Abstract：
BACKGROUND: Snakebite envenomation (SBE) is the world\'s most lethal neglected tropical disease. Bothrops jararaca is the species that causes the greatest number of SBEs in the South and Southeastern of Brazil. The main symptoms are local (inflammation, edema, hemorrhage, and myonecrosis) and systemic (hemorrhage, hemostatic alterations with consumptive coagulopathy, and death) effects. Species of the genus Siparuna, Siparunaceae, are used in folk and traditional medicine to treat SBE. However, limited information is available concerning Brazilian Siparuna species against SBE.
OBJECTIVE: To investigate the correlation between the compounds present in the extracts of five Siparuna species as potential agents against proteolytic activity, plasma coagulation, and phospholipase A2 (PLA2) activity caused by B. jararaca venom, using data obtained by UHPLC-MS/MS, biological activity, and multivariate statistics.
METHODS: The ethanol extracts from leaves of S. ficoides, S. decipiens, S. glycycarpa, S. reginae, and S. cymosa were fractionated by liquid-liquid extraction using different solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and n-butanol), affording their respective extracts, totaling 25 samples that were assayed through in vitro plasma coagulation and proteolytic activity assays. Moreover, the extracts were analyzed by UHPLC-MS/MS, using electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) in negative and positive ionization modes. The data was processed in MZmine v. 2.53 and evaluated by multivariate statistical tests (PLS) using the software UnscramblerX v. 10.4. These data were also used to build molecular networks (GNPS), and some ions of interest could be annotated using the library of molecules on the GNPS platform.
RESULTS: A total of 19 extracts inhibited B. jararaca-induced plasma coagulation, with emphasis on S. cymosa and S. reginae (800 s). The inhibition of the proteolytic activity was also promising, ranging from 16% (S. glycycarpa) to 99% (S. cymosa, S. decipiens, and S. reginae). In addition, most extracts from S. cymosa and S. reginae inhibited 70-90% of PLA2 activity. Based on data from positive mode APCI analyses, it was possible to obtain a statistic model with reliable predictive capacity which exhibited an average R2 of 0.95 and a Q2 of 0.88, indicating a robust fit. This process revealed five ions, identified as the alkaloids: coclaurine (1), stepholidine (2) O-methylisopiline (3), nornantenine (4) and laurolitsine (5). This is the first study to evidence the potential antivenom of alkaloids from Siparuna species.
CONCLUSIONS: Altogether, our results give support to the popular use of Siparuna extracts in SBE accidents, suggesting their potential as an alternative or complementary strategy against envenoming by B. jararaca venom. The predicted ions in the chemometric analysis for the assayed activities can also be correlated with the blocking activity and encourage the continuation of this study for possible isolation and testing of individual compounds on the used models.
OBJECTIVE: To investigate the correlation between the compounds present in the extracts of five Siparuna species as potential agents against proteolytic activity, plasma coagulation, and phospholipase A2 (PLA2) activity caused by B. jararaca venom, using data obtained by UHPLC-MS/MS, biological activity, and multivariate statistics.
METHODS: The ethanol extracts from leaves of S. ficoides, S. decipiens, S. glycycarpa, S. reginae, and S. cymosa were fractionated by liquid-liquid extraction using different solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and n-butanol), affording their respective extracts, totaling 25 samples that were assayed through in vitro plasma coagulation and proteolytic activity assays. Moreover, the extracts were analyzed by UHPLC-MS/MS, using electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) in negative and positive ionization modes. The data was processed in MZmine v. 2.53 and evaluated by multivariate statistical tests (PLS) using the software UnscramblerX v. 10.4. These data were also used to build molecular networks (GNPS), and some ions of interest could be annotated using the library of molecules on the GNPS platform.
RESULTS: A total of 19 extracts inhibited B. jararaca-induced plasma coagulation, with emphasis on S. cymosa and S. reginae (800 s). The inhibition of the proteolytic activity was also promising, ranging from 16% (S. glycycarpa) to 99% (S. cymosa, S. decipiens, and S. reginae). In addition, most extracts from S. cymosa and S. reginae inhibited 70-90% of PLA2 activity. Based on data from positive mode APCI analyses, it was possible to obtain a statistic model with reliable predictive capacity which exhibited an average R2 of 0.95 and a Q2 of 0.88, indicating a robust fit. This process revealed five ions, identified as the alkaloids: coclaurine (1), stepholidine (2) O-methylisopiline (3), nornantenine (4) and laurolitsine (5). This is the first study to evidence the potential antivenom of alkaloids from Siparuna species.
CONCLUSIONS: Altogether, our results give support to the popular use of Siparuna extracts in SBE accidents, suggesting their potential as an alternative or complementary strategy against envenoming by B. jararaca venom. The predicted ions in the chemometric analysis for the assayed activities can also be correlated with the blocking activity and encourage the continuation of this study for possible isolation and testing of individual compounds on the used models.
摘要：
背景：蛇咬伤毒害（SBE）是世界上最致命的被忽视的热带病。Bothropsjararaca是导致巴西南部和东南部SBE数量最多的物种。主要症状是局部的（炎症，水肿,出血,和心肌坏死）和全身性（出血，伴有消耗性凝血病的止血改变，和死亡）影响。Siparuna属的物种，Siparunaceae，用于民间和传统医学治疗SBE。然而,关于巴西Siparuna物种对抗SBE的信息有限。
目的：为了研究5种西帕鲁纳物种提取物中存在的化合物作为抗蛋白水解活性的潜在药物之间的相关性，血浆凝固术,和磷脂酶A2（PLA2）活性引起的B。jararaca毒液，使用UHPLC-MS/MS获得的数据，生物活性,和多元统计。
方法：从S.ficoides叶的乙醇提取物，S、蜕膜，S、甘草，S.reginae，和S.cymosa通过使用极性增加的不同溶剂（己烷，二氯甲烷,乙酸乙酯,和正丁醇)，提供各自的提取物，通过体外血浆凝血和蛋白水解活性测定总共25个样品。此外，提取物通过UHPLC-MS/MS分析，在负和正电离模式下使用电喷雾电离（ESI）和大气压化学电离（APCI）。在MZminev.2.53中处理数据，并使用软件UnscricblerXv.10.4通过多变量统计测试（PLS）进行评估。这些数据还用于构建分子网络(GNPS)，并且一些感兴趣的离子可以使用GNPS平台上的分子文库进行注释。
结果：总共有19种提取物抑制了贾拉氏芽孢杆菌诱导的血浆凝固，重点是S.cymosa和S.reginae（800秒）。蛋白水解活性的抑制也是有希望的，范围从16%(S.甘草)至99%(S.cymosa,S、蜕膜，和S.reginae）。此外,大多数来自S.cymosa和S.reginae提取物抑制70-90％的PLA2活性。根据积极模式APCI分析的数据，有可能获得具有可靠预测能力的统计模型,其显示平均R2为0.95,Q2为0.88,表明稳健拟合.这个过程揭示了五个离子，被鉴定为生物碱：考卡林(1)，去甲吡啶(2)O-甲基赖米(3)，nornantenine(4)和月桂碱(5)。这是第一项证明Siparuna物种生物碱潜在抗蛇毒血清的研究。
结论：总而言之，我们的结果支持Siparuna提取物在SBE事故中的流行使用，表明它们作为B.jararaca毒液的替代或补充策略的潜力。化学计量分析中测定活性的预测离子也可以与阻断活性相关，并鼓励继续进行这项研究，以在所用模型上分离和测试单个化合物。
目的：为了研究5种西帕鲁纳物种提取物中存在的化合物作为抗蛋白水解活性的潜在药物之间的相关性，血浆凝固术,和磷脂酶A2（PLA2）活性引起的B。jararaca毒液，使用UHPLC-MS/MS获得的数据，生物活性,和多元统计。
方法：从S.ficoides叶的乙醇提取物，S、蜕膜，S、甘草，S.reginae，和S.cymosa通过使用极性增加的不同溶剂（己烷，二氯甲烷,乙酸乙酯,和正丁醇)，提供各自的提取物，通过体外血浆凝血和蛋白水解活性测定总共25个样品。此外，提取物通过UHPLC-MS/MS分析，在负和正电离模式下使用电喷雾电离（ESI）和大气压化学电离（APCI）。在MZminev.2.53中处理数据，并使用软件UnscricblerXv.10.4通过多变量统计测试（PLS）进行评估。这些数据还用于构建分子网络(GNPS)，并且一些感兴趣的离子可以使用GNPS平台上的分子文库进行注释。
结果：总共有19种提取物抑制了贾拉氏芽孢杆菌诱导的血浆凝固，重点是S.cymosa和S.reginae（800秒）。蛋白水解活性的抑制也是有希望的，范围从16%(S.甘草)至99%(S.cymosa,S、蜕膜，和S.reginae）。此外,大多数来自S.cymosa和S.reginae提取物抑制70-90％的PLA2活性。根据积极模式APCI分析的数据，有可能获得具有可靠预测能力的统计模型,其显示平均R2为0.95,Q2为0.88,表明稳健拟合.这个过程揭示了五个离子，被鉴定为生物碱：考卡林(1)，去甲吡啶(2)O-甲基赖米(3)，nornantenine(4)和月桂碱(5)。这是第一项证明Siparuna物种生物碱潜在抗蛇毒血清的研究。
结论：总而言之，我们的结果支持Siparuna提取物在SBE事故中的流行使用，表明它们作为B.jararaca毒液的替代或补充策略的潜力。化学计量分析中测定活性的预测离子也可以与阻断活性相关，并鼓励继续进行这项研究，以在所用模型上分离和测试单个化合物。
