Oxymatrine (Matrine N-oxide)   中文名称: 苦参素

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

一种名为氧化苦参碱的生物碱提取自苦参根，已被证明具有抗炎、抗纤维化、抗肿瘤和保护心脏的特性。氧化苦参碱的潜在信号通路包括：δ-阿片受体-Bcl-2、CD40、核因子红细胞2相关因子2/血红素加氧酶-1信号通路、二甲基精氨酸二甲氨基水解酶/不对称二甲基精氨酸代谢通路、Janus激酶/信号转录转导子和激活子、Toll 样受体 9/TRAF6、δ-阿片受体激活的 B 细胞核因子 kappa 轻链增强子和 δ-阿片受体-Bcl-2 [1]。氧化苦参碱以时间和剂量依赖性方式显着抑制 PC-3 和 DU145 细胞系的生长。另一方面，氧化苦参碱治疗并没有抑制 PNT1B 健康人前列腺细胞的增殖 [2]。

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氧化苦参碱是一种生物碱，来源于中药苦参。氧化苦参碱已被证明具有抗炎、抗病毒和抗癌特性。本研究旨在探讨氧化苦参碱对人前列腺癌症细胞的抗癌作用及其潜在的分子机制。MTT分析表明，氧化苦参碱以时间和剂量依赖的方式显著抑制前列腺癌症细胞的增殖。此外，流式细胞术和末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记分析表明，氧化苦参碱治疗可能以剂量依赖性方式诱导前列腺癌症细胞凋亡。此外，蛋白质印迹分析显示，在癌症衰竭细胞中，p53和bax的表达显著增加，Bcl-2的表达显著降低，且呈剂量依赖性。[2]

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氧化苦参碱（OMT）作为苦参的主要活性成分，具有抗氧化、抗炎、抗肿瘤和抗病毒等多种药理活性，目前广泛用于治疗病毒性肝炎；然而，其对细小病毒感染的影响尚未有报道。在本研究中，我们研究了OMT对感染犬微小病毒（MVC）的Walter Reed犬细胞/3873D的细胞存活率、病毒DNA复制、病毒基因表达、细胞周期和凋亡的影响。发现OMT浓度低于4 mmol/L（无细胞毒性）时，可以抑制MVC DNA复制，并在mRNA和蛋白质水平上降低病毒基因表达，这与MVC感染早期细胞周期S期阻滞的抑制有关。此外，OMT显著提高了细胞存活率，减少了MVC感染的细胞凋亡，并降低了活化半胱氨酸天冬氨酸蛋白酶3的表达。我们的研究结果表明，OMT在对抗细小病毒感染方面具有潜在的应用[4]。

小鼠肿瘤的体积和重量呈剂量依赖性显着减少。氧化苦参碱在体内诱导细胞凋亡，从而减缓前列腺癌细胞的增殖[2]。氧化苦参碱显着减少实验大鼠肝组织中胶原蛋白的形成和沉积。当CCl4诱导SD大鼠肝纤维化时，氧化苦参碱可通过上调Smad 7的表达、下调Smad 3和CBP的表达来调节TGFβ-Smad通路的纤维化信号转导[3]。

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氧化苦参碱在体内减少前列腺癌症细胞增殖[2]
为了研究氧化苦参碱对体内肿瘤生长的影响，使用PC-3皮下异种移植物将三种浓度的氧化苦参碱腹腔注射到裸鼠体内。结果表明，小鼠肿瘤的体积（图4A）和重量（图4B）以剂量依赖的方式显著降低。TUNEL检测表明，凋亡细胞的数量以剂量依赖的方式显著增加（图4C）。根据体外分析，凋亡相关蛋白p53和bcl-2的表达以剂量依赖的方式降低，bax的表达增加（图4D）。因此，氧化苦参碱可能通过促进体内细胞凋亡来减少前列腺癌症细胞的生长。

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氧化苦参碱治疗的大鼠肝组织中胶原沉积和实质重排显著减少。与肝硬化模型大鼠相比，这些大鼠的半定量组织学评分（2.43+/-0.47 microm2 vs 3.76+/-0.68 microm2，P<0.05）和胶原蛋白的平均面积显著降低（94.41+/-37.26 microm2 vs 290.86+/-89.37 microm2，P<0.05）。治疗动物Smad 3 mRNA的基因表达显著降低。治疗组大鼠Smad 3 mRNA的A值低于模型组大鼠（0.034+/-0.090 vs 0.167+/-0.092，P<0.05）。相反，受试动物Smad 7 mRNA的A值显著增加（0.175+/-0.065 vs 0.074+/-0.012，P<0.05）。与模型大鼠相比，治疗组大鼠CBP mRNA的表达明显降低，其a值降低（0.065+/-0.049 vs 0.235+/-0.025，P<0.001）。 结论：氧化苦参碱能有效减少实验大鼠肝组织胶原的产生和沉积。氧化苦参碱可以促进SD大鼠CCl4诱导的肝纤维化中Smad 7的表达，抑制Smad 3和CBP的表达，可以调节TGF-β-Smad通路的纤维化信号转导[3]。

oxymatrine/氧化苦参碱是一种从槐属植物根中提取的生物碱成分，已被证明具有抗炎、抗纤维化和抗肿瘤作用，并具有保护心肌损伤等能力。氧化苦参碱临床应用中涉及的潜在信号通路可能包括TGF-β/Smad、toll样受体4/活化B细胞核因子κ轻链增强子、toll状受体9/TRAF6、Janus激酶/信号转导和转录激活因子、磷脂酰肌醇-3激酶/Akt、δ阿片受体-arrestin-Bcl-2、CD40、表皮生长因子受体、核因子红细胞介素-2相关因子2/血红素加氧酶-1信号通路和二甲基精氨酸二甲基氨基水解酶/不对称二甲基精胺代谢通路。在这项工作中，研究人员总结了最近对氧化苦参碱相关信号通路的研究，为进一步研究其临床应用提供了线索和参考[1]。

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低分子量（Hirt）DNA的分离和Southern Blot[4]
将WRD细胞（每孔1×10^6个）接种并培养在60mm培养皿中，然后用MVC（MOI）感染 = 10) 在37°C下培养1小时。细胞用PBS洗涤，分别在含有4 mmol/L oxymatrine/OMT（无毒浓度）的新鲜培养基中生长24、36和48小时。用PBS洗涤细胞两次，在2%十二烷基硫酸钠（SDS）中裂解，然后用蛋白酶K（0.5mg/mL）处理。收获Hirt DNA，用1%琼脂糖凝胶分离，并转移到Hybond N+膜上。根据制造商的方案，使用DIG High Prime DNA标记和检测起始试剂盒II将印迹与pI MVC基因组探针杂交，探针范围从nt 1到nt 5402。在ChemiDoc™MP成像系统（BIO-RAD）中检测到信号。Hirt DNA检测是在关的实验室进行的，所使用的方法在之前已有描述（Zhang等人，2017）[4]。

细胞增殖试验[2]
将DU145、PC-3和PNT1B细胞系接种到96孔板中，孵育过夜，并用oxymatrine/氧化苦参碱（0、2、4、6和8mg/ml）处理。使用MTT法测定细胞存活率。将细胞（3×104个细胞/孔）接种到96孔板中，在37°C的5%CO2中孵育过夜。随后，用不同浓度的氧化苦参碱（0、2、4、6和8mg/ml）孵育细胞。加入MTT（10 ml；5 mg/ml），将混合物在37°C的黑暗中孵育2小时。使用酶标仪在490 nm的波长下测量吸光度。

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流式细胞术分析[2]
用不同浓度的oxymatrine/氧化苦参碱（0、4和8mg/ml）处理人前列腺癌症细胞系。用氧化苦参碱处理48小时后，将细胞胰蛋白酶消化并以1000 x g离心，用PBS洗涤沉淀物两次。重新悬浮细胞并用PBS洗涤三次。根据制造商的说明，使用膜联蛋白V-异硫氰酸荧光素/碘化丙啶（膜联蛋白V-FITC/IP）细胞凋亡检测试剂盒检测凋亡细胞。

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蛋白质印迹分析[2]
经过oxymatrine/氧化苦参碱处理后，使用十二烷基硫酸钠聚丙烯酰胺电泳凝胶提取和分离蛋白质。然后将蛋白质转移到聚偏二氟乙烯膜上。将膜封闭并与以下一抗一起孵育：小鼠抗人p53单克隆抗体（1:1000稀释）、小鼠抗人bcl-2单克隆抗体（1:1 000稀释）、小鼠反人bax单克隆抗体（1∶1000稀释）和小鼠抗人GAPDH单克隆抗体（1:5000稀释），在4°C下过夜。用Tris缓冲盐水和吐温洗涤后，将膜与辣根过氧化物酶偶联的山羊抗小鼠二抗（1:10000稀释）一起孵育，并使用增强的化学发光检测试剂进行可视化。

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

49.74

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (7.87 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (7.87 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (7.87 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 100 mg/mL (378.27 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。