CN116535417B - 一种海洋真菌来源的含氯混源萜化合物及其制备方法和应用 - Google Patents
一种海洋真菌来源的含氯混源萜化合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种海洋真菌来源的含氯混源萜化合物及其制备方法和应用。该含氯混源萜化合物Amestolknoids C由真菌Talaromyces amestolkiae SCNU‑F0041发酵获得。试验表明，所述含氯混源萜化合物具有显著的抑制新型冠状病毒(SARS‑CoV‑2)活性的作用，为研究开发新的抗新型冠状病毒的药物提供了新的选择和途径。
Description
技术领域
本发明属于生物医药技术领域。更具体地，涉及一种海洋真菌来源的含氯混源萜类化合物及其制备方法和应用。
背景技术
新型冠状病毒感染(COVID-19)是由严重急性呼吸系统综合症冠状病毒(SARS-CoV-2)引起的一种高传染性的急性呼吸系统疾病。新型冠状病毒感染以发热、干咳、乏力等为主要表现，少数患者伴有鼻塞、流涕、腹泻等上呼吸道和消化道症状。重症病例多在1周后出现呼吸困难，严重者会快速转为急性呼吸窘迫综合征、脓毒症休克、难以纠正的代谢性酸中毒和出凝血功能障碍及多器官功能衰竭等。
红树林是生长于热带和亚热带潮间带海陆交错的一种多样化耐盐植物，由于其营养物质含量丰富，该生态系统总蕴藏着大量微生物群落，使其适应中等盐碱度和多变的环境。微生物多样性是红树林生态系统的重要特征之一，其中细菌真菌约占微生物总量的90％以上，红树林来源的真菌作为海洋真菌的第二大生态群落，代表着新天然产物的巨大潜力，产生了许多结构独特且生物活性多样的次级代谢产物，针对红树林内生真菌进行开发，有助于获得新的药源，且可以利用现代微生物发酵工程技术进行再生产，无原材料后顾之忧，不会破坏生态平衡，易实现产业化。因此，从红树林真菌寻找抗新型冠状病毒活性药物成为值得关注的热点。
发明内容
本发明要解决的技术问题是克服现有新型冠状病毒防治技术的缺陷和不足，提供一种海洋真菌来源的含氯混源萜化合物Amestolknoids C及其制备方法，以及其在抗新型冠状病毒药物中的应用。本发明提供了一种海洋真菌来源的含氯混源萜化合物Amestolknoids C，实验证明该含氯混源萜化合物Amestolknoids C对新型冠状病毒(SARS-CoV-2)具有显著的抗新型冠状病毒活性的效果且细胞毒性低，可用于抗新型冠状病毒的防治中，特别是用于制备抗新型冠状病毒的药物。
本发明的首要目的是提供一种海洋真菌来源的含氯混源萜化合物AmestolknoidsC。
本发明的另一目的是提供所述的含氯混源萜化合物Amestolknoids C的制备方法。
本发明的再一目的是提供所述含氯混源萜化合物Amestolknoids C、或其药学上可接受的盐、或其前药化合物在制备抗新型冠状病毒药物中的应用。
本发明的再一目的是提供一种抗新型冠状病毒的药物。
本发明的上述目的通过以下技术方案实现：
本发明提供了一种海洋真菌来源的含氯混源萜化合物Amestolknoids C，其化学结构如(I)所示：
本发明还提供了含氯混源萜化合物Amestolknoids C的制备方法：
S1：真菌Talaromyces amestolkiae SCNU-F0041的种子培养：
培养基组成按重量比为：马铃薯300g/L，葡萄糖20g/L，琼脂15g/L，氯霉素0.1g/L；制成试管斜面；挑取菌株接入斜面培养基，25～30℃培养3天；
S2：真菌Talaromyces amestolkiae SCNU-F0041的发酵培养：
发酵培养所用液体培养基的组成重量比为：蔗糖30g/L，蛋白胨3g/L，K2HPO41g/L，MgSO4·7H2O 0.5g/L，海盐5g/L，FeSO4 0.01g/L；将S1中培养所得的种子菌株接入液体培养基中，25～28℃静置30～50天，进行发酵培养；
S3：粗提和分离纯化：
将S2中培养所得的菌体与菌液分离，将分离所得的菌体用甲醇浸提多次，浓缩提取液，将分离所得的菌液用乙酸乙酯萃取多次，浓缩萃取液；将收集浓缩的浸膏合并，利用硅胶柱层析进行分离；分别用10％、20％、30％、40％、50％、60％、80％和100％的乙酸乙酯-石油醚梯度淋洗，收集20～30％乙酸乙酯-石油醚洗脱液，洗脱液浓缩后再次用硅胶柱层析分离技术进一步纯化，即可得到所述含氯混源萜化合物Amestolknoids C。
本发明所述的真菌Talaromyces amestolkiae SCNU-F0041是从中国广东省阳江红树林自然保护区采集的红树林植物秋茄(Kandelia)的健康树叶中分离得到了一株具有治疗新型冠状病毒活性的内生真菌。经ITS rRNA鉴定，该内生真菌为篮状菌属(Talaromyces amestolkiae)真菌，故将其命名为Talaromyces amestolkiae SCNU-F0041。2022年9月6日发表于《molecules》上的论文《New Steroid and Isocoumarin from theMangrove Endophytic Fungus Talaromyces sp.SCNU-F0041》公开了真菌Talaromycesamestolkiae SCNU-F0041，该真菌保藏于华南师范大学化学学院天然产物研究室。
本发明研究发现，含氯混源萜化合物Amestolknoids C对于新型冠状病毒具有显著的抑制作用，其抗病毒半数有效剂量EC50为2.5μM，可用于抗新型冠状病毒的防治。
因此，所述含氯混源萜化合物Amestolknoids C、或其药学上可接受的盐、或其前药化合物在制备抗新型冠状病毒药物中的应用也在本发明的保护范围内。
优选地，所述新型冠状病毒为SARS-CoV-2、B.1.1.7变异株(Alpha)、B.1.351变异株(Beta)、P.1变异株(Gamma)、B.1.617.2变异株(Delta)、B.1.1.529变异株(Omicron)。
所述含氯混源萜化合物药学上可接受的盐为其无机酸盐、无机碱盐或复盐。
所述无机酸盐的酸选自于盐酸、氢溴酸、硫酸、硝酸、乙酸、丙酸、丙二酸、丁酸、乳酸、甲磺酸、乙磺酸、苯磺酸、对甲苯磺酸、马来酸、苯甲酸、琥珀酸、苦味酸、酒石酸、柠檬酸、富马酸中任意一种或几种。
所述无机碱盐的碱选自于氢氧化钠、氢氧化钾、氢氧化钙、碳酸钠、碳酸钾、碳酸氢钠、碳酸氢钾、三甲胺、三乙胺、吡啶中的任意一种或几种。
所述含氯混源萜化合物Amestolknoids C的前药化合物是指可在体内转变成所述含氯混源萜化合物Amestolknoids C或其盐的物质。
本发明还进一步提供了一种抗新型冠状病毒的药物，包含所述含氯混源萜化合物、或其药学上可接受的盐、或其前药化合物。
优选地，所述药物还包括药学上可接受的辅料，制成不同剂型。
所述药学上可接受的辅料包括但不限于稀释剂、润滑剂、粘合剂、崩解剂、稳定剂或溶剂。
所述稀释剂包括但不限于淀粉、微晶纤维素、蔗糖、糊精、乳糖、糖粉或葡萄糖。
所述润滑剂包括但不限于硬脂酸镁、硬脂酸、氯化钠、油酸钠、月桂醇硫酸钠或泊洛沙姆。
所述粘合剂包括但不限于水、乙醇、淀粉浆、糖浆、羟丙基甲基纤维素、羧甲基纤维素钠、海藻酸钠或聚乙烯吡咯烷酮。
所述崩解剂包括但不限于淀粉泡腾混合物即碳酸氢钠和枸橼酸、酒石酸或低取代羟丙基纤维素。
所述稳定剂包括但不限于多糖如金合欢胶、琼脂、藻酸、纤维素醚或羧甲基甲壳酯。
所述溶剂包括但不限于水或经过平衡的盐溶液。
优选的，所述药物剂型包括但不限于注射剂、口服剂、喷剂、吸入剂。
所述注射剂包括但不限于小水针、输液或冻干粉针。
所述口服制剂为固体口服制剂或液体口服制剂。其中，固体口服制剂包括但不限于普通片剂、分散片、肠溶片、颗粒、胶囊、滴丸或散剂；所述液体口服制剂包括但不限于口服液或乳剂。
所述药物还可以是喷剂、吸入剂，用于吸入法或吹入法的药物包括在药学上可接受的含水溶剂或有机溶剂、或其溶液和悬浮液，以及散剂。可以做成雾化剂吸入，或借助设备喷入。
优选地，所述药物中混源萜化合物或其药学上可接受的盐或其前药化合物占所述药物质量的1％～90％。
本发明要求保护的含氯混源萜化合物Amestolknoids C、或其药学上可接受的盐、或其前药化合物应用于制备抗新型冠状病毒的药物，包括但不限于，使用本发明的化合物、或其药学上可接受的盐、或其前药化合物用于预防或治疗新型冠状病毒引发的疾病，减轻新型冠状病毒引发的疾病症状或者缓解新型冠状病毒引发的疾病的发展或发作的药品的用途。
本发明要求保护的含氯混源萜化合物Amestolknoids C、或其药学上可接受的盐、或其前药化合物除了对人类治疗有益以外，还可应用于兽医治疗宠物、引进品种的动物和农场的动物，包括哺乳动物，啮齿类动物等。
所述抗新型冠状病毒药物为具有抑制新型冠状病毒株SARS-CoV-2感染人ACE2高表达293T细胞(hACE2-293T)活性的药物。
本发明具有以下有益效果：
本发明所示的含氯混源萜化合物Amestolknoids C是新的混源萜衍生物，实验证明本发明的式(I)所示含氯混源萜化合物Amestolknoids C可通过真菌发酵获得，绿色环保，易于实现产业化。实验证明本发明的所示含氯混源萜化合物Amestolknoids C具有显著的抗新型冠状病毒(SARS-CoV-2)活性的效果，可用于抗新型冠状病毒的防治中，为研究开发新的抗新型冠状病毒的药物提供了新的选择和途径，具有很好的研究开发、应用前景。
具体实施方式
以下结合具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
菌株背景：以下实施例涉及的海洋红树林内生真菌Talaromyces amestolkiaeSCNU-F0041是从中国广东省阳江红树林自然保护区采集的红树林植物秋茄(Kandelia)的健康树叶中分离得到的一株具有治疗新型冠状病毒活性的内生真菌。经ITS rRNA鉴定，该内生真菌为篮状菌属(Talaromyces amestolkiae)真菌，故将其命名为Talaromycesamestolkiae SCNU-F0041。
实施例1：含氯混源萜化合物Amestolknoids C的分离和鉴定
本发明通过对真菌Talaromyces amestolkiae SCNU-F0041进行发酵培养，从其培养物中分离得到了含氯混源萜化合物Amestolknoids C。所述含氯混源萜化合物Amestolknoids C的制备方法，包括如下步骤：
1、含氯混源萜化合物Amestolknoids C的分离，具体步骤如下：
(1)真菌Talaromyces amestolkiae SCNU-F0041的种子培养：
挑真菌Talaromyces amestolkiae SCNU-F0041接入斜面培养基，25～30℃培养3天。
所述斜面培养基的重量比配方为：马铃薯300g/L；葡萄糖20g/L；琼脂15g/L；氯霉素0.1g/L；制成试管斜面。
(2)真菌Talaromyces amestolkiae SCNU-F0041的发酵培养：
将(1)中培养所得种子菌株接入液体培养基中，25～28℃静置30～50天，进行发酵培养。
发酵培养所用液体培养基的重量比配方为：蔗糖30g/L；蛋白胨3g/L；K2HPO41g/L；MgSO4·7H2O 0.5g/L；海盐5g/L；FeSO4 0.01g/L。
(3)含氯混源萜化合物Amestolknoids C的粗提和分离纯化
将(2)中培养所得菌体与菌液分离，然后将菌体用甲醇浸提，浸提三次后浓缩提取液，菌液用乙酸乙酯萃取，萃取三次后浓缩萃取液；将收集浓缩的浸膏合并，用硅胶柱层析进行分离；分别用10％、20％、30％、40％、50％、60％、80％和100％的乙酸乙酯-石油醚梯度淋洗，收集20～30％乙酸乙酯-石油醚洗脱液，洗脱液浓缩后再次用CH2Cl2/MeOH(v/v,180/1)为洗脱剂采用200-300目硅胶柱层析分离技术进一步纯化，即可得到所述含氯混源萜化合物Amestolknoids C。
所获含氯混源萜化合物Amestolknoids C的理化常数如下：
Amestolknoids C：[α]25 D+125(c0.1,MeOH)；HRESIMS：477.1319[M-H]+(理论计算值477.1394)确定分子式为C24H27ClO8；NMR数据见表1.
表1Amestolknoids C的NMR数据(CDCl3，150MHz/600MHz，ppm)
实施例2：含氯混源萜化合物Amestolknoids C在细胞水平上的抗新型冠状病毒活性检测的活性测试
1、测试病毒株：新型冠状病毒株SARS-CoV-2(BA.2GDPCC 2.00299)
细胞系：hACE2-293T(高表达人源血管紧张素转化酶2的293T细胞系)
2、检测方法：
含氯混源萜化合物Amestolknoids C抗病毒半数有效剂量(50％ EffectiveConcentration，EC50)：DMSO，浓度为1.5、3、6、9、12μM对应化合物提前1h饱和细胞，病毒感染1h后，换含对应浓度药物的无病毒培养基维持48h；收集细胞上清，用QIAGEN公司病毒核酸提取试剂盒(QIAGEN，#74104)提取病毒RNA；再用新型冠状病毒2019-nCoV核酸检测试剂盒(荧光PCR法)(广州达安基因股份有限公司，DA0932)，qPCR检测病毒感染后化合物不同剂量组与溶剂组(DMSO)Ct值，再用以下公式计算出新型冠状病毒RNA水平抑制率。
对应给药组抑制率(％)＝2^(溶剂对照组Ct值-给药组Ct值)×100％，用EXCEL2013的Forecast公式计算，当抑制率等于50％时，对应含氯混源萜化合物Amestolknoids C的浓度，作为EC50。三次重复实验取平均值。其活性结果显示含氯混源萜化合物Amestolknoids C具有显著的抗新型冠状病毒活性，EC50为2.5μM。
实施例3：含氯混源萜化合物Amestolknoids C细胞毒半数细胞活性抑制剂量检测
MTT法进行测试，含氯混源萜化合物Amestolknoids C梯度剂量加入到hACE2-293T细胞上清中，维持48h后加入MTT孵育4h，吸出培养基，加入DMSO检测490nm吸光度值与DMSO溶剂对照组进行比较，计算抑制率(％)＝(1-给药组490nm吸光度值/溶剂对照组490nm吸光度值)×100％，用EXCEL 2013的Forecast公式计算，当抑制率等于50％时，对应含氯混源萜化合物Amestolknoids C的浓度，作为CC50。三次重复实验取平均值。结果显示含氯混源萜化合物Amestolknoids C显示较弱细胞毒活性(CC50＝36μM)，具有较高的治疗指数SI＝14.4，显示出较大的成为抗新型冠状病毒药物的潜力。
显然，本发明的上述实施例仅仅是为清楚地说明本发明所作的举例，而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。
Claims (9)
1.一种海洋真菌来源的含氯混源萜化合物Amestolknoids C，其特征在于，其化学结构如式(I)所示：
2.权利要求1所述含氯混源萜化合物Amestolknoids C的制备方法，其特征在于：
S1：真菌Talaromyces amestolkiae SCNU-F0041的种子培养：
培养基组成按重量比为：马铃薯300g/L，葡萄糖20g/L，琼脂15g/L，氯霉素0.1g/L；制成试管斜面；挑取菌株接入斜面培养基，25～30℃培养3天；
S2:真菌Talaromyces amestolkiae SCNU-F0041的发酵培养：
发酵培养所用液体培养基的组成按重量比为：蔗糖30g/L，蛋白胨3g/L，K2HPO4 1g/L，MgSO4·7H2O 0.5g/L，海盐5g/L，FeSO4 0.01g/L；将S1中培养所得的种子菌株接入液体培养基中，25～28℃静置30～50天，进行发酵培养；
S3:粗提和分离纯化：
将S2中培养所得的菌体与菌液分离，将分离所得的菌体用甲醇浸提多次，浓缩提取液，将分离所得的菌液用乙酸乙酯萃取多次，浓缩萃取液；将收集浓缩的浸膏合并，利用硅胶柱层析进行分离；分别用10％、20％、30％、40％、50％、60％、80％和100％的乙酸乙酯-石油醚梯度淋洗，收集20～30％乙酸乙酯-石油醚洗脱液，洗脱液浓缩后再次用硅胶柱层析分离技术进一步纯化，即可得到所述含氯混源萜化合物Amestolknoids C。
3.权利要求1所述含氯混源萜化合物Amestolknoids C、或其药学上可接受的盐在制备抗新型冠状病毒药物中的应用。
4.根据权利要求3所述应用，其特征在于，所述新型冠状病毒为SARS-CoV-2、B.1.1.7变异株、B.1.351变异株、P.1变异株、B.1.617.2变异株或B.1.1.529变异株。
5.根据权利要求3所述应用，其特征在于，所述含氯混源萜化合物Amestolknoids C药学上可接受的盐为其无机酸盐、无机碱盐或复盐。
6.一种抗新型冠状病毒的药物，其特征在于，包含权利要求1所述含氯混源萜化合物Amestolknoids C、或其药学上可接受的盐。
7.根据权利要求6所述药物，其特征在于，还包括药学上可接受的辅料，制成不同剂型。
8.根据权利要求7所述药物，其特征在于，所述药学上可接受的辅料包含稀释剂、润滑剂、粘合剂、崩解剂、稳定剂或溶剂。
9.根据权利要求7所述药物，其特征在于，所述剂型包括注射剂、口服剂、喷剂或吸入剂。
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