Natural alkaloid; iNOS; TGF-β/Smad; anti-inflammatory; antifibrosis; antitumor; antiviral; bocavirus minute virus of canines (MVC)

An alkaloid called oxymatrine, which is taken from the roots of Sophora flavescens, has been demonstrated to have anti-inflammatory, anti-fibrotic, anti-tumor, and heart-protective properties. Potential signaling pathways for oxymatrine include the following: delta-opioid receptor-Bcl-2, CD40, nuclear factor erythroid-2-related factor 2/heme oxygenase-1 signaling pathway, dimethyl arginine dimethylaminohydrolase/asymmetric dimethylarginine metabolic pathway, Janus kinase/signal transducer and activator of transcription, toll-like receptor 9/TRAF6, delta-opioid receptor-activated B cell nuclear factor kappa light chain enhancer, and delta-opioid receptor-Bcl-2 [1]. The growth of PC-3 and DU145 cell lines was dramatically suppressed by oxymatrine in a time- and dose-dependent manner. On the other hand, oxymatrine therapy did not suppress the proliferation of PNT1B healthy human prostate cells [2].

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Oxymatrine is an alkaloid, which is derived from the traditional Chinese herb, Sophora flavescens Aiton. Oxymatrine has been shown to exhibit anti‑inflammatory, antiviral, and anticancer properties. The present study aimed to investigate the anticancer effects of oxymatrine in human prostate cancer cells, and the underlying molecular mechanisms of these effects. An MTT assay demonstrated that oxymatrine significantly inhibited the proliferation of prostate cancer cells in a time‑ and dose‑dependent manner. In addition, flow cytometry and a terminal deoxynucleotidyl transferase‑mediated dUTP‑biotin nick end‑labeling assay suggested that oxymatrine treatment may induce prostate cancer cell apoptosis in a dose‑dependent manner. Furthermore, western blot analysis demonstrated a significant increase in the expression of p53 and bax, and a significant decrease in that of Bcl‑2, in prostrate cancer cells in a dose‑dependent manner. [2]

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Oxymatrine (OMT), as the main active component of Sophoraflavescens, exhibits a variety of pharmacological properties, including anti-oxidative, anti-inflammatory, anti-tumor, and anti-viral activities, and currently is extensively employed to treat viral hepatitis; however, its effects on parvovirus infection have yet to be reported. In the present study, we investigated the effects of OMT on cell viability, virus DNA replication, viral gene expression, cell cycle, and apoptosis in Walter Reed canine cells/3873D infected with minute virus of canines (MVC). OMT, at concentrations below 4 mmol/L(no cellular toxicity), was found to inhibit MVC DNA replication and reduce viral gene expression at both mRNA and protein levels, which was associated with the inhibition of cell cycle S-phase arrest in early-stage of MVC infection. Furthermore, OMT significantly increased cell viability, decreased MVC-infected cell apoptosis, and reduced the expression of activated caspase 3. Our results suggest that OMT has potential application in combating parvovirus infection[4].

The dose-dependent reduction in the volume and weight of the mouse tumors was substantial. Oxymatrine induces apoptosis in vivo, which slows the proliferation of prostate cancer cells [2]. The formation and deposition of collagen in the liver tissue of experimental rats is significantly reduced by oxymatrine. When CCl4 induces liver fibrosis in SD rats, oxymatrine can regulate the TGFβ-Smad pathway's fibrosis signal transduction by upregulating the expression of Smad 7 and downregulating the expression of Smad 3 and CBP [3].

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Oxymatrine reduces prostate cancer cell proliferation in vivo [2]
In order to investigate the effect of oxymatrine on tumor growth in vivo, three concentration levels of oxymatrine were intraperitoneally injected into nude mice, using PC-3 subcutaneous xenografts. The results suggested that the volume (Fig. 4A) and weight (Fig. 4B) of tumors in mice significantly decreased in a dose-dependent manner. A TUNEL assay suggested that the number of apoptotic cells increased significantly in a dose-dependent manner (Fig. 4C). In accordance with the in vitro analyses, the expression of apoptosis-associated proteins, p53 and bcl-2 decreased and that of bax increased, in a dose-dependent manner (Fig. 4D). Oxymatrine may therefore reduce prostate cancer cell growth by promoting cell apoptosis in vivo.

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A significant reduction of collagen deposition and rearrangement of the parenchyma was noted in the liver tissue of Oxymatrine-treated rats. The semiquantitative histological scores (2.43 +/- 0.47 microm2 vs 3.76 +/- 0.68 microm2, P < 0.05) and average area of collagen in those rats were significantly decreased when compared with hepatic cirrhosis model rats (94.41 +/- 37.26 microm2 vs 290.86 +/- 89.37 microm2, P < 0.05). The gene expression of Smad 3 mRNA was considerably decreased in the treated animals. The A value of Smad 3 mRNA was lower in the treated rats than the model rats (0.034 +/- 0.090 vs 0.167 +/- 0.092, P < 0.05). Contrarily, the A value of Smad 7 mRNA was increased considerably in the treated animals (0.175 +/- 0.065 vs 0.074 +/- 0.012, P < 0.05). There was an obvious decrease in the expression of CBP mRNA in treated rats as illuminated by a reduction of its A value when compared with model rats (0.065 +/- 0.049 vs 0.235 +/- 0.025, P < 0.001). Conclusion: Oxymatrine is effective in reducing the production and deposition of collagen in the liver tissue of experimental rats. Oxymatrine could promote the expression of Smad 7 and inhibit the expression of Smad 3 and CBP in CCl4-induced hepatic fibrosis in SD rats, could modulate the fibrogenic signal transduction of TGFbeta-Smad pathway[3].

Oxymatrine, an alkaloid component extracted from the roots of Sophora species, has been shown to have antiinflammatory, antifibrosis, and antitumor effects and the ability to protect against myocardial damage, etc. The potential signaling pathways involved in the clinical application of oxymatrine might include the TGF-β/Smad, toll-like receptor 4/nuclear factor kappa-light-chain-enhancer of activated B cells, toll-like receptor9/TRAF6, Janus kinase/signal transduction and activator of transcription, phosphatidylinositol-3 kinase/Akt, delta-opioid receptor-arrestinl-Bcl-2, CD40, epidermal growth factor receptor, nuclear factor erythroid-2-related factor 2/heme oxygenase-1 signaling pathways, and dimethylarginine dimethylaminohydrolase/asymmetric dimethylarginine metabolism pathway. In this work, researchers summarize the recent investigations of the signaling pathways related to oxymatrine to provide clues and references for further studies on its clinical application [1].

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Isolation of Low Molecular Weight (Hirt) DNA and Southern Blot [4]
WRD cells (1 × 106 per well) were seeded and cultured in 60 mm dishes, then infected with MVC (MOI = 10) at 37 °C for 1 h. Cells were washed with PBS and grown in fresh medium containing 4 mmol/L of oxymatrine/OMT (non-toxic concentration) for 24, 36, and 48 h respectively. The cells were washed twice with PBS and lysed in 2% sodium dodecyl sulfate (SDS) followed by proteinase K (0.5 mg/mL) treatment. Hirt DNA was harvested and separated by 1% agarose gel and transferred to Hybond N+ membrane. The blots were hybridized with the pI-MVC genome probe, spanning from nt 1 to nt 5402, using the DIG High Prime DNA Labeling and Detection Starter Kit II according to the manufacturer’s protocol. Signals were detected in the ChemiDoc™ MP imaging system (BIO-RAD). Hirt DNA detection was carried out in Guan’s lab, and the method used was described previously (Zhang et al.2017)[4].

Cell proliferation assay [2]
DU145, PC-3 and PNT1B cell lines were seeded into 96-well plates, incubated overnight and treated with oxymatrine (0, 2, 4, 6 and 8 mg/ml). Cell viability was determined using an MTT assay. Cells (3×104 cells/well) were seeded into 96-well plates and incubated overnight at 37°C in 5% CO2. Subsequently, the cells were incubated with different concentrations of oxymatrine (0, 2, 4, 6 and 8 mg/ml). MTT (10 ml; 5 mg/ml) was added and the mixture was incubated in darkness at 37°C for 2 h. Absorbance was measured at a wavelength of 490 nm using a microplate reader.

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Flow cytometric analysis [2]
Human prostate cancer cell lines were treated with different concentrations of oxymatrine (0, 4 and 8 mg/ml). Following treatment with oxymatrine for 48 h, cells were trypsinized and centrifuged at 1,000 x g and the pellet was washed twice using PBS. Cells were resuspended and washed with PBS three times. Apoptotic cells were detected using an annexin V-fluorescein isothiocyanate/propidium iodide (annexin V-FITC/IP) cell apoptosis detection kit, according to the manufacturer’s instructions.

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Western blot analysis [2]
Following oxymatrine treatment, proteins were extracted and separated using a sodium dodecyl sulfate polyacrylamide electrophoresis gel. Proteins were then transferred to polyvinylidene difluoride membranes. Membranes were blocked and incubated with the following primary antibodies: Mouse anti-human p53 monoclonal antibody (1:1,000 dilution), mouse anti-human bcl-2 monoclonal antibody (1:1,000 dilution), mouse anti-human bax monoclonal antibody (1:1,000 dilution) and mouse anti-human GAPDH monoclonal antibody (1:5,000 dilution) overnight at 4°C. Following washing with Tris-buffered saline and Tween, membranes were incubated with a goat anti-mouse secondary antibody conjugated with horseradish peroxidase (1:10,000 dilution) and visualized using an enhanced chemiluminescent detection reagent.

In vivo xenografts [2]
BALB/c homozygous (nu/nu) nude mice (aged 6–8, weeks; weight, 18–20 g), bred in-house, were maintained in a specific pathogen-free environment. PC-3 cells (3×106) were suspended in 100 μl PBS and subcutaneously injected into the left axilla of recipient mice. On day five, 24 tumor-bearing mice were randomly divided into three groups: The control group was treated with PBS, and two groups were treated with different concentrations of oxymatrine (50 mg/kg and 100 mg/kg body weight). Oxymatrine was administered to the mice, using daily intraperitoneal injections. Tumor volume was calculated using the formula A × B2 × π/6, where A was the length of the longest aspect of the tumor, and B was the length of the tumor perpendicular to A. Following five weeks of treatment the mice were sacrificed by cervical dislocation and tumor weight was measured.

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One hundred healthy male SD rats were randomly divided into three groups: normal group (n = 20), treatment group of oxymatrine (n = 40) and CCl4-induced fibrosis group (n = 40). Experimental hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride (CCl4 soluted in liquid paraffin with the concentration of 300 g/L, the dosage of injection was 3 mL/kg, twice per week for 8 wk). The treated rats received oxymatrine via celiac injection at a dosage of 10 mg/kg twice a week at the same time. The deposition of collagen was observed with H&E and Masson staining. The concentration of serum TGF-beta1 was assayed with ELISA. The gene expression of Smads and CBP (CREB binding protein) was detected with in situ hybridization (ISH) and immunohistochemistry (IH), respectively. All the experimental figures were scanned and analyzed with special figure-analysis software.[3]

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Mice: 50 mg/kg and 100 mg/kg; i.p.

Mice: BALB/c homozygous (nu/nu) nude mice are used in the study. 24 tumor-bearing mice are randomLy divided into three groups: The control group is treated with PBS, and two groups are treated with different concentrations of oxymatrine (50 mg/kg and 100 mg/kg body weight). Oxymatrine is administered to the mice, using daily intraperitoneal injections. Rats: One hundred healthy male SD rats (weight 140-160 g) are used in the study. All 100 rats are randomLy divided into three groups: Control (n=20), Treatment (n=40) and Model group (n=40). For the model group, 300 g/L CCl4 soluted in liquid paraffin is injected subcutaneously at a dosage of 3 mL/kg twice per week. The treated rats receive oxymatrine celiac injections at 10 mg/kg twice a week besides the injection of CCl4

Interactions
The combination of Radix Angelicae sinensis (Oliv.) Diels and Radix Sophora flavescens Ait. was extensively used in traditional Chinese medicine to treat inflammatory diseases, such as acne, heart disease, and hepatitis. Sodium ferulate (SF) and oxymatrine (OMT) were effective component of Radix Angelicae sinensis (Oliv.) Diels and Radix Sophora flavescens Ait., respectively. In this study, /the authors/ investigated the synergistic anti-inflammatory effect of the combination of SF and OMT, and its modulation on inflammation-associated mediators in RAW 264.7 cells. In vivo, the anti-inflammatory effects of the combination of SF and OMT were evaluated with the xylene-induced mouse ear edema model and the carrageenan-induced rat paw edema model. In vitro, chemokines and cytokines mRNA expressions in lipopolysaccharide (LPS)-activated RAW 264.7 cells were determined by real-time PCR (RT-PCR) microarray analysis. The levels of interleukin-11 (IL-11), C-reactive protein (CRP) and interferon-gamma (INF-gamma) in the supernatant of LPS-stimulated RAW 264.7 cells were measured by enzyme-linked immune-sorbent assay (ELISA). The combination of SF and OMT could significantly inhibit the edema in the xylene-induced mouse ear edema and carrageenan-induced rat paw edema, but no effect was found when each drug was used alone according to above doses. The combination exhibited a better effect in down-regulating mRNA expressions of inflammation-associated mediators in LPS-stimulated RAW 264.7 cells than SF or OMT alone. The ELISA results showed that the combination synergistically inhibited LPS-induced IL-11, CRP and INF-gamma production in a dose-dependent manner. The combination of SF and OMT showed synergistic anti-inflammatory effect, and the activity was probably related to its modulation on inflammation-associated mediators, especially IL-11, CRP and INF-gamma.
Sodium ferulate (SF) and Oxymatrine (OMT) were compounds extracted from Chinese herbs, and have been used in clinical treatment of heart and hepatic diseases, respectively, in China for many years. The objective of this study was to examine the analgesic effect and the mechanism of the combined treatment of SF and OMT. Using the animal pain models by applying Acetic Acid Writhing Test and Formalin Test, the combination of SF and OMT showed significant analgesic effect in dose-dependent manner. In vitro, the combined treatment inhibited the increase in intracellular calcium concentration evoked by capsaicin in the dorsal root ganglion neurons. Importantly, a synergistic inhibitory effect of SF and OMT on the capsaicin-induced currents was demonstrated by whole-cell patch-clamp. Our results suggest that SF and OMT cause significant analgesic effect which may be related to the synergistic inhibition of transient receptor potential vanilloid-1.
/The aim of this was/ to study the effect of oxymatrine-baicalin combination (OB) against HBV replication in 2.2.15 cells and alpha smooth muscle actin (alpha SMA) expression, type I, collagen synthesis in HSC-T6 cells. The 2.2.15 cells and HSC-T6 cells were cultured and treated respectively. HBsAg and HBeAg in the culture supernatants were detected by ELISA and HBV DNA levels were determined by fluorescence quantitative PCR. Total RNA was extracted from HSC-T6 cells and reverse transcribed into cDNA. The cDNAs were amplified by PCR and the quantities were expressed in proportion to beta actin. The total cellular proteins extracted from HSC-T6 cells were separated by electrophoresis. Resolved proteins were electrophoretically transferred to nitrocellulose membrane. Protein bands were revealed and the quantities were corrected by beta actin. In the 2.2.15 cell culture system, the inhibitory rate against secretion of HBsAg and HBeAg in the OB group was significantly stronger than that in the oxymatrine group (HBsAg, P = 0.043; HBeAg, P = 0.026; respectively); HBV DNA level in the OB group was significantly lower than that in the oxymatrine group (P = 0.041). In HSC-T6 cells the mRNA and protein expression levels of alpha SMA in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P = 0.013; protein, P = 0.042; respectively); The mRNA and protein expression levels of type I collagen in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P < 0.01; protein, P < 0.01; respectively). /The authors concluded that/ OB combination has a better effect against HBV replication in 2.2.15 cells and is more effective against alpha SMA expression and type I collagen synthesis in HSC-T6 cells than oxymatrine in vitro.
Oxymatrine is proven to protect ischemic and reperfusion injury in liver, intestine and heart, this effect is via anti-inflammation and anti-apoptosis. Whether this protective effect applies to ischemic injury in brain, /the authors/ therefore investigate the potential neuroprotective role of oxymatrine and the underlying mechanisms. Male, Sprague-Dawley rats were randomly assigned to four groups: permanent middle cerebral artery occlusion (pMCAO), high dose (pMCAO+oxymatrine 120 mg/kg), low dose (pMCAO+oxymatrine 60 mg/kg) and sham operated group. /The authors/ used a permanent middle cerebral artery occlusion model and administered oxymatrine intraperitoneally immediately after cerebral ischemia and once daily on the following days. At 24 hr after MCAO, neurological deficit was evaluated using a modified six point scale; brain water content was measured; NF-kappaB expression was measured by immunohistochemistry, Western blotting and RT-PCR. Infarct volume was analyzed with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining at 72 hr. Compared with pMCAO group, neurological deficit in high dose group was improved (P < 0.05), infarct volume was decreased (P < 0.001) and cerebral edema was alleviated (P < 0.05). Consistent with these indices, immunohistochemistry, Western blot and RT-PCR analysis indicated that NF-kappaB expression was significantly decreased in high dose group. Low dose of oxymatrine did not affect NF-kappaB expression in pMCAO rats. Oxymatrine reduced infarct volume induced by pMCAO, this effect may be through the decreasing of NF-kappaB expression.

[1]. Potential Signaling Pathways Involved in the Clinical Application of Oxymatrine. Phytother Res. 2016 Jul;30(7):1104-12.

[2]. Oxymatrine inhibits the proliferation of prostate cancer cells in vitro and in vivo. Mol Med Rep. 2015 Jun;11(6):4129-34.

[3]. Effect of Oxymatrine on the TGFbeta-Smad signaling pathway in rats with CCl4-induced hepatic fibrosis. World J Gastroenterol. 2008 Apr 7;14(13):2100-5.

[4]. Oxymatrine Inhibits Bocavirus MVC Replication, Reduces Viral Gene Expression and Decreases Apoptosis Induced by Viral Infection. Virol Sin. 2019 Feb;34(1):78-87.

Therapeutic Uses
Anti-Arrhythmia Agents; Antiviral Agents
The aim of this study was/ to evaluate the efficacy and safety of capsule oxymatrine in the treatment of chronic hepatitis B. A randomized double-blind and placebo-controlled multicenter trial was conducted. Injection of oxymatrine was used as positive-control drug. A total of 216 patients with chronic hepatitis B entered the study for 24 weeks, of them 108 received capsule oxymatrine, 36 received injection of oxymatrine, and 72 received placebo. After and before the treatment, clinical symptoms, liver function, serum hepatitis B virus markers, and adverse drug reaction were observed. Among the 216 patients, six were dropped off, and 11 inconsistent with the standard were excluded. Therefore, the efficacy and safety of oxymatrine in patients were analysed. In the capsule treated patients, 76.47% became normal in ALT level, 38.61% and 31.91% became negative both in HBV DNA and in HBeAg. In the injection treated patients, 83.33% became normal in ALT level, 43.33% and 39.29% became negative both in HBV DNA and in HBeAg. In the placebo treated patients, 40.00% became normal in ALT level, 7.46% and 6.45% became negative both in HBV DNA and in HBeAg. The rates of complete response and partial response were 24.51% and 57.84% in the capsule treated patients, and 33.33% and 50.00% in the injection treated patients, and 2.99% and 41.79% in the placebo treated patients, respectively. There was no significance between the two groups of patients, but both were significantly higher than the placebo. The adverse drug reaction rates of the capsule, injection and placebo were 7.77%, 6.67% and 8.82%, respectively. There was no statistically significant difference among them. /It was concluded that/ oxymatrine is an effective and safe agent for the treatment of chronic hepatitis B.

C15H24N2O2

264.36

264.183

C, 68.15; H, 9.15; N, 10.60; O, 12.10

16837-52-8

114850

White to off-white solid powder

208 °C

0mmHg at 25°C

1.637

-0.35

0

2

0

19

400

4

C1C[C@@H]2[C@H]3CCC[N+]4([C@H]3[C@@H](CCC4)CN2C(=O)C1)[O-]

XVPBINOPNYFXID-VNSSVHEPSA-N

InChI=1S/C15H24N2O2/c18-14-7-1-6-13-12-5-3-9-17(19)8-2-4-11(15(12)17)10-16(13)14/h11-13,15H,1-10H2/t11-,12+,13+,15+,17+/m0/s1

(4R,41R,7aS,13aR,13bR)-10-oxododecahydro-1H,5H-dipyrido[2,1-f:3,2,1-ij][1,6]naphthyridine 4(41H)-oxide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (378.27 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)