CN112618614B - 一种具有抗抑郁症的刺梨活性提取物及应用 - Google Patents
一种具有抗抑郁症的刺梨活性提取物及应用 Download PDFInfo
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Abstract
本发明涉及一种具有抗抑郁症的刺梨活性提取物及应用。该活性提取物是通过刺梨果或刺梨果渣采用化学提取或膜分离技术得到,其中所述的化学提取是将鲜榨刺梨果渣或刺梨果经酒精回流提取、过滤、浓缩去乙醇,加水分散水不溶沉淀并过滤,依次用水和石油醚或正己烷洗涤沉淀,去除水溶性物质和油脂,干燥得到。该活性提取物含三萜、黄酮成分,研究发现抗抑郁活性显著,能够用于制备抗抑郁症的药物、辅助药物,具有较高的应用价值和市场前景。本发明既对刺梨产业高值化利用、可持续发展具有重要意义,又为抗抑郁症的药品提供了中间体原料,制备方法简单易行,成本低,可产业化生产。
Description
技术领域
本发明涉及刺梨果或刺梨果渣的提取物,特别是一种具有抗抑郁症的刺梨活性提取物及应用。
背景技术
抑郁症是一种精神紊乱性疾病,在临床上表现为慢性、反复性发作,主要症状为情绪低落、食欲不振和心境功能障碍等消极表现。抑郁症还具有发病率高、致残率高、自杀率高等特点,严重影响患者的身心健康和生活质量,甚至可危及生命。抑郁症患者的主要表现为:心情沉重,对工作与生活丧失基本兴趣,精神疲乏不堪,对生活中的事情总感到力不从心。抑郁症患者的生活态度极度悲观,时常感到非常绝望,且有很强烈的自杀倾向,随着生活节奏的加快、激烈的竞争,加上噪音污染、环境污染及人际关系复杂等,使人们长期处于紧张、焦虑、和不安等状态,导致抑郁症的发病率正逐年飙升,这将严重影响社会公共秩序、降低现代社会人们的生活质量。因此,对抑郁症的治疗成为人们广泛关注的重要问题,得到全球医药工作者的普遍重视。
抑郁症的发病机制和原因都没有明确的说法。于是就有了许多关于抑郁症病因的假说,主要包括以下三个:遗传因素、生化因素及心理-社会因素假说。其中,第一个观点认为:与抑郁症患者的血缘关系越近,则患抑郁症的风险也就越高。而第二个观点则认为,随着多巴胺(DA)、去甲肾上腺素(NE)及5-羟色胺(5-HT)等神经递质浓度的改变,就会引发抑郁症。而最后一个观点则这样认为:如果人们长期保持一种不愉快的情绪,那发生抑郁症的几率就会升高。
临床上对抑郁症患者的治疗方法主要包括心理治疗和药物治疗。心理疗法主要是指通过医生对病患进行正确积极的疏导,使抑郁症患者渐渐摒弃自身的负面、消极情绪,进而对周围世界产生正面、积极的看法,达到改善抑郁症患者精神状态的目的。药物治疗主要包括中药治疗及西药治疗。中药治疗主要依据抑郁症患者常见的肝气郁结等现象,利用中药药物为其疏肝理气,从而缓解抑郁症的症状,达到治疗的效果。西药治疗是当下最主要的抑郁症治疗方法。
目前社会上常见的西药药物有三环类抗抑郁药,如丙咪嗪、阿米替林等;选择性5-羟色胺重摄取抑制剂,如氟西汀、帕罗西汀、舍曲林等;5-羟色胺/去甲肾上腺素重摄取双重抑制剂,如万拉法新、度洛西汀等。但这些药物均存在起效慢、疗效低、毒副作用大等弊端。因此,从天然产物或者药食用植物资源中挖掘新型的抗抑郁药物,寻找和开发起效迅速、副作用小的的抗抑郁新药已经刻不容缓。
刺梨(Rosa roxbughii Tratt)为蔷薇科落叶灌木植物。刺梨果被誉“维 C 之王”、“中国登义果”以及“炎黄圣果”,是第三代水果(3G)代表的珍贵水果资源。刺梨已被列入卫健委批准的食品新资源名单品种,现为普通食品;1994 和 2003 版《贵州省中药材、民族药材质量标准》均将刺梨收录其中,刺梨具有药用和食用的双重属性。目前刺梨的药理作用主要包括以下几个方面:抗氧化、延缓衰老、调节机体免疫功能、抗肿瘤、降血糖、解毒镇静、解酒护肝、治疗口腔溃疡、治疗胃溃疡及促消化、抗动脉粥样硬化、抗肾纤维化及辐射防护等,但是目前还没有关于刺梨具有抗抑郁活性的研究。
发明内容
本发明的目的在于,从刺梨果或刺梨果渣中利用化学分离或者膜分离技术提取出一种具有抗抑郁症的刺梨活性提取物及并将其应用到抗抑郁的药物中。为刺梨产业的高值化、可持续发展利用提供新的途径,为实现上述目的,所采取的技术方案为:一种具有抗抑郁症的刺梨活性提取物,所述提取物含有总三萜和总黄酮,其中总三萜的含量为30%-45%;总黄酮的含量为1%~3%。
上述总三萜包括蔷薇酸、2-氧代坡模醇酸、刺梨苷 F1和刺梨酸;所述总黄酮包括槲皮素、儿茶素、二氢杨梅素和芦丁。
上述总三萜的含量为蔷薇酸、2-氧代坡模醇酸、刺梨苷F1和刺梨酸的含量总和,总黄酮的含量为槲皮素、儿茶素、二氢杨梅素和芦丁的含量总和。
上述总三萜的含量是以熊果酸为对照品,利用香草醛-冰醋酸-高氯酸显色比色法测定得到;所述总黄酮的含量是以槲皮素、儿茶素、二氢杨梅素和芦丁为对照品,利用高效液相色谱法测定得到。
上述的提取物的制备方法包括以下步骤:取刺梨果或刺梨果渣,用重量为刺梨果或刺梨果渣重量的5-10倍的50-95%乙醇水溶液回流提取2-4次,每次1-2小时,过滤,合并滤液,回收乙醇,在滤液中加体积为滤液体积的0.5-1倍的水进行分散,抽滤,水洗沉淀2-4次,然后用石油醚或正己烷洗沉淀2-4次,冷冻干燥或真空减压干燥沉淀得提取物。
上述的提取物的另一种制备方法包括以下步骤:取刺梨果或刺梨果渣,用重量为刺梨果或刺梨果渣重量的5-10倍的50-95%乙醇水溶液回流提取2-4次,每次1-2小时,过滤,合并滤液,回收乙醇,采用0.45μm中空纤维膜过滤,水洗沉淀2-4次,然后用石油醚或正己烷洗沉淀2-4次,冷冻干燥或真空减压干燥沉淀得提取物。
上述提取物具有抗抑郁活性,可用于制备抗抑郁症的药物、药物中间体。
上述提取物制备药物,直接使用或者以组合物的形式使用;所述组合物包括0.1–99%的提取物,其余为载体;所述药物的剂型为硬胶囊、片剂、口服液、颗粒剂、软胶囊或滴丸剂。
其中,刺梨活性提取物鉴定方法为:向提取物样品中加入1mL 70%的甲醇内标提取液,斡旋5 min混匀,离心(1200 r/min, 4℃)10min,取上清液用微孔滤膜(0.22µm)过滤,保存于进样瓶中,用于LC-MS/MS检测,图1为提取物的LC-MS离子流图。
色谱质谱采集条件:使用Thermo Fisher Scientific公司生产的DionexUltimate 3000 RSLC (HPG)液相色谱仪与Thermo Scientific Q Exactive Focus质谱仪进行检测,HESI-II离子源,Thermo Fisher Hypersil GOLD a Q (100*2.1 mm, 1.9μm)色谱柱。离子源参数设置为Spray Voltage(3.0kV(+)/2.5kV (-));Capillary Temperature(320℃);Sheath Gas(35 arb);AUX Gas(10 arb);Sweep Gas(0 arb);Probe HeaterTemperature(350℃);S-Lens(60)。流动相中A相为乙腈(0.1%甲酸)、B相为0.1%甲酸水,梯度洗脱0~50min A为5%~95%,流速为0.3 mL/min,柱温为40℃。质谱扫描参数设置Scan mode(Full MS-ddms2),Full MS scan range(100 to 1500m/z),Spectrum data type(Profile),Resolution(Full MS: 70,000,MS/MS: 17,500),AGC target(Full MS:1e6,MS/MS:2e5),Maximum IT(Full MS: 100 ms,MS/MS: 50 ms),Isolation width(1.5m/z),Dynamic exclution (5 s),NCE (Stepped NCE):20, 40, 60。对得到的提取物进行质谱分析,如图2-6所示。图2-6分别为刺梨果渣活性提取物中槲皮素、刺梨酸、蔷薇酸、2-氧代坡模醇酸、刺梨苷 F1的质谱图。
与现有技术相比,本发明提供的提取物是从刺梨果或者刺梨果渣中提取富集制备而成,并对提取物中总三萜和总黄酮做了定性分析,确定了对急性酒精性肝损伤具有辅助保护作用的具体成分为总三萜:蔷薇酸、2-氧代坡模醇酸、刺梨苷 F1和刺梨酸;总黄酮包括槲皮素、儿茶素、二氢杨梅素和芦丁。且每个成分的具体含量不同,体现的活性不同,当总三萜的含量为30%-45%;总黄酮的含量为1%~3%表现出很好的抗抑郁活性。
为了进一步验证本发明所制备的刺梨活性提取物抗抑郁活性,通过动物试验,表明了刺梨活性提取物具有抗抑郁活性,从抑郁症模型小鼠旷场实验总路程统计结果表明与模型组相比,刺梨活性提取物给药组的总路程显著增加(P<0.05);从抑郁症模型小鼠悬尾实验不动时间统计结果表明与模型组比较,刺梨活性提取物给药组在悬尾实验中的不动时间显著降低(P < 0.001);从刺梨活性提取物对抑郁症小鼠糖水偏好的影响实验结果表明刺梨活性提取物能够提高小鼠对蔗糖的偏爱(P<0.01),结果与阿米替林组相似(P<0.01),差异有统计学意义;刺梨活性提取物对抑郁症模型小鼠海马区域的HE染色结果表明刺梨活性提取物各剂量给药组小鼠海马CA1区域病理损伤较模型组明显减轻;从刺梨活性提取物对抑郁症模型小鼠海马神经元的形态学改变实验结果表明刺梨活性提取物各剂量给药组小鼠海马神经元排列尚整齐,凋亡神经元细胞数较少,说明刺梨活性提取物对海马神经元具有保护作用。以上各组实验结果揭示了刺梨活性提取物抗抑郁活性效果显著。
附图说明
图1刺梨活性提取物LC-MS离子流图;
图2为刺梨活性提取物中槲皮素的质谱图;
图3为刺梨活性提取物中刺梨酸的质谱图;
图4为刺梨活性提取物中蔷薇酸的质谱图;
图5为刺梨活性提取物中2-氧代坡模醇酸的质谱图;
图6为刺梨活性提取物中刺梨苷 F1的质谱图;
图7刺梨活性提取物对抑郁症模型小鼠旷场实验总路程图;
图8刺梨活性提取物对抑郁症模型小鼠悬尾不动时间统计柱形图;
图9刺梨活性提取物对抑郁症模型小鼠海马区域HE染色结果图;
图10刺梨活性提取物对抑郁症模型小鼠海马神经元的形态学图。
具体实施方式
实施例1
一种具有抗抑郁症的刺梨活性提取物,所述提取物含有总三萜和总黄酮,其中总三萜的含量为30%;总黄酮的含量为3%。
所述的刺梨活性提取物中总三萜包括蔷薇酸、2-氧代坡模醇酸、刺梨苷 F1和刺梨酸;所述总黄酮包括槲皮素、儿茶素、二氢杨梅素和芦丁。
其中所述总三萜的含量为蔷薇酸、2-氧代坡模醇酸、刺梨苷 F1和刺梨酸的含量总和,总黄酮的含量为槲皮素、儿茶素、二氢杨梅素和芦丁的含量总和。
上述总三萜的含量是以熊果酸为对照品,利用香草醛-冰醋酸-高氯酸显色比色法测定得到;所述总黄酮的含量是以槲皮素、儿茶素、二氢杨梅素和芦丁为对照品,利用高效液相色谱法测定得到。
上述的提取物的制备方法包括以下步骤:取刺梨果或刺梨果渣,用重量为刺梨果或刺梨果渣重量的5倍的95%乙醇水溶液回流提取2次,每次2小时,过滤,合并滤液,回收乙醇,在滤液中加体积为滤液体积的0.5倍的水进行分散,抽滤,水洗沉淀4次,然后用石油醚或正己烷洗沉淀2次,冷冻干燥或真空减压干燥沉淀得提取物。
实施例2
一种具有抗抑郁症的刺梨活性提取物,所述提取物含有总三萜和总黄酮,其中总三萜的含量为45%;总黄酮的含量为1%。
所述的刺梨活性提取物中总三萜包括蔷薇酸、2-氧代坡模醇酸、刺梨苷 F1和刺梨酸;所述总黄酮包括槲皮素、儿茶素、二氢杨梅素和芦丁。
其中所述总三萜的含量为蔷薇酸、2-氧代坡模醇酸、刺梨苷 F1和刺梨酸的含量总和,总黄酮的含量为槲皮素、儿茶素、二氢杨梅素和芦丁的含量总和。
上述总三萜的含量是以熊果酸为对照品,利用香草醛-冰醋酸-高氯酸显色比色法测定得到;所述总黄酮的含量是以槲皮素、儿茶素、二氢杨梅素和芦丁为对照品,利用高效液相色谱法测定得到。
上述的提取物的另一种制备方法包括以下步骤:取刺梨果或刺梨果渣,用重量为刺梨果或刺梨果渣重量的10倍的50%乙醇水溶液回流提取2次,每次1小时,过滤,合并滤液,回收乙醇,采用0.45μm中空纤维膜过滤,水洗沉淀4次,然后用石油醚或正己烷洗沉淀2次,冷冻干燥或真空减压干燥沉淀得提取物。
实施例3
一种具有抗抑郁症的刺梨活性提取物,所述提取物含有总三萜和总黄酮,其中总三萜的含量为40%;总黄酮的含量为2%。
所述的刺梨活性提取物中总三萜包括蔷薇酸、2-氧代坡模醇酸、刺梨苷 F1和刺梨酸;所述总黄酮包括槲皮素、儿茶素、二氢杨梅素和芦丁。
其中所述总三萜的含量为蔷薇酸、2-氧代坡模醇酸、刺梨苷 F1和刺梨酸的含量总和,总黄酮的含量为槲皮素、儿茶素、二氢杨梅素和芦丁的含量总和。
上述总三萜的含量是以熊果酸为对照品,利用香草醛-冰醋酸-高氯酸显色比色法测定得到;所述总黄酮的含量是以槲皮素、儿茶素、二氢杨梅素和芦丁为对照品,利用高效液相色谱法测定得到。
上述的提取物的另一种制备方法包括以下步骤:取刺梨果或刺梨果渣,用重量为刺梨果或刺梨果渣重量的8倍的75%乙醇水溶液回流提取3次,每次1.5小时,过滤,合并滤液,回收乙醇,采用0.45μm中空纤维膜过滤,水洗沉淀3次,然后用石油醚或正己烷洗沉淀3次,冷冻干燥或真空减压干燥沉淀得提取物。
实施例4
本发明提取物的应用,将提取物制备成抗抑郁症的口服液。一种具有抗抑郁症的刺梨活性提取物,所述提取物含有总三萜和总黄酮,其中总三萜的含量为30%;总黄酮的含量为3%。取100 mg的活性提取物溶解于1000mL纯水中,过滤(0.45μm),滤液在无菌条件下装入30mL口服液瓶中。
实施例5
本发明提取物的应用,将提取物制备成抗抑郁症的含片。一种具有抗抑郁症的刺梨活性提取物,所述提取物含有总三萜和总黄酮,其中总三萜的含量为40%;总黄酮的含量为2%。按照重量为刺梨活性提取物50mg/片计,按照片剂的常规方法制备。
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,任何未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、变换材质、等同变化与修饰,均仍属于本发明技术方案的范围内。
为了进一步验证本发明刺梨活性提取物抗抑郁活性,选用实施例1制备的刺梨活性提取物(总三萜的含量为30%;总黄酮的含量为3%)开展药理实验,实验方法与结果如下。
1.试剂与仪器
1.1试剂和供试样品
尼氏染色液(G1434)、HE染色液(G1120)、柠檬酸盐缓冲液、PBS购买于索莱宝生物科技有限公司。供试样品:刺梨果渣活性提取物(总三萜30%,总黄酮2.3%)。
1.2 仪器
LeicaTP1020自动阻止脱水机:Leica包埋机:LeicaCM2245轮转式石蜡切片机:光学显微镜:德国Leica公司产品;电热鼓风干燥机:上海博讯实业公司;光学显微镜:德国Leica公司;悬尾实验视频分析系统(ZH-XWT)、强迫游泳实验箱(ZH)、旷场实验箱(ZH-OFT)购买于安徽正华生物仪器设备有限公司。
2.试验方法
2.1主要实验用液的配制
2.1.1主要溶液的配制
1)PBS液:称取KCl 0.20 g,NaCl 8.0 g,KH2PO4 0.20 g,Na2HPO4·12H2O 3.48 g,加双蒸水定容至1L。
2)4%甲醛溶液:将40%甲醛溶液用PBS液稀释至4%,调pH值为7.4左右。
3)柠檬酸盐缓冲液:购买的1包柠檬酸盐缓冲液用双蒸水定容至2L。
2.2 实验分组及给药
1) 模型与制备
所有小鼠自由饮水与进食,在光暗周期为12h的安静环境适应性饲养一周后,采用Willner P方法改良为慢性轻度不可预见应激动物模型(CUMS)结合孤养进行实验。接受应激处理的小鼠在21天内随机接受不同刺激,每日1种,同种刺激不能连续出现。应激的应激操作方法如下:倾斜鼠笼(45°,24h),禁水、禁食(24h),冰水游泳(4℃,5min),夹尾(1min),热应激(45℃,5min),闪光刺激(频率 3次/分),潮湿垫料(24h),昼夜颠倒(24h)。
2) 分组及给药
实验前2天每只鼠进行1h糖水偏爱度实验,根据糖水偏爱度的结果选取无显著差异的小鼠分别称取体重。实验前1天进行开场实验,剔除中央活动总路程结果差异较大的小鼠,随机将60只小鼠随机分为空白对照组;模型组;阿米替林组(15mg/kg);梨果渣活性提取物低、中、高(50mg/kg、100mg/kg和200mg/kg)口服给药组,每组10只。造模结束后,正常组和模型组灌胃等体积蒸馏水,阿米替林组和梨果渣活性提取物低、中、高各剂量给药组连续给药21天。
2.3行为学检测及及标本制备
给药结束后做小鼠强迫游泳、旷场实验及悬尾实验。行为学实验结束后第二天每组随机抽取3只小鼠脱臼处死,开胸暴露心脏,经0.1M PBS液及预冷的4%多聚甲醛(0.1MPBS配置)透心灌注后取脑,放入4%多聚甲醛溶液中固定一周,石蜡包埋后5μm连续切片,做常规HE染色和尼氏染色。剩余小鼠脱臼处死取小鼠海马及前额叶组织放入EP管中,﹣80℃低温保存备用。
3 数据处理
4 试验结果
4.1刺梨果渣活性提取物对抑郁症模型小鼠旷场实验总路程(mm)统计结果
如图7所示,与空白组相比,模型组小鼠在旷场实验中总路程降低(P<0.05);与模型组相比,刺梨果渣活性提取物给药组的总路程显著增加(P<0.05)。
4.2刺梨果渣活性提取物对抑郁症模型小鼠悬尾实验不动时间统计结果
图8为刺梨果渣活性提取物对抑郁症小鼠悬尾实验不动实验统计结果,如图8所示,空白组比较,模型组小鼠悬尾实验的不动时间都有显著增加(P < 0.001);与模型组比较,刺梨果渣活性提取物给药组在悬尾实验中的不动时间显著降低(P < 0.001)。
4.3刺梨果渣活性提取物对抑郁症小鼠糖水偏好的影响
如表2显示,与空白对照组比较,模型组小鼠的蔗糖偏好明显降低(P<0.01),差异有统计学意义;与模型组小鼠相比,刺梨果渣活性提取物能够提高小鼠对蔗糖的偏爱(P<0.01),结果与阿米替林组相似(P<0.01),差异有统计学意义。
组别 | 剂量(mg/kg) | 糖水偏好分数 |
空白组 | 0.720土0.08<sup>**</sup> | |
模型组 | 0.506土0.09 | |
阿米替林组 | 15 | 0.858土0.11<sup>**</sup> |
活性提取物低剂量组 | 50 | 0.882土0.05<sup>**</sup> |
活性提取物中剂量组 | 100 | 0.896土0.06<sup>**</sup> |
活性提取物高剂量 | 200 | 0.886土0.04<sup>**</sup> |
注:与模型组相比,P **<0.01。
4.4刺梨果渣活性提取物对抑郁症模型小鼠海马区域的HE染色结果
如图9所示,HE染色结果显示,在光学显微镜下发现空白对照组小鼠海马CA1区域神经元数量多且排列整齐,基质丰富,细胞保持有原有的生长形态,细胞核完整,染色均匀;而模型组海马CA1区域神经元细胞减少且排列疏松,细胞间隙增大,细胞核固缩,被染成深蓝色,存在大量细胞凋亡;阳性药组及刺梨果渣活性提取物各剂量给药组小鼠海马CA1区域病理损伤较模型组明显减轻。
4.5刺梨果渣活性提取物对抑郁症模型小鼠海马神经元的形态学改变
如图10所示,空白组小鼠尼氏染色海马神经元呈颗粒状,排列紧密、整齐,凋亡神经元细胞数较少;模型组小鼠尼氏染色海马神经元分散稀疏、排列散乱、凋亡神经元细胞数较多,存活细胞数显著减少;阳性药及刺梨果渣活性提取物各剂量给药组小鼠海马神经元排列尚整齐,凋亡神经元细胞数较少;提示刺梨果渣活性提取物对海马神经元具有保护作用。
Claims (3)
1.一种具有抗抑郁症的刺梨活性提取物,其特征在于:所述提取物含有总三萜和总黄酮,其中总三萜的含量为30%-45%;总黄酮的含量为1%~3%;
其中所述总三萜包括蔷薇酸、2-氧代坡模醇酸、刺梨苷F1和刺梨酸;所述总黄酮包括槲皮素、儿茶素、二氢杨梅素和芦丁;所述总三萜的含量为蔷薇酸、2-氧代坡模醇酸、刺梨苷F1和刺梨酸的含量总和,总黄酮的含量为槲皮素、儿茶素、二氢杨梅素和芦丁的含量总和;所述总三萜的含量是以熊果酸为对照品,利用香草醛-冰醋酸-高氯酸显色比色法测定得到;所述总黄酮的含量是以槲皮素、儿茶素、二氢杨梅素和芦丁为对照品,利用高效液相色谱法测定得到;所述的提取物的制备方法包括以下步骤:取刺梨果,用重量为刺梨果重量的5-10倍的50-95%乙醇水溶液回流提取2-4次,每次1-2小时,过滤,合并滤液,回收乙醇,在滤液中加体积为滤液体积的0.5-1倍的水进行分散,抽滤,水洗沉淀2-4次,然后用石油醚或正己烷洗沉淀2-4次,冷冻干燥或真空减压干燥沉淀得提取物。
2.根据权利要求1所述的提取物的制备方法,其特征在于:取刺梨果,用重量为刺梨果重量的5-10倍的50-95%乙醇水溶液回流提取2-4次,每次1-2小时,过滤,合并滤液,回收乙醇,采用0.45μm中空纤维膜过滤,水洗沉淀2-4次,然后用石油醚或正己烷洗沉淀2-4次,冷冻干燥或真空减压干燥沉淀得提取物。
3.根据权利要求1所述的提取物的应用,其特征在于:所述提取物应用于制备抗抑郁症的药物;直接使用或者以组合物的形式使用;所述组合物包括0.1–99%的提取物,其余为载体;所述药物的剂型为硬胶囊、片剂、口服液、颗粒剂、软胶囊或滴丸剂。
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