CN114796398A - 一种铁皮石斛花提取物在制备改善溃疡性结肠炎药物中的应用 - Google Patents
一种铁皮石斛花提取物在制备改善溃疡性结肠炎药物中的应用 Download PDFInfo
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Abstract
本发明涉及一种中药材铁皮石斛的花的提取物作为治疗溃疡性结肠炎的药物或功能食品中的用途。铁皮石斛花提取物由中药材铁皮石斛的花经醇水提取制备而成，得率为13.71‑18.39%。本发明所得到的铁皮石斛花提取物可用于治疗或改善溃疡性结肠炎，该提取物可抑制反映溃疡性结肠炎的炎症细胞浸润从而干预葡聚糖硫酸钠（DSS）诱导的小鼠溃疡性结肠炎。
Description
技术领域
本发明涉及一种铁皮石斛花提取物作为治疗溃疡性结肠炎的药物用途。
背景技术
溃疡性结肠炎(ulcerative colitis,UC)是一种慢性炎症性疾病，其特征在于结肠和直肠粘膜的弥漫性炎症[1]。血性腹泻是该疾病的典型症状，且伴有体重减轻、腹痛、里急后重等症状[2]。该疾病发病率在全世界逐年上升，且病程漫长，严重影响患者生活质量[3]。UC的治疗药物包括5-氨基水杨酸药[4]、类固醇[5]、免疫抑制剂[1]、生物制剂[6]和中医药制剂[7]。一些患者可能需要进行结肠切除术以治疗难治性溃疡性结肠炎或结肠肿瘤[8]。UC的病因复杂且尚未明确，涉及遗传因素、环境因素、微生物因素、肠屏障因素、免疫因素和线粒体因素[9]。环境因素会刺激高度遗传易感性个体，在肠道微生物菌群的参与下，肠屏障失调，激活免疫系统诱发免疫应答。遗传、环境饮食等因素虽然增加了UC的易感性，但上皮屏障功能障碍，固有免疫，适应性免疫等因素在UC疾病发病机制中具有关键作用。
肠屏障由粘液层，上皮细胞和驻留免疫细胞的粘膜组成。UC患者中可观察到肠屏障功能障碍和肠通透性增加[10]。粘液在预防细菌渗透的肠道炎症发挥重要作用，MUC2基因缺陷小鼠发展为结肠炎及结直肠癌的风险明显增加，这与直接接触肠上皮的细菌有关[11]。与内粘液层接触的肠上皮，形成了抵御细菌入侵的第二道防线。上皮细胞包括肠上皮细胞，杯状细胞，神经内分泌细胞，Paneth细胞和微折叠细胞。Paneth细胞是在小肠隐窝底部发现的上皮细胞，可以感应腔内微生物群和抗原，并分泌抗菌肽以促进先天免疫。上皮细胞被细胞间紧密连接所密封，紧密连接起着屏障的作用，调节大分子在腔环境与宿主之间的运输[12]。
肠道的固有免疫系统由肠黏膜屏障，先天免疫细胞，先天性免疫分子等组成。固有免疫不需要接触抗原，并且反应快速，只需要几分钟至四天。先天免疫细胞(树突状细胞)(DC)巨噬细胞、自然杀伤((NK)细胞)和非免疫细胞(肠上皮细胞、成纤维细胞)都可以感受到肠道菌群对微生物的保守结构作出快速且有效的反应，并诱发相关的免疫应答。这种对微生物抗原的感应是由模式识别受体(PRR)介导的。PRRs家族可以归类为跨膜受体，包括Toll样受体(TLR)和C型凝集素受体，负责识别细胞外和内体来源的PAMPS；胞质受体，包括视黄酸诱导型基因I(RG-1)样受体和核苷酸结合寡聚域(NOD)样受体(NLR)，参与细胞内感染监测和自发性损伤相关分子模式(DAMPS)的识别[13]。PRR信号级联反应导致核因子(NF-κB)活化和促炎性介质产生，从而保证机体对病原体产生有效免疫应答，维护肠黏膜免疫动态平衡[14]。
与先天免疫不同，适应性免疫具有高度特异性，赋予机体持久免疫力。适应性免疫的关键参与者是T细胞，通常适应性免疫免疫系统的组成部分相互配合，并与先天免疫系统的分子和细胞互相作用，产生有效的免疫应答清除入侵的病原体。抗原刺激后，原始T细胞扩增并分化为不同的亚群，例如Th1，Th2，Thl7和Treg细胞。Thl细胞可消除细胞内的病原体；Th2细胞可保护人体免受有害寄生虫的侵害，并调节过敏反应；Th7细胞去除细胞外细菌和真菌：Treg细胞促进组织修复[15,16]。一旦T细胞应答紊乱和T细胞亚群失衡将会导致细胞因子和趋化因子的过度释放，从而导致炎症的发生[17]。
在正常情况下，免疫系统対共生细菌的反应低，仅在面对病原体时才启动保护性免疫反应。炎性肠病的先天免疫异常导致适应性免疫应答(Th1/Th2调节失衡和Th17/Treg转化失衡)。炎性细胞因子反过来会増加先天免疫损伤(肠道上皮细胞亡，连接蛋白表达降低，Paneth细胞分泌的抗菌肽减少等)，减弱肠道屏障功能，并加剧炎症，形成恶性循环。因此对先天性和适应性免疫系统两者之间的相互作用和“桥梁”的探索，尤其是与们与肠道菌群的关系，将为探讨炎症性肠道疾病的发病机制和药物研发开辟新的视野。
铁皮石斛花为兰科植物铁皮石斛(Dendrobium officinale Kimura etMigo)的新鲜或干燥花。铁皮石斛具有益胃生津，滋阴清热的功效。用于热病津伤，口干烦渴，胃阴不足，食少干呕，病后虚热不退，阴虚火旺，目暗不明，筋骨痿软[18]。目前对铁皮石斛的研究主要集中在降糖和增强免疫方面，尚未见报道铁皮石斛花及其提取物在改善溃疡性结肠炎方面的作用及其在治疗此疾病药物或功能食品中用途。
参考文献
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发明目的
本项目发明的目的在于发现铁皮石斛花提取物可用于治疗溃疡性结肠炎病症的医药新用途。
技术方案
铁皮石斛花提取物在制备改善溃疡性结肠炎药物或功能食品中的应用。
所述的铁皮石斛花提取物经醇水提取制备而成，所述的铁皮石斛花提取物得率为13.71-18.39％。
所述的铁皮石斛花提取物由如下方法制得；铁皮石斛花，以10倍于生药体积的95％乙醇回流提取3次，每次3h，再以10倍于生药体积的75％乙醇回流提取2h，过滤，合并提取液，减压去除乙醇，得总浸膏。
所述的铁皮石斛花提取物其特征在于按重量百分比计，所述的铁皮石斛花提取物含黄酮1.10-2.31％，多糖13.45-14.01％。
具体来说：
本项发明是经过研究证明铁皮石斛花提取物可改善DSS诱导小鼠溃疡性结肠炎模型，通过观察HE染色后结肠组织病理切片。实验结果表明：本发明中的铁皮石斛花提取物可改善模型小鼠结肠组织炎症程度，显示出对小鼠溃疡性结肠炎模型的干预作用，可用于治疗溃疡性结肠炎疾病的新医药用途。
有益效果
1、目前，没有任何研究报道铁皮石斛花提取物或其中某类单体成分可用于治疗溃疡性结肠炎，本发明人通过体内实验证明该铁皮石斛花提取物对于葡聚糖硫酸钠诱导的小鼠溃疡性结肠炎具有很好的改善能力，可以用于制备治疗相应疾病的药物或功能食品。
2、本发明中的铁皮石斛花提取物是个复杂体系，实验结果证明提取物整体有效，尚不能确定活性成分具体类型。就本发明的实验结果表明，在实施中所制备的提取物给药组中，HE染色病理切片显示，铁皮石斛花提取物给药组结肠炎程度明显改善，提示铁皮石斛花提取物具有保护结肠组织的作用。
3、本发明涉及实验材料来自原植物，原植物分别范围广，成本低，具有广泛的实用价值。
附图说明
图1铁皮石斛花提取物对葡聚糖硫酸钠诱导的小鼠结肠形态改变(A)以及结肠长度变化(B)；Control空白；Model模型；Mes美沙拉嗪；TQW-L铁皮石斛花提取物低剂量组；TQW-H铁皮石斛花提取物高剂量组；。*p<0.05，**p<0.01，***p<0.001；
图2各组结肠组织的炎症程度(HE染色)，其中Control空白；Model模型；Mes美沙拉嗪；TQW-L铁皮石斛花提取物低剂量组；TQW-H铁皮石斛花提取物高剂量组，(×200，比例尺＝50μm)；
图3各组结肠部组织病理学炎症评分。Control空白；Model模型；Mes美沙拉嗪；TQW-L铁皮石斛花提取物低剂量组；TQW-H铁皮石斛花提取物高剂量组。*p<0.05，**p<0.01，***p<0.001。
具体实施方式
实施例1
一、铁皮石斛花提取物的制备
铁皮石斛花为兰科植物铁皮石斛(Dendrobium officinale Kimura etMigo)的新鲜或干燥花，购于安徽亳州。乙醇等试剂均为分析纯。
铁皮石斛花以10倍于生药体积的95％乙醇回流提取3次，每次3h，再以10倍于生药体积的75％乙醇回流提取2h，过滤，合并提取液，减压去除乙醇，得到铁皮石斛花提取物。以蒸馏水稀释铁皮石斛花提取物，配置成药液用于活性研究。
二、铁皮石斛花提取物化学成分结构确认
1、铁皮石斛花中黄酮的含量测定方法如下：
①标准溶液、样品溶液的制备：称取1mg芦丁，置于10mL容量瓶中，加适量蒸馏水溶解并稀释至刻度，摇匀备用。称取5mg于105℃干燥至恒重的铁皮石斛花提取物，置于25mL容量瓶中，加适量蒸馏水溶解并稀释至刻度，摇匀备用。
②标准曲线的绘制：准确量取0.1mg/mL芦丁标准液0mL、1.0mL、2.0mL、3.0mL、4.0mL、5.0mL于10.0mL容量瓶中，加入体积分数5％NaNO2溶液0.3mL，摇匀，静置6min后，加入体积分数10％Al(NO3)3溶液0.3mL，摇匀，放置6min后加入1mol/L NaOH溶液4.0mL，摇匀，静置1.5min，用体积分数30％乙醇溶液定容至10.0mL。以0号管为空白对照于510nm波长测吸光值。以溶液浓度C为纵坐标，吸光度A为纵坐标绘制标准曲线，得线性回归方程为C＝0.0016A+0.0098，R2＝0.9993。
③样品中黄酮含量测定：吸取样品液1mL，按标准曲线制备步骤操作，测定吸收度。计算铁皮石斛花中黄酮的含量为1.10-2.31％。
2、铁皮石斛花中多糖的含量测定方法如下：
①标准溶液、样品溶液的制备：称取10mg于105℃干燥至恒重的标准葡萄糖，置于10mL容量瓶中，加适量蒸馏水溶解并稀释至刻度，摇匀备用。称取5mg于105℃干燥至恒重的铁皮石斛花提取物，置于25mL容量瓶中，加适量蒸馏水溶解并稀释至刻度，摇匀备用。
②标准曲线的绘制：精密吸取葡萄糖贮备液10mL于100mL容量瓶，用水稀释至刻度，得0.1mg/mL葡萄糖标准液。分别吸取葡萄糖标准液0、0.2、0.4、0.6、0.8、1.0mL于10mL具塞刻度试管中，用蒸馏水补至1.0mL，加入6％苯酚1mL，摇匀，然后立即加入5mL浓硫酸，摇匀静置5min，于90℃水浴加热20min，然后冰浴冷却5min，于490nm波长处测吸光度。以溶液浓度C为纵坐标，吸光度A为纵坐标绘制标准曲线，得线性回归方程为C＝66.091A-0.0205，R2＝0.9991。
③样品中多糖含量测定：吸取样品液1mL，按标准曲线制备步骤操作，测定吸收度。计算铁皮石斛花中多糖的含量为13.45-14.01％。
三、铁皮石斛花提取物改善葡聚糖硫酸钠所致小鼠溃疡性结肠炎模型
选取实例中所制备的铁皮石斛花提取物进行下述体内药效学研究。饮用含4％葡聚糖硫酸钠的水溶液7天复制小鼠溃疡性结肠炎模型。
1材料与仪器成年雌性C57BL/6小鼠，6-8周龄，18-20g。由南京市必凯动物繁殖场提供。娃哈哈纯净水。葡聚糖硫酸钠(MP Biomedicals)。
2实验方法雌性C57BL/6小鼠，分空白组，模型组，阳性药组，铁皮石斛花提取物低剂量和高剂量组，每组各20只。饮用含4％葡聚糖硫酸钠的水溶液7天复制小鼠溃疡性结肠炎模型，然后饮用纯净水1天，空白组实验期间一直饮用纯净水。
空白组，模型组每天灌胃生理盐水，阳性药组灌胃200mg/kg/d美沙拉嗪。给药组取实施例中所制备的铁皮石斛花提取物，灌胃100mg/kg,400mg/kg铁皮石斛花提取物，连续灌胃8天。8天后小鼠摘眼球取血，血液流入1.5ml离心管中，静置后，4℃，3000rpm离心20min，-80℃保存备用。取出结肠组织，放入4％甲醛中固定，逐级酒精脱水，二甲苯透明，浸蜡，石蜡包埋后，常规切片，HE染色，观察结肠组织损伤变化。
所有数据均用均数±SD(x±s)表示。应用GraphPad Prism 8统计软件处理，统计采用单因素方差分析(one-way ANOVA)，P<0.05表示差异有统计学意义。
病理组织切片经HE染色，结果显示模型组小鼠结肠屏障结构损伤严重，伴有大量炎性细胞浸润，溃疡性结肠炎形成，说明实验小鼠溃疡性结肠炎模型制备成功。经实施所制备的铁皮石斛花提取物给药组小鼠结肠组织结构较完整，炎性细胞浸润轻。经阳性药物美沙拉嗪治疗后，阳性对照组小鼠结肠屏障结构较清晰，较多炎性细胞浸润，病变程度较模型组减轻。给药各组及阳性药与模型组相比，炎症程度均减轻，表明铁皮石斛花提取物对溃疡性结肠炎小鼠症状具有调节作用。
五、讨论
本实验结果表明，本研究中所制备的提取物给药组中，其结肠长度与模型组小鼠相比，给药组小鼠结肠长度显著增加，病理切片结果显示给药组溃疡性结肠炎程度明显改善，说明铁皮石斛花提取物对溃疡性结肠炎进程有一定的保护作用，证实了铁皮石斛花提取物改善溃疡性结肠炎的药物或功能食品的新用途。
Claims (4)
1.一种铁皮石斛花提取物在制备改善溃疡性结肠炎药物或功能食品中的应用。
2.根据权利要求1所述的应用，其特征在所述的铁皮石斛花提取物经醇水提取制备而成。
3.根据权利要求2所述的的应用，其特征在于所述的铁皮石斛花提取物由如下步骤制得；铁皮石斛花以10倍于生药体积的水回流提取3次，每次3 h，再以10倍于生药体积的75 %乙醇回流提取2 h，过滤，合并提取液，减压去除乙醇，得到铁皮石斛花的提取物。
4.根据权利要求3所述的应用，其特征在于按重量百分比计，所述的铁皮石斛花提取物含有黄酮1.10-2.31 %，多糖13.45-14.01 %。
Priority Applications (1)
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| CN105030763A (zh) * | 2015-06-16 | 2015-11-11 | 中国药科大学 | 蟛蜞菊内酯在制备抗溃疡性结肠炎药物中的应用 |
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| CN105030763A (zh) * | 2015-06-16 | 2015-11-11 | 中国药科大学 | 蟛蜞菊内酯在制备抗溃疡性结肠炎药物中的应用 |
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| XIANG ZHENG 等: "Benefit Effect of Dendrobium officinale Ultrafine Powder on DSSInduced Ulcerative Colitis Rats by Improving Colon Mucosal Barrier", EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE, vol. 2021, pages 10 * |
| 刘志龙 等: "铁皮石斛提取物对 DSS 诱导的溃疡性结肠炎 小鼠的抗氧化及抗炎作用", vol. 28, no. 28, pages 214 - 220 * |
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