CN114835668A - 青龙衣中一种环醚型二芳基庚烷的制备方法和应用 - Google Patents
青龙衣中一种环醚型二芳基庚烷的制备方法和应用 Download PDFInfo
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Abstract
本发明属于医药技术领域,涉及一种从青龙衣中提取的二芳基庚烷类新化合物,并公开了制备该化合物的方法及应用。具体制备流程是将青龙衣经醇提法、有机溶剂萃取法、吉拉德试剂的纯化与富集、硅胶柱色谱分离以及制备HPLC分离等步骤,从中得到1个环醚型二芳基庚烷化合物,命名为3',4''‑环氧‑1‑(4'‑甲氧基苯基)‑7‑(2'',6''‑二羟基‑3''‑甲氧基苯基)‑3‑庚酮,分子式为C21H24O6。体外抗肿瘤活性研究表明其对人宫颈癌细胞Hela、人肝癌细胞HepG‑2具有一定的抑制作用。本发明采用的制备二芳基庚烷化合物的方法操作明确可控,获得的化合物杂质少、纯度高,具有开发为抗肿瘤药物的应用前景。
Description
技术领域
本发明涉及医药技术领域,具体涉及一种从青龙衣中分离得到的环醚型二芳基庚烷化合物及其制备方法和在制备抗肿瘤药物上的应用。
背景技术
恶性肿瘤已经成为严重危害人类健康的重大常见疾病,肝癌是世界第三大恶性肿瘤,宫颈癌是女性恶性肿瘤发病率排名第四的癌症,这严重威胁人民群众的身体健康,并增加患者的经济负担。中医药在发挥增效与减毒作用、改善肿瘤患者临床症状、提高生存质量、稳定肿瘤病灶方面优势更加明显。
青龙衣为胡桃科胡桃属植物胡桃楸Juglans mandshurica Maxim.的未成熟外果皮,别名青胡桃皮、核桃青皮。青龙衣中含有醌类、萜类、黄酮类、有机酸类以及酯类等多种化学成分,《中药大辞典》记载其味辛、苦,性涩、平,以清热解毒、祛风疗癣、止痛止痢等功效入药,因此在临床上广泛使用。现代研究表明,青龙衣提取物可通过抑制肿瘤细胞生长和增殖,诱导肿瘤细胞凋亡,抑制肿瘤细胞迁移和侵袭等机制发挥抗肿瘤作用。
青龙衣在民间应用虽已多年,但加工带来的大量青皮多数仍被当作废弃物堆在田间地头,造成巨大的浪费和严重的污染。青龙衣来源广阔,具有良好的开发前景,应继续挖掘其潜在价值,研究其化学成分以及药理活性,以期为临床应用奠定物质基础。
发明内容
本发明属于医药技术领域,涉及一种从青龙衣中提取的二芳基庚烷类新化合物,并公开了一种制备该化合物的方法及其应用,以进一步提高青龙衣利用率并解决环境污染的问题。
为了达成上述目的,本发明的解决方案是:
一种从废弃青龙衣中分离得到的环醚型二芳基庚烷,其结构式如下所示:
分子式为C21H24O6。
本发明还提供了该环醚型二芳基庚烷制备方法:依次通过醇提法、有机溶剂萃取、吉拉德试剂的纯化与富集、硅胶色谱柱以及制备HPLC分离等步骤得到。具体制备步骤如下:
(1)醇提法:将新鲜青龙衣低温烘干,取10 kg粉碎成粗粉,加入8倍量体积的95%乙醇,加热回流提取3次,将所得提取液合并,回收溶剂,减压干燥,得浸膏;
(2)有机溶剂萃取法:将步骤(1)中得到的浸膏,加入5倍量的纯净水稀释,再用与稀释液等体积的二氯甲烷对稀释溶液进行5次萃取,合并二氯甲烷萃取液,浓缩干燥得到二氯甲烷浸膏;
(3)吉拉德试剂的纯化与富集:取经步骤(2)中获得的二氯甲烷浸膏,反复捏溶于乙酸-无水乙醇(1:12,重量比)溶液体系中,加入吉拉德试剂P后,加热回流,反应完毕后回收乙醇至无醇味,加水稀释,用等体积乙醚萃取5次,分取水层用盐酸酸化,再用等体积乙醚萃取5次,得到浸膏;
(4)硅胶柱色谱分离:取步骤(3)所得浸膏进行硅胶柱色谱分离。采用体积比为100:0→0:100的二氯甲烷-甲醇混合溶剂进行梯度洗脱,其中将体积比为20:1的二氯甲烷-甲醇混合溶剂洗脱部分减压回收得到分离粗品;
(5)制备HPLC分离:将上述步骤(4)中所得粗品采用甲醇溶解进入制备型HPLC,流动相为体积比为55:45的甲醇和水混合溶液,洗脱流速为1.5 mL/min,保留时间tR=23.4min~24.3min阶段内收集馏分后,回收干燥,即得如式Ⅰ所示的化合物。
本发明的有益效果在于:本发明首次提供了以废弃的青龙衣为原料,制备及鉴定环醚型二芳基庚烷3',4''-环氧-1-(4'-甲氧基苯基)-7-(2'',6''-二羟基-3''-甲氧基苯基)-3-庚酮的方法,该化合物结构新颖并且通过抗肿瘤活性测定发现其对两种常见肿瘤细胞均具有较好的肿瘤细胞抑制率,为进一步开发利用青龙衣提供了有价值的药效物质基础。同时以废弃的青龙衣为原料,不仅能提高青龙衣资源的有效利用率,还能减轻废弃青龙衣所带来的环境污染。
附图说明
图1为本发明化合物的正性HR-ESI-MS谱图;
图2为本发明化合物的1H-NMR谱图;
图3为本发明化合物的13C-NMR谱图;
图4为本发明化合物的DEPT谱图;
图5为本发明化合物的HSQC谱图;
图6为本发明化合物的HMBC谱图;
图7为本发明化合物的1H-1H COSY谱图;
图8为本发明化合物的HMBC谱及1H-1H COSY谱主要相关关系图。
具体实施方式
下文清楚、完整地介绍了本发明,并提供了具体的实施实例,以便本技术领域的人员更好地理解本发明方案。这些实施例只做示范作用,并不限制本发明的适用范围。基于本发明中的实施例,本领域普通技术人员可以对本发明技术方案的细节和方式加以改进或替换,但在不脱离本发明原理、没有实质创新性改造的情况下,这些改进和替换均落入本发明的保护范围内。
实施例1:
3',4''-环氧-1-(4'-甲氧基苯基)-7-(2'',6''-二羟基-3''-甲氧基苯基)-3-庚酮的制备方法:
(1)醇提法:将新鲜青龙衣低温烘干,取10 kg粉碎成粗粉,加入8倍量体积的95%乙醇,加热回流提取3次,将所得提取液合并,回收溶剂,减压干燥,得浸膏688.8 g;
(2)有机溶剂萃取法:将步骤(1)中得到的浸膏,加入5倍量的纯净水稀释,再用与稀释液等体积的二氯甲烷对稀释溶液进行5次萃取,合并二氯甲烷萃取液,浓缩干燥得到二氯甲烷浸膏238.6 g;
(3)吉拉德试剂的纯化与富集:取经步骤(2)中获得的二氯甲烷浸膏,反复捏溶于5000 mL的乙酸-无水乙醇(1:12,重量比)溶液体系中,加入吉拉德试剂P 150 g后,加热回流3.5 h,反应完毕后回收乙醇至无醇味,加水稀释至3000 mL用等体积乙醚萃取5次,分取水层用0.5% 盐酸酸化,再用等体积乙醚萃取5次,得到浸膏16 g;
(4)硅胶柱色谱分离:取步骤(3)所得乙醚浸膏用200mL甲醇加热溶解,加入100g的80~100目硅胶混合均匀,干燥后研磨成拌样硅胶,待用;取300g的200~300目硅胶进行柱色谱分离,柱径高比为1:8;依次采用体积比为100:0、50:1、30:1、20:1的二氯甲烷-甲醇混合溶剂梯度洗脱,每一个比例洗脱6个柱体积,其中体积比为30:1的二氯甲烷-甲醇混合溶剂洗脱部分减压回收溶剂得到分离粗品2.4 g;
(5)制备HPLC分离:将上述步骤(4)中所得粗品采用甲醇溶解进入制备型HPLC,流动相为体积比为55:45的甲醇和水混合溶液,洗脱流速为1.5 mL/min,保留时间tR=23.4min~24.3min阶段内收集馏分后,回收干燥即得成品21.2 mg,纯度为96.5%。
实施例2
本发明化合物其质谱、NMR波谱数据如下:
HR-ESI-MS m/z:371.1495 [M-H]-,表明本化合物的分子量为372。1H-NMR (CD3OD,600 MHz) δ: 1.48 (2H, m, H-5)、1.63 (1H, m, H-6a)、1.71 (1H, m, H-4a)、1.77 (1H,m, H-6b)、2.18 (1H, dt, J = 19.4, 8.2 Hz, H-4b)、2.28 (1H, m, H-2a) 、2.33 (1H,m, H-7a)、2.35 (1H, m, H-2b) 、2.61 (1H, dd, J = 16.0, 8.2 Hz, H-1a)、2.92 (1H,dd, J = 16.0, 10.2 Hz, H-1b)、3.13 (1H, dt, J = 13.0, 5.2 Hz, H-7b)、3.80 (3H,s, H-7')、3.86 (3H, s, H-7'')、5.50 (1H, d, J = 1.6 Hz, H-2')、6.03 (1H, s, H-5'')、6.62 (1H, dd, J = 8.0, 1.6 Hz, H-6')、6.83 (1H, d, J = 8.0 Hz, H-5');13C-NMR (CD3OD,150 MHz) δ: 18.7 (C-5)、23.9 (C-6)、26.5 (C-1)、29.8 (C-7)、40.6 (C-2)、45.7 (C-4)、55.4(C-7')、60.3(C-7'')、112.3 (C-5')、112.8 (C-2')、114.4 (C-5'')、121.1 (C-6')、125.6 (C-1'')、134.2 (C-1')、140.7 (C-3'')、146.5 (C-4')、146.6 (C-4'')、148.5 (C-6'')、149.3 (C-2'')、149.5 (C-3')、211.5 (C-3)。
效果实施例——体外抑制肿瘤细胞增殖作用
(1)材料和方法
对实施例1中制备得到的化合物进行了体外抗肿瘤试验,试验所采用的细胞为人宫颈癌细胞Hela、人肝癌细胞HepG-2,上述瘤株均购自赛尔试剂公司,采用常规的MTT法进行测试。
具体方法如下:
取对数生长期生长状态良好的Hela、HepG-2细胞,用含10%胎牛血清的培养液(DMEM)配成单个细胞悬液,以每孔104个细胞接种到96孔板,每孔体积100 μl。边缘36孔用100 μL PBS填充。接种好的细胞于37℃、5% CO2培养箱内,培养24小时后给药,待测不同浓度药物分别为5、10、20、40、80、160 μM,空白对照组为100 μL的DMEM,加10 μL无药溶剂作为对照组,每组设3个复孔,继续孵育48 h后,每孔加入5 mg/mL 的MTT 20 μL,37℃继续培养4小时后离心,吸弃96孔板中孔内培养上清液,每孔加入150 μL DMSO溶液,置摇床上低温振荡10分钟,使结晶物充分融解。选择570 nm波长,用酶联免疫检测仪读取各孔光吸收值(OD值),记录结果。计算受试药物对肿瘤细胞生长抑制率。细胞生长抑制率%=[1-(药物处理孔平均OD值-调零孔OD值)/(细胞对照孔平均OD值-调零孔OD值)]×100%。使用Logit法计算药物的IC50值。结果如下表2所示:
(2)结果
经线性回归计算IC50值显示本发明涉及的环醚型二芳基庚烷化合物对人宫颈癌Hela、人肝癌细胞HepG-2作用IC50值分别为58.11±2.73 μM、37.64±1.66 μM,顺铂作为阳性对照药对人宫颈癌Hela、人肝癌细胞HepG-2作用IC50值分别为18.62±2.06 μM、5.45±1.22 μM。
结果表明本发明化合物对人宫颈癌Hela、人肝癌细胞HepG-2的生长均有一定的抑制作用,具有制备临床抗肿瘤药物的前景。
Claims (5)
2.权利要求1所述环醚型二芳基庚烷类化合物的制备方法,其特征在于,以青龙衣为原料,依次经醇提法、有机溶剂萃取法、吉拉德试剂的纯化与富集、硅胶柱色谱分离以及制备HPLC分离步骤得到。
3.权利要求2所述环醚型二芳基庚烷类化合物的制备方法,其特征在于,通过以下具体步骤制备:
(1)醇提法:将新鲜青龙衣低温烘干,取10 kg粉碎成粗粉,加入8倍量体积的95%乙醇,加热回流提取3次,将所得提取液合并,回收溶剂,减压干燥,得浸膏;
(2)有机溶剂萃取法:将步骤(1)中得到的浸膏,加入5倍量的纯净水稀释,再用与稀释液等体积的二氯甲烷对稀释溶液进行5次萃取,合并二氯甲烷萃取液,浓缩干燥得到二氯甲烷浸膏;
(3)吉拉德试剂的纯化与富集:取经步骤(2)中获得的二氯甲烷浸膏,反复捏溶于乙酸-无水乙醇(1:12,重量比)溶液体系中,加入吉拉德试剂P后,加热回流,反应完毕后回收乙醇至无醇味,加水稀释,用等体积乙醚萃取5次,分取水层用盐酸酸化,再用等体积乙醚萃取5次,得到浸膏;
(4)硅胶柱色谱分离:取步骤(3)所得浸膏进行硅胶柱色谱分离,采用体积比为100:0→0:100的二氯甲烷-甲醇混合溶剂进行梯度洗脱,其中将体积比为20:1的二氯甲烷-甲醇混合溶剂洗脱部分减压回收得到分离粗品;
(5)制备HPLC分离:将上述步骤(4)中所得粗品采用甲醇溶解进入制备型HPLC,流动相为体积比为55:45的甲醇和水混合溶液,洗脱流速为1.5 mL/min,保留时间tR=23.4min~24.3min阶段内收集馏分后,回收干燥,即得如式Ⅰ所示的化合物。
4.权利要求1所述的环醚型二芳基庚烷类化合物3',4''-环氧-1-(4'-甲氧基苯基)-7-(2'',6''-二羟基-3''-甲氧基苯基)-3-庚酮在制备抗肿瘤药物方面的应用。
5.权利要求4所述的应用,其特征在于,所述的肿瘤为宫颈癌、肝癌。
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