CN104892617B - 白鲜皮活性成分促进5‑羟色胺受体增量表达的新功能 - Google Patents

白鲜皮活性成分促进5‑羟色胺受体增量表达的新功能 Download PDF

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CN104892617B
CN104892617B CN201510265214.6A CN201510265214A CN104892617B CN 104892617 B CN104892617 B CN 104892617B CN 201510265214 A CN201510265214 A CN 201510265214A CN 104892617 B CN104892617 B CN 104892617B
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Abstract

白鲜皮是一种重要的中药材。本申请介绍白鲜皮活性成分的一种新功能，通过其对大鼠原代培养神经元的作用实验，我们发现白鲜皮活性成分的加入能够促进5‑羟色胺受体基因5HTR1A的过量表达，由于5‑羟色胺能神经元对情绪、记忆等行为有重要的影响，上述结果表明白鲜皮活性成分的使用可促进人体快感、长时程记忆等功能的变化。

Description

白鲜皮活性成分促进5-羟色胺受体增量表达的新功能
技术领域
本申请属于药材活性成分新功能的技术领域，尤其涉及于一种分离于中药材白鲜皮的活性成分可促进海马神经元5-羟色胺受体增量表达的新功能。
背景技术
5-羟色胺又叫血清素，是一种抑制性神经递质，其广泛分布于各脑区，尤以大脑皮层居多。5-羟色胺是受到研究最为广泛的神经递质，其参与人的情绪、记忆、精力等多种生命过程，其表达及功能的异常与多种神经系统疾病的发生密切相关，最主要的如精神分裂症和抑郁狂躁型忧郁症(bipolar disorders). 5HTR1A是一种重要的5-羟色胺受体亚型，在脑中分布广泛，其参与到多种神经细胞的5-羟色胺的信号输入过程，并在下游偶联多种信号转导途径，以完成以递质传递为基础的抑制性神经活动。有研究表明，5HTR1A的减量表达与精神分裂症和抑郁狂躁型忧郁症的发生存在病例-对照实验中的关联性。
白鲜(Dictamnus dascarpus Turcz.)，别名：藓皮、野花椒根皮、臭根皮、北鲜皮，为芸香科白鲜属多年生草本植物，植物本身有特异香味。白鲜属植物全世界约 6 种，主要分布于欧亚大陆，我国白鲜属植物主要有白鲜和新疆白鲜。白鲜皮为白鲜的干燥根皮，为我国传统中草药，历代本草及《中国药典》2010 版一部收载记录其功能主治有清热燥湿、祛风解毒。用于湿热疮毒，黄水淋漓，湿疹，风疹，疥癣疮癞，风湿热痹，黄疸尿赤。白鲜皮中的化学成分较为复杂， 目前，从白鲜皮中已分离鉴定出的化学成分主要包括柠檬苦素类、生物碱、黄酮类、内酯类、倍半萜及其苷类等。其中柠檬苦素类和生物碱类物质被认为是生物活性物质。现代药理学研究表明，白鲜皮提取物具有抗菌、抗肿瘤、抗炎与抗变态反应、抗溃疡、抗冠状动脉粥样硬化、保护肝脏和神经等作用，临床上主要用治疗慢性支气管炎、皮肤脱皮症、风湿性关节炎、外出血、荨麻疹等。 但白鲜皮提取物对中枢神经系统的功能作用目前尚未见报道。
发明内容
白鲜皮为常用中药材，其购自安徽省中医院药房。
白鲜皮活性成分的分离过程为：
将5-10 kg的白鲜皮依次用95%的乙醇、70%乙醇10倍量回收提取三次，每次一小时，将提取液合并，浓缩得浸膏并加水混悬后，依次用石油醚、二氯甲烷及乙酸乙酯进行萃取，得到二氯甲烷提取成分约50g-100g，即为所述白鲜皮的活性成分。
所述白鲜皮活性成分的识别过程为：
取上述活性成分经硅胶柱层析分离得到四个分离峰，将第一个分离峰继续进行Sedhadex LH-20柱色谱分析并进行重结晶。将所得峰分别进行质谱和核磁共振分析，其中一个分离峰根据EI-MS推测其分子质量为 199，为奇数，可能为生物碱类化合物。1H-NMR(CDCl3 , 600 MHz)波谱给出 δ7.47 (1H, t, J = 7.2 Hz，H-6), 7.71(1H, t, J =7.2Hz, H-7), 8.29 (1H, d, J =8.4 Hz，H-5), 8.04 (1H, d, J = 8.4 Hz, H-8)为偶合的苯环信号，4.48 (3H, s, -OCH3）为一个含氧取代的甲基信号, 7.11(1H, brs, H-3),7.66 (1H, brs, H-2)两个烯碳质子信号。经检索，光谱数据与文献报道的白鲜碱(dictamnine)一致, 故确定该化合物为白鲜碱(dictamnine)。因此本申请所述白鲜皮主要活性成分应为白鲜碱。
白鲜皮活性成分的生理活性检测：
将出生24h内的SD大鼠处死并取海马组织原代培养。将海马组织放在200目筛网上，用眼科剪剪小块过筛，经过1000rpm离心8min后倒掉上清液，加高糖DMEM培养基并加入10%胎牛血清，巴氏管吹打后种6孔板。培养条件为37oC5%水平的二氧化碳。每隔三天更换培养基。在培养至第7天时，向其中三孔的原代培养细胞加入终浓度为10μM的上述白鲜皮提取物，另外三孔加入同样体积的无菌水作为对照组。继续培养至14天时，回收细胞。使用本领域常规的技术手段提取细胞中总mRNA、反转录为cDNA，并使用Cycle480（Roche）荧光定量PCR仪（使用SybrGreen 染料）对5HTR1A的表达量进行定量分析。所使用的PCR条件为：95oC预变性 5min，然后是95oC10s， 60oC 10s， 72oC 30s 扩增共40个循环。所使用的引物为：上游GGCAACAACACCACAACG; 下游TGACCGCCAAGGAGCCGATG。所获得的定量数值为5HTR1A表达量与内参基因GAPDH的相对比值。
实验组和对照组所获得的mRNA相对定量结果如图1所示。
从图1中可以看出，经过白鲜皮活性成分的处理，5HTR1A基因的表达量有了明显的提高，其表达量为对照组的1.7倍。
本申请首次证明了白鲜皮活性成分对大鼠中枢神经系统5-羟色胺能神经元的调节作用，该结果意味着该成分可以参与到人和哺乳动物的情感、记忆及精力等神经活动中，并对该活动施加影响，同时为精神分裂症等精神疾病的治疗提供了潜在的解决方案。
附图说明
图1 白鲜皮提取物处理所引起的大鼠海马神经元5HTR1A基因的表达变化。
Control指对照组；substances’ addition指白鲜皮提取物处理组；纵坐标为相对mRNA水平。
具体实施方式
实施例1
白鲜皮活性成分的分离过程为：
将5 kg的白鲜皮依次用95%的乙醇、70%乙醇10倍量回收提取三次，每次一小时，将提取液合并，浓缩得浸膏并加水混悬后，依次用石油醚、二氯甲烷及乙酸乙酯进行萃取，得到二氯甲烷提取成分约50g，即为所述白鲜皮的活性成分。
实施例2
将10kg的白鲜皮依次用95%的乙醇、70%乙醇10倍量回收提取三次，每次一小时，将提取液合并，浓缩得浸膏并加水混悬后，依次用石油醚、二氯甲烷及乙酸乙酯进行萃取，得到二氯甲烷提取成分约100g，即为所述白鲜皮的活性成分。
实施例3
将8 kg的白鲜皮依次用95%的乙醇、70%乙醇10倍量回收提取三次，每次一小时，将提取液合并，浓缩得浸膏并加水混悬后，依次用石油醚、二氯甲烷及乙酸乙酯进行萃取，得到二氯甲烷提取成分约80g，即为所述白鲜皮的活性成分。
实施例4
所述白鲜皮活性成分的识别过程为：
取上述活性成分经硅胶柱层析分离得到四个分离峰，将第一个分离峰继续进行Sedhadex LH-20柱色谱分析并进行重结晶。将所得峰分别进行质谱和核磁共振分析，其中一个分离峰根据EI-MS推测其分子质量为 199，为奇数，可能为生物碱类化合物。1H-NMR(CDCl3 , 600 MHz)波谱给出 δ7.47 (1H, t, J = 7.2 Hz，H-6), 7.71(1H, t, J =7.2Hz, H-7), 8.29 (1H, d, J =8.4 Hz，H-5), 8.04 (1H, d, J = 8.4 Hz, H-8)为偶合的苯环信号，4.48 (3H, s, -OCH3）为一个含氧取代的甲基信号, 7.11(1H, brs, H-3),7.66 (1H, brs, H-2)两个烯碳质子信号。经检索，光谱数据与文献报道的白鲜碱(dictamnine)一致, 故确定该化合物为白鲜碱(dictamnine)。因此所述白鲜皮主要活性成分应为白鲜碱。
实施例5
白鲜皮活性成分的生理活性检测：
将出生24h内的SD大鼠处死并取海马组织原代培养。将海马组织放在200目筛网上，用眼科剪剪小块过筛，经过1000rpm离心8min后倒掉上清液，加高糖DMEM培养基并加入10%胎牛血清，巴氏管吹打后种6孔板。培养条件为37oC5%水平的二氧化碳。每隔三天更换培养基。在培养至第7天时，向其中三孔的原代培养细胞加入终浓度为10μM的上述白鲜皮提取物，另外三孔加入同样体积的无菌水作为对照组。继续培养至14天时，回收细胞。使用本领域常规的技术手段提取细胞中总mRNA、反转录为cDNA，并使用Cycle480（Roche）荧光定量PCR仪（使用SybrGreen 染料）对5HTR1A的表达量进行定量分析。所使用的PCR条件为：95oC预变性 5min，然后是95oC10s， 60oC 10s， 72oC 30s 扩增共40个循环。所使用的引物为：上游GGCAACAACACCACAACG; 下游TGACCGCCAAGGAGCCGATG。所获得的定量数值为5HTR1A表达量与内参基因GAPDH的相对比值。

Claims (1)

1.一种白鲜皮的活性成分在制备用于促使大鼠海马原代培养神经元的5-羟色胺受体基因5HTR1A增量表达的药物中的用途，所述白鲜皮的活性成分为白鲜碱。
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