CN113304177A - 一种破壁灵芝孢子粉提取物及其崩解片剂的制备方法与应用 - Google Patents
一种破壁灵芝孢子粉提取物及其崩解片剂的制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种破壁灵芝孢子粉提取物及其崩解片剂的制备方法与应用，通过对破壁灵芝孢子粉进行筛选，经过萃取、过滤去除孢子粉外壁等杂质及有害物质。低温萃取所留下的有益天然化合物，即灵芝多糖、灵芝三萜以及微量元素成分，制备得到破壁灵芝孢子粉提取物，然后基于破壁灵芝孢子粉提取物制备崩解片剂。本发明制备的破壁灵芝孢子粉提取物对人体细胞具有增强免疫的调控作用，对于肿瘤的生长以及转移有着一定的抑制作用，从而使免疫细胞能够增强对于肿瘤的杀伤效果，改善患者预后。
Description
技术领域
本发明涉及生物医药领域，尤其涉及一种有益于生物免疫力的天然多糖类化合物的制取方法及其在动物及人体内的免疫调节作用，具体为一种破壁灵芝孢子粉提取物及其崩解片剂的制备方法与应用。
背景技术
随着人体细胞的老化，细胞的正常功能会受到影响，正常功能衰退并且导致了细胞癌变。近年来，世界各国的医学专家都在试图寻找研发延长癌症病人生存期的药物。在手术、放化疗的基础上，如何延长接受治疗后的癌症病人生存是摆在医学专家面前的难题。目前临床上认为免疫治疗对于消灭残癌，减少肿瘤复发，改善机体的免疫功能状态具有比较好的发展前途。它治疗肿瘤的基本原则主要包括提高机体的免疫功能，用单克隆抗体等免疫手段，结合药物或者毒素进行治疗，调节机体的免疫状态使其恢复正常。而免疫治疗中的非特异性免疫治疗具有比较好的发展前景。手术、放化疗、免疫治疗均存在对于年老，体弱病人中副作用较多的困难，而使用免疫增强剂作为补充治疗手段能够明显减少手术后的恢复时间、放化疗的不良反应以及副作用；增强免疫治疗的疗效以及减少不良免疫反应。从以往被动的治疗，转变为多种方法联合，积极主动进行治疗，能够起到更好的治疗效果。
为了避免肝脏的首过效应和有效药物快速发挥作用，一些水溶性药物可以考虑采用舌下给药或者含服途径给药。现行中国药典对于舌下崩解片的规定：“主药和辅料应易溶于水”，“崩解片按照崩解测定法应5分钟内溶化。”本发明考虑到崩解用辅料存在吸水膨胀，使其短时间内难以溶解于水的情况，采用水溶性辅料等工艺，使本发明制备的崩解片剂在舌下崩解时不会溶化过快，能够保证崩解片的缓慢溶出系指药物灵芝多糖能够在10-20分钟内匀速溶出。
综上所述，本发明的目的是提供一种合适的水溶性多糖药物制备工艺，符合现行中国药典的相关规定，解决大生产工艺中存在的问题。使灵芝多糖能够在舌下含服的10-20分钟内缓慢崩解，使药物缓释降低其副作用、提高其生物利用度，更加适合肿瘤体弱病人。
发明内容
本发明目的在于针对现有技术的不足，提出一种破壁灵芝孢子粉提取物及其崩解片剂的制备方法与应用，破壁灵芝孢子粉提取物对人体细胞具有免疫调控作用，增强人体细胞免疫力、减轻免疫治疗副作用。
本发明的目的是通过以下技术方案来实现的：一种破壁灵芝孢子粉提取物的制备方法，该方法包括以下步骤：
(1)首先将破壁灵芝孢子粉进行筛选并且去除其中的杂质。称取除杂后的孢子粉后在4℃环境下加入超纯水，1g孢子粉加入10ml超纯水；
(2)将孢子粉与超纯水混合所得的样本进行充分震荡涡旋制成悬液。在4℃下以超声萃取装置在50MHz功率，启动1.5s，间歇1.5s模式下进行超声萃取30分钟。期间萃取反应釜以冰水浴进行冷却维持4℃环境。
(3)萃取循环进行一次后，进行10000×g离心，获得滤液以及沉淀，收集上层清液作为粗提取母液。沉淀称量重量后加入10倍质量的超纯水，以步骤(2)的方式进行第二次萃取循环之后进行10000×g离心，合并获得的滤液得到总粗提取母液。总粗提取母液过70目筛子除去其中悬浮杂质后进行-80℃真空冻干。获得的冻干粉即为灵芝孢子粉水提取物粉剂。
进一步地，所述灵芝孢子粉水提取物粉剂以吸光度及HPLC色谱法测定水提取物中的灵芝多糖含量并进行安全监测后，在GMP标准下水剂或冰冻关封，制成口服液或冻干粉。
进一步地，所述灵芝孢子粉提取物中，1g提取物中含有0.75g的天然灵芝多糖、0.24g的天然灵芝三萜和0.01g的天然微量元素。
进一步地，所述崩解片剂每片总计100.605mg，其组分如下：破壁灵芝孢子粉提取物15mg，山梨醇40mg，乳糖20mg，甘露醇20mg，柠檬酸1mg，三氯蔗糖0.07mg，亮蓝0.015mg，乙二胺四乙酸二钠0.02mg，
按照上述成分，先将水溶性多糖与柠檬酸混合，然后，按照等量递加原则与山梨醇进行混合，混合后的粉末入搅拌机制粒混匀，再加入乳糖和甘露醇组分，搅拌混合均匀。
将乙二胺四乙酸二钠、三氯蔗糖溶解于质量分数为70％的乙醇水溶液，加入亮蓝，进行粘合。搅拌5分钟后制成软材，过24目筛子制粒。烘干后，干颗粒20目筛整粒。以厚度7毫米、硬度50N进行冲压浅凹片。
制粒在GMP标准下冰冻关封。制成崩解片，成品检验合格后，印字包装。
本发明还提供了一种破壁灵芝孢子粉提取物的崩解片剂在调控人体细胞增强免疫以及抑制肿瘤的生长和转移方面的应用。
本发明的有益效果：本发明通过对破壁灵芝孢子粉进行筛选，经过萃取、过滤去除孢子粉外壁等杂质及有害物质。低温萃取所留下的有益天然化合物，即灵芝多糖、灵芝三萜以及微量元素成分。这些有益的化合物以及微量元素对人体细胞具有增强免疫的调控作用，对于肿瘤的生长以及转移有着一定的抑制作用。将其与传统化疗药物(顺铂、阿糖胞苷、环磷酰胺)、免疫疗法制剂(durvalumab、atezolimumab等)联用可以显著减少化疗药物的副作用，具有协同作用，使化疗以及免疫治疗的效果更佳明显，具有更好的促使机体激活增强免疫功能，减少肿瘤细胞的免疫逃逸，并且使已经因为肿瘤发生而耗竭的免疫细胞再生，再恢复，从而使免疫细胞能够增强对于肿瘤的杀伤效果，改善患者预后。
本发明所述的灵芝孢子粉提取物，对于非小细胞肺癌、骨肉瘤、黑色素瘤等有效，能促使全身以及免疫系统情况改善。经过动物安全性试验、急性、亚急性毒性试验阴性；异常毒性、长期毒性试验阴性。在小鼠以及大鼠体内能够增加效应T淋巴细胞、减少抑制性的调节T细胞，改善荷瘤小鼠脾指数、体重减轻以及毛发脱落等情况。能够明显增加荷瘤小鼠的活动能力、进食能力。
附图说明
图1为灵芝多糖崩解片主要成分中灵芝多糖的结构式示意图；
图2为本发明实施例2中灵芝孢子粉提取物的抗肿瘤作用示意图；
图3为灵芝孢子粉崩解片基于PD-L1阻断的免疫调节效应示意图。
具体实施方式
以下结合附图对本发明具体实施方式作进一步详细说明。
实施例1：将已经加工过的破壁灵芝孢子粉进行筛选并且去除其中的杂质。称取除杂后的孢子粉后在4℃环境下加入10倍质量的超纯水(1g孢子粉加入10ml超纯水)进行低温萃取。将孢子粉与超纯水混合所得的样本进行充分震荡涡旋制成悬液。在4℃低温下以超声萃取装置在50MHz功率，启动1.5s，间歇1.5s模式下进行超声萃取30分钟。期间萃取反应釜以冰水浴进行冷却维持4℃低温环境。
萃取循环进行一次后，进行10000×g高速离心，获得滤液以及沉淀，收集上层清液作为粗提取母液。沉淀称量重量后加入10倍的超纯水按照上述超声萃取条件进行第二次低温萃取循环。合并获得的滤液得到总粗提取母液。弃去离心获得的残渣。总粗提取母液过70目筛子除去其中悬浮杂质后进行-80℃低温真空冻干。
如图1所示，获得的冻干粉即为灵芝孢子粉水提取物粉剂。所述灵芝孢子粉水提取物粉剂，其组分包括灵芝多糖、灵芝三萜、微量元素等。以吸光度及HPLC色谱法测定水提取物中的灵芝多糖含量并进行安全监测后，在GMP标准下水剂或冰冻关封，制成口服液或冻干粉。所述灵芝孢子粉提取物中，1g提取物中含有0.75g的天然灵芝多糖、0.24g的天然灵芝三萜和0.01g的天然微量元素。
本发明还提供了一种破壁灵芝孢子粉提取物的崩解片剂的制备方法，本发明崩解片剂中的生理活性药物是上述提取的水溶性灵芝多糖，来自破壁灵芝孢子粉。本发明所述的崩解片配方中山梨醇占比例约为60％。山梨醇因为其易吸水性特点，需要以适宜比例进行加入，避免溶解速度过慢或者粘冲的问题。
其他辅料为：矫味剂(三氯蔗糖、青苹果粉末香精或柠檬酸)，着色剂(亮蓝、果绿、胭脂红、葡萄紫)，粘合剂(聚维酮水溶液、淀粉浆、羟丙基甲基纤维素溶液)，润滑剂(硬脂酸镁、滑石粉)，稳定剂(维生素C、乙二胺四乙酸二钠)。
在上述配方下，冲粘问题得到解决，并且溶解时间在要求范围内。甜味剂能掩盖苦味改善口感，对于老年体弱癌症病人时常存在的口苦问题能够有效的应对。抗氧化剂和络合剂的组分能够有效的起到抗氧化作用，保护灵芝多糖等有效成分。
所述崩解片剂每片总计100.605mg，其组分如下：破壁灵芝孢子粉提取物15mg，山梨醇40mg，乳糖20mg，甘露醇20mg，柠檬酸1mg，三氯蔗糖0.07mg，亮蓝0.015mg，已二酸四乙胺二钠0.02mg，维生素C1mg，硬脂酸镁0.5mg，滑石粉3mg。
按照上述成分，先将水溶性多糖与柠檬酸混合，然后，按照等量递加原则与山梨醇进行混合，混合后的粉末入搅拌机制粒混匀，再加入乳糖和甘露醇组分，搅拌混合均匀。
将乙二胺四乙酸二钠、三氯蔗糖溶解于70％醇水溶液，加入亮蓝，进行粘合。搅拌5分钟后制成软材，过24目筛子制粒。烘干后，干颗粒20目筛整粒。以厚度7毫米、硬度50N进行冲压浅凹片。
制粒在GMP标准下冰冻关封。制成崩解片，成品检验合格后，印字包装。
产品检验：崩解仪在37度测定溶化试验，结果为：0分钟溶出度0％，10分钟溶出度80％溶出度，20分钟100％溶出度。
实施例2：如图2所示，将4×106个肿瘤细胞接种于裸鼠皮下，待成瘤后随即分为6组，每组8只。分别给予腹腔注射生理盐水(对照组)，顺铂30mg/kg，灵芝孢子粉提取物崩解片溶液3mg/kg，10mg/kg，30mg/kg，100mg/kg。进行2周处理后，取皮下肿瘤进行肿瘤体积大小的测量。可见对照组肿瘤体积最大；顺铂组肿瘤体积最小，灵芝孢子粉提取物组的肿瘤大小随着剂量的提升，肿瘤大小逐渐减小，可以证明其对于肿瘤的治疗作用。
实施例3：如图3所示，PD-L1作为肿瘤细胞表面代表免疫逃逸能力的重要标志物，降低PD-L1的表达水平有助于减少肿瘤细胞的免疫逃逸，提升机体的免疫系统对于肿瘤细胞的杀伤，对改善肿瘤患者的预后有很大帮助。在人骨肉瘤细胞系Human osteosarcoma细胞系，小鼠骨肉瘤K7M2细胞系的体外培养体系中，分别加入不同浓度的灵芝孢子粉崩解片谁溶液(1mg/ml，2mg/ml，3mg/ml，4mg/ml，5mg/ml)，进行24小时培养后，提取培养体系中的总蛋白，进行SDS-PAGE蛋白质电泳及免疫印迹检测试验。可见PD-L1随着灵芝孢子粉崩解片水溶液的浓度提升，其表达量明显下降。
上述实施例用来解释说明本发明，而不是对本发明进行限制，在本发明的精神和权利要求的保护范围内，对本发明作出的任何修改和改变，都归入本发明的保护范围。
Claims (5)
1.一种破壁灵芝孢子粉提取物的制备方法，其特征在于，该方法包括以下步骤：
(1)首先将破壁灵芝孢子粉进行筛选并且去除其中的杂质。称取除杂后的孢子粉后在4℃环境下加入超纯水，1g孢子粉加入10ml超纯水；
(2)将孢子粉与超纯水混合所得的样本进行充分震荡涡旋制成悬液。在4℃下以超声萃取装置在50MHz功率，启动1.5s，间歇1.5s模式下进行超声萃取30分钟。期间萃取反应釜以冰水浴进行冷却维持4℃环境。
(3)萃取循环进行一次后，进行10000×g离心，获得滤液以及沉淀，收集上层清液作为粗提取母液。沉淀称量重量后加入10倍质量的超纯水，以步骤(2)的方式进行第二次萃取循环之后进行10000×g离心，合并获得的滤液得到总粗提取母液。总粗提取母液过70目筛子除去其中悬浮杂质后进行-80℃真空冻干。获得的冻干粉即为灵芝孢子粉水提取物粉剂。
2.根据权利要求1所述的一种破壁灵芝孢子粉提取物的制备方法，其特征在于，所述灵芝孢子粉水提取物粉剂以吸光度及HPLC色谱法测定水提取物中的灵芝多糖含量并进行安全监测后，在GMP标准下水剂或冰冻关封，制成口服液或冻干粉。
3.根据权利要求1所述的一种破壁灵芝孢子粉提取物的制备方法，其特征在于，所述灵芝孢子粉提取物中，1g提取物中含有0.75g的天然灵芝多糖、0.24g的天然灵芝三萜和0.01g的天然微量元素。
4.一种依据权利要求1-3任一项制备的破壁灵芝孢子粉提取物的崩解片剂的制备方法，其特征在于，所述崩解片剂每片总计100.605mg，其组分如下：破壁灵芝孢子粉提取物15mg，山梨醇40mg，乳糖20mg，甘露醇20mg，柠檬酸1mg，三氯蔗糖0.07mg，亮蓝0.015mg，乙二胺四乙酸二钠0.02mg，
按照上述成分，先将水溶性多糖与柠檬酸混合，然后，按照等量递加原则与山梨醇进行混合，混合后的粉末入搅拌机制粒混匀，再加入乳糖和甘露醇组分，搅拌混合均匀。
将乙二胺四乙酸二钠、三氯蔗糖溶解于质量分数为70％的乙醇水溶液，加入亮蓝，进行粘合。搅拌5分钟后制成软材，过24目筛子制粒。烘干后，干颗粒20目筛整粒。以厚度7毫米、硬度50N进行冲压浅凹片。
制粒在GMP标准下冰冻关封。制成崩解片，成品检验合格后，印字包装。
5.一种基于权利要求4所述制备的破壁灵芝孢子粉提取物的崩解片剂在调控人体细胞增强免疫以及抑制肿瘤的生长和转移方面的应用。
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| CN114431316A (zh) * | 2022-02-16 | 2022-05-06 | 上海中医药大学 | 基于中药食材及蔬果可提高免疫力的茶饮及其制备方法 |
| CN114681497A (zh) * | 2022-04-25 | 2022-07-01 | 广州白云山汉方现代药业有限公司 | 一种灵芝孢子油对铂类药物增效的应用 |
| CN115778989A (zh) * | 2022-12-16 | 2023-03-14 | 福建仙芝楼生物科技有限公司 | 一种抑制肿瘤转移的灵芝提取物 |
| CN115778989B (zh) * | 2022-12-16 | 2023-12-15 | 福建仙芝楼生物科技有限公司 | 一种抑制肿瘤转移的灵芝提取物 |
| WO2024192715A1 (zh) * | 2023-03-22 | 2024-09-26 | 北京同仁堂国药有限公司 | 一种灵芝孢子粉在制备治疗黑色素瘤的药物中的应用 |
| CN116650542A (zh) * | 2023-06-20 | 2023-08-29 | 哈尔滨医科大学 | 黄芪多糖联合灵芝孢子粉在制备治疗骨肉瘤药物中的应用 |
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