CN110041401A - 肯塔森纳霉素及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一类肯塔森纳霉素及其制备方法和应用，所述的肯塔森纳霉素源于安徽仙人岕山蜜蜂的内生放线菌Kitasatospora sp.的液体发酵产物。实验研究结果表明，本发明所述肯塔森纳霉素可以调控巨噬细胞分泌IFN‑β，可进一步用于制备抗病毒及抗肿瘤药物。
Description
技术领域
本发明属于生物制药技术领域，具体涉及一类肯塔森纳霉素及其制备方法和应用。
背景技术
昆虫的生物多样性非常丰富，目前已报道的昆虫越有100多万种，并且分布广泛，地球上的各个角落几乎都有它们的足迹。而在这些昆虫体内分布着为数众多的共生微生物，它们在昆虫体内有着非常重要的作用：(1)促进宿主昆虫的生长：如酵母类内共生菌可为宿主提供脂肪酸、维生素B、固醇等营养物质；蟑螂的内共生菌能够产生尿酸降解酶等来代谢蟑螂体内的氮素废弃物；蚜虫共生菌能为蚜虫提供分解植物细胞壁的酶、必需的营养物质或若虫发育所必须的共生素蛋白。(2)促进宿主昆虫的发育：如某些昆虫共生菌能够合成胚胎和胚后发育所需的蛋白质,促进胚胎腹节分化、提高若虫的存活率等。(3)影响宿主昆虫的种群：当种群密度过大时,在自然选择压力下,昆虫共生菌会转变成致病菌,淘汰体质较差的昆虫并同时提高整个种群的自然竞争能力。
昆虫共生菌同时也是重要的活性次生代谢产物的源泉,与昆虫种类研究相比,目前对昆虫共生菌研究偏少,对其代谢产物研究更少,但昆虫物种的多样性和分布的复杂性造成了昆虫体内共生菌的多样性,这也直接导致其次生代谢产物结构和活性的多样性。
发明内容
本发明首次从安徽仙人岕山蜜蜂体内获得了一种新的内生放线菌Kitasatosporasp.，从其发酵产物中发现了一类新的活性物质肯塔森纳霉素(kitacinnamycins)，体外活性实验表明，该化合物可以促进巨噬细胞分泌IFN-β，从而产生抗病毒及抗肿瘤的作用。
本发明具体技术方案如下：
一类肯塔森纳霉素，具有如下结构式：
其中，R1代表C1-C6的烷基，R2代表C1-C6的烷基、C1-C6的烷基醇、-(CH2)n(CO)mR3，n代表0-5的整数，m代表0或者1，R3代表H、-OH、
优选的，所述R1代表-CH3、-CH2CH3，R2代表-CH3、-CH2CH3、-CH3OH、-CH2CH3OH、-R3或者-COR3,R3代表H、-OH、
更优选的，R1代表-CH3、-CH2CH3，R2代表或者-COR3,R3代表H、-OH或者
R1代表-CH3，R2代表-CH3；
R1代表-CH3，R2代表--CH2CH3；
R1代表-CH2CH3，R2代表-CH3。
本发明所述的肯塔森纳霉是利用北里孢菌属Kitasatospora sp.菌种通过液体发酵提取得到，优选的，北里孢菌属Kitasatospora sp.菌种选自保藏于中国普通微生物菌种保藏管理中心(北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101)，保藏编号为CGMCC No.16924的菌株。
本发明另一目的在于提供所述的肯塔森纳霉素的制备方法，包括以下步骤：
(1)将北里孢菌属Kitasatospora sp.菌种发酵培养(优选26～30℃培养10天)；优选的，将Kitasatospora sp.菌种(优选生物保藏编号为CGMCC No.16924的北里孢菌属Kitasatospora sp.菌种)先接种于种子培养基，在26～30℃培养1.5～2.5天，作为种子液，将种子液接种于SCAS培养液，在26～30℃培养10天；所述的SCAS培养液的配方为40g/L可溶性淀粉，5g/L络蛋白水解物，0.5g/L磷酸二氢钾，0.5g/L七水合硫酸镁，0.01g/L七水合硫酸铁，溶剂为水。
所述的种子培养基为TSB培养基(胰蛋白胨15g/L，大豆蛋白胨5g/L，氯化钠5g/L，溶剂为水)、38#培养基(酵母浸出粉4g/L，麦芽浸出粉5g/L，葡萄糖4g/L，溶剂为水)。
所述的种子液与SCAS培养液的体积比为2.5～3.5:10，转速为220～240rpm/min。
(2)将步骤(1)所得发酵液过滤，浓缩得浸膏F1；
(3)将浸膏F1悬浮于水，依次用石油醚和乙酸乙酯萃取，所得乙酸乙酯萃取液经浓缩得浸膏F2；
(4)对浸膏F2进行正相硅胶柱层析，依次用体积比为100：1、100：2、100：4、100：8、100：16的二氯甲烷-甲醇进行等度洗脱，得到10个出峰组分；
(5)将第二出峰组分经HPLC分离纯化制得式(I)所示化合物。
步骤(5)中，HPLC分离纯化过程如下：semi-HPLC半制备反相高效液相色谱：ODS-2Hypersil colum，5μm，250mm×10mm，流动相为：乙腈-水体积比＝1-16：4，以2.5mL/min流速梯度洗脱15min。泵的型号可以为Hitachi pump L-7100，紫外灯的型号可以为UVdetector L-7400。
本发明的另一目的为提供一种北里孢菌属Kitasatospora sp.菌种，本发明上述制备方法中使用的Kitasatospora sp.系从安徽仙人岕山蜜蜂体内中分离、纯化得到，特征为：在ISP4平板上，培养初期基内菌丝为白色，易产生单个菌落，一周后开始产生灰色孢子，并逐渐加深，划线不连续气生菌丝白色。该菌种已经保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为：CGMCC No.16924，保藏日期2018年12月18日，分类命名：北里孢菌Kitasatospora sp.，地址：北京市朝阳区北辰西路1号院3号，邮编100101。
有益效果：
本发明首次从安徽仙人岕山蜜蜂体内获得内生放线菌Kitasatospora sp.CGMCC16924，并从其发酵产物中首次发现了一类肯塔森纳霉素(kitacinnamycins)，体外活性实验表明，这类化合物可以调控巨噬细胞分泌IFN-β，其中可提高IFN-β的肯塔森纳霉素F可进一步用于制备抗病毒及抗肿瘤药物。
附图说明
图1为本发明肯塔森纳霉素A-L的HPLC色谱图。
图2为本发明肯塔森纳霉素调控巨噬细胞分泌IFN-β的实验结果图。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1：Kitasatospora sp.CGMCC16924的分离、纯化与鉴定
将来自安徽仙人岕山的蜜蜂在饥饿条件下放养24h，然后在无菌条件下于超净台中用75％酒精对其消毒后，然后用无菌水漂洗3次，再加少量无菌水在灭菌研钵中研磨，用无菌水对研磨液梯度稀释成3个梯度10-1，10-2，10-3，分别取各梯度稀释液200μl涂布于高氏一号培养基(可溶性淀粉20g、KNO3 1g、NaCl 0.5g、K2HPO4·3H2O 0.5g、MgSO4·7H2O 0.5g、FeSO4·7H2O 0.01g、琼脂15-25g、蒸馏水1L，PH7.4～7.6)中，置于28℃恒温箱中培养，待菌落长出(约十天后)，用竹签挑取单菌落用四区划线法接到高氏平板中进行纯化培养，得到蜜蜂内生放线菌。在ISP4平板上，培养初期基内菌丝为白色，易产生单个菌落，一周后开始产生灰色孢子，并逐渐加深。经过形态及16S rRNA鉴定该菌为拟无枝酸菌属Kitasatosporasp.(accession number:MK351795)。将该菌命名为Kitasatospora sp.NA04385，并于2018年12月18日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为：CGMCCNo.16924。
实施例2：Kitasatospora sp.CGMCC 16924的液体发酵
活化Kitasatospora sp.CGMCC 16924，将新鲜的孢子接种到1L的摇瓶中，每瓶含有TSB培养基(胰蛋白胨15g，大豆蛋白胨5g，氯化钠5g，蒸馏水1L)200mL，置于摇床上28℃培养2天，作为种子液。以每瓶20mL的接种量接种到100瓶含有200mL SCAS培养液(40g可溶性淀粉，5g络蛋白水解物，0.5g磷酸二氢钾，0.5g七水合硫酸镁，0.01g七水合硫酸，水1L)的摇瓶中，28℃、230rpm/min培养10天。
实施例3：肯塔森纳霉素的提取与分离
实施例2中所得发酵液用4层纱布过滤，滤液用树脂进行吸附后经甲醇洗脱，真空浓缩干燥得粗浸膏F1。将粗浸膏F1悬浮于水，用石油醚萃取后取水相再用乙酸乙酯萃取，所得乙酸乙酯萃取液经浓缩得浸膏F2 5.8g；对浸膏F2进行正相硅胶柱层析，以CH2Cl2/MeOH作为流动相梯度洗脱(v/v100:1，100:2，100:4，100:8，100:16)，8个柱体积，获得10个出峰组分。将第二出峰组分经过semi-HPLC(色谱柱：Allsphere ODS-2.5mm column)，乙腈-水体积比＝1-16：4，以2.5mL/min流速梯度洗脱15min，制得本发明所述kitacinnamycin A-L(100mg)。泵的型号可以为Hitachi pump L-7100，紫外灯的型号可以为UV detector L-7400。HPLC色谱图如图1所示(其中A-L分别对应肯塔森纳霉素Kitacinnamycin A-L)。
实施例4：肯塔森纳霉素的结构鉴定
本发明所述肯塔森纳霉素的结构是基于它们的质谱、核磁共振谱确定的。
光谱学数据如下：
Kitacinnamycin A:UV(MeOH)λmax(logε):266(4.43),296(4.39)；[α]D20(c0.03MeOH)-66.7；HR-ESI-MS(m/z):1447.6492[M+Na]+(calcd for C67H96N10O24Na,1447.6491)；
Kitacinnamycin B:UV(MeOH)λmax(logε):266(4.44),296(4.40)；[α]D20(c0.03MeOH)-66.7；HR-ESI-MS(m/z):1461.6642[M+Na]+(calcd for C68H98N10O24Na,1461.6648)；
Kitacinnamycin C:HR-ESI-MS(m/z):1312.6447[M+Na]+(calcd forC63H91N11O18Na,1312.6436)；
Kitacinnamycin D:HR-ESI-MS(m/z):1326.6580[M+Na]+(calcd forC64H93N11O18Na,1326.6592)；
Kitacinnamycin E:UV(MeOH)λmax(logε):262(4.20),296(4.16)；[α]D20(c0.05MeOH)-40.0；HR-ESI-MS(m/z):1123.5435[M+Na]+(calcd for C55H76N10O14Na,1123.5435)；
Kitacinnamycin F:UV(MeOH)λmax(logε):262(4.11),296(4.07)；[α]D20(c0.05MeOH)-40.0；HR-ESI-MS(m/z):1137.5588[M+Na]+(calcd for C56H78N10O14Na,1137.5591)；
Kitacinnamycin G:UV(MeOH)λmax(logε):248(4.27),288(4.28)；[α]D20(c0.05MeOH)-40.0；HR-ESI-MS(m/z):1093.5688[M+Na]+(calcd for C55H78N10O12Na,1093.5693)；
Kitacinnamycin H:UV(MeOH)λmax(logε):248(4.16),288(4.15)；[α]D20(c0.05MeOH)-40.0；HR-ESI-MS(m/z):1107.5845[M+Na]+(calcd for C56H80N10O12Na,1107.5849)；
Kitacinnamycin I:UV(MeOH)λmax(logε):248(3.84),288(3.87)；[α]D20(c0.1MeOH)-20.0；HR-ESI-MS(m/z):1109.5656[M+Na]+(calcd for C55H78N10O13Na,1109.5642)；
Kitacinnamycin J:UV(MeOH)λmax(logε):248(3.97),288(3.98)；[α]D20(c0.1MeOH)-20.0；HR-ESI-MS(m/z):1123.5792[M+Na]+(calcd for C56H80N10O13Na,1123.5799)；
Kitacinnamycin K:UV(MeOH)λmax(logε):273(4.15),296(4.11)；[α]D20(c0.05MeOH)-133.3；HR-ESI-MS(m/z):1085.5670[M+H]+(calcd for C55H76N10O13H,1085.5666)；
Kitacinnamycin L:UV(MeOH)λmax(logε):273(4.23),296(4.24)；[α]D20(c0.05MeOH)-133.3；HR-ESI-MS(m/z):1121.5640[M+Na]+(calcd for C56H78N10O13Na,1121.5642).
肯塔森纳霉素的1H和13C NMR数据见以下列表：
表1.1H and 13C NMR data for kitacinnamycin A and B
Measured in DMSO-d6,600MHz
表2.1H and 13C NMR data for kitacinnamycin C and D
Measured in DMSO-d6,600MHz
表3.1H and 13C NMR data for kitacinnamycin E and F
Measured in DMSO-d6,600MHz
表4.1H and 13C NMR data for kitacinnamycin G and H
Measured in DMSO-d6,600MHz
表5 1H and 13C NMR data for kitacinnamycin I and J
Measured in DMSO-d6,600MHz
表6.1H and 13C NMR data for kitacinnamycin K and L
Measured in DMSO-d6,600MHz。
Kitacinnamycin A-L结构如下：
实施例5：活性试验
实验材料：人单核巨噬细胞THP-1，IFN-βELISA试剂盒，1640培养基，Poly(dA:dT)(dsDNA naked-complexed with transfection reagent)，2'3'-cGAMP。
实验方法：
IFN-β的测定根据ELISA试剂盒(达科为)描述的方法进行。流程如下：从已平衡至室温的密封袋中取出所需板条，其它板条密封放回4℃。除空白孔外，分别将标本或不同浓度标准品(100μl/孔)加入相应孔中，除空白孔外，同时再加入生物素化抗体工作液(50μl/孔)。混匀后用封板胶纸封住反应孔，37℃培养箱孵育90min。洗板4次。除空白孔外，加入HRP标记的二抗(100μl/孔)。用封板胶纸封住反应孔，37℃培养箱孵育30min。洗板4次。加入显色剂100μl/孔，避光37℃培养箱孵育10-15min。加入终止液100μl/孔，混匀后即刻测量OD450值(30min内)。每个标准品和标本的OD值应减去零孔的OD值。以标准品浓度作横坐标，OD值作纵坐标，以平滑线连接各标准品的坐标点，通过标本的OD值可以在标准曲线上计算出其浓度。
具体操作步骤如下：
THP-1培养于含10％胎牛血清的1640培养基中，待细胞长至对数生长期时，采用PMA(500nM)分化THP-1 3小时，分别加入Poly(dA:dT)与肯塔森纳霉素A、B、E、F、G、H、I、J、K共同孵育6小时；收集细胞培养上清，ELISA法测定IFN-β的浓度。
实验结果如图2所示。STING是天然免疫信号通路中的重要接头蛋白，定位于内质网，可直接结合几种环化二核苷酸，其中cGAMP是由DNA受体cGAS结合外源DNA后合成的，由此介导一型干扰素IFN-β的表达，产生抗病毒的作用。单独加入Poly dA:dT，IFN-β的表达量是500pg/ml,而分别加入10μM kitacinnamycin A,B,E,G,H能够使IFN-β的表达量轻微下降，加入I,J,K则使IFN-β的表达量显著下降。kitacinnamycin F能显著增加IFN-β的表达，达到1000pg/ml，可进一步用于制备抗病毒及抗肿瘤药物。
Claims (10)
1.一类肯塔森纳霉素，其特征在于具有如下结构式：
其中，R1代表C1-C6的烷基，R2代表C1-C6的烷基、C1-C6的烷基醇、-(CH2)n(CO)mR3，n代表0-5的整数，m代表0或者1，R3代表H、-OH、
2.根据权利要求1所述的肯塔森纳霉素，其特征在于所述R1代表-CH3、-CH2CH3，R2代表-CH3、-CH2CH3、-CH3OH、-CH2CH3OH、-R3或者-COR3,R3代表H、-OH、
3.根据权利要求1所述的肯塔森纳霉素，其特征在于：
R1代表-CH3、-CH2CH3，R2代表或者-COR3,R3代表H、-OH或者
R1代表-CH3，R2代表-CH3；
R1代表-CH3，R2代表-CH2CH3；
R1代表-CH2CH3，R2代表-CH3。
4.根据权利要求1-3任一项所述的肯塔森纳霉素的制备方法，其特征在于利用北里孢菌属Kitasatospora sp.菌种通过液体发酵提取得到。
5.根据权利要求4所述的制备方法，其特征在于所述菌种为生物保藏编号CGMCCNo.16924的北里孢菌属Kitasatospora sp.菌种。
6.根据权利要求4或5所述的制备方法，其特征在于包括以下步骤：
(1)将北里孢菌属Kitasatospora sp菌种发酵培养；
(2)将步骤(1)所得发酵液过滤、浓缩得到浸膏F1；
(3)将浸膏F1悬浮于水，依次用石油醚和乙酸乙酯萃取，所得乙酸乙酯萃取液经浓缩得浸膏F2；
(4)对浸膏F2进行正相硅胶柱层析，依次用体积比为100：1、100：2、100：4、100：8、100：16的二氯甲烷-甲醇进行梯度洗脱，得到10个出峰组分
(5)将第二出峰组分经HPLC分离纯化制得式(I)所示化合物。
7.根据权利要求4所述的制备方法，其特征在于步骤(1)26-30℃培养10天。
8.根据权利要求4所述的制备方法，其特征在于步骤(5)中HPLC色谱条件为：流动相：乙腈-水体积比＝1-16：4梯度洗脱，2.5mL/min流速梯度洗脱15min。
9.一种北里孢菌属Kitasatospora sp.菌种，其特征在于生物保藏编号CGMCCNo.16924。
10.根据权利要求1-3任一项所述的肯塔森纳霉素在制备调控IFN-β表达药物中的应用。
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