CN117298177B - 一种天然免疫调节剂及其制备方法 - Google Patents
一种天然免疫调节剂及其制备方法 Download PDFInfo
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- CN117298177B CN117298177B CN202311595881.1A CN202311595881A CN117298177B CN 117298177 B CN117298177 B CN 117298177B CN 202311595881 A CN202311595881 A CN 202311595881A CN 117298177 B CN117298177 B CN 117298177B
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- valienamine
- fatty acid
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Classifications
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- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
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Abstract
本发明涉及天然药物技术领域,具体涉及一种天然免疫调节剂及其制备方法,所述天然免疫调节剂包括微生物发酵制剂、脂肪酸‑井冈霉烯胺‑己糖苷化合物或其药学上可接受的盐、溶剂化物或水合物。本发明还提供了该天然免疫调节剂的制备方法,生物活性实验显示本发明一种天然免疫调节剂,具有免疫调节作用,可抑制TNF‑α和IL‑17的表达,可用于免疫相关疾病的预防和/或治疗,同时具有较好的减毒效果。
Description
技术领域
本发明涉及天然药物技术领域,具体涉及一种天然免疫调节剂及其制备方法。
背景技术
免疫***平衡对身体健康至关重要,当其被打破时容易患免疫性疾病。肿瘤和感染与机体免疫低下相关;免疫反应过强时又可能引起器官移植免疫排斥、过敏和自身免疫病。免疫调节剂可通过调节机体的免疫反应治疗免疫功能紊乱所引起的免疫性疾病。对于肿瘤和感染等与免疫力低下相关的疾病,可采用增强机体免疫力的调节剂进行干预。而对于器官移植免疫排斥、过敏和自身免疫病等与机体免疫反应过强相关的疾病,可采用免疫抑制剂改善疾病症状和预后。雷公藤是卫茅科雷公藤属多年生藤本植物,具有清热解毒、消肿、消积、杀虫、止血等功效,具有显著的免疫抑制活性,20世纪50年代,开始有中医将雷公藤用于治疗类风湿性关节炎、慢性肾炎、红斑狼疮和银屑病等自身免疫病。雷公藤有效成分包括雷公藤内酯醇、雷公藤内酯酮、雷公藤红素、雷公藤甲素等。虽然具有显著的药理活性,但对正常细胞有较强的杀伤作用、水溶性差、治疗窗窄,极大地限制了在临床上的应用。因此,提高或保留雷公藤的药理活性,同时降低其对正常细胞的毒性,提升其治疗指数,开发出新的高效、低毒的免疫调节剂是亟需解决的技术问题。
发明内容
针对以上技术问题,本发明提出了一种天然免疫调节剂及其制备方法,采用微生物对雷公藤等具有免疫调节功效的中药进行发酵,具有增效、解毒等效果;哈茨木霉菌可将纤维素分解为葡萄糖,供嗜水气单胞菌和蕈状芽孢杆菌利用,转化为具有免疫调节功能的氨基糖,氨基糖以糖苷键与井冈霉烯胺结合,再引入脂肪酸链,得到的脂肪酸-井冈霉烯胺-己糖苷具有更强的生物亲和力,可提高免疫调节效果。
本发明是通过以下技术方案实现的:
一种天然免疫调节剂,包括微生物发酵制剂和脂肪酸-井冈霉烯胺-己糖苷。
进一步地,所述微生物发酵制剂的制备方法,包括以下步骤:
(1) 将雷公藤、白芍、山茱萸按质量比5:1:1的比例混合洗净,于55°C下干燥粉碎,过80目筛后与水混匀,121°C灭菌30min后,加入K2HPO4、KH2PO4、NaCl和MgSO4,得到发酵培养基;
(2) 将哈茨木霉菌、嗜水气单胞菌和蕈状芽孢杆菌共同接种于步骤(1)所得发酵培养基中,接种量均为5%,发酵温度26-30°C,摇床转速150-200r/min下,摇床培养96h,得到发酵液;
(3) 将步骤(2)所得的发酵液3000r/min下离心10min,固液分离得到沉淀和上清液I,将沉淀置于无菌管中,加入无水甲醇,于30°C、500W超声提取3-4h,冷却至室温,以12000r/min离心10min,分离得到上清液II,重复提取3次,混合每次上清液II,经过0.22μm微孔滤膜过滤,得到滤液,将滤液冷冻干燥,研磨成粉末,得到所述微生物发酵制剂。
进一步地,步骤(1)所述K2HPO4、KH2PO4、NaCl和MgSO4的加入量分别为0.7 g/L、0.3g/L、0.5 g/L和0.5g/L。
进一步地,步骤(2)所述哈茨木霉菌保藏地址为中国普通微生物菌种保藏管理中心,保藏编号为CGMCC 5.1213;嗜水气单胞菌保藏地址为中国普通微生物菌种保藏管理中心,保藏编号为CGMCC 1.2017;蕈状芽孢杆菌保藏地址为中国普通微生物菌种保藏管理中心,保藏编号为CGMCC 1.10168;以上所述菌株均为已公开菌株。
进一步地,步骤(3)所述沉淀与无水甲醇的质量体积比为2g:15mL。
进一步地,所述脂肪酸-井冈霉烯胺-己糖苷包括脂肪酸-井冈霉烯胺-己糖苷化合物或其药学上可接受的盐、溶剂化物或水合物。
进一步地,所述脂肪酸-井冈霉烯胺-己糖苷化合物的结构式如式1所示:
式1。
进一步地,式1中所述R1基团为CH3(CH2)4CH=CHCH2CH=CH(CH2)7—、CH3(CH2)4CH=CHCH2CH=CHCH2CH=CH(CH2)7—和CH3(CH2)4(CH=CH-CH2)4(CH2)2—中的至少一种。
进一步地,所述脂肪酸-井冈霉烯胺-己糖苷化合物的制备方法,包括以下步骤:
I. 将井冈霉烯胺与脂肪酸,按摩尔比1:1的比例混合加入反应釜中,加入去离子水,用碳酸氢钠调节pH至7.5-8,温度维持在40-50°C,不断搅拌30min,得到反应物I;
II. 向步骤I所得反应物I中加入步骤(3)制得的上清液I和植酸,用1mol/L的NaOH调节pH至10-11,搅拌并加热至100°C,加热维持4h,于60°C下浓缩至原体积的20%,然后喷雾干燥制成粉末;
III. 将步骤II所得粉末与乙酸乙酯按照1:3的质量比混合,搅拌1h后过滤,将滤液在绝对压强为0.08MPa下浓缩至干,得到粗提物;
IV. 将步骤III制得的粗提物利用硅胶柱层析,采用环己烷和乙酸乙酯体积比为70:30的洗脱液进行洗脱后经中低压液相ODS柱层析,采用甲醇和水体积比为90:10的洗脱液洗脱,洗脱后在绝对压强为0.08MPa下浓缩至干,洗脱时洗脱液流速为3mL/min,得到所述脂肪酸-井冈霉烯胺-己糖苷化合物。
进一步地,步骤I所述井冈霉烯胺与去离子水的质量比为1:10。
进一步地,步骤II所述反应物I、上清液I和植酸的体积比为20:60:1。
进一步地,所述脂肪酸为亚油酸、α-亚麻酸、四花生烯酸中的至少一种。
进一步地,所述药学上可接受的盐为式1化合物与有机碱或无机碱形成的盐。
更进一步的,所述形成的盐为钠盐、钾盐、钙盐、铁盐、镁盐、锌盐、铝盐、钡盐或铵盐。
进一步地,所述药学上可接受的溶剂化物为式1化合物在溶剂中溶解后形成的溶液。
进一步地,所述药学上可接受的水合物为式1化合物与水分子以共价键相结合形成的水合物。
本发明还提供了所述天然免疫调节剂的制备方法,其特征在于,包括以下步骤:将微生物发酵制剂、脂肪酸-井冈霉烯胺-己糖苷按照5:1的质量比在混合机中混合均匀,制得一种天然免疫调节剂。
进一步地,所述天然免疫调节剂可以是包括注射剂如静脉乳剂、口服固体制剂如片剂、胶囊剂、颗粒剂等各种临床可接受剂型。
进一步地,所述天然免疫调节剂含有效剂量的微生物发酵制剂、脂肪酸-井冈霉烯胺-己糖苷。
与现有技术相比,本发明具有以下有益效果:
本发明采用哈茨木霉菌、嗜水气单胞菌、蕈状芽孢杆菌对雷公藤等具有免疫抑制功效的中药进行发酵,经微生物作用后,中药细胞壁中的纤维素、木质素等物质被降解,活性成分得以释放;中药活性成分酶解为小分子物质,有利于机体吸收,增强药效。同时,微生物发酵后,中药的毒性降低,可降低有效成分如雷公藤甲素、雷公藤红素的细胞毒性,减少毒副作用。同时中药中的纤维素、蛋白质等成分可供微生物利用,促进微生物的生长繁殖,中药与微生物协同作用、相辅相成,通过微生物的生长代谢和生物转化来发酵中药,可提高药效,降低中药毒性和毒副作用,发酵产物表现出高于药性基质相加的药效。此外,哈茨木霉菌可产生纤维素酶,将纤维素分解为葡萄糖,供嗜水气单胞菌和蕈状芽孢杆菌利用,一方面,可促进二者生长繁殖,另一方面,嗜水气单胞菌可将葡萄糖转化为氨基半乳糖,蕈状芽孢杆菌可将葡萄糖转化为氨基葡萄糖,氨基半乳糖和氨基葡萄糖均为氨基糖,具有免疫调节的功能,氨基糖与井冈霉烯胺通过糖苷键结合,产物具有抗炎、调节免疫的功效,引入脂肪酸链,得到的脂肪酸-井冈霉烯胺-己糖苷具有更强的生物亲和力,可提高免疫调节效果。
附图说明
为了更清楚地说明本发明或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1 为本发明实施例3所述亚油酸-井冈霉烯胺-己糖苷化合物的核磁共振光谱图,其中,A为1H-NMR图,B为13C-NMR图;
图2 为本发明实施例3所述亚油酸-井冈霉烯胺-己糖苷化合物的结构式图;
图3 为本发明实施例5和对比例1-2所述天然免疫调节剂对各组小鼠结肠组织TNF-α含量的影响;
图4 为本发明实施例5和对比例1-2所述天然免疫调节剂对各组小鼠结肠组织IL-17含量的影响;
图5为本发明实施例5和对比例1-2所述天然免疫调节剂对人正常肝细胞L02细胞活力的影响图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,以下结合具体实施例,对本发明进一步详细说明,但本发明并不仅限于以下的实施例。
需要说明的是,无特殊说明外,本发明中涉及到的化学试剂均通过商业渠道购买,菌株为已公开菌株。
实施例1:本实施例提供了一种微生物发酵制剂,所述微生物发酵制剂的制备方法,包括以下步骤:
(1) 将雷公藤、白芍、山茱萸按质量比5:1:1的比例混合洗净,于55°C下干燥粉碎,过80目筛后与水混匀,121°C灭菌30min后,加入K2HPO4、KH2PO4、NaCl和MgSO4,加入量分别为0.7 g/L、0.3 g/L、0.5 g/L和0.5g/L,得到发酵培养基;
(2) 将哈茨木霉菌、嗜水气单胞菌和蕈状芽孢杆菌共同接种于步骤(1)所得发酵培养基中,接种量为5%,发酵温度30°C,摇床转速200r/min下,摇床培养96h,得到发酵液;
(3) 将步骤(2)所得的发酵液3000r/min下离心10min,固液分离得到沉淀和上清液I,将沉淀置于无菌管中,加入无水甲醇,沉淀与无水甲醇的质量体积比为2g:15mL,于30°C、500 W超声提取4h,冷却至室温,以12000r/min离心10min,分离得到上清液II,重复提取3次,混合每次上清液II,经过0.22μm微孔滤膜过滤,得到滤液,将滤液冷冻干燥,研磨成粉末,得到所述微生物发酵制剂。
实施例2:本实施例提供了一种微生物发酵制剂,所述微生物发酵制剂的制备方法,包括以下步骤:
(1) 将雷公藤、白芍、山茱萸按质量比5:1:1的比例混合洗净,于55°C下干燥粉碎,过80目筛后与水混匀,121°C灭菌30min后,加入K2HPO4、KH2PO4、NaCl和MgSO4,加入量分别为0.7 g/L、0.3 g/L、0.5 g/L和0.5g/L,得到发酵培养基;
(2) 将哈茨木霉菌、嗜水气单胞菌和蕈状芽孢杆菌共同接种于步骤(1)所得发酵培养基中,接种量为5%,发酵温度26°C,摇床转速150r/min下,摇床培养96h,得到发酵液;
(3) 将步骤(2)所得的发酵液3000r/min下离心10min,固液分离得到沉淀和上清液I,将沉淀置于无菌管中,加入无水甲醇,沉淀与无水甲醇的质量体积比为2g:15mL,于30°C、500 W超声提取3h,冷却至室温,以12000r/min离心10min,分离得到上清液II,重复提取3次,混合每次上清液II,经过0.22μm微孔滤膜过滤,得到滤液,将滤液冷冻干燥,研磨成粉末,得到所述微生物发酵制剂。
实施例3:本实施例提供了一种脂肪酸-井冈霉烯胺-己糖苷化合物,所述脂肪酸-井冈霉烯胺-己糖苷化合物的制备方法,包括以下步骤:
I. 将井冈霉烯胺与脂肪酸,按摩尔比1:1的比例混合加入反应釜中,加入去离子水,用碳酸氢钠调节pH至8,温度维持在50°C,不断搅拌30min,得到反应物I;
II. 向步骤I所得反应物I中,加入实施例1步骤(3)制得的上清液I和植酸,用1mol/L的NaOH调节pH至11,搅拌并加热至100°C,加热维持4h,于60°C下浓缩至原体积的20%,然后喷雾干燥制成粉末;
III. 将步骤II所得粉末与入乙酸乙酯按照1:3的质量比混合,搅拌1 h后过滤,将滤液在绝对压强为0.08MPa下浓缩至干,得到粗提物;
IV. 将步骤III制得的粗提物利用硅胶柱层析,采用环己烷和乙酸乙酯体积比为70:30的洗脱液进行洗脱后经中低压液相ODS柱层析,采用甲醇和水体积比为90:10的洗脱液洗脱,洗脱后取一部分通过超导核磁共振仪分析,剩余的在绝对压强为0.08MPa下浓缩至干,洗脱时洗脱液流速为3mL/min,得到所述脂肪酸-井冈霉烯胺-己糖苷化合物。
本实施例步骤I所述井冈霉烯胺与去离子水的质量比为1:10,脂肪酸为亚油酸;步骤II所述反应物I、上清液I和植酸的体积比为20:60:1;图1为本实施例制得的脂肪酸-井冈霉烯胺-己糖苷化合物的核磁共振图谱,确定化合物分子式为C32H56N2O8,结构式如图2所示。
实施例4:本实施例提供了一种脂肪酸-井冈霉烯胺-己糖苷化合物,所述脂肪酸-井冈霉烯胺-己糖苷化合物的制备方法,包括以下步骤:
I. 将井冈霉烯胺与脂肪酸,按摩尔比1:1的比例混合加入反应釜中,加入去离子水,用碳酸氢钠调节pH至7.5,温度维持在40°C,不断搅拌30min,得到反应物I;
II. 向步骤I所得反应物I中,加入实施例1步骤(3)制得的上清液I和植酸,用1mol/L的NaOH调节pH至10,搅拌并加热至100°C,加热维持4h,于60°C下浓缩至原体积的20%,然后喷雾干燥制成粉末;
III. 将步骤II所得粉末与入乙酸乙酯按照1:3的质量比混合,搅拌1 h后过滤,将滤液在绝对压强为0.08MPa下浓缩至干,得到粗提物;
IV. 将步骤III制得的粗提物利用硅胶柱层析,采用环己烷和乙酸乙酯体积比为70:30的洗脱液进行洗脱后经中低压液相ODS柱层析,采用甲醇和水体积比为90:10的洗脱液洗脱,洗脱后在绝对压强为0.08MPa下浓缩至干,洗脱时洗脱液流速为3mL/min,得到所述脂肪酸-井冈霉烯胺-己糖苷化合物。
本实施例步骤I所述井冈霉烯胺与去离子水的质量比为1:10,脂肪酸为α-亚麻酸;步骤II所述反应物I、上清液I和植酸的体积比为20:60:1。
实施例5:本实施例提供了一种天然免疫调节剂,包括生物发酵制剂和脂肪酸-井冈霉烯胺-己糖苷化合物,本实施例还提供了所述天然免疫调节剂的制备方法,包括以下步骤:将实施例1制得的微生物发酵制剂和实施例3制得的脂肪酸-井冈霉烯胺-己糖苷化合物按照5:1的质量比在混合机中混合均匀,制得一种天然免疫调节剂。
对比例1:本对比例提供了一种天然免疫调节剂,包括中药制剂和脂肪酸-井冈霉烯胺-己糖苷化合物。
所述中药制剂的制备方法包括以下步骤:将雷公藤、白芍、山茱萸按质量比5:1:1的比例混合洗净,于55°C下干燥粉碎过80目筛,得到中药粉末,置于无菌管中,加入无水甲醇,中药粉末与无水甲醇的质量体积比为2g:15mL,于30°C、500 W超声提取4h,冷却至室温,以12000r/min离心10min,分离得到上清液II,重复提取3次,混合每次上清液II,经过0.22μm微孔滤膜过滤,得到滤液,将滤液冷冻干燥,研磨成粉,制得中药制剂。
所述脂肪酸-井冈霉烯胺-己糖苷化合物的制备方法与实施例3相同。
本对比例还提供了所述天然免疫调节剂的制备方法,包括以下步骤:将中药制剂和脂肪酸-井冈霉烯胺-己糖苷化合物按照5:1的质量比在混合机中混合均匀,制得一种天然免疫调节剂。
对比例2:本对比例提供了一种天然免疫调节剂,包括脂肪酸-井冈霉烯胺反应物和微生物发酵制剂。
所述脂肪酸-井冈霉烯胺反应物的制备方法,包括以下步骤:将井冈霉烯胺与脂肪酸,按摩尔比1:1的比例混合加入反应釜中,加入去离子水,用碳酸氢钠调节pH至7.5,温度维持在40°C,不断搅拌30min,然后于60°C下浓缩至原体积的20%,喷雾干燥制成粉末,制得脂肪酸-井冈霉烯胺反应物。
所述微生物发酵制剂的制备方法与实施例1相同。
本对比例还提供了所述天然免疫调节剂的制备方法,包括以下步骤:将微生物发酵制剂和脂肪酸-井冈霉烯胺反应物按照5:1的质量比在混合机中混合均匀,制得一种天然免疫调节剂。
实验例1:将实施例5和对比例1-2的天然免疫调节分别与去离子水混合,配置成0.1g/mL的溶液。选取50只SPF级昆明小鼠,雄性,7-9周龄,体重(20±2)g,将其中40只小鼠作为模型组,用无菌蒸馏水饲喂1周后,用3wt%的DSS溶液替代无菌蒸馏水,连续7 d,每天更换新的DSS溶液,期间,另外10只小鼠作为空白组每天饲喂无菌蒸馏水,然后将模型组的40只小鼠分为实施例5组、对比例1组、对比例2组和CK组,每组10只,每天固定时间分别灌服实施例5、对比例1、对比例2制得的天然免疫调节剂配置的溶液以及等体积无菌生理盐水,空白组小鼠在相同时间灌服等体积无菌生理盐水,连续16d,每天1次,试验期间各组小鼠自由饮食饮水。小鼠第15d灌胃结束开始禁食,在第16d灌胃结束2h后处死。取鼠结肠组织,用生理盐水将肠腔冲洗干净,并取约0.1g结肠组织置于液氮保存。每组随机挑取5只小鼠的结肠组织,检测前按质量比结肠组织:PBS=1:9匀浆;制备好的匀浆液3000r/min离心10 min,吸取上清液,用ELISA试剂盒检测小鼠TNF-α、IL-17指标,具体操作步骤严格按照试剂盒说明书进行。结果如图3-图4所示。
由图3-图4可知,与空白组相比,CK组小鼠结肠TNF-α、IL-17含量极显著升高。与CK组相比,实施例5和对比例1-2组细胞因子TNF-α、IL-17含量极显著降低。**表示与空白组相比有极显著差异,##表示与CK组相比有极显著差异。
图3-图4结果显示,实施例5和对比例1-2的天然免疫调节剂对结肠组织中促炎因子TNF-α、IL-17含量有不同程度的下调,以实施例5组效果最佳。CK组小鼠结肠组织中促炎因子TNF-α、IL-17含量显著升高,表明CK组小鼠结肠已发生严重的炎症反应,天然免疫调节剂可改善各细胞因子异常变化,趋于恢复平衡。表明本发明一种天然免疫调节制剂具有抑制TNF-α、IL-17表达的效果,可作为免疫调节剂用于防治免疫相关疾病,例如类风湿性关节炎等自身免疫病。
实验例2:将人正常肝细胞L02置于37℃、含5% CO2的细胞培养箱中培养,以含有10% 胎牛血清的RPMI-1640培养基为完全培养基,每60h传代一次,将对数生长期的人正常肝细胞L02以每孔10000个/100μL接种于96孔板,将96孔板的孔平均分为4组,分别加入实施例5和对比例1-2制得的天然免疫调节剂,每孔浓度为2μg/mL,同时设置不加天然免疫调节剂的空白对照组。将各组置于37℃、含5%CO2的细胞培养箱中培养48h。使用CCK-8试剂盒测定细胞吸光度值,按照公式C(%)=[(Ac-A0)/(Ab-A0)]*100%计算细胞活力,C为细胞活力,Ac为试验组吸光度值,Ab为对照组吸光度值,A0为背景吸光度值。结果如图5所示。**表示与空白对照组相比有极显著差异,*表示与对照组相比有显著差异。
由图5结果可知,实施例5组的细胞活力较空白对照组没有显著变化,对比例1-2组的细胞活力较对照组显著降低,减毒效果较差,表明本发明一种天然免疫调节剂细胞毒性较小,更加安全可靠。
所属领域的普通技术人员应当理解:以上任何实施例的讨论仅为示例性的,并非旨在暗示本发明的范围(包括权利要求)被限于这些例子;在本发明的思路下,以上实施例或者不同实施例中的技术特征之间也可以进行组合,步骤可以以任意顺序实现,并存在如上所述的本发明的不同方面的许多其它变化,为了简明它们没有在细节中提供。
本发明旨在涵盖落入所附权利要求的宽泛范围之内的所有这样的替换、修改和变型。因此,凡在本发明的精神和原则之内,所做的任何省略、修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.一种天然免疫调节剂,其特征在于,包括微生物发酵制剂和脂肪酸-井冈霉烯胺-己糖苷;
所述微生物发酵制剂的制备方法,包括以下步骤:
(1) 将雷公藤、白芍、山茱萸按质量比5:1:1的比例混合洗净,于55°C下干燥粉碎,过80目筛后与水混匀,121°C灭菌30min后,加入K2HPO4、KH2PO4、NaCl和MgSO4,加入量分别为0.7g/L、0.3 g/L、0.5 g/L和0.5g/L,得到发酵培养基;
(2) 将哈茨木霉菌、嗜水气单胞菌和蕈状芽孢杆菌共同接种于步骤(1)所得发酵培养基中,接种量均为5%,发酵温度26-30°C,摇床转速150-200r/min下,摇床培养96h,得到发酵液;
(3) 将步骤(2)所得的发酵液3000r/min下离心10min,固液分离得到沉淀和上清液I,将沉淀置于无菌管中,加入无水甲醇,沉淀与无水甲醇的质量体积比为2g:15mL,于30°C、500W超声提取3-4h,冷却至室温,以12000r/min离心10min,分离得到上清液II,重复提取3次,混合每次上清液II,经过0.22μm微孔滤膜过滤,得到滤液,将滤液冷冻干燥,研磨成粉末,得到所述微生物发酵制剂;
所述脂肪酸-井冈霉烯胺-己糖苷包括脂肪酸-井冈霉烯胺-己糖苷化合物或其药学上可接受的盐、溶剂化物或水合物;
所述脂肪酸-井冈霉烯胺-己糖苷化合物的结构式如式1所示:
式1;
式1中所述R1基团为CH3(CH2)4CH=CHCH2CH=CH(CH2)7—、
CH3(CH2)4CH=CHCH2CH=CHCH2CH =CH(CH2)7—、
CH3(CH2)4(CH=CH-CH2)4 (CH2)2—中的至少一种;
所述脂肪酸-井冈霉烯胺-己糖苷化合物的制备方法,包括以下步骤:
I. 将井冈霉烯胺与脂肪酸,按摩尔比1:1的比例混合加入反应釜中,加入去离子水,用碳酸氢钠调节pH至7.5-8,温度维持在40-50°C,不断搅拌30min,得到反应物I;
II. 向步骤I所得反应物I中,加入步骤(3)制得的上清液I和植酸,用1mol/L的NaOH调节pH至10-11,搅拌并加热至100°C,加热维持4h,于60°C下浓缩至原体积的20%,然后喷雾干燥制成粉末;
III. 将步骤II所得粉末与入乙酸乙酯按照1:3的质量比混合,搅拌1h后过滤,将滤液在绝对压强为0.08MPa下浓缩至干,得到粗提物;
IV. 将步骤III制得的粗提物利用硅胶柱层析,采用环己烷和乙酸乙酯体积比为70:30的洗脱液进行洗脱后经中低压液相ODS柱层析,采用甲醇和水体积比为90:10的洗脱液洗脱,洗脱后在绝对压强为0.08MPa下浓缩至干,洗脱时洗脱液流速为3mL/min,得到所述脂肪酸-井冈霉烯胺-己糖苷化合物。
2.根据权利要求1所述的一种天然免疫调节剂,其特征在于,步骤I所述井冈霉烯胺与去离子水的质量比为1:10,脂肪酸为亚油酸、α-亚麻酸、四花生烯酸中的至少一种;步骤II所述反应物I、上清液I和植酸的体积比为20:60:1。
3.根据权利要求2所述的一种天然免疫调节剂,其特征在于,所述药学上可接受的盐为式1化合物与有机碱或无机碱形成的盐,所述形成的盐为钠盐、钾盐、钙盐、铁盐、镁盐、锌盐、铝盐、钡盐或铵盐;所述药学上可接受的溶剂化物为式1化合物在溶剂中溶解后形成的溶液;所述药学上可接受的水合物为式1化合物与水分子以共价键相结合形成的水合物。
4.一种如权利要求1-3任一项所述的天然免疫调节剂的制备方法,其特征在于,包括以下步骤:将微生物发酵制剂、脂肪酸-井冈霉烯胺-己糖苷按照5:1的质量比在混合机中混合均匀,制得一种天然免疫调节剂。
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