CN114766672A - 一种小叶苦丁茶老叶片提取物及其在制备缓解酒精诱导肝损伤食品方面的应用 - Google Patents
一种小叶苦丁茶老叶片提取物及其在制备缓解酒精诱导肝损伤食品方面的应用 Download PDFInfo
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- Medicines Containing Plant Substances (AREA)
Abstract
本发明提供了一种小叶苦丁茶老叶片提取物及其在制备缓解酒精诱导肝损伤食品方面的应用，涉及食品加工技术领域。所述小叶苦丁茶老叶片提取物的制备工艺包括原料预处理、破碎、提取、浓缩、干燥、粉碎等步骤。本发明制备的小叶苦丁茶老叶片提取物中富含毛蕊花糖苷、金石蚕苷、甘露醇、野漆树苷、连翘酯苷E、右旋奎宁酸、茵芋苷等54种有效成分，可用于制备具有缓解酒精诱导的肝损伤的功能性食品。
Description
技术领域
本发明涉及食品加工技术领域，尤其涉及一种小叶苦丁茶老叶片提取物及其在制备缓解酒精诱导肝损伤食品方面的应用。
背景技术
小叶苦丁茶是木樨科女贞属小叶女贞的鲜叶加工而成的茶，俗称茶丁、富丁茶、皋卢茶，是我国一种传统的纯天然保健饮料佳品。其成品茶清香有苦味、而后甘凉，具有清热消暑、明目益智、生津止渴、利尿强心、润喉止咳、降压减肥、抑癌防癌、抗衰老、活血脉等多种功效。从中医的角度讲，苦丁茶具有散风热、清头目、除烦渴的作用，用来治疗头痛、牙痛、目赤、热病烦渴、痢疾等药用效果非常明显。
发明内容
本发明公开了一种通过破碎、提取、浓缩、干燥、粉碎的小叶苦丁茶老叶片提取物，通过HPLC测定提取物中的含量，分析得出其中含有毛蕊花糖苷、金石蚕苷、甘露醇、野漆树苷、连翘酯苷E、右旋奎宁酸、茵芋苷、芹菜素-7-O-β-D-吡喃葡萄糖苷、橄榄苦苷、木犀草苷、芦丁、红景天苷、腺嘌呤、柠檬酸、5-羟基马鞭草苷、紫地黄苷C、忍冬苷、对羟基肉桂酸、槲皮素-3-葡萄糖醛酸苷、香豆素、维采宁II、金丝桃苷、L-亮氨酸、桑黄素、L-苯丙氨酸、罗汉果黄素、木通苯乙醇苷B、苍术苷A、对甲氧基苯甲醛、L-酪氨酸、芹菜素、羟基酪醇、山柰酚-3-O-芸香糖苷、大车前苷、白果内酯、鸟嘌呤、2-金刚烷酮、三七素、原儿茶醛、木犀草素、原儿茶酸、樟脑、隐绿原酸、莽草酸、咖啡酸、1-咖啡酰奎宁酸、马鞭草苷、连翘苷、马钱子苷、6-姜酚、丹参素、香兰素、乙酰哈巴苷等54种有效成分。所述小叶苦丁茶老叶片提取物的制备工艺包括以下步骤：
(1)原料预处理：取新鲜、叶片完整的小叶苦丁茶进行经蒸汽杀青、烘干处理，得到小叶苦丁茶老叶片；
(2)破碎：用粉碎机对小叶苦丁茶老叶片进行粉碎处理并过10目筛；
(3)提取：称取粉碎过的原材料放入过滤袋中，加水入蒸馏水混匀，先在常温下浸泡，然后将原料加热并保温，停止加热后对原料进行超声处理，处理完成后抽滤，将滤渣重复提取2～3次，合并滤液；
(4)浓缩：将滤液进行旋蒸浓缩至黏稠状取出；
(5)干燥：将浓缩液预冻后进行冷冻干燥，得到固体浓缩提取物；
(6)粉碎：将固体浓缩提取物用粉碎并过300目筛，得到小叶苦丁茶老叶片提取物。
进一步地，所述步骤(3)中茶叶与水的料液比为1g：10～20mL。
进一步地，所述步骤(3)中常温浸泡时间为3～5h。
进一步地，所述步骤(3)中原料加热温度为90～95℃，保温时间为1～2h，且在保温过程中，每保温15min搅拌5min。
进一步地，所述步骤(3)中超声处理频率为30～40KHZ，超声处理时间为10～20min。
进一步地，所述步骤(4)中浓缩温度为50～70℃。
进一步地，所述步骤(5)中固体浓缩提取物的水分含量＜6％。
本发明还提供了一种根据上述方法制备而成的小叶苦丁茶老叶片提取物。
本发明的另一个目的在于提供所述小叶苦丁茶老叶片提取物在制备缓解酒精诱导的肝损伤食品方面的应用。
本发明的小叶苦丁茶老叶片提取物主要成分包括毛蕊花糖苷、金石蚕苷、甘露醇、野漆树苷等，各组分具体功效如下：
金石蚕苷：散瘀止血，消肿止痛，用于吐血，咯血，衄血，便血；外用治疗外伤出血，抗菌消炎抗病毒作用；毛蕊花糖苷：具有消炎、增强免疫、抗缺氧、抗癌的作用，用于含量测定/鉴定/药理实验等，具有调节免疫、抗氧化、增强体力、抗疲劳、等药理作用；野漆树苷：抗肿瘤；抗高血压；黄嘌呤氧化酶抑制剂；抗氧化剂；甘露醇：在医药上是良好的利尿剂，降低颅内压、眼内压及治疗肾药、脱水药、食糖代用品、也用作药片的赋形剂及固体、液体的稀释剂。
与现有技术相比，本发明的有益技术效果：
本发明所述小叶苦丁茶老叶片提取物中富含毛蕊花糖苷、金石蚕苷、甘露醇、异麦角甾苷、野漆树苷等功效成分，可溶于水直接饮用，或与其他成分制成功能性饮料饮用，长期饮用可缓解长期饮酒造成的酒精肝损伤，此外该产品具有抗菌消炎、增强免疫、抗氧化、增强体力、抗疲劳及利尿等效果。同时，本发明材料来源广泛，成本低廉，制备工艺简单，具有良好的应用前景。
附图说明
下面结合附图说明对本发明作进一步说明。
图1为给药4周时对酒精肝损伤小鼠肝脏的影响；
图2为给药4周后血清中ALT和AST的含量变化；
图3为给药4周后血清中TG的含量变化。
具体实施方式
以下结合实施例对本发明提供的技术方案进行进一步说明。
实施例1
一种小叶苦丁茶老叶片提取物的制备工艺：
(1)原料预处理：取新鲜、叶片完整的小叶苦丁茶进行经蒸汽杀青、烘干处理，得到小叶苦丁茶老叶片；
(2)破碎：用粉碎机对小叶苦丁茶老叶片进行粉碎处理并过10目筛；
(3)提取：称取粉碎过的原材料放入过滤袋中，按料液比为1g：20mL比例水入蒸馏水混匀，在常温下浸泡5h，然后将原料加热至95℃并保温2h，每加热15min，搅拌5min，停止加热后40KHZ超声15min，倒出提取液，抽滤，滤渣重复提取3次，合并滤液；
(4)浓缩：提取液用50℃水温水浴加热，旋转蒸馏仪浓缩至黏稠状倒出，倒出；
(5)干燥：将浓缩液置于-5℃温度保存，待冻凝固后，进行冷冻干燥，得到固体浓缩提取物；
(6)粉碎：将固体浓缩提取物用粉碎并过300目筛，得到小叶苦丁茶老叶片提取物。
测试例1
(1)实验材料：4-6周龄SPF级雄性KM小鼠46只，体重20～25g。实验试剂有红星二锅头(购于贵阳市白云区绿地新都会德旺佳超市)；联苯双酯滴丸；4％多聚甲醛。
(2)实验方法：
酒精肝损伤模型制作
60只小鼠随机分为两组，正常组10只，造模组50只。按每周所测得的体重正常组每日灌胃无菌水1次；造模组先灌无菌水3天，以适应刺激，后灌56度红星二锅头3周：第一周以0.005ml/g每日灌胃1次，第二周以0.008ml/g每日灌胃1次,第三周以0.010ml/g每日灌胃1次，建立急性酒精肝损伤模型。
动物分组与处理
造模后的小鼠还剩45只，正常组10只，造模组10只。将造模组的36只小鼠随机分为模型组、阳性药组(联苯双酯滴丸)、苦丁茶提取物低剂量组和苦丁茶提取物高剂量组4组，每组9只。给药组每天灌胃给药1次,空白对照组和模型组每次给予等容量的无菌水，连续灌胃。分别于小鼠给药2、4周时，对小鼠进行眼球取血和组织分离。分组情况如下：
表1：实验动物分组
| 组别 | 数量(只) | 小叶苦丁茶提取物浓度 |
| 正常组 | 9 | — |
| 模型组 | 9 | — |
| 阳性药组 | 9 | 150mg/kg |
| 低剂量组 | 9 | 150mg/kg |
| 高剂量组 | 9 | 300mg/kg |
实验动物相关指标检测：
1)一般情况观察：实验过程中，每日观察实验小鼠精神状态、活动度、皮毛色泽、饮水量、摄食量、排尿排便情况、有无撕咬伤口及死亡等情况。如有小鼠死亡，应记录小鼠死亡数量及可能原因。
2)血清生化指标测定：为避免进食影响检测结果，末次给药后，禁食过夜(自由饮水)16小时，从实验小鼠眼球取血，取血时应避免小鼠皮毛掉入。室温下静置自然凝血，置于离心机中4000rpm离心15分钟，分离血清。每只小鼠的血清分别做好标记，置于-80C超低温冰箱保存备用。分别测定实验小鼠血清中谷草转氨酶(AST)、谷丙转氨酶(ALT)、甘油三酯(TG)的含量水平。
3)小鼠肝指数测定：在小鼠解剖前，称量记录小鼠的体重。取血后立即开腹摘取小鼠肝脏组织，滤纸轻轻擦干表面残留血液，观察小鼠脏器外观并拍照保存，拍照之后立即置于4℃生理盐水中漂洗，除去残留血液，滤纸轻轻擦干，称量记录小鼠的肝脏湿重。根据以下公式计算小鼠肝脏指数：
小鼠肝指数＝肝脏湿重/体重×100％
小鼠脏器采集：
取血后立即开腹摘取小鼠肝脏组织后，另外将小鼠的心、肝、脾、肺取出称重并记录。
数据分析：
采用统计软件Excel及SPSS23.0分析，计量资料用均数±标准差(x±s)表示，多组间数值比较用单因素方差分析，以P<0.05为差异有统计学意义。
(3)实验结果
1)小叶苦丁茶提取物对糖尿病小鼠体型特征及精神状况的影响
在造模过程中，正常对照组小鼠精神状态、体型特征良好，活动自如，毛色光泽，正常进食进饮，排尿量正常，无受伤。造模小鼠在造模过程中共出现17只小鼠死亡，其中有6只解剖发现其肝组织出现病变，有2只小鼠在灌胃酒精后死亡，其余9只死亡小鼠体形消瘦，解剖后观察有肠胀气情况发生，有的出现了酒精肝损伤。造模3周时，大多数小鼠出现体重变轻的现象。
在实验过程中，各酒精肝模型小鼠精神状态较正常对照组小鼠差，反应稍有迟缓；其中以模型对照组最为显著。随着药物干预时间增加，苦丁茶提取物各剂量组和阳性药组酒精肝模型小鼠较模型组小鼠精神稍好，垫料相对洁净。在给药3天后，各酒精肝模型小鼠出现变得屁股肿大的现象；给药5天后，阳性药组小鼠有4只屁股上有伤口，低剂量组小鼠有四只在尾部有伤口，高剂量组小鼠有1只屁股有伤口，1只腿部有伤口；给药8天后，这些伤口都在逐渐恢复，但是高剂量组、低剂量组、阳性药和模型组还是各死了一只。给药2周后，高剂量组小鼠有4只在屁股上又出现了伤口，但未发生死亡情况。
2)小叶苦丁茶提取物对酒精肝损伤小鼠体重及各脏器重量的影响
在给药2周、4周后分别采取小鼠各脏器并称重记录。从表中可以看到，各组之间小鼠体重及各脏器重量没有统计学差异，说明余庆小叶苦丁茶提取物对酒精肝损伤小鼠体重及各脏器重量无显著的影响。
表2：给药2周小鼠体重及各脏器重量
表3：给药4周小鼠体重及各脏器重量
| 组别 | 体重 | 心 | 脾 | 肺 | 肾 |
| 正常组 | 51.40±1.41 | 0.27±0.06 | 0.15±0.03 | 0.29±0.04 | 0.68±0.04 |
| 模型组 | 48.15±8.95 | 0.23±0.54 | 0.11±0.04 | 0.26±0.04 | 0.54±0.12 |
| 低剂量组 | 48.96±3.96 | 0.25±0.03 | 0.11±0.01 | 0.26±0.04 | 0.58±0.09 |
| 高剂量组 | 42.88±3.94 | 0.20±0.01 | 0.20±0.12 | 0.25±0.01 | 0.55±0.04 |
| 阳性药组 | 45.22±5.39 | 0.24±0.05 | 0.19±0.17 | 0.25±0.01 | 0.54±0.03 |
3)苦丁茶提取物对酒精肝损伤小鼠肝脏外观的影响
由图1可知，在灌胃酒精且同时灌胃阳性对照药和小叶苦丁茶提取物的第4周时，对小鼠进行解剖采样观察肝脏表面的病理变化。观察结果为正常组肝表面深红色，无白色病变斑点；模型组肝脏表面颜色呈浅粉红色，有较多白色病变斑点，有轻微的纤维化；阳性药组肝组织表面颜色呈粉红色，无白色病变斑点少；低剂量组肝组织表面颜色呈粉红色，有轻微的白色病变斑点，且不明显；高剂量组肝组织表面颜色呈深粉红色，无白色病变斑点。
4)苦丁茶提取物对小鼠TG、ALT和AST的影响
在灌胃酒精且同时灌胃阳性对照药和小叶苦丁茶提取物的第4周时，对小鼠进行眼球采血，并收集血清检测ALT、AST和TG的含量。由图2可知，在灌胃至第4周后，与模型组比较，血清中ALT和AST的含量在模型组、联苯双酯滴丸、低剂量组及高剂量组中均与正常组比较均达到极显著升高(P<0.01)；与模型组比较，血清中ALT和AST的含量又同时在联苯双酯滴丸和高剂量组中极显著低于模型组(P<0.01)，在低剂量组中的含量与模型组没有达到显著性差异(P>0.05)；而且血清中ALT和AST的含量在联苯双酯滴丸和高剂量组间差异不显著(P>0.05)。说明高剂量灌胃提取物在一定程度可缓解酒精肝损伤，其效果与市面上常用护肝药联苯双酯滴丸相差不大。
由图2可知，给药4周后，与正常组比较，模型组和低剂量组小鼠血清中TG的含量极显著高于正常组(P<0.01),而联苯双酯滴丸和高剂量组小鼠血清中TG的含量显著高于正常组(P<0.05)；与模型组比较，而联苯双酯滴丸和高剂量组在小鼠血清中的含量极显著低于模型组(P<0.01)，与其他组比较差异不显著，且联苯双酯滴丸和高剂量组之间差异不显著(P<0.05)。由此说明灌胃该提取物可降低因酒精肝损伤导致的血液中TG含量的增高，且效果与联苯双酯滴差异不大。
综合以上，得出本发明通过酒精肝损伤动物模型，并灌胃本发明小叶苦丁茶提取物，经检测酒精肝损伤相关指标，灌胃动物行为观察，体重变化，解剖病理观察等一系列结果证明，本发明提取物可在一定程度缓解酒精肝损伤，为该产品进一步开发护肝，保肝产品提供了实验基础。
本文中应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想；同时，对于本领域的一般技术人员，依据本发明的思想，在具体实施方式及应用范围上均会有改变之处。综上所述，本说明书内容不应理解为对本发明的限制。
Claims (9)
1.一种小叶苦丁茶老叶片提取物的制备工艺，其特征在于，包括以下步骤：
(1)原料预处理：取新鲜、叶片完整的小叶苦丁茶进行经蒸汽杀青、烘干处理，得到小叶苦丁茶老叶片；
(2)破碎：用粉碎机对小叶苦丁茶老叶片进行粉碎处理并过10目筛；
(3)提取：称取粉碎过的原材料放入过滤袋中，加水入蒸馏水混匀，先在常温下浸泡，然后将原料加热并保温，停止加热后对原料进行超声处理，处理完成后抽滤，将滤渣重复提取2～3次，合并滤液；
(4)浓缩：将滤液进行旋蒸浓缩至黏稠状取出；
(5)干燥：将浓缩液预冻后进行冷冻干燥，得到固体浓缩提取物；
(6)粉碎：将固体浓缩提取物用粉碎并过300目筛，得到小叶苦丁茶老叶片提取物。
2.根据权利要求1所述的一种小叶苦丁茶老叶片提取物的制备工艺，其特征在于，所述步骤(3)中茶叶与水的料液比为1g：10～20mL。
3.根据权利要求1所述的一种小叶苦丁茶老叶片提取物的制备工艺，其特征在于，所述步骤(3)中常温浸泡时间为3～5h。
4.根据权利要求1所述的一种小叶苦丁茶老叶片提取物的制备工艺，其特征在于，所述步骤(3)中原料加热温度为90～95℃，保温时间为1～2h，且在保温过程中，每保温15min搅拌5min。
5.根据权利要求1所述的一种小叶苦丁茶老叶片提取物的制备工艺，其特征在于，所述步骤(3)中超声处理频率为30～40KHZ，超声处理时间为10～20min。
6.根据权利要求1所述的一种小叶苦丁茶老叶片提取物的制备工艺，其特征在于，所述步骤(4)中浓缩温度为50～70℃。
7.根据权利要求1所述的一种小叶苦丁茶老叶片提取物的制备工艺，其特征在于，所述步骤(5)中固体浓缩提取物的水分含量＜6％。
8.一种小叶苦丁茶老叶片提取物，其特征在于，由权利要求1-7任一项所述方法制备而成。
9.根据权利要求9所述的小叶苦丁茶老叶片提取物，其特征在于，可用于制备缓解酒精诱导肝损伤的食品。
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