflammatory protein-1α; and monocyte chemoattractant protein-1] in A549 cells. OSC also reduced the formation of reactive oxygen species and enhanced the activities of antioxidant enzymes in RSV-infected cells. The anti-oxidative effect of OSC on RSV-infected cells was dependent on NF-E2-related factor 2 activation. In vivo, OSC significantly alleviated RSV-triggered mouse lung injury. Overall, these results indicated that OSC attenuates RSV-enhanced pulmonary damage by inhibiting oxidative stress and inflammation.

Oxysophocarpine reduces oxygen-glucose deprivation-induced microglial activation and injury.

Microglial over-activation and apoptosis are associated with ischemic brain diseases. These processes may be hindered by oxysophocarpine (OSC) that generates anti-inflammatory and anti-apoptotic activities. However, the precise roles of OSC in microglial inflammation and apoptosis induced by oxygen-glucose deprivation/reoxygenation (OGD/R) remain unclear. In this study, we found that OSC reduced OGD/R-induced inflammation in BV-2 microglia. OSC elevated cell viability and prevented the release of lactate dehydrogenase. OSC downregulated cyclooxygenase 2 and inducible nitric oxide synthase and reduced the levels of inflammatory mediators, including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, monocyte chemoattractant protein-1, prostaglandin E2, and nitric oxide. OSC inhibited the expression of Toll-like receptor 4 (TLR4) and myeloid differentiation protein 88 (MyD88) and blocked the activation of nuclear factor (NF)-κB. In addition, OSC suppressed OGD/R-elicited BV-2 cell apoptosis, as indicated as follows: The restored mitochondrial membrane potential and the reduced caspase-3 activity; the decrease of Bax and cleaved caspase-3 and the increase of Bcl-2; the enhanced phosphorylation of Akt and mTOR. These results implied that OSC impedes OGD/R-induced inflammation and apoptosis of microglial cells. Therefore, OSC may be potentially used for ischemic stroke therapy.

The Anticonvulsant and Neuroprotective Effects of Oxysophocarpine on Pilocarpine-Induced Convulsions in Adult Male Mice.

Epilepsy is one of the prevalent and major neurological disorders, and approximately one-third of the individuals with epilepsy experience seizures that do not respond well to available medications. We investigated whether oxysophocarpine (OSC) had anticonvulsant and neuroprotective property in the pilocarpine (PILO)-treated mice. Thirty minutes prior to the PILO injection, the mice were administrated with OSC (20, 40, and 80 mg/kg) once. Seizures and electroencephalography (EEG) were observed, and then the mice were killed for Nissl and Fluoro-jade B (FJB) staining. The oxidative stress was measured at 24 h after convulsion. Western blot analysis was used to examine the expressions of the Bax, Bcl-2, and Caspase-3. In this study, we found that pretreatment with OSC (40, 80 mg/kg) significantly delayed the onset of the first convulsion and status epilepticus (SE) and reduced the incidence of SE and mortality. Analysis of EEG recordings revealed that OSC (40, 80 mg/kg) significantly reduced epileptiform discharges. Furthermore, Nissl and FJB staining showed that OSC (40, 80 mg/kg) attenuated the neuronal cell loss and degeneration in hippocampus. In addition, OSC (40, 80 mg/kg) attenuated the changes in the levels of Malondialdehyde (MDA) and strengthened glutathione peroxidase and catalase activity in the hippocampus. Western blot analysis showed that OSC (40, 80 mg/kg) significantly decreased the expressions of Bax, Caspase-3 and increased the expression of Bcl-2. Collectively, the findings of this study indicated that OSC exerted anticonvulsant and neuroprotective effects on PILO-treated mice. The beneficial effects should encourage further studies to investigate OSC as an adjuvant in epilepsy, both to prevent seizures and to protect neurons in brain.

Extraction and separation of oxysophocarpine from Sophora alopecuroides by inverse three-phase membrane cycle method / 中草药

Objective: To establish the HPLC method for determination of oxysophocarpine (OSC) and optimize the extraction and purification technology of OSC from Sophora alopecuroides by inverse phase membrane. Methods: Based on single-factor test, the influence of aqueous phase and organic phase volume ratio, the concentration of sodium hydroxide and hydrochloric acid, and the extraction cycle time were investigated using orthogonal design method. Results: OSC was determined by Shim-pack VP-ODS chromatographic column (250 mm×4.6 mm, 5 μm), mobile phase was methanol-0.2% phosphoric acid aqueous solution (7:93), gradient elution, flow rate was 1 mL/min, column temperature was 30℃, and detection wavelength was 221 nm. The ratio of aqueous phase and organic phase volume was 1:1, hydrochloric acid concentration was 0.03 mol/L, sodium hydroxide concentration was 0.5 mol/L, water pump flow rate was 6 mL/min, and cycle time was 60 min. The extraction rate of OSC 98.21% in 60 min was under the best experimental conditions. OSC had good linearity relationship within the range of 0.01-0.7 mg/mL, r2=0.9978, and the respective average recovery rate was 97.47%, RSD=1.95%. Conclusion: This extraction technology is simple operation, with low organic solvent consumption, and can be used for alkaloids extraction.

Human microsomal cyttrochrome P450-mediated reduction of oxysophocarpine, an active and highly toxic constituent derived from Sophora flavescens species, and its intestinal absorption and metabolism in rat.

Oxysophocarpine (OSC), an active and toxic quinolizidine alkaloid, is highly valued in Sophora flavescens Ait. and Subprostrate sophora Root. OSC is used to treat inflammation and hepatitis for thousands of years in China. This study aims to investigate the CYP450-mediated reduction responsible for metabolizing OSC and to evaluate the absorption and metabolism of OSC in rat in situ. Four metabolites were identified, with sophocarpine (SC) as the major metabolite. SC formation was rapid in human and rat liver microsomes (HLMs and RLMs, respectively). The reduction rates in the liver are two fold higher than in the intestine, both in humans and rats. In HLMs, inhibitors of CYP2C9, 3A4/5, 2D6, and 2B6 had strong inhibitory effects on SC formation. Meanwhile, inhibitors of CYP3A and CYP2D6 had significant inhibition on SC formation in RLMs. Human recombinant CYP3A4/5, 2B6, 2D6, and 2C9 contributed significantly to SC production. The permeability in rat intestine and the excretion rates of metabolites were highest in the duodenum (p<0.05), and the absorbed amount of OSC in duodenum and jejunum was concentration-dependent. The metabolism could be significantly decreased by CYP3A inhibitor ketoconazole. In conclusion, the liver was the main organ responsible for OSC metabolism. First-pass metabolism via CYP3A4/5, 2B6, 2D6, and 2C9 may be the main reason for the poor OSC bioavailability.

pKa determination of oxysophocarpine by reversed - phase high performance liquid chromatography.

BACKGROUND: In this study, a RP - HPLC method was applied for determination of pKa value by using the dependence of the capacity factor (k) on the pH of the mobile phase for oxysophocarpine (OSP). FINDINGS: The effect of the mobile phase composition on the ionization constant was studied by measuring the pKa value at different MeOH concentrations, ranging from 10 to 20% (v/v). Based on all pH - k curves plotted and pH values at inflection point calculated, experimental pKa value obtained for oxysophocarpine was 6.5. CONCLUSION: This method was successfully applied to realize low sample consumption, rapid sample throughput, high sensitivity and precision.

Determination of Five Kinds of Alkaloids in Sophorae Flavescentis Radix by HPLC / 中国中医药信息杂志

Objective To determine the content of sophocarpine, matrine, oxysophocarpine, sophoridine, oxymatrine in Sophora Flavescentis Radix from different areas. Methods Agilent ZORBAX NH2 column (4.6 mm×150 mm, 5 μm) was used with mobile phase of acetonitrile-ethanol-3% phosphate (84∶10∶6), at a flow rate of 1 mL/min. The wavelength of detection was 210 nm. Results The linear range of sophocarpine, matrine, oxysophocarpine, sophoridine and oxymatrine were 0.022 88-0.114 4 μg (r=0.999 7), 0.083 2-0.416 0 μg (r=0.999 7), 0.376 2-1.836 0 μg (r=0.999 8), 0.104 4-0.522 μg (r=0.999 2), 0.491 2-2.456 μg (r=0.999 9), respectively. The average recovery were 101.63% (RSD=2.08%), 98.29%(RSD=1.87%), 101.89% (RSD=1.97%), 99.87% (RSD=2.06%), 102.66% (RSD=1.34%), respectively. Conclusion The method is simple, rapid and accurate, and suitable for the quality control of Sophorae Flavescentis Radix.