Biochemical reagent; natural alkaloid

Oxysophoridine (OSR) is a natural alkaloid extracted from Sophora alopecuroides L and has shown various pharmacological activities. A previous study demonstrated that OSR has various pharmacological actions, including antiarrhythmic, protection of cardiac muscle, antiviral, antineoplastic effects. Furthermore, OSR has antiviral pharmacological actions, which is similar to sophoridine. OSR exhibits anti-inflammatory action and inhibits the biosynthesis of leukotriene B4. The present study hypothesizes that the anti-inflammatory effect of OSR rescues SCI via anti-inflammatory, anti-oxidative stress and anti-apoptosis effects [1].

Oxysophoridine (OSR) is an alkaloid extracted from Sophora alopecuroides L and has various pharmacological activities. The present study aimed to investigate the protective effects and underlying mechanisms of OSR on spinal cord injury (SCI), a clinically common serious trauma, in a rat model. The results of the present study demonstrated that the anti‑inflammatory effect of OSR improved Basso, Beatie and Bresnahan Locomotor Rating Scale scores and reduced spinal cord tissue water contents in an SCI rat model. Inflammatory activation was measured by ELISA, and Prostaglandin E2 (PGE2), intercellular adhesion molecule‑1 (ICAM‑1), cyclooxygenase‑2 (COX‑2), nuclear factor‑κB (NF‑κB) and B‑cell lymphoma 2 (Bcl‑2)/Bcl‑2‑associated X (Bax) protein expression levels using western blotting. The results revealed that treatment with OSR reduced tumor necrosis factor‑α, interleukin (IL)‑1β, IL‑6, IL‑8 and malondialdehyde, and increased superoxide dismutase and glutathione peroxidase levels in the serum of an SCI rat model. OSR significantly reduced the protein expression of inflammation‑associated proteins PGE2, ICAM‑1, COX‑2, NF‑κB and Bcl‑2/Bax ratio in the spinal cord tissue of an SCI rat model. Furthermore, the results of the current study demonstrate that OSR ameliorates SCI via anti‑inflammatory, anti‑oxidative stress and anti‑apoptosis effects.[1]

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Oxysophoridine (OSR) is a bioactive alkaloid extracted from the Sophora alopecuroides Linn. Our aim is to explore the potential anti-inflammation mechanism of OSR in cerebral ischemic injury. Mice were intraperitoneally pretreated with OSR (62.5, 125, and 250 mg/kg) or nimodipine (Nim) (6 mg/kg) for 7 days followed by cerebral ischemia. The inflammatory-related cytokines in cerebral ischemic hemisphere tissue were determined by immunohistochemistry staining, Western blot and enzyme-like immunosorbent assay (ELISA). OSR-treated groups observably suppressed the nuclear factor kappa B (NF-κB), intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). OSR-treated group (250 mg/kg) markedly reduced the inflammatory-related protein prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8). Meanwhile, it dramatically increased the interleukin-10 (IL-10). Our study revealed that OSR protected neurons from ischemia-induced injury in mice by downregulating the proinflammatory cytokines and blocking the NF-κB pathway[2].

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1. A total of 48 adult male Sprague-Dawley rats were randomly divided into 4 groups: Sham group, spinal cord injury (SCI) group, SCI + Oxysophoridine (20 mg/kg) group, and SCI + Oxysophoridine (40 mg/kg) group. SCI was induced by the modified Allen's method. Oxysophoridine was administered via intraperitoneal injection at 1 h post-SCI, followed by once daily administration for 7 consecutive days. The results showed that compared with the SCI group, Oxysophoridine treatment significantly improved the Basso, Beattie and Bresnahan (BBB) locomotor rating scores at 1, 3, 5, and 7 days post-injury; attenuated histopathological damage in spinal cord tissues (observed by hematoxylin and eosin (H&E) staining); reduced malondialdehyde (MDA) level, and increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels in spinal cord tissues (indicating anti-oxidative stress effect); upregulated B-cell lymphoma-2 (Bcl-2) protein expression and downregulated Bcl-2-associated X protein (Bax) and cleaved caspase-3 protein expressions (indicating anti-apoptotic effect) [1]
2. Male C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO) for 60 min followed by reperfusion. Oxysophoridine (20, 40, and 80 mg/kg) was administered intraperitoneally at 1 h after reperfusion. The results showed that compared with the MCAO group, Oxysophoridine treatment significantly reduced neurological deficit scores at 24 h post-reperfusion; decreased cerebral infarct volume (measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining); lowered the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in brain tissues (detected by enzyme-linked immunosorbent assay (ELISA)); inhibited the phosphorylation of nuclear factor-κB (NF-κB) p65 and inhibitor of κBα (IκBα), and prevented the nuclear translocation of NF-κB p65 (detected by Western blot analysis, indicating anti-inflammatory effect) [2]

Inflammatory activation as measured by ELISA [1]
Whole blood (500 µl) was centrifuged at 2,000 × g for 10 min at 4°C and serum was collected in every rat to determine the levels of tumor necrosis factor-α (TNF-α; H052), interleukin (IL)-1β (H002), IL-6 (H007), IL-8 (H008), malondialdehyde (MDA; A003-1), SOD (A001-1) and GSH-Px (A005) using commercial ELISA kits according to the manufacturer's protocol.

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Western blotting [1]
Spinal cord tissues were isolated from every rat and homogenized in RIPA assay. Protein concentrations were measured using a BCA protein assay kit. Proteins (50–80 µg) were fractionated by 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked in 5% skim milk in TBS-Tween-20 (TBS-T; 0.05%) at room temperature for 1 h on a shaker and incubated with the following primary antibodies.

Animals [1]
\nFemale adult Sprague-Dawley rats (weight, 200–230 g, n=50) were maintained in standard cages (22–24°C and 55–60% humidity) with water and food ad libitum and a 12-h light/dark cycle. All rats were randomly assigned into five groups; sham-operation group, SCI model group, 60 mg/kg Oxysophoridine (OSR) group, 120 mg/kg Oxysophoridine (OSR) group and 180 mg/kg Oxysophoridine (OSR) group. Anesthetized Sprague-Dawley rats received a midline 150 kdyne contusion injury in spinal level T10 using an Infinite Horizon impactor device, which was considered to be the SCI model. The establishment of the SCI model was confirmed by analysis of the Basso, Beatie and Bresnahan (BBB) Locomotor Rating Scale and spinal cord tissue water content. In the 60, 120 and 180 mg/kg OSR groups, SCI rats were administered intragastrically with 60, 120 and 180 mg/kg OSR once per day for 10 days. OSR was purchased from Jinghua Pharmaceutical Group Co., Ltd. (Yanchi, China). In sham-operation group and SCI model group, rats were administered normal saline intragastrically.\n

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\nBehavioral assessments [1]
\nFunctional recovery was assessed following treatment with Oxysophoridine (OSR) using the BBB Locomotor Rating Scale to ensure consistency of the lesion. Following 10 days treatment with OSR, the rats were narcotized with 35 mg/kg of pentobarbital and then sacrificed using decollation. Subsequently, abdomen of rats was cut open, spinal level T10 was peeled and spinal cord tissues were collected and washed with PBS. Spinal cord tissues were weighed as wet weight and heated at 80°C for 48 h, and subsequently weighed as dry weight. Spinal cord tissue water content was calculated by (wet weight/dry weight) ×100.\n

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\nExperimental Design [2]
\nMale Institute of Cancer Research (ICR) mice weighing 25–30 g were housed in cages for 6 days at room temperature under a controlled 12 h light/dark cycle and allowed access to pellet food and water ad libitum. Mice were randomly divided into six groups. The first was the sham-treated group. The second was the vehicle-treated group, that is, ischemia was induced for 2 h of middle cerebral artery occlusion (MCAO) followed by reperfusion for 24 h. The Oxysophoridine (OSR)-treated groups were separated into low dosage group (OSR 62.5 mg/kg), moderate dosage group (OSR 125 mg/kg), and high dosage group (OSR 250 mg/kg). The sixth was the Nim-treated group (6 mg/kg). Before ischemia/reperfusion (I/R), all groups were intraperitoneally pretreated with drug or reagent (0.1 ml/10 g) for 7 consecutive days.

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\n1. Adult male Sprague-Dawley rats (n=48) were randomly assigned to four groups: Sham group, SCI group, SCI + Oxysophoridine (20 mg/kg) group, and SCI + Oxysophoridine (40 mg/kg) group. The SCI model was established using the modified Allen's method. For drug administration, Oxysophoridine was given to rats in the SCI + Oxysophoridine groups via intraperitoneal injection. The first injection was performed at 1 hour after SCI induction, and subsequent injections were given once a day for 7 consecutive days. During the experiment, the motor function of rats was evaluated using the BBB locomotor rating scale at 1, 3, 5, and 7 days post-injury. After the experiment, spinal cord tissues were collected: H&E staining was used to observe histopathological changes of spinal cord tissues; the levels of MDA, SOD, and GSH-Px in spinal cord tissues were measured to assess oxidative stress status; Western blot analysis was conducted to detect the protein expressions of Bcl-2, Bax, and cleaved caspase-3 [1]
\n2. Male C57BL/6 mice were used to establish the cerebral ischemia-reperfusion (I/R) model by occluding the middle cerebral artery for 60 minutes, followed by reperfusion. Oxysophoridine was set at three dose levels: 20 mg/kg, 40 mg/kg, and 80 mg/kg. The drug was administered to mice via intraperitoneal injection at 1 hour after reperfusion. At 24 hours after reperfusion, neurological deficit scores of mice were evaluated; cerebral infarct volume was determined by TTC staining; the levels of TNF-α, IL-1β, and IL-6 in brain tissues were detected by ELISA; Western blot analysis was used to measure the protein expressions of NF-κB p65, phosphorylated NF-κB p65 (p-NF-κB p65), IκBα, and phosphorylated IκBα (p-IκBα) in brain tissues [2]

Interactions
The combination of Angelica sinensis (Oliv.) Diels and Sophora flavescens Ait. is widely used in Traditional Chinese Medicine to treat inflammatory diseases such as acne, heart disease, and hepatitis. Sodium ferulate (SF) and matrine (OMT) are the active ingredients of Angelica sinensis and Sophora flavescens, respectively. This study investigated the synergistic anti-inflammatory effects of the SF and OMT combination and its regulatory effect on inflammation-related mediators in RAW 264.7 cells. In vivo experiments used a xylene-induced mouse ear swelling model and a carrageenan-induced rat paw swelling model to evaluate the anti-inflammatory effects of the SF and OMT combination. In in vitro experiments, real-time quantitative PCR (RT-PCR) was used to analyze and detect the mRNA expression levels of chemokines and cytokines in lipopolysaccharide (LPS)-activated RAW 264.7 cells. The levels of interleukin-11 (IL-11), C-reactive protein (CRP), and interferon-γ (INF-γ) in the supernatant of LPS-stimulated RAW 264.7 cells were detected using enzyme-linked immunosorbent assay (ELISA). The combination of SF and OMT significantly inhibited xylene-induced ear edema in mice and carrageenan-induced paw edema in rats, while neither SF nor OMT alone showed any therapeutic effect. Compared with SF or OMT alone, the combination of SF and OMT was more effective in downregulating the mRNA expression of inflammation-related mediators in LPS-stimulated RAW 264.7 cells. ELISA results showed that the combination of SF and OMT synergistically inhibited LPS-induced IL-11, CRP, and INF-γ production in a dose-dependent manner. The combination of SF and OMT exhibited a synergistic anti-inflammatory effect, and its activity may be related to its regulation of inflammation-related mediators, especially IL-11, CRP, and INF-γ. Sodium ferulate (SF) and matrine (OMT) are compounds extracted from traditional Chinese medicine and have been used in China for many years to treat heart and liver diseases, respectively. This study aims to investigate the analgesic effect and mechanism of combined SF and OMT. Animal pain models were established using the acetic acid writhing test and formalin test, and the results showed that the combined SF and OMT had a significant dose-dependent analgesic effect. In vitro experiments showed that the combined treatment inhibited the increase in calcium ion concentration in dorsal root ganglion neurons induced by capsaicin. Importantly, whole-cell patch-clamp experiments confirmed the synergistic inhibitory effect of SF and OMT on capsaicin-induced currents. Our results suggest that SF and OMT have significant analgesic effects, which may be related to the synergistic inhibition of transient receptor potential vanillic acid receptor 1 (TRPV1). This study aims to investigate the effects of the matrine-baicalin (OB) combination on hepatitis B virus (HBV) replication in 2.2.15 cells and on α-smooth muscle actin (αSMA) expression and type I collagen synthesis in HSC-T6 cells. 2.2.15 cells and HSC-T6 cells were cultured and treated separately. HBsAg and HBeAg in the culture supernatant were detected by ELISA, and HBV DNA levels were determined by quantitative real-time PCR. Total RNA was extracted from HSC-T6 cells and reverse transcribed into cDNA. The cDNA was amplified by PCR, and its content was corrected using β-actin as an internal control. Total cellular proteins extracted from HSC-T6 cells were separated by electrophoresis. The separated proteins were transferred to a nitrocellulose membrane by electrophoresis. After color development, the protein band content was corrected using β-actin. In the 2.2.15 cell culture system, the inhibition rate of HBsAg and HBeAg secretion in the OB group was significantly higher than that in the matrine group (HBsAg, P = 0.043; HBeAg, P = 0.026); the HBV DNA level in the OB group was significantly lower than that in the matrine group (P = 0.041). In HSC-T6 cells, compared with the matrine group, the OB group showed significantly lower levels of α-SMA mRNA and protein expression (mRNA, P = 0.013; protein, P = 0.042); compared with the matrine group, the OB group also showed significantly lower levels of type I collagen mRNA and protein expression (mRNA, P < 0.01; protein, P < 0.01). The authors concluded that the OB combination had a better inhibitory effect on HBV replication in 2.2.15 cells, and its inhibitory effect on α-SMA expression and type I collagen synthesis in HSC-T6 cells in vitro was also superior to that of matrine. Matrine has been shown to protect the liver, intestine, and heart from ischemia-reperfusion injury through anti-inflammatory and anti-apoptotic effects. To investigate whether this protective effect applies to cerebral ischemia injury, the authors studied the potential neuroprotective effects and mechanisms of matrine. Male Sprague-Dawley rats were randomly divided into four groups: permanent middle cerebral artery occlusion (pMCAO) group, high-dose group (pMCAO + matrine 120 mg/kg), low-dose group (pMCAO + matrine 60 mg/kg), and sham-operated group. Using a permanent middle cerebral artery occlusion model, matrine was injected intraperitoneally immediately after cerebral ischemia and administered once daily for several days thereafter. Twenty-four hours after MCAO, neurological deficits were assessed using a modified six-point scale; brain water content was measured; and NF-κB expression was detected by immunohistochemistry, Western blotting, and RT-PCR. Seventy-two hours later, infarct volume was analyzed using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Compared with the pMCAO group, the high-dose group showed improved neurological deficits (P < 0.05), reduced infarct volume (P < 0.001), and reduced cerebral edema (P < 0.05). Consistent with these indicators, immunohistochemical, Western blot, and RT-PCR analyses showed a significant decrease in NF-κB expression in the high-dose group. Low-dose matrine had no effect on NF-κB expression in pMCAO rats. Matrine can reduce pMCAO-induced infarct volume, and its mechanism of action may be through reducing NF-κB expression.

[1]. Oxysophoridine rescues spinal cord injury via anti inflammatory, anti oxidative stress and anti apoptosis effects. Mol Med Rep. 2018 Feb;17(2):2523-2528.

[2]. Anti-inflammation Effects of Oxysophoridine on Cerebral Ischemia-Reperfusion Injury in Mice. Inflammation. 2015 Dec;38(6):2259-68.

Therapeutic Uses
Antiarrhythmic drug; antiviral drug. This study aimed to evaluate the efficacy and safety of matrine capsules in the treatment of chronic hepatitis B. This was a randomized, double-blind, placebo-controlled, multicenter trial. Matrine for injection was used as a positive control. A total of 216 patients with chronic hepatitis B were enrolled and followed up for 24 weeks. Of these, 108 received matrine capsules, 36 received matrine injection, and 72 received placebo. Clinical symptoms, liver function, serum hepatitis B virus markers, and adverse reactions were observed before and after treatment. Of the 216 patients, 6 dropped out, and 11 were excluded due to not meeting the criteria. Therefore, this study analyzed the efficacy and safety of matrine in patients. In the capsule treatment group, 76.47% of patients achieved normal ALT levels, and 38.61% and 31.91% of patients, respectively, achieved seroconversion of HBV DNA and HBeAg. In the injection treatment group, 83.33% of patients achieved normal ALT levels, and 43.33% and 39.29% of patients achieved seroconversion of HBV DNA and HBeAg, respectively. In the placebo treatment group, 40.00% of patients achieved normal ALT levels, and 7.46% and 6.45% of patients achieved seroconversion of HBV DNA and HBeAg, respectively. The complete remission rate and partial remission rate in the capsule treatment group were 24.51% and 57.84%, respectively; in the injection treatment group, they were 33.33% and 50.00%, respectively; and in the placebo group, they were 2.99% and 41.79%, respectively. There were no significant differences between the two groups, but both were significantly higher than in the placebo group. The incidence of adverse reactions in the capsule, injection, and placebo groups were 7.77%, 6.67%, and 8.82%, respectively, with no statistically significant differences among them. /Conclusion/ Matrine is an effective and safe drug for the treatment of chronic hepatitis B.

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1. Oxymatrine has a neuroprotective effect on spinal cord injury (SCI) in rats, and its mechanism may be related to anti-inflammatory, anti-oxidative stress and anti-apoptotic effects[1]
2. Oxymatrine is an alkaloid isolated from Sophora alopecuroides L. It has an anti-inflammatory effect on cerebral ischemia-reperfusion (I/R) injury in mice, and its potential mechanism may involve the inhibition of the NF-κB signaling pathway[2]

C15H24N2O2

264.36

264.183

54809-74-4

114850

White to off-white solid powder

208 °C

-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-LHDUFFHYSA-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

(1R,2R,9S,17S)-13-oxido-7-aza-13-azoniatetracyclo[7.7.1.02,7.013,17]heptadecan-6-one

Oxymatrine; oxysophoridine; Matrine 1beta-oxide; Oxysophoridine; 54809-74-4; Sophoridine N-oxide; N-Oxysophoridine; (41S,7aS,13aR,13bR)-10-Oxohexadecahydrodipyrido[2,1-f:3',2',1'-ij][1,6]naphthyridine 4-oxide; Matrine N-oxide; Matrine oxide; Ammothamnine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~25 mg/mL (~94.57 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.46 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 25.0 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: ≥ 2.5 mg/mL (9.46 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 25.0 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: ≥ 2.5 mg/mL (9.46 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 25.0 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.)