CN110960565B - 一种黑沙蒿根提取物在制备治疗过敏性鼻炎药物中的应用 - Google Patents
一种黑沙蒿根提取物在制备治疗过敏性鼻炎药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种黑沙蒿根提取物及其制备方法和应用。所述黑沙蒿根提取物的制备方法包括以下步骤：黑沙蒿根经干燥、粉碎后，加入乙醇溶液超声提取；然后将各次的提取液合并，并浓缩于燥后得到提取物A；(2)将提取物A用蒸馏水溶解后依次用石油醚、乙酸乙酯和正丁醇萃取；收集乙酸乙酯层萃取物，减压浓缩，干燥，即得。本发明的黑沙蒿根提取物含总黄酮50‑90％，其中至少含有圣草酚、异樱花素、柚皮素、羟基芫花素、芫花素、金合欢素、香叶木素、黄豆黄素、异鼠李素、桑色素、槲皮素的含量之和大于2％。能显著降低豚鼠过敏型鼻炎症状，并能减低豚鼠血清中组胺、卵清蛋白特异性IgE、IL‑2/4/10、IFN‑γ、ICam‑1等炎性因子的水平，且能减少豚鼠鼻腔鼻黏膜嗜酸粒细胞浸润。
Description
技术领域
本发明涉及一种药物，具体是一种黑沙蒿根提取物及其制备方法和应用。
背景技术
随着工业化程度的提高，城市空气污染的日益加重，过敏性鼻炎发病率呈逐年上升趋势。过敏性鼻炎是易感机体暴露于变应原后主要由IgE介导的鼻黏膜非感染性慢性炎性疾病，以鼻粘膜病变为主，分季节性鼻炎和常年变态反应性鼻炎，症状表现为发作性鼻痒、鼻塞、喷嚏及流清涕，控制不佳可诱发中耳炎、过敏性结膜炎、鼻息肉、慢性鼻窦炎，严重者引起哮喘。过敏性鼻炎患者由于长期受到该疾病的困扰，不仅身体上受到影响，精神上亦具有一定程度的损害，严重降低了患者日常工作以及生活的质量。过敏性鼻炎成为当今医学界亟待解决和攻克的重点课题之一。
目前治疗过敏性鼻炎的方案主要包括避免接触变应原、药物治疗、免疫疗法、手术疗法、替代疗法等。各种疗法均具有各自优缺点。目前还未发现根治的方法，最大限度降低发作期给人体带来的不适已成为主要目的，而临床以药物治疗为主，现有临床药物虽短期疗效确切但停药后疗效难以维持成为制约瓶颈。西药对缓解症状有效，但其依赖性和毒副作用会对机体造成新的伤害，且有停药后病情反复的缺点；免疫与基因疗法费用高、疗程长难以广泛普及；手术治疗有创伤，且疗效不能保证，患者很难接受。
相比较西医而言，中医药主要是对过敏性鼻炎进行辩证论治，大多是根据疾病发生发展的病因病机及患者本身的症状来采用相对应的分型施治，以祛风散寒，通鼻窍，补脾益肺，温肾纳气为主。目前药物应用中，黄芪、防风、白术、苍耳子、辛夷是治疗过敏性鼻炎比较核心的药物。中医药是通过局部的用药以及全面的调理来实现最终的治标治本，与其他疗法相比，具有安全可靠，毒副作用小，疗效持续、稳定、复发率低等优势。但由于大多数中药类药物在配方上不尽合理，导致治疗周期长、易复发、治疗效果上不尽人意，给患者造成了极大的负担。
黑沙蒿(Artemisia ordosica Krasch.)为菊科蒿属植物，又名鄂尔多斯蒿、油蒿、籽蒿、哈拉-啥巴嘎(蒙语)。黑沙蒿味辛苦，性微温，全草可入药。诸多文献揭示黑沙蒿根对于治疗鼻部症状有据可依。据《中国沙漠地区药用植物》记载，黑沙蒿根可治鼻出血：鲜黑沙蒿根，去外皮，折断用鼻嗅之(如嗅之过久，能引起鼻腔肿胀)；休克晕倒用鲜根闻之即能苏醒。《内蒙古中草药》记载其具有止血，治鼻衄、吐血、功能性子宫出血的功效。《中华本草》记载其可祛风除湿、解毒消肿，主治风湿性关节炎、感冒头痛、咽喉肿痛、痈肿疮疖；蒙医作消炎、止血、祛风、清热药。
黑沙蒿化学成分复杂多样：枝叶富含多种必需氨基酸、粗蛋白、脂肪及无机离子等；种子含有丰富的亚油酸和VE；全草含有多种黄酮类化合物以及烷烃、醛、酮、酯、单萜和倍半萜等挥发性成分，在药理作用方面具有抗氧化、抗免疫、降血糖、降低胆周醇、抗癌、抗炎、抗菌等活性。
目前黑沙蒿的研究主要集中于全草化学成分的提取和分离，公开报道的黑沙蒿全草化学成分以精油和黄酮类化合物为主，黄酮类化合物以野樱素、异野樱素、圣草素-7-甲醚、 5，7，2’，4’-四羟基-6，5’-二甲氧基黄酮等为主，精油则以香木兰醇、反式-β-罗勒烯等为主；黑沙蒿提取物生物活性主要有镇痛和抗炎活性、降血糖作用、抗惊厥作用、抗肿瘤作用、抗氧化活性、利尿作用和抗寄生虫活性等，根据所做的资料检索，黑沙蒿根提取物在抗过敏性鼻炎方面的研究尚未见报道。
专利CN102265906B申请了一种黑沙蒿杀菌剂及其制备方法，用于防治果树、蔬菜及花卉上的霜霉病和灰霉病。国内外专利均未涉及黑沙蒿根为原料的抗过敏性鼻炎提取物的制备方法及应用。
超声波萃取具有如下优点：1.提取效率高：超声波独具的物理特性能促使植物细胞组织破壁或变形，使中药有效成分提取更充分，提取率比传统工艺显著提高达50-500％；2. 提取时间短：超声波强化中药提取通常在24-40分钟即可获得最佳提取率，提取时间较传统方法大大缩短2/3以上，药材原材料处理量大；3.提取温度低：超声提取中药材的最佳温度在40-60℃，对遇热不稳定、易水解或氧化的药材中有效成分具有保护作用，这个温度同时也是超声波发挥最佳效果的温度；4.适应性广：超声提取中药材不受成分极性、分子量大小的限制，适用于绝大多数种类中药材和各类成分的提取；5.提取药液杂质少，有效成分易于分离、纯化；6.提取工艺运行成本低，综合经济效益显著；7.操作简单易行，设备维护、保养方便。
发明内容
本发明的目的在于提供一种黑沙蒿根提取物及其制备方法和应用，以解决上述背景技术中提出的问题。
为实现上述目的，本发明提供如下技术方案：
一种黑沙蒿根提取物的制备方法，包括以下步骤：
(1)称取0.5-2份黑沙蒿根，经干燥、粉碎后，加入黑沙蒿根8-13倍重量的质量分数为60-95％的乙醇溶液超声提取，提取时间60-120min，料液比20∶1-50∶1ml/g，超声功率200-600W；然后将各次的提取液合并，并置于55-70℃下浓缩干燥30-40min，得到提取物A；
(2)将提取物A用其8-13倍重量的蒸馏水溶解，得到提取物A溶解液；然后将提取物A溶解液依次用等体积的石油醚、乙酸乙酯和正丁醇萃取，得到石油醚层萃取物、乙酸乙酯层萃取物、正丁醇层萃取物及水溶物；收集乙酸乙酯层萃取物，减压浓缩，85-105℃下干燥，即得黑沙蒿根提取物。
作为本发明进一步的方案：步骤(1)称取1份黑沙蒿根，经干燥、粉碎后，加入黑沙蒿根10倍重量的质量分数为95％的乙醇溶液超声提取，提取时间100min，料液比 35∶1ml/g，超声功率400W；然后将各次的提取液合并，并置于60℃下浓缩干燥35min，得到提取物A。
作为本发明进一步的方案：步骤(2)将提取物A用其10倍重量的蒸馏水溶解，得到提取物A溶解液；然后将提取物A溶解液依次用等体积的石油醚、乙酸乙酯和正丁醇各萃取3次，得到石油醚层萃取物、乙酸乙酯层萃取物、正丁醇层萃取物及水溶物；收集乙酸乙酯层萃取物，减压浓缩，98℃下干燥，即得黑沙蒿根提取物。
一种根据上述制备方法制备得到的黑沙蒿根提取物。
一种根据上述制备方法制备得到的黑沙蒿根提取物在制备治疗过敏性鼻炎药物中的应用。
与现有技术相比，本发明的有益效果是：
本发明利用超声萃取技术，以黑沙蒿根为原料；经过提取、萃取制备得到一种天然的能对抗过敏性鼻炎的黄酮类组份；本发明的黑沙蒿根提取物含总黄酮50-90％，其中至少含有圣草酚、异樱花素、柚皮素、羟基芫花素、芫花素、金合欢素、香叶木素、黄豆黄素、异鼠李素、桑色素、槲皮素的含量之和大于2％。以61.25mg/kg/d的剂量灌胃给药10天，能显著降低豚鼠过敏型鼻炎症状，并能减低豚鼠血清中组胺、卵清蛋白特异性IgE、 IL-2/4/10、IFN-γ、ICam-1等炎性因子的水平，且能减少豚鼠鼻腔鼻黏膜嗜酸粒细胞浸润，产生显著的对抗过敏性鼻炎的效果。本发明生产工艺简单，抗过敏性鼻炎效果好，无毒副作用，使用方便，用于治疗过敏性鼻炎效果良好。
附图说明
图1为各给药组豚鼠抓鼻行为情况。
图2为各给药组豚鼠打喷嚏行为情况。
图3为正常组豚鼠鼻腔鼻黏膜嗜酸粒细胞的浸润情况。
图4为模型组豚鼠鼻腔鼻黏膜嗜酸粒细胞的浸润情况。
图5为阳性对照组豚鼠鼻腔鼻黏膜嗜酸粒细胞的浸润情况。
图6为石油醚层萃取物组豚鼠鼻腔鼻黏膜嗜酸粒细胞的浸润情况。
图7为乙酸乙酯层萃取物组豚鼠鼻腔鼻黏膜嗜酸粒细胞的浸润情况。
图8为正丁醇层萃取物组豚鼠鼻腔鼻黏膜嗜酸粒细胞的浸润情况。
图9为水层组豚鼠鼻腔鼻黏膜嗜酸粒细胞的浸润情况。
具体实施方式
下面结合具体实施方式对本专利的技术方案作进一步详细地说明。
实施例1
一种黑沙蒿根提取物的制备方法，包括以下步骤：
(1)称取0.5份黑沙蒿根，经干燥、粉碎后，加入黑沙蒿根8倍重量的质量分数为60％的乙醇溶液超声提取，提取时间60min，料液比20∶1ml/g，超声功率200W；然后将各次的提取液合并，并置于55℃下浓缩干燥30min，得到提取物A；
(2)将提取物A用其8倍重量的蒸馏水溶解，得到提取物A溶解液；然后将提取物A溶解液依次用等体积的石油醚、乙酸乙酯和正丁醇各萃取2次，得到石油醚层萃取物、乙酸乙酯层萃取物、正丁醇层萃取物及水溶物；收集乙酸乙酯层萃取物，减压浓缩，85℃下干燥，即得黑沙蒿根提取物。
实施例2
一种黑沙蒿根提取物的制备方法，包括以下步骤：
(1)称取2份黑沙蒿根，经干燥、粉碎后，加入黑沙蒿根13倍重量的质量分数为75％的乙醇溶液超声提取，提取时间120min，料液比50∶1ml/g，超声功率600W；然后将各次的提取液合并，并置于70℃下浓缩干燥40min，得到提取物A；
(2)将提取物A用其13倍重量的蒸馏水溶解，得到提取物A溶解液；然后将提取物 A溶解液依次用等体积的石油醚、乙酸乙酯和正丁醇各萃取5次，得到石油醚层萃取物、乙酸乙酯层萃取物、正丁醇层萃取物及水溶物；收集乙酸乙酯层萃取物，减压浓缩，105℃下干燥，即得黑沙蒿根提取物。
实施例3
一种黑沙蒿根提取物的制备方法，包括以下步骤：
(1)称取1份黑沙蒿根，经干燥、粉碎后，加入黑沙蒿根10倍重量的质量分数为95％的乙醇溶液超声提取，提取时间100min，料液比35∶1ml/g，超声功率400W；然后将各次的提取液合并，并置于60℃下浓缩干燥35min，得到提取物A。
(2)将提取物A用其10倍重量的蒸馏水溶解，得到提取物A溶解液；然后将提取物 A溶解液依次用等体积的石油醚、乙酸乙酯和正丁醇各萃取3次，得到石油醚层萃取物、乙酸乙酯层萃取物、正丁醇层萃取物及水溶物；收集乙酸乙酯层萃取物，减压浓缩，98℃下干燥，即得黑沙蒿根提取物。
黄酮类含量测定：以芦丁为对照品，利用NaNO2-Al(NO3)3-NaOH显色方法测定总黄酮含量。黑沙蒿根提取物中总黄酮含量为50-90％。
采用Exion LC+X500R高效液相色谱质谱联用系统，LC Conditions：A Phase：水(含0.02％甲酸和2mM甲酸铵)；B Phase：甲醇∶乙腈(1∶1；v∶v)；梯度洗脱；70min；流速：0.3mL/mim；进样体积：2uL；色谱柱：Kinetex C18(100×2.1mm，2.6mm)； MS mode-100-1250m/z；MS/MS mode-10MS/MS，each 50-1200m/z，IDA。所得含量其中至少含有圣草酚、异樱花素、柚皮素、羟基芫花素、芫花素、金合欢素、香叶木素、黄豆黄素、异鼠李素、桑色素、槲皮素的含量之和大于2％。
实验例
此天然黄酮主要用于治疗过敏性鼻炎，经对豚鼠药理实验表明：10天喂养该制剂使豚鼠抓鼻和喷嚏次数明显降低，显著对抗过敏性鼻炎症状，并能显著降低豚鼠血清炎性生理因子水平。
一、实验内容：建立卵清蛋白(OVA)致豚鼠过敏性鼻炎模型；ELISA法检测血清组胺、卵清蛋白特异性IgE、IL＝2/4/10、IFN＝γ、ICam＝1等炎性因子的水平；光学电子显微镜下观察豚鼠鼻腔鼻黏膜嗜酸粒细胞的浸润情况。
二、实验方法和实验结果
1、OVA诱导豚鼠AR模型的建立
以OVA0.3mg和佐剂Al(OH)330mg加0.9％氯化钠溶液1mL制成混悬液，每只豚鼠腹腔注射1mL，隔日1次，共7次，进行基础致敏；随后以2％OVA(2mg/mL)滴鼻，每侧50μL，每日1次，共7次，进行局部激发致敏。正常组用等量生理盐水代替OVA及Al(OH)3腹腔注射和滴鼻攻击。末次激发后，将豚鼠放入观察笼中观察30min，记录各组豚鼠的喷嚏数、鼻痒程度和鼻分泌物量。以记分方式进行造模效果评定，总积分达到5分即造模成功。计分标准如表1。
表1 AR症状计分标准
2、将致敏豚鼠分为6组：模型组(OVAcontrol，OVA)、阳性对照组(positivecontrol， PC，给药氯雷他定)、石油醚层萃取物组(petroleumether，PE，给药石油醚层萃取物)、乙酸乙酯层萃取物组(ethylestate，EE，给药乙酸乙酯层萃取物)、正丁醇层萃取物组 (n-butylalcohol，BA，给药正丁醇层萃取物)、水层组(distilledwater，DW，给药水溶物)，每组8只。各组均于造模成功后第2天开始灌胃给药(给药量61，25mg/kg/d)，每日1次，连续给药10天，阳性对照组(给与氯雷他定，46.50mg/kg/d)，正常组(normalcontrol，NC)和模型组给予等量生理盐水。
3、豚鼠行为学观察
实验结果见图1-2。与模型组相比，各给药组豚鼠抓鼻和打喷嚏行为数明显下降，其中阳性对照组接近正常组，而乙酸乙酯层萃取物组缓解效果最为显著。
4、ELISA法检测豚鼠血清histamine、IL-2、IL-4、IL-10、IFN-γ及ICAM-1水平
末次给药后，10％水合氯醛麻醉豚鼠，腹主动脉取血置于抗凝管，血样常温下静置2h 后，在4℃离心机3500r离心10min，取上层血清于-20℃保存。检测前从-20℃冰箱中取出血清样本，4℃复融，根据ELISA试剂盒说明书的操作步骤检测豚鼠血清中histamine、 IL-2、IL-4、IL-10、IFN-γ及ICam-1的水平。
具体检测步骤如下：设置空白孔和标准孔，分别加入50μL不同浓度的标准品、10μL的样品以及40μL样品稀释液。轻轻振动，混匀，加覆膜，37℃孵育0.5h。弃去孔内液体，洗涤5次。除空白孔外向每孔中加入酶标试剂50μL，37℃孵育30min。弃去孔内液体，洗涤5次，轻轻甩干。随后向每孔中加显色A溶液50μL和显色B溶液50μL，37℃避光显色10min。之后向每孔中加入终止溶液50μL终止反应。在450nm波长处用酶标仪检测各孔的吸光度。
检测完成后，根据标准曲线，计算histamine、IL-2、IL-4、IL-10、IFN-γ及ICam-1的含量。应用SPSS统计学软件进行分析，计量资料采用
表示，组间差异采用两样本t检验。P＜0.05为差异有统计学意义。结果见表2。
表2 检测豚鼠血清histamine、IL-2、IL-4、IL-10、IFN-γ及ICAM-1水平
注：*表示与正常组相比，+表示与模型组相比，p＜0.01，**/++；p＜0.05，*/+。
模型组AR豚鼠血清中histamine、IFN-γ、IL-2、IL-4、IL-10和VCAM-1水平明显高于正常组且具有显著性差异(P＜0.05)。与模型组比较，乙酸乙酯层萃取物组各细胞因子水平均降低(P＜0.05)，尤其histamine、IFN-γ、IL-4降低明显(P＜0.01)。表明乙酸乙酯层萃取物对豚鼠AR具有较强活性。
5、AR豚鼠鼻黏膜嗜酸性粒细胞浸润情况观察
取血完成后，去除豚鼠头部皮肤组织，剪去上颚，剪下鼻腔。将豚鼠鼻腔用PBS冲洗干净以后，放入4％的多聚甲醛中固定2天。然后放入15％的甲酸溶液中脱钙处理3天。脱钙后再放入4％多聚甲醛进行固定，4℃过夜，用不同梯度的乙醇脱水，二甲苯透明，常规石蜡包埋切片，切片厚度4-6μm，将切片粘附于预先经3-氨丙基-3-7氧基甲硅烷(APES) 处理的载玻片上，60℃烘箱烘烤1h后于37℃烘箱保存。
将石蜡切片放入二甲苯中进行脱蜡处理，再放入高浓度到低浓度酒精再到蒸馏水中水化。水化后的切片放入苏木精水溶液中染色，冲洗，盐酸酒精分化，冲洗，放入75％和90％的酒精中脱水各，再用0.5％的伊红染液染色。随后用100％酒精脱水，二甲苯透明处理。向处理后的切片滴上树胶，盖上盖玻片封固。HE染色，在光学电子显微镜观察豚鼠鼻腔鼻黏膜嗜酸粒细胞的浸润情况。
如图3-9所示，正常组切片可见腺体分布均匀，结构完整，粘膜完好，排列整齐，未见明显的嗜酸性粒细胞浸润；模型组腺体增多且有明显的嗜酸性粒细胞浸润现象产生，粘膜破裂排列紊乱；阳性对照组与模型组对比嗜酸性粒浸润明显减少，粘膜排列较为整齐；乙酸乙酯层萃取物组明显观察到嗜酸性粒细胞浸润减少，粘膜较为完好，水层组可观察到嗜酸性粒细胞，但乙酸乙酯层萃取物组效果更明显。石油醚层萃取物组和正丁醇层萃取物组嗜酸性粒细胞浸润明显，粘膜上皮细胞排列较为紊乱，石油醚层萃取物组粘膜破裂清晰可见。
上面对本专利的较佳实施方式作了详细说明，但是本专利并不限于上述实施方式，在本领域的普通技术人员所具备的知识范围内，还可以在不脱离本专利宗旨的前提下作出各种变化。
Claims (1)
1.一种黑沙蒿根提取物在制备治疗过敏性鼻炎药物中的应用，其特征在于，所述黑沙蒿根提取物的制备方法包括以下步骤：
(1)称取1份黑沙蒿根，经干燥、粉碎后，加入黑沙蒿根10倍重量的质量分数为95％的乙醇溶液超声提取，提取时间100min，超声功率400W；然后将各次的提取液合并，并置于60℃下浓缩干燥35min，得到提取物A；
(2)将提取物A用其10倍重量的蒸馏水溶解，得到提取物A溶解液；然后将提取物A溶解液依次用等体积的石油醚、乙酸乙酯和正丁醇各萃取3次，得到石油醚层萃取物、乙酸乙酯层萃取物、正丁醇层萃取物及水溶物；收集乙酸乙酯层萃取物，减压浓缩，98℃下干燥，即得黑沙蒿根提取物。
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