CN116270863B - 一种具有抗炎消肿和治疗关节炎作用的中药喷雾剂及其制备方法 - Google Patents
一种具有抗炎消肿和治疗关节炎作用的中药喷雾剂及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种具有抗炎消肿和治疗关节炎作用的中药喷雾剂及其制备方法，属于药物制剂技术领域。本发明的中药喷雾剂的原料药包括藏菖蒲、人工麝香、川木香、草乌、诃子肉、黄葵子、决明子、乳香、冰片、大黄、草红花、骨碎补、宽筋藤；通过提取原料药中有效成分，制备成喷雾剂，该喷雾剂具有显著的抗炎消肿作用，能够快速治疗急性炎症导致的红肿和关节炎以及类风湿性关节炎，方便患者随时使用与携带，并且药效显著。
Description
技术领域
本发明涉及一种具有抗炎消肿和治疗关节炎作用的中药喷雾剂及其制备方法，属于药物制剂技术领域。
背景技术
对于急性期关节疼痛，痛风，肩周炎的治疗目前主要以西药为主，主要有安康信(依托考昔片)和优立通(非布司他)等西药以及青鹏软膏等。其中西药安康信和优立通毒副作用明显，作用靶点单一导致药效有限；而青鹏软膏主要用于活血化瘀，消炎止痛；而对于急性期关节疼痛、痛风、风湿、类风湿关节炎的作用有限，无法有效缓解病灶。
发明内容
[技术问题]
现有技术中治疗关节炎以及类风湿性关节炎的药物药效有限，且毒副作用明显，很难有效的达到治疗目的，并且市售药物中很少有对关节炎有治疗作用的中药。
[技术方案]
为解决上述技术问题，本发明提供了一种具有抗炎消肿和治疗关节炎作用的中药喷雾剂及其制备方法，通过采用不同浓度乙醇提取中药复方药：藏菖蒲、人工麝香、川木香、草乌、诃子肉、黄葵子、决明子、乳香、冰片、大黄、草红花、骨碎补、宽筋藤中有效成分，制备成喷雾剂，达到治疗急性炎症的红肿和关节炎以及类风湿性关节炎的问题。该喷雾剂具有显著的抗炎消肿作用，能够快速治疗急性炎症导致的红肿和关节炎以及类风湿性关节炎，方便患者随时使用与携带，并且药效显著。
本发明的目的是提供一种具有抗炎消肿和治疗关节炎作用的中药喷雾剂，所述中药喷雾剂包括以下重量份的原料药：藏菖蒲4～6份，人工麝香0.05～0.2份，川木香4～8份，草乌3～7份，诃子肉3～8份，黄葵子4～6份，决明子5～8份，乳香4～5份，冰片2～3份，大黄3～7份，草红花2～3份，骨碎补4～7份，宽筋藤4～6份。
在一种实施方式中，所述中药喷雾剂包括以下重量份的原料药：藏菖蒲5～6份，人工麝香0.05～0.1份，川木香4～5份，草乌5～7份，诃子肉5～8份，黄葵子4～5份，决明子5～8份，乳香4～5份，冰片2.5～3份，大黄4～5份，草红花2.5～3份，骨碎补5～7份，宽筋藤4～5份。
在一种实施方式中，所述中药喷雾剂包括以下重量份的原料药：藏菖蒲5份，人工麝香0.1份，川木香5份，草乌5份，诃子肉5份，黄葵子5份，决明子5份，乳香5份，冰片2.5份，大黄5份，草红花2.5份，骨碎补5份，宽筋藤5份。
本发明所采用的原料药在本配方中的药理作用：
藏菖蒲：主要起抗菌消炎止痛作用。
人工麝香：主要起活血散结，消肿止痛作用。
川木香：主要起行气止痛作用。
草乌：主要起麻醉止痛、抗炎杀菌，促进组织细胞再生，能加快伤处愈合的作用。
黄葵子：主要起消肿、生肌的作用。
诃子肉：主要起抗菌作用。
乳香：主要起活血行气、消肿生肌的作用。
冰片：主要起促进药物透皮吸收、消肿止痛的作用。
大黄：主要起泻火解毒，凉血消肿，治热毒痈肿疔疖的作用。
草红花：主要起活血通经、散瘀止痛、活血通经的作用。
骨碎补：主要起活血散瘀、消肿止痛的作用。
宽筋藤：主要起舒筋活络的作用。
在一种实施方式中，所述中药喷雾剂还包括提取溶剂和辅料；所述提取溶剂为30～70％的乙醇溶液；优选为30～50％的乙醇溶液；所述辅料包括络合剂、抗氧化剂、表面活性剂和透皮剂。
在一种实施方式中，所述络合剂包括乙二胺四乙酸(EDTA)、乙二胺四乙酸二钠盐(EDTA-2Na)、乙二胺四乙酸四钠盐(EDTA-4Na)或次氨基三乙酸二钠(NTA)中的一种或几种。
在一种实施方式中，所述抗氧化剂为柠檬酸。
在一种实施方式中，所述表面活性剂包括十二烷基硫酸钠、硬脂酸钠、十二烷基苯磺酸钠或十二烷基硫酸钠中的一种或几种。
本发明的另一目的是提供一种由上述所述中药喷雾剂的制备方法，所述方法包括如下步骤：
(1)取上述原料药粉碎置于30～70％的乙醇水溶液中，浸泡，过滤，静置，再过滤，得中药提取液；
(2)在步骤(1)制备的中药提取液中加入络合剂、抗氧化剂、表面活性剂和透皮剂，静置，过滤，即得中药喷雾剂溶液，装入喷雾剂瓶中即得中药喷雾剂。
在一种实施方式中，步骤(1)所述乙醇水溶液为30～50％的乙醇水溶液；优选为30％。
在一种实施方式中，步骤(1)所述静置的时间为24～36h。
在一种实施方式中，步骤(1)所述浸泡的时间为3～6天。
在一种实施方式中，步骤(2)所述静置时间为12～24h。
在一种实施方式中，步骤(2)所述络合剂的使用量为中药提取液的0.1～0.2wt％。
在一种实施方式中，步骤(2)所述抗氧化剂的使用量为中药提取液的2～5wt％。
在一种实施方式中，步骤(2)所述表面活性剂的使用量为中药提取液的2～5wt％。
在一种实施方式中，步骤(2)所述透皮剂的使用量为中药提取液的1～3wt％。
在一种实施方式中，所述原料药与乙醇水溶液的料液比为1:10。
本发明的第三个目的是提供一种具有治疗急性炎症导致的红肿热痛的药物，所述药物包含上述所述的中药喷雾剂中的原料药。
本发明的第四个目的是一种具有治疗关节炎的药物，所述药物包含上述所述中药喷雾剂的原料药；所述关节炎包括急性关节炎和类风湿性关节炎。
本发明的有益效果：
(1)本发明将中药原料药的乙醇提取物制备成喷雾剂，用于对急性期关节疼痛，痛风，肩周炎等的治疗，并且30％乙醇提取物显著效果；方便患者随时使用与携带，将中药喷雾剂喷至二甲苯诱导的小鼠耳廓肿胀急性炎症模型，1小时后小鼠耳廓肿胀度显著减轻，效果显著；
(2)本发明的中药喷雾剂在长期的应用实践中发现，中药喷雾剂具有清热消炎，镇痛，舒筋活络，干黄水功效，用于急性期关节疼痛，痛风，肩周炎，急性腰扭伤，颈椎病，以及无创口的急性期跌打损伤具有显著效果，并且效果显著优于市售青鹏软膏。
附图说明
图1为本发明实施例1制备的不同浓度乙醇中药提取液对细胞活力的影响数据图；
图2为本发明实施例1制备的不同浓度乙醇中药提取液在RAW264.7细胞炎症模型中的抗炎作用数据图；
图3为本发明实施例1制备的不同浓度乙醇中药提取液对炎症通路TLR4/p65-NFκB蛋白的抑制作用数据图；
图4为本发明实施例2制备的中30％乙醇提取液制备的中药喷雾剂治疗大鼠关节炎效果图。
具体实施方式
下面结合实施例对本发明进一步说明但不限于本发明。
1、灵配方喷雾剂外观性状检测标准
溶液型喷雾剂应澄清透明，不应有沉淀析出。
2、每喷喷量
检查法：取供试品1瓶，弃去若干喷次，擦净，精密称定，喷射1次，擦净，再精密称定；前后两次重量之差为1个喷量；分别测定标示喷次前(初始3个喷量)、中(n/2喷起4个喷量，n为标总喷次)、后(最后3个喷量)，共1个喷量；计算上述10个喷量的平均值；再重复测试3瓶；每一次喷出液体量均应为标示喷量的80％-120％。
3、每喷主药含量
检查法：取供试品1瓶，弃去若干喷次，用溶剂洗净喷口，充分干燥后，喷射10次或20次(注意喷射每次间隔5秒并缓缓振摇)，收集于一定量的吸收溶剂中，转移至适宜量瓶中并稀释至刻度，摇匀，测定；所得结果除以10或20,即为平均每喷主药含量，每喷主药含量应为标示含量的80％-120％。
4、每瓶总喷次
每瓶总喷次大于450次。
5、完全培养基组分
采用DMEM高糖培养基，加入10％Gibico血清，1％青霉素链霉素双抗制成完全培养基进行细胞培养。
实施例1本发明中药喷雾剂原料药的提取溶剂筛选实验
以55.1份计，1份以100克(g)计；称取藏菖蒲500g，人工麝香10g，川木香500g，草乌500g，诃子肉500g，黄葵子500g，决明子500g，乳香500g，冰片250g，大黄500g，草红花250g，骨碎补500g，宽筋藤500g；粉碎过40目筛；
分别采用10％乙醇、30％乙醇、50％乙醇、70％乙醇和90％乙醇按照酊剂的浸渍法，置于有盖容器中，密盖，搅拌或振摇，浸泡5日后，过滤收集提取液，加溶剂至规定量(根据料液比为1:10，g/ml)后，静置24小时，过滤，即得不同浓度乙醇中药提取液。
性能测定实验
1、检测不同浓度乙醇中药提取液中多糖化合物、皂苷类化合物和黄酮类化合物含量，结果如表1所示。
表1不同浓度乙醇提取液中各化合物含量占比
2、不同浓度乙醇中药提取液对细胞活力影响的测定
将上述制备的不同浓度乙醇中药提取液，进行MTT实验：
具体是将稳定传代的小鼠单核巨噬细胞RAW264.7细胞以8000/孔的密度接种于96孔板中，贴壁12h后，加入不同乙醇浓度提取的提取液，处理24h后，加入MTT试剂于37℃孵育4h后，每孔加入100μL DMSO于37℃孵育4h，通过酶标仪于570nm波长处检测吸光度，计算细胞存活率。
结果如图1所示，和对照组相比，10％乙醇中药提取液在10-200μg/mL范围内对细胞活力影响不大，30％乙醇中药提取液在10-200μg/mL范围内可以明显促进细胞增殖；50％乙醇中药提取液在500ng-1μg/mL范围内可以促进细胞增殖，10-50μg/mL范围内对细胞活力具有显著影响；70％乙醇中药提取液在1-50μg/mL范围内，对细胞活力无显著影响，当浓度达到100μg/mL时可以显著抑制细胞增殖；90％乙醇中药提取液在500ng-1μg/mL浓度范围内，可以显著促进细胞增殖，当浓度在10-50μg/mL浓度时可以显著抑制细胞增殖。
3、不同浓度乙醇中药提取液在RAW264.7细胞炎症模型中的抗炎作用
实验过程如下：
(1)体外巨噬细胞炎症模型建立：将处于对数生长期的Raw246.7细胞以每孔2×105的密度接种于六孔板中，待贴壁后；每个处理组加入等体积的试剂和药物；
对照组：只加普通的完全培养基；
模型组：只加LPS不加药物；
治疗组：加LPS和不同浓度乙醇中药提取液；于37℃细胞培养箱中孵育24h；其中：脂多糖(Lipopolysaccharide，LPS)浓度为1μg/ml；不同浓度乙醇中药提取液分别为10％乙醇提取液和30％乙醇提液浓度为20μg/ml和200μg/ml；50％乙醇提液浓度为500ng/ml和10μg/ml；70％乙醇提液浓度为10μg/ml和50μg/ml；；90％乙醇提液浓度为1μg/ml和10μg/ml。
(2)qRT-PCR测定炎症因子IL-1β、TNF-α、IL-6和iNOS：
Trizol法提取细胞RNA，用逆转录试剂盒合成cDNA，得到的cDNA样本用特异性的引物进行PCR扩增。IL-1β：上游序列(Forward primer)GCTGAAAGCTCTCCACCTCAATG)，下游序列(Reverse primer)(TGTCGTTGCTTGGTTCTCCTTG)，TNF-α：上游序列(CATCTTCTCAAAATTCGAGTGAC)，下游序列(TGGGAGTAGACAAGGTACAAC)。
测定不同处理对LPS诱导Raw264.7细胞24小时的炎症因子IL-1β、TNF-α、IL-6和iNOS的作用；结果如图2所示：
从图2A可知，中药10％的乙醇提取液在对细胞无毒性的情况下，对炎症因子IL-1β、TNF-α、IL-6和iNOS的表达无显著抑制作用；从图2B可知，中药30％的乙醇提取液在对细胞无毒性的情况下，200μg/ml浓度对炎症因子IL-1β、TNF-α、IL-6和iNOS均具有显著的抑制作用；从图2C可知，中药50％的乙醇提取液在对细胞无毒性的情况下，仅仅对炎症因子iNOS具有显著的抑制作用。从图2D可知，中药70％的乙醇提取液在对细胞无毒性的情况下，仅对IL-1β炎症因子具有显著的抑制作用。从图2E可知，中药90％的乙醇提取液在对细胞无毒性的情况下，对炎症因子IL-1β、TNF-α、IL-6和iNOS的表达均无显著抑制作用。
由以上结果可见，中药30％的乙醇提取液具有减轻LPS诱导的Raw264.7细胞炎症的作用，能够有效的抑制IL-1β、TNF-α、IL-6和iNOS表达水平，显著优于50％乙醇提取液组和70％乙醇提取液组。
4、Western blotting分析不同浓度乙醇中药提取液对炎症相关通路蛋白的抑制作用
实验前准备：
(1)电泳液配制：在1000mL蒸馏水中加入18.87g的甘氨酸、3.04g Tris，1.01g SDS粉末。最后用盐酸或者氢氧化钠使缓冲液的pH调至8.3左右；
(2)电泳转膜液的配制：在800mL的蒸馏水中加入14.3g甘氨酸、3.01g Tris，200mL甲醇，固定溶液的pH；
(3)TBST溶液的配制：1000m L蒸馏水加入8.7g氯化钠、1.11g Tris和0.5mL吐温-20，最后pH调至7.5左右；
实验具体步骤如下：实验分为三组
对照组：只加普通的完全培养基；
模型组：只加LPS不加药物；
治疗组：加LPS和不同浓度乙醇中药提取液；于37℃细胞培养箱中孵育24h；培养结束后，进行后续实验：
1)进行细胞蛋白提取；采用试剂盒提取蛋白；
2)进行蛋白定量：用BCA试剂盒测定皮肤组织中蛋白浓度；主要操作步骤如下，先制作BSA 标准曲线，然后根据待测蛋白提取液的吸光值确定提取蛋白的浓度；
3)SDS-PAGE电泳凝胶制备：根据目的蛋白分子量选择分离胶浓度；
4)电泳与转膜：取出20μL进行8％-10％的SDS-PAGE电泳，先80V电压电泳30min，再100V电压电泳100min。电泳结束后，将胶与硝酸纤维素膜(NC膜)按照顺序依次置于电转三明治夹子中，加入电转液。然后置于-20℃冰箱中中5-10min后，取出放置于电转槽中，在80-100V(根据具体情况)电压电转50-110min；
5)封闭与一抗孵育：电转结束后，先用新配制的TBST清洗三次后，再用加入TBST配制的5％的脱脂奶粉室温下封闭1-2h。然后加入按照要求稀释好的一抗抗体，在4℃冰箱中孵育过夜；
6)二抗孵育与显色：一抗孵育后，用新鲜配制的TBST洗膜3次，每次3-5min，然后加入按一定比例配制的特定的二抗，在室温条件下孵育1.5-2h，洗膜后按照所给的说明书方法进行条带显色；
7)拍照与半定量分析：条带显色后拍照，然后用Image Lab软件对条带中目的蛋白的灰度进行灰度值分析，以目的蛋白与内参Actin蛋白灰度比值代表目的蛋白表达水平。空白对照组设为100％；
结果如图3所示：由图3B可以看出中药30％和70％的乙醇提取液，无论是高浓度还是低浓度都可以显著抑制TLR4受体活化；由图3C可以看出中药30％和70％的乙醇提取液，高浓度可以显著抑制p65-NFκB的活化；由图3D和E可以看出，青鹏中药30％和70％的乙醇提取液，无论是高浓度还是低浓度都可以显著抑制炎症因子IL-1β和TNF-α的表达。
以上结果表明中药30％(200ug/ml)和70％(50ug/ml)乙醇提取液，可以通过抑制TLR4/p65-NFκB通过活化，进而抑制炎症因子IL-1β和TNF-α的表达。
实施例2
一种具有抗炎消肿和治疗关节炎作用的中药喷雾剂的制备方法，所述方法包括如下步骤：
以55.1份重量计，1份以1克(g)计；包括：藏菖蒲5g，人工麝香0.1g，川木香5g，草乌5g，诃子肉5g，黄葵子5g，决明子5g，乳香5g，冰片2.5g，大黄5g，草红花2.5g，骨碎补5g，宽筋藤5g；
具体步骤如下：
(1)取藏菖蒲，人工麝香，川木香，草乌，诃子肉，黄葵子，决明子，乳香，冰片，大黄，草红花，骨碎补，宽筋藤粉末分别溶于551mL的30％、50％和70％的乙醇中，浸泡5日后，过滤，静置24小时，过滤，即得中药提取液；
(2)取中药溶液质量分数为0.1％的乙二胺四乙酸二钠(EDTA-2Na)，质量分数为2％的柠檬酸、质量分数为2％的十二烷基硫酸钠和质量分数为1％月桂氮酮；分别加入步骤(2)制备的不同浓度乙醇的中药溶液中，静置24小时，过滤，即得中药喷雾剂溶液；将中药喷雾剂溶液装入50mL喷雾剂瓶中即得中药喷雾剂。
中药喷雾剂性能测定
1、根据2020版药典第四部制剂通则中喷雾剂制备原则要求，本发明制备的中药喷雾剂澄清透明，无沉淀析出；每喷喷量和每喷主药含量如表2所示，结果表明中药喷雾剂符合质量标准。
表2中药喷雾剂每喷量和每喷含药量
2、中药喷雾剂治疗小鼠耳廓肿胀
将实施例1和实施例2制备的不同浓度乙醇中药提取液和中药喷雾剂；中药30％乙醇提取液和30％乙醇提取液制备的中药喷雾剂；中药50％乙醇提取液和50％乙醇提取液制备的中药喷雾剂；中药70％乙醇提取液和70％乙醇提取液制备的中药喷雾剂，应用于二甲苯诱导的小鼠耳廓肿胀模型。
每组3只小鼠，左侧耳廓给药治疗，右侧耳廓作模型对照，给药量为0.48ml，给药1小时后，检测抑制率。
结果如表3，从表3数据可知，30％乙醇提取液制备的中药喷雾剂以及50％乙醇提取液制备的中药喷雾剂对小鼠耳廓肿胀具有显著的抑制效果。
表3.中药喷雾剂对小鼠耳廓肿胀模型抑制率
3、中药喷雾剂治疗大鼠关节炎
将实施例2中制备的中药30％乙醇提取液喷雾剂应用于大鼠关节炎的治疗。
取16只大鼠，分为4组：分别为正常对照组，关节炎模型组，关节炎+中药喷雾剂组，关节炎+市售青鹏软膏组；
大鼠CIA关节炎模型构建：将牛Ⅱ型胶原(2mg/mL)与不完全弗氏佐剂等体积混合，置于冰上用注射器充分乳化，乳液以滴入水面不扩散为乳化终点。第1天，进行初次免疫，在每只需造模大鼠的尾根部1cm处及足底处多点注射共200μL的Ⅱ型胶原乳剂，第8天，再次注射同等体积的胶原乳剂进行加强免疫。加强免疫7天后，观察大鼠四肢脚爪及关节状况，测量足爪厚度。加强免疫7天后按照上述方式开始给药，每次给药0.48mL，给药30天后利用Micro-CT检测大鼠足爪、膝关节股骨密度。
结果如图4和表4，由图4可以看出，模型组大鼠脚踝表明出现明显的骨质缺损和骨畸形，而中药喷雾剂组显著改善了骨缺损和骨畸形的问题。并且由拍摄图片可以看出，中药喷雾剂组和青鹏软膏组显著改善了大鼠踝关节的肿胀程度。
由表4数据可以得出，30％乙醇提取液制备的中药喷雾剂组，对关节炎大鼠股骨密度和骨体积分数具有显著改善，并且对关节炎模型大鼠脚踝肿胀具有显著的抑制作用，且效果显著优于市售青鹏软膏。
表4.中药喷雾剂对关节炎大鼠骨密度、骨体积分数和脚踝肿胀程度的影响
*与模型组相比p<0.05；**与模型组相比p<0.01。
Claims (7)
1.一种具有抗炎消肿和治疗关节炎作用的中药喷雾剂，其特征在于，所述中药喷雾剂的原料药由以下重量份组成：藏菖蒲4~6份，人工麝香0.05~0.2份，川木香4~8份，草乌3~7份，诃子肉3~8份，黄葵子4~6份，决明子5~8份，乳香4~5份，冰片2~3份，大黄3~7份，草红花2~3份，骨碎补4~7份，宽筋藤4~6份；所述中药喷雾剂还包括提取溶剂和辅料；所述提取溶剂为30%的乙醇水溶液；所述辅料包括络合剂、抗氧化剂、表面活性剂和透皮剂。
2.根据权利要求1所述的中药喷雾剂，其特征在于，所述中药喷雾剂的原料药由以下重量份组成：藏菖蒲5~6份，人工麝香0.05~0.1份，川木香4~5份，草乌5~7份，诃子肉5~8份，黄葵子4~5份，决明子5~8份，乳香4~5份，冰片2.5~3份，大黄4~5份，草红花2.5~3份，骨碎补5~7份，宽筋藤4~5份。
3.根据权利要求1所述的中药喷雾剂，其特征在于，所述中药喷雾剂的原料药由以下重量份组成：藏菖蒲5份，人工麝香0.1份，川木香5份，草乌5份，诃子肉5份，黄葵子5份，决明子5份，乳香5份，冰片2.5份，大黄5份，草红花2.5份，骨碎补5份，宽筋藤5份。
4.一种由权利要求1~3任一所述的中药喷雾剂的制备方法，其特征在于，所述方法包括如下步骤：
（1）取原料药粉碎置于30%的乙醇水溶液中，浸泡，过滤，静置，再过滤，得中药提取液；
（2）在步骤（1）制备的中药提取液中加入络合剂、抗氧化剂、表面活性剂和透皮剂，静置，过滤，即得中药喷雾剂溶液，装入喷雾剂瓶中即得中药喷雾剂。
5.根据权利要求4所述的制备方法，其特征在于，步骤（1）所述原料药与乙醇水溶液的料液比为1:10。
6.根据权利要求4所述的制备方法，其特征在于，步骤（2）所述络合剂的使用量为中药提取液的0.1~0.2wt%；所述抗氧化剂的使用量为中药提取液的2~5wt%；所述表面活性剂的使用量为中药提取液的2~5wt%；所述透皮剂的使用量为中药提取液的1~3wt%。
7.权利要求1~3任一所述的中药喷雾剂在制备用于治疗类风湿性关节炎或急性炎症导致的红肿热痛的药物中的应用。
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