CN105641148B - 枳壳挥发油提取物作为制备抗抑郁药物的应用 - Google Patents
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Abstract
枳壳挥发油提取物作为制备抗抑郁药物的应用，所述枳壳挥发油提取物从枳壳药材中提取，主要成分为柠檬烯。所述枳壳挥发油提取物的提取工艺为：取枳壳药材，浸泡1～3h，采用水蒸气蒸馏法，加6～10倍体积的水提取5～9h即得。优选工艺为：取枳壳药材，浸泡3h，采用水蒸气蒸馏法，加8倍体积的水提取7h，即得。所述枳壳挥发油提取物可以制备成多种制剂，并可添加药学上可接受的辅料及载体应用于临床抗抑郁新药的开发。本发明提供的枳壳挥发油提取物作为制备抗抑郁药物的应用，通过体外药效实验，证明枳壳挥发油提取物对缺氧损伤SH‑SY5Y神经细胞具有保护作用，具有显著的抗抑郁作用，为抗抑郁方面的新药开发奠定基础。
Description
技术领域
本发明涉及中医药技术领域，尤其涉及枳壳挥发油提取物作为制备抗抑郁药物的应用。
背景技术
抑郁症是一种以持续精神低落为主要症状的精神情志障碍性疾病。临床对抑郁症的治疗主要依靠西药抗抑郁剂，并辅助以心理治疗，但此治疗方法存在起效慢、疗程长、副作用大、药价昂贵等缺点。近年来，中医药在治疗抑郁症实验研究方面取得了丰富的经验和较好的疗效。经大量文献调研可知，许多中药复方、单味药及中药活性成分均具有抗抑郁作用。因此，近年来中医中药在抑郁症的治疗方面日益被人们所关注。
枳壳最早载于《神农本草经》，列为中品。枳壳为芸香科植物酸橙Citrus aurantium L.及其栽培变种的干燥未成熟果实，性微寒，味苦、辛、酸，入肺、脾、大肠经。功能理气宽中，行滞消胀。主治胸肋气滞、胀满疼痛、食积不化、痰饮内停、胃下垂等。
枳壳类药材化学成分比较复杂，目前枳壳药材中已知的成分主要包括挥发油、黄酮类、香豆素类和生物碱等成分。枳壳挥发油中所含的主要化合物为柠檬烯，能够显著抑制胃肠平滑肌收缩，也具有一定的镇痛、抗菌、中枢抑制及溶胆石作用。目前，针对枳壳挥发油抗抑郁症的研究未见报道。
体外细胞实验，由于细胞直接来源于机体组织，生物性状尚未发生大的变化，在一定程度上能够反映体内的状态，具有操作简便、周期短、节约成本等优势，广泛应用于药效学研究领域。SH-SY5Y细胞（神经母细胞瘤细胞）在抗抑郁、治疗帕金森病等神经系统疾病研究方面应用较为普遍，SH-SY5Y细胞氧化损伤模型常用于抗抑郁药物的筛选研究。
发明内容
针对上述问题，本发明提供一种枳壳挥发油提取物作为制备抗抑郁药物的应用，该挥发油提取物从枳壳药材中提取，对缺氧损伤SH-SY5Y神经细胞具有较好的保护作用，可应用于抑郁症的治疗研究。
为实现本发明的上述目的，本发明提供枳壳挥发油提取物作为制备抗抑郁药物的应用，所述枳壳挥发油提取物的主要成分为柠檬烯。
所述枳壳挥发油提取物的提取工艺为：取枳壳药材，浸泡1～3h，采用水蒸气蒸馏法，加6～10倍重量的水提取5～9h，即得。
所述枳壳挥发油提取物的提取工艺为：取枳壳药材，浸泡3h，采用水蒸气蒸馏法，加8倍重量的水提取7h，即得。
所用的枳壳为芸香科植物酸橙Citrus aurantium L.及其栽培变种的干燥未成熟果实。
所述枳壳挥发油提取物能制备多种制剂，并可添加药学上可接受的辅料及载体应用于临床抗抑郁新药的开发。药物剂型可以为：糖衣片剂、薄膜衣片剂、肠溶衣片剂、硬胶囊剂、软胶囊剂、口服液、口含剂、颗粒剂、冲剂、蜜丸剂、散剂、丹剂、注射剂、栓剂、硬膏剂、糖浆剂、针粉剂或缓释胶囊剂等。
与现有技术相比本发明的有益效果。
本发明提供的枳壳挥发油提取物作为制备抗抑郁药物的应用，通过体外药效实验，证明枳壳挥发油提取物有显著的抗抑郁作用，为该药物抗抑郁方面的新药开发奠定基础。
具体实施方式
下面结合具体实施例进一步详细说明本发明，但不应将此理解为本发明上述主题的范围仅限于下述的实施例，凡基于本发明上述内容实现的技术均属于本发明的范围。
一 实验材料。
1 实验细胞株。
人骨髓神经母细胞瘤细胞株SH-SY5Y（购自中国科学院上海生命科学研究院细胞资源中心）。
2 药材与试剂。
盐酸帕罗西汀片（中美史克，批号：13030713）；枳壳药材（由辽宁华润本溪三药有限公司提供）；DMEM/F12培养基（美国GIBCO公司）；胎牛血清（FCS）（杭州四季青生物工程材料有限公司）；水为超纯水。
3 仪器与设备。
US AUTOFLOW型CO2培养箱（德国NUAIRE公司）；AE31型倒置相差显微镜（Motic公司）；SUNRISE酶标仪（瑞士TECAN公司）；LEGEND MICRO 17R型低温高速离心机（ThermoFisher Scientific）。
二 实验方法与结果。
1 枳壳挥发油提取工艺研究：取枳壳药材，精密称取50.0g，采用水蒸气蒸馏法，按下述条件进行提取试验。
1.1正交试验设计。
选取浸泡时间、加水倍数、提取时间三个因素，对主要影响因素各取三个水平，进行L9(34)正交试验考察。以挥发油得率为考察指标，优选其最佳提取工艺。正交试验L9（34）因素水平表见表1，正交试验设计表及直观分析结果见表2，方差分析结果见表3。
表1 因素水平表。
表2 正交实验设计表及直观分析结果。
表3 以挥发油为指标正交试验方差分析。
注：F0.05＝19.00；*为P＜0.05。
以挥发油含量为指标，直观分析及方差分析影响枳壳挥发油提取工艺的因素顺序为A>B>C，筛选出最佳提取条件为A2B2C3，即浸泡3h，加8倍量的水提取7h。
2 药物活性研究。
2.1实验方法：（1）MTT法药效检测。取对数生长期、生长状态良好的SH-SY5Y细胞，经PBS清洗，用0.25%胰蛋白酶消化，待细胞趋于变圆用含15%进口胎牛血清的DMEM培养液冲洗吹打细胞，制成单细胞悬液。以每毫升1×105个细胞接种于96孔培养板中，每孔100µL（除空白调零孔外），将培养板置于CO2培养箱中继续培养，24 h后模型组和给药组加入含H2O2（终浓度为100µmol·L-1）的PBS溶液，构建氧化损伤抑郁模型，空白对照组加无H2O2的PBS溶液，造模2h后空白对照组和模型组加入含10 %血清的培养基100 µL培养，各给药组分别加入含10 %血清的含药培养基100 µL。设空白调零组（只加培养基，酶标仪调零用）、空白对照组、模型组（只加细胞）、枳壳挥发油组和盐酸帕罗西汀组,设5个复孔。在37℃、5% CO2培养箱中继续培养24h后，每孔吸取细胞上清液50µL，置-20℃保存，留作测试剂盒用，每孔补加50 µL培养液，继续培养3 h，每孔加入20µL（5 mg/mL）MTT，继续培养4 h后吸净孔内上清液，每孔加入150 μL DMSO用酶标仪振摇10min，使紫色结晶完全溶解，在492 nm处扫描，测定吸光值（OD），结果见表4。其中存活率=OD试验组均值/OD空白对照组均值×100％。
表4 枳壳挥发油对SH-SY5Y神经细胞的保护作用。
| 组别 | 给药浓度（mg·mL<sup>-1</sup>） | OD值（s） | 存活率（%） |
| 空白对照组 | - | 1.0732±0.0845 | 100 |
| 模型组 | - | 0.6184±0.0655<sup>**</sup> | 57.62 |
| 枳壳挥发油组 | 0.5 | 0.7954±0.0306<sup>#</sup> | 74.11 |
| 盐酸帕罗西汀 | 0.01 | 0.8530±0.1132<sup>##</sup> | 79.48 |
注：与空白组比较，*P<0.05，**P<0.01；与模型组比较，#P<0.05，##P<0.01。
由表4可知，枳壳挥发油对SH-SY5Y神经细胞有很好的保护作用，其作用效果等效于盐酸帕罗西汀，证明枳壳挥发油具有较强的抗抑郁作用，可以开发为以枳壳挥发油为原料的中药抗抑郁药物，便于临床制成不同剂型给药，扩大了枳壳的应用范围，为其抗抑郁新药的开发提供理论依据。
Claims (2)
1.枳壳挥发油提取物在制备保护缺氧损伤SH-SY5Y神经细胞药物的应用；
所述枳壳挥发油提取物的主要成分为柠檬烯，是从枳壳药材中提取的；
所述枳壳挥发油提取物的提取工艺为：取枳壳药材，浸泡1～3h，采用水蒸气蒸馏法，加6～10倍量的水提取5～9h，即得枳壳挥发油提取物。
2.如权利要求1所述的应用，其特征在于，所述保护缺氧损伤SH-SY5Y神经细胞药物的剂型为：糖衣片剂、薄膜衣片剂、肠溶衣片剂、口服液、冲剂、蜜丸剂、散剂、丹剂、硬膏剂、糖浆剂或缓释胶囊剂。
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