CN115006511B - 一种清肺汤冲剂及其制备方法 - Google Patents
一种清肺汤冲剂及其制备方法 Download PDFInfo
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- CN115006511B CN115006511B CN202210804849.9A CN202210804849A CN115006511B CN 115006511 B CN115006511 B CN 115006511B CN 202210804849 A CN202210804849 A CN 202210804849A CN 115006511 B CN115006511 B CN 115006511B
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Abstract
本发明公开了一种清肺汤冲剂，原料包括：罗汉果、陈皮、生姜、枸杞、枇杷叶、百合、玄参、麦冬、芦根、菊花、甘草、薄荷、艾草、羊栖菜、螺旋藻、海参及辅料适量。本发明的清肺汤冲剂原料配伍合理科学，可接受度高，不仅具有止咳、化痰、润肺的功效，而且具有较强的抗炎、保护肺组织的生物活性，能有效修复肺组织损伤，非常适合因空气污染（雾霾、汽车尾气等）、感冒、支气管炎、肺炎等原因而引起咳嗽的人群饮用。本发明还公开了一种清肺汤冲剂制备方法，通过酶解、喷雾干燥即可，工艺步骤简单，条件温和，可操作性强，适合工业化生产。
Description
技术领域
本发明涉及一种清肺汤冲剂，尤其是涉及一种清肺汤冲剂及其制备方法。
背景技术
随着城市现代化的迅速发展，建筑施工、汽车尾汽、工业燃料燃烧等原因造成空气质量不断地下降，导致雾霾天气不断侵袭。而雾霾中含有的大量PM2.5会对人体呼吸系统造成影响，容易引起急性上呼吸道感染、肺炎等疾病。
目前为对抗雾霾一般都是从日常饮食入手，以期通过食用富含维生素和具有润肺功效的食物来抗毒排毒来消除PM2.5对人体的侵害。而通过日常饮食来对抗雾霾，实际效果存疑。
申请公布号CN107950961A，申请公布日2018年4月24日的中国专利公开了一种抗雾霾清肺汤的制备方法，该汤中的成分的重量比为：雪花梨200-400克；百合干20-50克；冰糖40-80克；荸荠80-150克；枸杞5-15克；红枣10-30克；银耳5-20克；水1.5L。该抗雾霾清肺汤所具有的功效是排毒、美容养颜及增强免疫力，并不能改善雾霾引发的肺组织损伤，实际抗雾霾效果较差。
发明内容
本发明是为了解决现有技术的雾霾清肺汤的制备方法所存在的上述问题，提供了一种原料配伍合理，可接受度高，不仅具有止咳、化痰、润肺的功效，而且具有较强的抗炎、保护肺组织的生物活性，能有效修复肺组织损伤的清肺汤冲剂。
本发明还提供了一种清肺汤冲剂，工艺步骤简单，条件温和，可操作性强，适合工业化生产。
为了实现上述目的，本发明采用以下技术方案：本发明的一种清肺汤冲剂，以重量计，其原料包括：20±1g罗汉果，20±1g陈皮，10±0.5g生姜，10±0.5g枸杞，20±1g枇杷叶，20±1g百合，15±0.75g玄参，20±1g麦冬，20±1g芦根，15±0.75g菊花，20±1g甘草，10±0.5g薄荷，10g±0.5艾草，35±1.75g羊栖菜，35±1.75g螺旋藻，54±2.7g海参，辅料适量。本发明清肺汤冲剂的原料均采用药食同源的原料，特增加了海洋植物因子(羊栖菜、螺旋藻)与海洋动物因子(海参)，以进一步提高食用效果。
作为优选，所述辅料包括填充剂和香精。
作为优选，所述填充剂为糊精，所述香精为薄荷香精。
一种清肺汤冲剂制备方法，包括以下步骤：
(1)按上述重量配比称取各原料，待用。
(2)将罗汉果、陈皮、生姜、枸杞、枇杷叶、百合、玄参、麦冬、芦根、菊花、甘草、薄荷及艾草混合后，加入纤维素酶、果胶酶和水，酶解得第一酶解液；将羊栖菜和螺旋藻混合后，加入纤维素酶、果胶酶、木瓜蛋白酶和水，酶解得第二酶解液；在海参中加入木瓜蛋白酶和水，酶解得第三酶解液。
(3)将第一酶解液、第二酶解液及第三酶解液混合后，过滤取滤液，加入辅料后喷雾干燥，即得清肺汤冲剂。
作为优选，步骤(1)中，所述辅料包括填充剂和香精。
作为优选，所述填充剂为糊精，所述香精为薄荷香精。
作为优选，步骤(2)中，第一酶解液的酶解条件为：料液比为(1～1.1)：10；以罗汉果、陈皮、生姜、枸杞、枇杷叶、百合、玄参、麦冬、芦根、菊花、甘草、薄荷及艾草生物的总质量为基准，纤维素酶的添加量为2～2.5％，果胶酶的添加量为1～1.5％，酶解pH为6～6.5，酶解温度为50～55℃，酶解时间为1.5～2h；第二酶解液的酶解条件为：料液比为(5～7)：100；木瓜蛋白酶的添加量为螺旋藻质量的1～1.5％，纤维素酶的添加量为羊栖菜质量的1～1.5％，果胶酶的添加量为羊栖菜质量的1.5～2％，酶解pH为5.5～6.0，酶解温度为50～55℃，酶解时间为2～3h；第一酶解液的酶解条件为：料液比为(1～1.5)：10；木瓜蛋白酶的添加量为海参质量的5～7％，酶解pH为6.0～7.0，酶解温度为50～60℃，酶解时间为24～30h。
作为优选，所述羊栖菜、螺旋藻酶解前进行脱腥处理，脱腥方法为：在羊栖菜、螺旋藻中加入适量酵母，37±2℃发酵。本发明中对羊栖菜及螺旋藻进行脱腥处理，以改善本发明冲剂的风味，提高可接受度。
作为优选，所述海参酶解前进行脱腥处理，脱腥方法为：在海参中加入适量酵母，37±2℃发酵。
因此，本发明具有如下有益效果：
(1)本发明的清肺汤冲剂原料配伍合理科学，可接受度高，不仅具有止咳、化痰、润肺的功效，而且具有较强的抗炎、保护肺组织的生物活性，能有效修复肺组织损伤，非常适合因空气污染(雾霾、汽车尾气等)、感冒、支气管炎、肺炎等原因而引起咳嗽的人群饮用；
(2)本发明的清肺汤冲剂制备方法工艺步骤简单，条件温和，可操作性强，适合工业化生产。
附图说明
图1是正常对照组、模型对照组、低剂量清肺汤冲剂组、高剂量清肺汤冲剂组中小鼠肺组织切片经海瑞氏苏木精-伊红(HE)染色后的显微结构图。
具体实施方式
下面通过具体实施方式对本发明做进一步的描述。
实施例1
(1)按罗汉果20g、陈皮20g、生姜10g、枸杞10g、枇杷叶20g、百合20g、玄参15g、麦冬20g、芦根20g、菊花15g、甘草20g、薄荷10g、艾草10g、羊栖菜35g、螺旋藻35g、海参54g的重量称取各原料。
(2)将罗汉果、陈皮、生姜、枸杞、枇杷叶、百合、玄参、麦冬、芦根、菊花、甘草、薄荷及艾草混合后，加水2L(料液比为1.05：10)，加入纤维素酶4.2g，加入果胶酶2.1g，在pH为6、温度为50℃的条件下酶解1.5h，灭酶，得第一酶解液；将羊栖菜与螺旋藻混合后加入适量酵母，37℃发酵2h，洗净后加水1L(料液比为7：100)，加入纤维素酶0.7g，加入果胶酶0.35g，加入木瓜蛋白酶0.35g，在pH为6、温度为50℃的条件下酶解2h，灭酶，得第二酶解液；在海参中加入2.5g酵母，37℃发酵4h，洗净后加水540mL(料液比为1：10)，加入木瓜蛋白酶0.35g，在pH为6.5、温度为55℃的条件下酶解5h，灭酶，得第三酶解液。
(3)将第一酶解液、第二酶解液及第三酶解液混合后，过滤取滤液，加入辅料(74g糊精、2.52mL薄荷香精)，喷雾干燥，即得清肺汤冲剂(182g)。
一、本发明得到的清肺汤冲剂的质量指标如下：
1.理化指标
铅(以Pb计)≤0.5mg/kg；砷(以As计)≤0.3mg/kg；铜(以Cu计)≤2.5mg/kg。
2.感官指标
棕黄色固体颗粒，均匀一致，无杂质，无结块，颗粒度≥85％(20-15目之间)，具有发酵特有的香味，略带腥味，舒适爽口，甜度适中；易溶于水，冲饮均匀，透明，无沉淀。
二、急性经口毒性实验
1.检测依据：
GB 15193.3-2014《食品安全国家标准急性经口毒性试验》。
2.检测环境：
屏障动物房，实验动物使用许可证号：SYXK(鲁)2021 0015，室温20℃～26℃；相对湿度40％～70％。
3.实验动物：
SPF级KM小鼠20只，雌雄各半(雌性动物未交配过、未妊娠)，体重为18～22g。质量合格证号：No.370726211100974553；由济南朋悦实验动物繁育有限公司提供，生产许可证号：SCXK(鲁)20190003。
动物饲养：饲料为鼠料，质量合格证号：No.120210927048；由江苏省协同医药生物工程有限责任公司提供，生产许可证号：苏饲证(2019)01008。
垫料为实验用玉米芯垫料，质量合格证号：No.120210802001；由江苏省协同医药生物工程有限责任公司提供，生产许可证号：苏饲证(2019)01008。
4.染毒途径：
灌胃给药。
5.样品制备：
称10.0165g样品加纯水配制成20mL样品溶液，混合均匀，标识备用(终浓度为500mg/mL)。
6.检测方法：
KM小鼠在本实验室屏障环境动物房中预饲养3天，以适应环境。试验前，KM小鼠禁食4h，自由饮水。
限量法，灌胃给予剂量10016mg/kg·bw，灌胃体积为20mL/kg·bw。给予受试物后继续禁食1h。给予受试物后，每天观察中毒症状或行为变化；在试验开始和结束时称取并记录KM小鼠体重，并且在观察期每周称取KM小鼠体重1次。
全面观察并记录KM小鼠变化发生时间、程度和持续时间。对中毒死亡KM小鼠和试验结束处死的KM小鼠进行大体解剖检查，出现大体解剖病理改变时做病理组织学观察。
7.检测结果：
KM小鼠在染毒14天内未见任何异常症状和死亡，试验观察结束后对受试动物进行大体解剖检查未见异常。该样品对KM小鼠的急性经口毒性LD50>10016mg/kg·bw。(详情见表1)。
8.检测结论：
本试验条件下，清肺汤冲剂原样对KM小鼠的急性经口毒性LD50>10016mg/kg·bw；根据不同动物之间的剂量换算关系，计算得小鼠剂量换算成大鼠剂量为6934mg/kg·bw；换算后清肺汤冲剂原样对大鼠的急性经口毒性LD50>6934mg/kg·bw。根据GB 15193.3-2014《食品安全国家标准急性经口毒性试验》的要求，该样品急性经口毒性试验属实际无毒。
表1急性经口毒性试验结果
三、生物活性评价实验
1.实验地点：
浙江海洋大学国家工程中心
2.实验动物：
雄性ICR小白鼠，体重18～20g
3.实验模型建立及动物实验：
ICR小鼠共70只，随机分为7组，每组10只。其中60只小鼠经20mg/kg的PM2.5吸入性造模(将PM2.5颗粒溶于无菌生理盐水中混合打均，配制成浓度为0.02mg/μL的PM2.5悬液，应用移液枪对小鼠进行悬液滴鼻操作，每只小鼠悬液滴鼻容量为20μL)，处理为1h；另外10只小鼠用同剂量的生理盐水进行吸入性操作，时间为1h。生理盐水处理小鼠作为正常对照组，PM2.5吸入性小鼠分为6组，分别为模型对照组、低剂量清肺汤冲剂组、高剂量清肺汤冲剂组、无海洋植物功效因子对照组、无海洋动物功效因子对照组及无海洋功效因子对照组。在小鼠造模当天，低剂量清肺汤冲剂组小鼠灌胃清肺汤冲剂25mg/kg，高剂量清肺汤冲剂组、无海洋植物功效因子对照组、无海洋动物功效因子对照组及无海洋功效因子对照组小鼠灌胃清肺汤冲剂100mg/kg，正常对照组和模型对照组灌胃相等量的生理盐水。小鼠均正常饮食饮水。动物实验周期为2周。实验结束后，禁食不禁水10h，取小鼠血液，离心分离获得血清，检测其炎症分子。取小鼠肺组织，进行组织形态学观察其病变，并用荧光定量PCR分析其炎症。
4.小鼠血清炎症因子检测：
小鼠禁食不禁水10h，摘眼球取血，7500×g离心15min，分离小鼠血清，用ELISA试剂盒检测小鼠血清中炎症因子的浓度。
5.小鼠肺组织HE染色：
取小鼠肺组织，10％的中性甲醛溶液中固定，经梯度乙醇脱水、二甲苯透明、石蜡包埋，以5μm厚度连续切片，切片经海瑞氏苏木精-伊红(HE)染色，二甲苯透明后用中性树胶封片，于光学显微镜下观察肺组织的显微结构变化，拍照。
6.荧光定量PCR分析：
取小鼠肺组织0.1g，加入800mL TRIzol，低速匀浆破碎细胞，提取总RNA。将RNA溶于0.3mL DEPC水，取适量稀释，于紫外可见分光光度计测定吸光度值A280和A260，并计算溶解后RNA的纯度和含量。取1μg总RNA在M-MLV逆转录酶的催化作用下逆转录成cDNA。cDNA于25mL的反应体系中扩增，各反应物用量参照Maxima SYBR Green qRT-PCR Master mix说明书要求，反应体系包括：cDNA模版6μL(稀释10倍)，Maxima SYBR Green qRT-PCR Mastermix 12.5μL，上、下游引物各0.3μL，超纯水5.9μL。反应条件为：95℃预变性10min，95℃变性15s，60℃退火20s，72℃延伸30s，共45个循环。扩增结束后进行熔解曲线分析，确保目的基因产物的专一性。目的基因mRNA表达量均以β-actin mRNA的量作为内参校正，将正常对照control组定为1个单位。各目的基因引物序列使用Primer Premier 5.0设计，如表2所示，由上海生工生物工程有限公司合成。
表1荧光定量PCR中引用表
7.实验结果：
如表3所示，与正常对照组比较，模型对照组中小鼠血清炎症因子水平均显著升高(P<0.01)。经清肺汤冲剂灌胃后，与模型对照组比较，高剂量组小鼠血清TNF-α、IL-1β、IL-6、IL-8、MMP-9、NO和COX-2的含量均显著降低(P<0.05，P<0.01)；低剂量组小鼠血清IL-6、IL-8和MMP-9均显著降低(P<0.05，P<0.01)，而TNF-α、IL-1β、NO和COX-2含量下降不显著。表明，清肺汤冲剂可以显著抑制PM2.5小鼠身体炎症反应。
表3小鼠血清炎症因子含量
注：##P<0.01vs正常对照组；*P<0.05，**P<0.01vs模型对照组；$P<0.05vs 100mg/kg清肺汤冲剂组。
如图1所示，正常对照组小鼠肺组织中，肺泡结构较完整，泡腔无明显水肿及炎性浸润现象，大小正常。模型对照组小鼠肺织织中，有可见的粘膜损伤性脱落，泡壁和泡膈结构破损显著，淋巴细胞增加、中性粒细胞浸润、肺泡间隔明显增宽。低剂量组和高剂量组小鼠肺组织中，炎性损伤程度较模型对照组减轻，泡壁和泡膈结构也得到一定的修复。表明，清肺汤冲剂可以有效地预防PM2.5对小鼠肺组织的损伤。
如表4所示，与正常对照组比较，模型对照组中小鼠肺组织炎症因子基因mRNA相对表达量均显著升高(P<0.01)。经清肺汤冲剂灌胃后，与模型对照组比较，高剂量组小鼠肺组织TNF-α、IL-1β、IL-6、IL-8、MMP-9、iNOS和COX-2的基因mRNA相对表达量均显著降低(P<0.05,P<0.01)；低剂量组小鼠血清IL-6和IL-8均显著降低(P<0.05)，而TNF-α、IL-1β、MMP-9、NO和COX-2含量下降不显著。表明，清肺汤冲剂可以显著抑制PM2.5对小鼠肺组织损伤引发的肺部炎症反应。
表4小鼠肺组织炎症因子基因mRNA相对表达量
注：##P<0.01vs正常对照组；*P<0.05，**P<0.01vs模型对照组；$P<0.05vs 100mg/kg清肺汤冲剂组。
8.实验结论：
本发明的清肺汤冲剂能有效地改善PM2.5引发的小鼠肺组织损伤，抑制肺部炎症因子基因的表达，从而抑制了小鼠机体炎症反应，具有较强的抗炎、清肺、保护肺组织的生物活性。
以上所述的实施例只是本发明的一种较佳的方案，并非对本发明作任何形式上的限制，在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。
Claims (6)
1.一种清肺汤冲剂，其特征在于，以重量计，其由以下原料制成：20±1g罗汉果，20±1g陈皮，10±0.5g生姜，10±0.5g枸杞，20±1g枇杷叶，20±1g百合，15±0.75g玄参，20±1g麦冬，20±1g芦根，15±0.75g菊花，20±1g甘草，10±0.5g薄荷，10±0.5g艾草，35±1.75g羊栖菜，35±1.75g螺旋藻，54±2.7g海参，辅料适量；所述清肺汤冲剂通过以下制备方法制得：
（1）按上述重量配比称取各原料，待用；
（2）将罗汉果、陈皮、生姜、枸杞、枇杷叶、百合、玄参、麦冬、芦根、菊花、甘草、薄荷及艾草混合后，加入纤维素酶、果胶酶和水，酶解得第一酶解液；将羊栖菜和螺旋藻混合后，加入纤维素酶、果胶酶、木瓜蛋白酶和水，酶解得第二酶解液；在海参中加入木瓜蛋白酶和水，酶解得第三酶解液；
（3）将第一酶解液、第二酶解液及第三酶解液混合后，过滤取滤液，加入辅料后喷雾干燥，即得清肺汤冲剂。
2.根据权利要求1所述的一种清肺汤冲剂，其特征在于，所述辅料包括填充剂和香精。
3.根据权利要求2所述的一种清肺汤冲剂，其特征在于，所述填充剂为糊精，所述香精为薄荷香精。
4.根据权利要求1所述的一种清肺汤冲剂，其特征在于，步骤（2）中，第一酶解液的酶解条件为：料液比为（1~1.1）：10；以罗汉果、陈皮、生姜、枸杞、枇杷叶、百合、玄参、麦冬、芦根、菊花、甘草、薄荷及艾草生物的总质量为基准，纤维素酶的添加量为2~2.5%，果胶酶的添加量为1~1.5%，酶解pH为6~6.5，酶解温度为50~55℃，酶解时间为1.5~2h；
第二酶解液的酶解条件为：料液比为（5~7）：100；木瓜蛋白酶的添加量为螺旋藻质量的1~1.5%，纤维素酶的添加量为羊栖菜质量的1~1.5%，果胶酶的添加量为羊栖菜质量的1.5~2%，酶解pH为5.5~6.0，酶解温度为50~55℃，酶解时间为2~3h；
第三酶解液的酶解条件为：料液比为（1~1.5）：10；木瓜蛋白酶的添加量为海参质量的5~7%，酶解pH为6.0~7.0，酶解温度为50~60℃，酶解时间为24~30h。
5.根据权利要求4所述的一种清肺汤冲剂，其特征在于，所述羊栖菜、螺旋藻酶解前进行脱腥处理，脱腥方法为：在羊栖菜、螺旋藻中加入适量酵母，37±2℃发酵。
6.根据权利要求4所述的一种清肺汤冲剂，其特征在于，所述海参酶解前进行脱腥处理，脱腥方法为：在海参中加入适量酵母，37±2℃发酵。
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