CN115212247B - 一种白刺花花制备物及其应用 - Google Patents
一种白刺花花制备物及其应用 Download PDFInfo
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- Medicines Containing Plant Substances (AREA)
Abstract
本发明属于医药制备技术领域，涉及一种白刺花花制备物及其应用。白刺花花制备物包括水提醇沉物和残渣醇提物，其中按100份质量份计，残渣醇提物占0‑100份；所述水提醇沉物和残渣醇提物的制备方法如下：取白刺花的花，用水提取，滤取滤液，得到水提液，花残渣备用；水提液再加乙醇至醇浓度为30‑70%，放置析出沉淀，再进行抽滤，沉淀干燥，得水提醇沉物；花残渣再用15‑80%的乙醇溶液提取，滤取滤液，得到乙醇提取液，回收干溶剂，得残渣醇提物。与现有技术相比，本发明所述白刺花花制备物在制备时去除了药材水提物中的醇沉溶液，仅用残渣醇提物或\和水提醇沉物，避免了残渣醇提物中的某些成分的药效受到干扰，提高了其治疗呼吸系统疾病的药效。
Description
技术领域
本发明属于医药制备技术领域，具体涉及到一种白刺花花制备物及其在制备治疗呼吸系统疾病药物上的应用。
背景技术
白刺花（Sophora davidii(Franch.) Skeels）又叫苦刺花、狼牙刺、马蹄刺，是豆科槐属灌木或小乔木。白刺花是民间常用中草药之一，其药用最早记载于《贵州草药》。白刺花的根、叶、花、果实均可入药，大多性味苦寒。其根具有清热解毒、利湿消肿、凉血止血的功效，可用于治疗痢疾、膀胱炎、血尿、水肿、喉炎、衄血。其果实具有理气消积的功效，可用于消化不良、胃痛、腹痛、表皮癌和白血病。其花，可药食两用，具有清热解毒、凉血消肿的功效，用于治疗疮痈肿毒。其叶具有凉血、解毒、杀虫的功效，可用于治疗衄血、便血、疔疮肿毒、疥癣、烫伤、阴道滴虫。白刺花全身是宝，且易于种植，并可用于河道固土。对白刺花进行医用价值的开发，具有重要的社会意义。
呼吸系统是人体与外界空气进行气体交换的一系列器官的总称，包括鼻、咽、喉、气管、支气管，及由大量的肺泡、血管、淋巴管、神经构成的肺，以及胸膜等组织。临床上常将鼻、咽、喉称为上呼吸道，气管以下的气道称为下呼吸道。呼吸系统疾病就是指组成呼吸系统的器官、组织所患的疾病，包括上呼吸道的疾病和下呼吸道的疾病。只要是发生在鼻、咽、喉部位的疾病都是上呼吸道疾病。上呼吸道疾病包括鼻炎、鼻窦炎、鼻咽癌、咽炎、喉炎等疾病。发生在气管、支气管、肺等部位的疾病是下呼吸道疾病。下呼吸道疾病包括了急性气管、支气管炎、肺炎、慢性支气管炎、慢性阻塞性肺疾病、支气管扩张等。
呼吸系统疾病，范围较为广泛。本课题组主要关注的有咳嗽、咽喉炎、肺炎、肺水肿、肺部感染、呼吸窘迫综合征、肺癌等。现有研究只对白刺花的果实进行了治疗喉炎的报道。而对白刺花的花未有关于治疗呼吸系统疾病的报道，尤其是对这些疾病（咳嗽、肺炎、肺水肿、肺部感染、呼吸窘迫综合征、肺癌）的更未有报道，同时，也没有以治疗呼吸系统疾病为导向对白刺花花的有效部位进行研究，对里面的化学成分进行分析。
发明内容
针对上述问题，通过实验与研究，本发明提供了一种白刺花花制备物及其在制备治疗呼吸系统疾病药物上的应用。
本发明所述的一种白刺花花制备物，包括水提醇沉物和残渣醇提物，其中按100份质量份计，残渣醇提物占0-100份；所述水提醇沉物和残渣醇提物的制备方法如下：取白刺花的花，用水提取，滤取滤液，得到水提液，花残渣备用；水提液再加乙醇至醇浓度为30-70%，放置析出沉淀，再进行抽滤，沉淀干燥，得水提醇沉物；花残渣再用15-80%的乙醇溶液提取，滤取滤液，得到乙醇提取液，回收干溶剂，得残渣醇提物。
本发明所述的一种白刺花花制备物，所述水提醇沉物和残渣醇提物的制备方法如下：取白刺花的花，用4-16倍量水提取1-3次，每次0.5-3小时，滤取滤液，合并后得到水提液，花残渣备用；水提液再加乙醇至醇浓度为40-60%，放置析出沉淀，再进行抽滤，沉淀干燥，得水提醇沉物；花残渣再用4-16倍量15-80%的乙醇溶液提取1-3次，每次0.5-3小时，滤取滤液，合并得到醇提液，回收干溶剂，得残渣醇提物。
本发明所述的一种白刺花花制备物，所述水提醇沉物和残渣醇提物的制备方法如下：取白刺花的花，用4-16倍量水提取1-3次，每次0.5-3小时，滤取滤液，得到水提取液，花残渣备用，回收干溶剂，得水提物；水提物再加乙醇至醇浓度为40-60%，放置析出沉淀，再进行抽滤，沉淀干燥得水提醇沉物；花残渣再用4-16倍量25-65%的乙醇溶液提取1-3次，每次0.5-3小时，回收干溶剂，得残渣醇提物。
本发明所述的一种白刺花花制备物，包括水提醇沉物和残渣醇提物，其中按100份质量份计，残渣醇提物占25-100份。
本发明所述的一种白刺花花制备物，包括水提醇沉物和残渣醇提物，其中按100份质量份计，残渣醇提物占100份。
本发明所述的一种白刺花花制备物，由残渣醇提物组成，残渣醇提物的制备方法如下：取白刺花的花，用4-16倍量水提取1-3次，每次0.5-3小时，过滤；花残渣再用4-16倍量25-65%的乙醇溶液提取1-3次，每次0.5-3小时，回收干溶剂，得残渣醇提物。
本发明所述的一种用于治疗呼吸系统疾病的白刺花花制备物。或者，本发明所述的一种白刺花花制备物的应用，应用于治疗或缓解呼吸系统疾病药物的制备；所述呼吸系统疾病为咳嗽、咽喉炎、肺炎、肺水肿、肺部感染、呼吸窘迫综合征、肺癌中的一种或多种。
本发明所述的一种白刺花花制备物的应用，用于功能食品或普通食品的制备。
与现有技术相比，本发明所述白刺花花制备物在制备时去除了药材水提物中的醇沉溶液，仅用残渣醇提物或\和水提醇沉物，避免了残渣醇提物中的某些成分的药效受到干扰，提高了其治疗呼吸系统疾病的药效。
附图说明
图1：大鼠肺组织切片情况，A为空白对照大鼠，B为模型大鼠，C为实施例2干预大鼠，D为对比例4干预大鼠。图2：白刺花花制备物的UPLC-MS特征图谱。上图（标有4）为水提后残渣再用50%乙醇提取的样品，即实施例2；下图（标有5）为直接用50%乙醇提取的样品，即对比例4。
具体实施方式
下面结合具体的实施例对本发明所述的白刺花花制备物及其应用做进一步说明，但是本发明的保护范围并不限于此。
实施例1
取阴干的白刺花的花15 g，用14倍量水回流提取1次，每次2小时，弃去水提取液，保留残渣；残渣再用12倍量25%的乙醇溶液回流提取1次，每次2小时，将乙醇提取液回收干溶剂即得。
实施例2
取阴干的白刺花的花15 g，用14倍量水回流提取1次，每次2小时，弃去水提取液，保留残渣；残渣再用12倍量50%的乙醇溶液回流提取1次，每次2小时，将乙醇提取液回收干溶剂即得。
实施例3
取阴干的白刺花的花15 g，用14倍量水回流提取1次，每次2小时，弃去水提取液，保留残渣；残渣再用12倍量65%的乙醇溶液回流提取1次，每次2小时，将乙醇提取液回收干溶剂即得。
实施例4
取阴干的白刺花的花15 g，用14倍量水回流提取1次，每次2小时，再加乙醇至醇浓度为50%，室温下放置12小时，再进行抽滤，沉淀干燥得水提醇沉物。白刺花的花经水提取后的残渣再用12倍量50%的乙醇溶液回流提取1次，每次2小时，将乙醇提取液回收干溶剂，即得残渣50%乙醇提取物。将水提醇沉物与残渣50%乙醇提取物按生药量1:3混合，得组合物。
实施例5
取阴干的白刺花的花15 g，用14倍量水回流提取1次，每次2小时，再加乙醇至醇浓度为50%，室温下放置12小时，再进行抽滤，沉淀干燥得水提醇沉物。白刺花的花经水提取后的残渣再用12倍量50%的乙醇溶液回流提取1次，每次2小时，将乙醇提取液回收干溶剂，即得残渣50%乙醇提取物。将水提醇沉物与残渣50%乙醇提取物按生药量3:1混合，得组合物。
实施例6
取阴干的白刺花的花15 g，用10倍量水回流提取3次，每次0.5小时，再加乙醇至醇浓度为40%，室温下放置12小时，再进行抽滤，沉淀干燥得水提醇沉物。白刺花的花经水提取后的残渣再用12倍量50%的乙醇溶液回流提取2次，每次1小时，将乙醇提取液回收干溶剂，即得残渣60%乙醇提取物。将水提醇沉物与残渣50%乙醇提取物按生药量1:2混合，得组合物。
实施例7
取阴干的白刺花的花15 g，用6倍量水回流提取2次，每次0.5小时，再加乙醇至醇浓度为60%，室温下放置6小时，再进行抽滤，沉淀干燥得水提醇沉物。白刺花的花经水提取后的残渣再用8倍量35%的乙醇溶液回流提取2次，每次0.5小时，将乙醇提取液回收干溶剂，即得残渣35%乙醇提取物。将水提醇沉物与残渣35%乙醇提取物按生药量1:1混合，得组合物。
实施例8
按实施例2方法制备白刺花花制备物干膏250 g，以作为原料；将原料粉碎过60目筛，加入预胶化淀粉60 g，混合均匀，得混合物。2）制粒：向上述混合物中加入70%的乙醇水溶液作润湿剂，制软材，过筛得湿颗粒，干燥，整粒，得干颗粒；3）压片：将干颗粒进行压片，得素片；4）包衣：将素片置于包衣锅内，启动包衣机，开启加热，用15%的虫胶乙醇溶液均匀喷洒在素片表面，溶剂受热挥发，包衣材料在素片表面形成隔离层；以同样的方式，以70%糖浆包糖衣层；最后，向装有包衣片的包衣锅中撒入川蜡粉，转动包衣锅，至包衣片表面光亮为止，出锅即得。
实施例9
按实施例2方法制备白刺花花制备物干膏250 g，以作为原料；将原料粉碎过60目筛，用小袋封装，制成袋装茶。
对比例1-3
取阴干的白刺花的花30 g，用14倍量水回流提取1次，每次2小时，将水提取液平分成两份。一份作为白刺花花水提取物，去除溶剂即得，为对比例1。另一份再加乙醇至醇浓度为50%，室温下放置12小时，再进行抽滤，溶液部分去除溶剂得对比例2，沉淀部分干燥得对比例3。
对比例4
取阴干的白刺花的花15 g，用14倍量50%的乙醇溶液回流提取1次，每次2小时，将乙醇提取液回收干溶剂即得。
对比例5
取阴干的白刺花的花15 g，用14倍量水回流提取1次，每次2小时，弃去水提取液，保留残渣；残渣再用12倍量15%的乙醇溶液回流提取1次，每次2小时，将乙醇提取液回收干溶剂即得。
对比例6
取阴干的白刺花的花15 g，用14倍量水回流提取1次，每次2小时，弃去水提取液，保留残渣；残渣再用12倍量80%的乙醇溶液回流提取1次，每次2小时，将乙醇提取液回收干溶剂即得。
氨水耐受实验
取84只昆明小鼠，体重20-25 g，适应性饲养7天，随机分成12组，即为：实施例1-5组、对比例1-6组、模型组，每组7只。其中11个给药组小鼠，连续6天灌胃，每天灌胃1次，给予相应药物（6 g生药量/kg/次），第7天早上、中午再分别给药一次。末次给药2小时后，将各组小鼠均置于容积4 L的上盖开有直径1 cm小孔的透明方盒中，用注射器从小孔注入0.3 ml的氨水，在小鼠卧倒后晃动盒子以观测死亡情况，并记录小鼠死亡时间（观察误差控制在10s内，若10 min后仍小鼠未死亡，算存活，停止测试）。实验结果见表1。
表1 小鼠氨水耐受时间结果
将小鼠置于氨水环境中，小鼠吸入后对鼻、喉和肺有刺激性引起咳嗽、气短等，并可因肺水肿而导致死亡，即为呼吸窘迫综合征。由表1可知，实施例1-5及对比例1和3-4均能显著提高了小鼠在氨水环境中的耐受时间，可能是在一定程度上抑制了肺水肿从而延长了存活时间。
止咳引咳实验
取36只雌性SD大鼠，重263~298 g，按体重随机分成6组，每组6只，即空白组、模型组、实施例2组、实施例5组、对比例3组、对比例6组。空白组、模型组灌胃给予生理盐水。实施例2组、实施例5组、对比例3组、对比例6组分别灌胃给予相应药物（1 g生药量/kg/次）。除空白组外，其它5组大鼠在灌胃给予相应试药前5 min内及2 h后均置于4 L的长方形透明盒中（该盒顶中央开有直径1 cm的圆孔），注入0.6 mL的氨水，给药前刺激2 min，给药2 h后刺激4 min，记录2 h时大鼠4 min内的咳嗽次数（以大鼠腹肌收缩同时张嘴呼吸为咳嗽1次）。大鼠咳嗽次数计数情况见表2。麻醉后腹主动脉取血，然后取肺组织用于切片染色，以观察大鼠肺部损伤情况，结果见图1。
表2大鼠咳嗽次数计数情况（mean ± sd，n = 6）
| 组别 | 大鼠咳嗽次数计数 |
| 空白组 | - |
| 模型组 | 58.0 ± 11.4 |
| 实施例2 | 35.0 ± 8.2** |
| 实施例5 | 42.6 ± 9.8* |
| 对比例3 | 45.8 ± 11.2* |
| 对比例4 | 49.1 ± 12.9 |
| 对比例6 | 53.4 ± 16.4 |
与模型组比，*表示P<0.05，**表示P<0.01
氨水引咳实验是测试药物止咳作用的经典模型。氨水刺激大鼠，除了会导致咳嗽外，还会导致其肺毛细血管弥漫性损伤、通透性增强、引起呼吸增快、呼吸困难等，从而出现呼吸窘迫综合征。本课题组进一步从氨水耐受实验中选择代表性组别，进行大鼠氨水引咳实验。研究结果表明，氨水引咳实验与氨水耐受实验的结果相一致。通过观察肺组织切片情况可发现，模型组大鼠的肺泡边缘增厚变粗，为水渗透浸润（肺水肿）所致，红色小点为肺毛细血管弥漫性损伤所致的血渗入（注意，给药C和D组的酱紫部分是由于切片洗片时不小心发生了卷曲所致，并非是血），并出现肺泡融合。D组（直接50%乙醇提取物）肺水肿较轻，但出血和肺泡融合较为严重。而C组（残渣50%乙醇提取物）能明显降低肺毛细血管弥漫性损伤和通透性，减轻水肿。
抗肺部感染实验
取108只昆明小鼠，体重19-24 g，适应性饲养7天，随机分成6组，即模型组、实施例2组、实施例5组、对比例3组、对比例4组、对比例6组。将金黄色葡萄球菌（ATCC4300）菌液10μL(6×109CFU·mL-1)缓慢滴入小鼠鼻腔内建立肺部感染小鼠模型。6组小鼠均予以造模。其中5个给药小鼠组，在造模型前及造模后连续4天灌胃，每天灌胃1次，给予相应药物（6 g生药量/kg/次）。模型组灌胃给予生理盐水。造模后统计小鼠死亡（每隔12h检查一次）及肺部感染情况。菌落计数：在造模给药4 d后于每组中随机选取3-5只小鼠颈椎脱臼处死，在无菌操作下取出部分肺脏，称质量、组织匀浆、稀释，固定于培养基表层，恒温箱培养中培养24 h后于显微镜下进行菌落计数。实验结果见表3和表4。
表3 肺部感染小鼠死亡情况（n = 18）
与模型组相比，实施例2、5及各对比例3-4组能较明显地降低小鼠的死亡率，其中以实施例2的效果最佳。其它各组的效果与小鼠氨水耐受实验保持了一致的趋势。
表4小鼠肺内菌落计数情况（mean ± sd，n = 5）
| 组别 | 小鼠肺内菌落计数（CFU·mg-1） |
| 模型组 | (7.5 ± 4.9)×106 |
| 实施例2 | (9.6 ± 5.2)×104** |
| 实施例5 | (2.7 ± 1.3)×105** |
| 对比例3 | (8.3 ± 4.9)×105* |
| 对比例4 | (6.8 ± 3.4)×105* |
| 对比例6 | (5.8 ± 3.7)×106 |
与模型组比，*表示P<0.05，**表示P<0.01
与模型组相比，实施例2、5以及对比例3-4能较显著抑制小鼠肺部金黄色葡萄球菌感染情况，其中以实施例2的效果最佳。
抗肺癌实验
在5 % CO2、37℃条件下，用含10%新生小牛血清的RPMI-1640培养液传代培养人肺癌A549细胞。取对数生长期A549细胞以5×104/孔接种于96孔板培养，常规条件培养24 h后，设置药物组和正常对照组，药物组分别加入经培养基稀释的生药浓度为10 mg·mL- 1的实施例1-5组、对比例1-6组，含DMSO的体积分数小于0.1%的供试液200 μL，正常对照组加入等体积的培养液，各组均设3个复孔，置5% CO2、37℃孵箱培养48 h，于每孔加入20 μl的CCK8，孵育2 h后，吸取并弃去孔内上清液，每孔再加入DMSO 150 μl，振荡待蓝色晶体溶解后，酶标仪于450 nm处检测每孔的吸光度（A）值，确定药物对肿瘤细胞的抑制率。细胞的增殖抑制率（％）= [(加药实验组吸光度－不加药空白组吸光度)/(不加药空白组吸光度－不含细胞和药物组吸光度)] ×100%。各组药物对A549细胞增殖的抑制率见表5。
表5 各组药物对A549细胞增殖的抑制率
| 组别 | 抑制率（%） |
| 实施例1 | 30.1 ± 8.9 |
| 实施例2 | 42.9 ± 10.4 |
| 实施例3 | 34.6 ± 10.6 |
| 实施例4 | 51.4 ± 11.1 |
| 实施例5 | 46.1 ± 9.2 |
| 对比例1 | 18.4 ± 4.9 |
| 对比例2 | 20.9 ± 5.4 |
| 对比例3 | 15.7 ± 4.3 |
| 对比例4 | 20.4 ± 6.1 |
| 对比例5 | 22.1 ± 6.8 |
| 对比例6 | 19.7 ± 4.7 |
实验结果表明，各实施例组及对比例组均对肺癌A549细胞的增殖均用一定的抑制作用，但大部分组的抑制率却不太显著。实施例1-5及对比例1、3-4均有较明显的抑制效果。但与氨水耐受实验、氨水引咳实验及肺部感染实验有所不同，实施例4-5的抗肺癌效果要明显比实施例1-3组强。这表明，在用于抗肺癌时残渣醇提物与水提醇沉物联合使用，能够起到协同增效的作用。
UPLC-MS特征图谱
取实施例2样品（水提后残渣再用50%乙醇提取的样品）和对比例4样品（直接用50%乙醇提取的样品）各6 mg，用50%甲醇超声溶解后，稀释至适宜浓度，12000 rmp离心10min，吸取上清液，用于UPLC-MS分析。质谱条件：电离源温度为100℃；锥孔气流速为50 L·h-1；去溶剂气温度为400℃，流速为800 L·h-1。正离子模式下，毛细管电压分别为3.0 kV，锥孔电压均为40 V；正离子模式下提取锥孔电压为80 V。采集质量数范围为100～1 000 Da；利用亮氨酸脑啡肽进行实时质量校正。串联质谱碰撞气为氩气，低碰撞能为6 eV，高碰撞能为20～60 eV。液相条件为：流动相A采用乙腈，流动相B为5mmol/L的甲酸铵溶液，柱温40℃，流速0.3 ml/min，梯度洗脱，具体梯度见表6。
表6 UPLC-MS梯度洗脱程序
| 时间（min） | 流动相A（乙腈） | 流动相B（5 mmol/L的甲酸铵溶液） |
| 0 | 5 | 95 |
| 7 | 12 | 88 |
| 25 | 22 | 78 |
| 30 | 22 | 78 |
| 50 | 71 | 29 |
| 55 | 80 | 20 |
| 61 | 86 | 14 |
| 62 | 100 | 0 |
| 62.3 | 5 | 95 |
| 65 | 5 | 95 |
一般来说，将药材直接用50%乙醇提取，得到的成分较全（若把成分按极性分为特大极性成分、大极性成分、中等极性成分、小极性成分、特小极性成分；特大极性成分如多糖和多肽类和特小极性成分如油脂和叶绿素等不会溶出。因这两者的代谢吸收不好，之前被中医药界视为无效成分，故用50%乙醇提俗称全提），从大极性成分到小极性成分都较多。而用水直接提取，药材中的特大极性成分到中等极性成分都会被水提走。然而，通过附图2可知，虽然实施例2（图2上）确实比对比例4（图2下）少了一小些大、中等极性成分，但无论从成分的数量和含量上来说，小极性成分却明显要比于对比例4多很多。
Claims (7)
1.一种白刺花花制备物，包括水提醇沉物和残渣醇提物，其中按100份质量份计，残渣醇提物占0-100份；所述水提醇沉物和残渣醇提物的制备方法如下：取白刺花的花，用水提取，滤取滤液，得到水提液，花残渣备用；水提液再加乙醇至醇浓度为40-60％，放置析出沉淀，再进行抽滤，沉淀干燥，得水提醇沉物；花残渣再用25-65％的乙醇溶液提取，滤取滤液，得到乙醇提取液，回收干溶剂，得残渣醇提物。
2.根据权利要求1所述的白刺花花制备物，其特征在于，所述水提醇沉物和残渣醇提物的制备方法如下：取白刺花的花，用4-16倍量水提取1-3次，每次0.5-3小时，滤取滤液，合并后得到水提液，花残渣备用；水提液再加乙醇至醇浓度为40-60％，放置析出沉淀，再进行抽滤，沉淀干燥，得水提醇沉物；花残渣再用4-16倍量25-65％的乙醇溶液提取1-3次，每次0.5-3小时，滤取滤液，合并得到醇提液，回收干溶剂，得残渣醇提物。
3.根据权利要求1所述的白刺花花制备物，其特征在于，所述水提醇沉物和残渣醇提物的制备方法如下：取白刺花的花，用4-16倍量水提取1-3次，每次0.5-3小时，滤取滤液，得到水提取液，回收干溶剂，得水提物，花残渣备用；水提物再加乙醇至醇浓度为40-60％，放置析出沉淀，再进行抽滤，沉淀干燥得水提醇沉物；花残渣再用4-16倍量25-65％的乙醇溶液提取1-3次，每次0.5-3小时，回收干溶剂，得残渣醇提物。
4.根据权利要求1所述的白刺花花制备物，其特征在于，包括水提醇沉物和残渣醇提物，其中按100份质量份计，残渣醇提物占25-100份。
5.根据权利要求1所述的白刺花花制备物，其特征在于，包括水提醇沉物和残渣醇提物，其中按100份质量份计，残渣醇提物占100份。
6.根据权利要求1所述的白刺花花制备物，由残渣醇提物组成，其特征在于，残渣醇提物的制备方法如下：取白刺花的花，用4-16倍量水提取1-3次，每次0.5-3小时，过滤；花残渣再用4-16倍量25-65％的乙醇溶液提取1-3次，每次0.5-3小时，回收干溶剂，得残渣醇提物。
7.如权利要求1所述的白刺花花制备物在制备用于治疗或缓解呼吸系统疾病药物中的应用，其特征在于，所述呼吸系统疾病为咳嗽、咽喉炎、肺炎、肺水肿、肺部感染、呼吸窘迫综合征、肺癌中的一种或多种。
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