CN106967031B - 一种新的二氢高异黄酮及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种新的二氢高异黄酮及其制备方法和应用，所述的新的二氢高异黄酮分离提取自黄精。实验证明，该二氢高异黄酮可显著抑制Hep‑G2细胞内的脂质堆积，并能显著降低该细胞内的甘油三酯(TG)和总胆固醇(TC)含量，其中在降低TG方面优于阳性对照药辛伐他汀，具有优异的降血脂活性。本发明新二氢高异黄酮的发现为研制新的降血脂药物提供了新的先导化合物，具有很好的开发应用前景。本发明制备工艺简单，稳定可靠，化合物的纯度高(98％以上)，易于工业化推广，为降血脂药物的研发开辟了新的途径，具有良好的社会和经济效益。
Description
技术领域
本发明涉及一种新的二氢高异黄酮及其制备方法和应用，属于医药领域。
背景技术
近年来，随着我国城乡居民生活习惯和饮食结构的改变，高血脂症发病率逐年上升并有发病年龄年轻化的趋势。因高血脂引起的心梗、脑梗、中风、偏瘫等致死人数以每年12％的速度递增。高血脂症的危害是隐匿、逐渐和全身性的，目前已成为危害人类健康的“头号杀手”。
目前临床上使用的降脂类药以合成药物为主，其中应用最广的是他汀类，主要用于治疗高胆固醇血症，可显著降低血液中的LDL-C浓度，但长期服用会引起肝功能损害、肌肉毒性，因此在肝功能不全的患者中的应用受到限制。与此相比，中药在治疗高血脂症具有独特优势：药源丰富，疗效确切，不良反应较小，又能通过多种途径和多靶点在体内发挥降脂作用。因此，从我国传统中草药中寻找出具有显著降血脂活性、化学结构明确且不良反应少的药物，对新型降血脂药物的研究与开发具有极大的意义。
黄精(Polygonatum sibiricum)，别名土灵芝、太阳草、鸡头参，为百合科(Liliaceae)黄精属多年生草本植物。药用其根茎，始载于《名医别录》，列为上品，具有补肾益精、滋阴润燥等功效。现代药理研究表明黄精在抗衰老、降血糖、降血脂、提高机体免疫力、抗病毒等方面均具有较好的药理活性。自20世纪80年代开始，国内外学者对黄精的化学成分进行了广泛研究，发现了多种化学成分，其中以多糖和甾体皂苷类含量较大，为其主要药效成分，研究较为深入，文献报道较多。除此之外，高异黄酮类成分也是黄精属植物中一类特征成分，其结构比异黄酮多一个碳原子，是黄酮化合物中特殊的一类。高异黄酮类在自然界分布较少，主要存在于百合科、豆科的少数属植物中，具有抗炎、抗诱变、免疫调节、细胞毒和雌激素等作用。目前从黄精中仅发现1种高异黄酮，属于微量成分，且针对黄精中高异黄酮成分的制备方法以及降血脂活性研究尚未见文献报道。
发明内容
为了解决上述问题，本发明的目的是提供一种从黄精中提取的新的二氢高异黄酮及其制备方法和应用，该方法能够有效解决快速从黄精中提取新二氢高异黄酮类化合物的问题，且提取的新二氢高异黄酮具有良好的降血脂作用。
为了实现上述目的，本发明所采用的技术方案是：
一种新的二氢高异黄酮，所述的二氢高异黄酮的化学结构式如下：
一种新的二氢高异黄酮，所述的二氢高异黄酮提取自黄精。
一种新的二氢高异黄酮的制备方法，具体为：
(1)粗提物的制备：取黄精新鲜块茎切片后，将黄精加入黄精重量8倍量的体积浓度95％的乙醇溶液中回流提取3次，提取温度为80～95℃，提取时间为1～2h，合并提取液，过滤，滤液减压浓缩至无醇味，得到浓缩物；在浓缩物中加入蒸馏水混悬，得到浓缩物相对密度为1.1～1.2的混悬液，再用与混悬液等体积的乙酸乙酯萃取3～5次，合并乙酸乙酯层，回收溶剂，得到乙酸乙酯萃取物；
(2)硅胶柱色谱初步分离：乙酸乙酯萃取物经硅胶柱色谱初步分离，依次用体积比为100:0～0:100的石油醚-乙酸乙酯系统进行梯度洗脱，收集洗脱比例为100:50和100:100的洗脱液，薄层色谱检识，硫酸-乙醇喷雾105℃加热显色，合并薄层显色为红色斑点的洗脱液，40～45℃减压浓缩，得到含有目标成分的总样；
(3)flash快速制备色谱及制备液相纯化：将上述含有目标成分的总样，采用反相ODS色谱柱，依次用体积浓度10～100％的甲醇水溶液进行梯度洗脱，收集体积浓度50％的甲醇水溶液洗脱部位，45～50℃减压浓缩，进一步经制备型高效液相分离，用体积浓度65％甲醇水溶液洗脱，流速为7ml/min，检测波长为210nm，按色谱图收集洗脱时间为37min的色谱峰流出液，45～50℃减压浓缩，得到黄褐色粉末，即为二氢高异黄酮。
一种所述的二氢高异黄酮在制备降血脂药物中的应用。
本发明的有益效果：
1、本发明从传统中药黄精中分离提取得到一种新的二氢高异黄酮类化合物，实验证明，该二氢高异黄酮可显著抑制Hep-G2细胞内的脂质堆积，并能显著降低该细胞内的甘油三酯(TG)和总胆固醇(TC)含量，其中在降低TG方面优于阳性对照药辛伐他汀，具有优异的降血脂活性。因此本发明新二氢高异黄酮的发现为研制新的降血脂药物提供了新的先导化合物，具有很好的开发应用前景。
2、本发明制备工艺简单，稳定可靠，化合物的纯度高(98％以上)，易于工业化推广，为降血脂药物的研发开辟了新的途径，具有良好的社会和经济效益。
具体实施方式
以下结合实施例对本发明的具体实施方式作进一步详细说明。
实施例1
黄精中总高异黄酮类化合物的提取方法，包括以下步骤：
(1)粗提物的制备：取黄精新鲜块茎切片后，将黄精加入黄精重量8倍量的体积浓度95％的乙醇溶液中回流提取3次，提取温度为95℃，提取时间为2h，合并提取液，过滤，滤液减压浓缩至无醇味，得到浓缩物；在浓缩物中加入蒸馏水混悬，得到浓缩物相对密度为1.1的混悬液，再用与混悬液等体积的乙酸乙酯萃取3次，合并乙酸乙酯层，回收溶剂(乙酸乙酯)，得到乙酸乙酯萃取物；
(2)硅胶柱色谱初步分离：乙酸乙酯萃取物经硅胶柱色谱初步分离，依次用体积比为100:0、100:5、100:10、100:20、100:50、100:100、0:100的石油醚-乙酸乙酯系统进行梯度洗脱，收集洗脱比例为100:50和100:100的洗脱液，薄层色谱检识(具体方法参考中国药典2010年版一部附录ⅥB法)，硫酸-乙醇喷雾105℃加热显色，合并薄层显色为红色斑点的洗脱液，40℃减压浓缩，得到含有目标成分的总样；
(3)flash快速制备色谱及制备液相纯化：将上述含有目标成分的总样，采用反相ODS色谱柱，依次用体积浓度10％、30％、50％、70％和100％的甲醇水溶液进行梯度洗脱，收集体积浓度50％的甲醇水溶液洗脱部位，50℃减压浓缩，进一步经制备型高效液相分离，用体积浓度65％甲醇水溶液洗脱，流速为7ml/min，检测波长为210nm，按色谱图收集洗脱时间为37min的色谱峰流出液，50℃减压浓缩，得到黄褐色粉末，命名为Polygonatin A。
化合物的纯度检测：
采用高效液相色谱检测新化合物的纯度。色谱条件如下，色谱柱：Kromasil 100-5，C-18(250×4.6mm，5μm)；流动相：乙腈/水梯度(5％→95％)；时间：65min；流速：1ml/min；检测波长：210nm；254nm；柱温：25℃；进样：10μl。经面积归一化法测定，Polygonatin A的纯度为98.37％。
化合物的结构鉴定：
所述的从黄精中分离得到的新成分Polygonatin A，其结构经高分辨质谱(HR-ESI-MS)、紫外光谱(UV)、红外光谱(IR)、核磁共振氢谱(1H-NMR)、碳谱(13C-NMR)、二维谱(1H-1H COSY、DEPT、HSQC、HMBC、NOESY)及圆二色谱鉴定了其化学结构：
Polygonatin A为黄褐色粉末，易溶于甲醇；HR-ESI-MS m/z:329.1026[M-H]-；结合1H-NMR谱和13C-NMR谱数据推出其分子式为C18H18O6，不饱和度为10，提示其可能具有2个或2个以上苯环结构。IR(KBr)vmax cm-1:在3379、1638和1600cm-1处显示羟基、不饱和羰基和苯环的特征吸收峰。UVλmax nm(logξ)202(4.67),225sh(4.46),288(4.29),330(3.56)，显示高黄酮类化合物的特征紫外吸收峰。
1H-NMR谱中芳香区在δ6.96(1H,d,J＝8.0Hz)，6.37(1H,d,J＝2.5Hz)和6.35(1H,dd,J＝8.0,2.5Hz)处的3个氢信号，根据其化学位移和偶合常数提示分子中存在一个ABX系统取代的苯环。此外，在δ6.01(1H,d,J＝2.0Hz)，5.98(1H,d,J＝2.0Hz)的2个氢信号分别为黄酮类A环C-8和C-6位间位偶合的典型氢信号，提示A环C-5位和C-7位均被取代。高场区在δ4.26(1H,dd,J＝11.5,4.5Hz,H-2α)，4.13(1H,dd,J＝11.5,7.5Hz,H-2β)，3.18(1H,dd,J＝13.5,4.5Hz,H-9)，2.98(1H,m,H-3)和2.60(1H,dd,J＝13.5,9.5Hz)处的氢信号，结合1H-1HCOSY图谱提示分子中存在一个(2)CH2—(3)CH—(9)CH2—片段，提示Polygonatin A为高异黄酮类化合物。此外，在δ3.79(3H,s)和3.72(3H,s)的氢信号提示分子中存在2个与苯环相连的甲氧基。13C-NMR和DEPT-135图谱显示18个碳信号，以上信息进一步证实Polygonatin A为一个高异黄酮类化合物。Polygonatin A的结构中C-7位被甲氧基取代，这一推测可被HMBC谱和NOESY谱证实：在HMBC图谱中，δ3.79处的甲氧基氢信号与δC 169.3(C-7)上的碳有远程相关信号；NOESY图谱中δ3.79处的甲氧基氢信号与C-6上的氢有NOE相关。此外，在HMBC图谱中H-9(δH 3.18和2.60)与C-1′(δC 118.0)，C-2′(δC 157.6)和C-6′(δC 132.6)有远程相关信号；H-3′(δH 6.37)与C-1′(δC 118.0)和C-5′(δC 105.7)有远程相关信号。在NOESY图谱中H-9与H-6′有NOE相关；δ3.72处的甲氧基氢信号与H-5′有相关信号。以上信息证实分子中B环为1,2,4-三取代，由此可确定Polygonatin A的平面结构。Polygonatin A的旋光值为-8.0(c 0.21,甲醇)，CD图谱在287-295nm区域显示负的cotton效应，结合文献[Gan LS,Chen JJ,Shi MF,et al,Nat.Prod.Commun.2013,8:597]，确定其C-3位手性碳为R构型。因此，Polygonatin A的结构可确定为
(3R)-5-hydroxy-7-methoxyl-3-(2′-hydroxy-4′-methoxybenzyl)-chroman-4-one，即(3R)-3-(2-羟基-4-甲氧基苄基)-5-羟基-7-甲氧基苯并二氢吡喃-4-酮。
表1.化合物Polygonatin A的1H-NMR(500MHz,CD3OD)和13C NMR谱数据(125MHz,CD3OD)(δin ppm,J in Hz)
为进一步说明本发明在医药领域中的作用，下面通过体外降血脂实验来说明。
1、Hep-G2细胞体外脂质堆积模型的建立
Hep-G2细胞使用胰酶消化后，均匀接种至96孔板中，待细胞生长至80％汇合后，用10％胎牛血清的DMEM培养基饥饿细胞12h。吸弃培养基，用含有1.2μM的脂肪酸(油酸)的DMEM培养基(10％胎牛血清)分组孵育细胞24h，取出后，吸弃培养基，PBS洗1～3次。4％多聚甲醛在4℃下固定12h。取出后，每孔加油红O染色液20μL染色15min，于光学显微镜下观察各组细胞脂质堆积情况。
2、Hep-G2细胞体外脂质堆积实验
Hep-G2细胞使用胰酶消化后，均匀接种至96孔板中，待细胞生长至80％汇合后，用10％胎牛血清的DMEM培养基饥饿细胞12h。吸弃培养基，实验分为正常组、模型组、阳性对照组、样品组。正常对照组仅加入DMEM培养基(10％胎牛血清)，模型组加入仅含有脂肪酸(1.2μM)的DMEM培养基(10％胎牛血清)，阳性对照组加入含有脂肪酸(1.2μM)及阳性药辛伐他汀(10μM)的DMEM培养基(10％胎牛血清)，样品组加入含有脂肪酸(1.2μM)及筛选样品Polygonatin A(10μM)的DMEM培养基(10％胎牛血清)，各组孵育细胞24h。吸弃培养基，PBS洗1～3次。4％多聚甲醛在4℃下固定12h。取出后，每孔加油红O染色液20μL染色15min。每孔加DMSO 100μL溶解，置于358nm下测定吸光度(OD值)。结果见表2。
表2 Hep-G2细胞脂质堆积实验结果
| 组别 | OD值 | 统计学意义 |
| 正常对照组 | 0.1799±0.0071 | / |
| 模型组 | 0.2524±0.1274 | / |
| 阳性对照组 | 0.2027±0.1076 | * |
| 样品组 | 0.1803±0.0021 | ** |
注：与模型组(model)比较*P<0.05；**P<0.01。
3、Hep-G2细胞中甘油三酯(TG)及总胆固醇(TC)含量测定：
Hep-G2细胞使用胰酶消化后，均匀接种至24孔板中，待细胞生长至80％汇合后，用10％胎牛血清的DMEM培养基饥饿细胞12h。吸弃培养基，实验分为正常组、模型组、阳性对照组、样品组。正常对照组仅加入DMEM培养基(10％胎牛血清)，模型组加入仅含有脂肪酸(1.2μM)的DMEM培养基(10％胎牛血清)，阳性对照组加入含有脂肪酸(1.2μM)及阳性药辛伐他汀(10μM)的DMEM培养基(10％胎牛血清)，样品组加入含有脂肪酸(1.2μM)及筛选样品Polygonatin A(10μM)的DMEM培养基(10％胎牛血清)，各组孵育细胞24h。取出，吸弃培养基，预冷PBS洗1～3次，每孔加100μL裂解液(蛋白裂解液：蛋白酶抑制剂按99：1配制)，置冰上35min使其充分裂解。按照试剂盒说明书方法使用甘油三酯(TG)测试盒(即GPO-PAP酶法)和总胆固醇(T-CHO)测试盒(即COD-PAP法)分别对甘油三酯(TG)及总胆固醇(TC)含量进行测定。结果见表3。
表3 Hep-G2细胞中甘油三酯(TG)及总胆固醇(TC)含量测定实验结果
| 组别 | TG含量 | TC含量 |
| 正常对照组 | 0.2848±0.0219 | 0.1382±0.0254 |
| 模型组 | 0.5148±0.0371 | 0.2591±0.0282 |
| 阳性对照组 | 0.3107±0.0096** | 0.1320±0.0154** |
| 样品组 | 0.2903±0.0331** | 0.1506±0.0580** |
注：与模型组(model)比较*P<0.05；**P<0.01。
以上实验表明，本发明制备出的Polygonatin A可显著抑制Hep-G2细胞内的脂质堆积，并能显著降低该细胞内的甘油三酯(TG)和总胆固醇(TC)含量，其中在降低TG方面优于阳性对照药辛伐他汀。本发明为研制新的降血脂药物提供了新的先导化合物，具有很好的开发应用前景。
以上所述仅为本发明最佳的实施例，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (4)
1.一种二氢高异黄酮，其特征在于，所述的二氢高异黄酮的化学结构式如下：
2.一种二氢高异黄酮，其特征在于，所述的二氢高异黄酮提取自黄精，化学结构式为：
3.一种权利要求1或2所述的二氢高异黄酮的制备方法，其特征在于，具体方法为：
(1)粗提物的制备：取黄精新鲜块茎切片后，将黄精加入黄精重量8倍量的体积浓度95％的乙醇溶液中回流提取3次，提取温度为80～95℃，提取时间为1～2h，合并提取液，过滤，滤液减压浓缩至无醇味，得到浓缩物；在浓缩物中加入蒸馏水混悬，得到浓缩物相对密度为1.1～1.2的混悬液，再用与混悬液等体积的乙酸乙酯萃取3～5次，合并乙酸乙酯层，回收溶剂，得到乙酸乙酯萃取物；
(2)硅胶柱色谱初步分离：乙酸乙酯萃取物经硅胶柱色谱初步分离，依次用体积比为100:0～0:100的石油醚-乙酸乙酯系统进行梯度洗脱，收集洗脱比例为100:50和100:100的洗脱液，薄层色谱检识，硫酸-乙醇喷雾105℃加热显色，合并薄层显色为红色斑点的洗脱液，40～45℃减压浓缩，得到含有目标成分的总样；
(3)flash快速制备色谱及制备液相纯化：将上述含有目标成分的总样，采用反相ODS色谱柱，依次用体积浓度10～100％的甲醇水溶液进行梯度洗脱，收集体积浓度50％的甲醇水溶液洗脱部位，45～50℃减压浓缩，进一步经制备型高效液相分离，用体积浓度65％甲醇水溶液洗脱，流速为7ml/min，检测波长为210nm，按色谱图收集洗脱时间为37min的色谱峰流出液，45～50℃减压浓缩，得到黄褐色粉末，即为二氢高异黄酮。
4.一种权利要求1或2所述的二氢高异黄酮在制备降血脂药物中的应用。
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