CN112168868A - 一种牡丹花瓣微粉组合物及其在制备防治肝损伤的功能食品中的应用 - Google Patents
一种牡丹花瓣微粉组合物及其在制备防治肝损伤的功能食品中的应用 Download PDFInfo
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- CN112168868A CN112168868A CN202011087355.0A CN202011087355A CN112168868A CN 112168868 A CN112168868 A CN 112168868A CN 202011087355 A CN202011087355 A CN 202011087355A CN 112168868 A CN112168868 A CN 112168868A
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Abstract
本发明涉及一种牡丹花瓣微粉组合物及其在制备防治肝损伤的功能食品中的应用。该组合物包含牡丹花瓣超细破壁微粉1‑8份,L‑半胱氨酸0.1‑1.0份、L‑丙氨酸0.1‑1.0份,二氢杨梅素0.1‑0.8份,枸杞提取物0.1‑3份。
Description
技术领域
本发明属于天然植物制备与应用领域,具体涉及一种牡丹花瓣微粉组合物及其在制备防治肝损伤的功能食品中的应用。
技术背景
牡丹(Paeonia suffruticosa Andr.)是毛茛科、芍药属植物,为多年生落叶灌木,别称鼠姑、富贵花,是我国的国花,“花中之王”[1-2],具有很高的观赏价值[3];千百年来深受中国人民的喜爱,是我国传统名贵花卉之一。现代研究表明牡丹花还有较高的可食用价值[4]。牡丹花瓣制成的鲜花饼[5],牡丹花炮制成的花茶,牡丹花酿成的花酒[7],深受人们的喜爱。在人口日益增加的今天,资源利用问题,是摆在我们面前不容忽视的问题。破壁率对于提高生物利用度,减少资源浪费是必不可少的手段[8-9]。现代社会人们压力越来越大,导致情绪过度紧张或抑郁引起肝郁等亚健康;还因饮食不节,长期或大量饮酒导致肝损伤.肝脏是人体最重要器官之一,保持其功能的正常是人体维持健康主要指标[10]。因此,本发明用于肝损伤的食疗具有重要意义。
发明内容
本发明提供一种牡丹花瓣超细破壁微粉,其特征在于所述牡丹花瓣超细破壁微粉的制备方法包括如下步骤:
(1)牡丹花瓣细粉的制备:将新鲜牡丹花瓣粉碎,冷冻干燥后,过80目筛,得牡丹花瓣细粉(简称“细粉”);
(2)牡丹花瓣超细破壁微粉的制备:取步骤(1)得到的牡丹花瓣细粉,使用超微破壁粉碎机进一步粉碎后,过300目筛,即得所述牡丹花瓣超细破壁微粉(简称“微粉”)。
步骤(1)、(2)中粉碎时的温度不高于10℃,优选在-15~10℃条件下粉碎。
本发明的另一实施方案提供上述牡丹花瓣超细破壁微粉的制备方法,其特征在于包括如下步骤:
(1)牡丹花瓣细粉的制备:将新鲜牡丹花瓣粉碎,冷冻干燥后,过80目筛,得牡丹花瓣细粉(简称“细粉”);
(2)牡丹花瓣超细破壁微粉的制备:取步骤(1)得到的牡丹花瓣细粉,使用超微破壁粉碎机进一步粉碎后,过300目筛,即得所述牡丹花瓣超细破壁微粉(简称“微粉”)。
步骤(1)、(2)中粉碎时的温度不高于10℃,优选在-15~10℃条件下粉碎。
本发明的另一实施方案提供上述牡丹花瓣超细破壁微粉在制备防治肝损伤的功能食品中的应用。所述功能性食品的形式优选功能饮料、咀嚼片、压片糖、固体饮料、异形片或胶囊等食品或药学可以接受的食用剂型或形式,用于预防或治疗饮酒、情志或其他各种原因引起的肝损伤等。
本发明的另一实施方案提供一种治疗肝损伤的组合物,其特征在于该组合物以上述牡丹花瓣微粉作为有效成分。该组合物优选包含牡丹花瓣超细破壁微粉1-8份,L-半胱氨酸0.1-1.0份、L-丙氨酸0.1-1.0份,二氢杨梅素0.1-0.8份,枸杞提取物0.1-3份。必要时,依据食品或药品外形特征及应用依从性可添加适量食药工业生产允许的辅料,如赋型剂娇味剂等(注:“份”表述为质量份即“克”或公斤)。
本发明的另一实施方案提供上述组合物在预防和/或治疗肝损伤中的应用。
本领域的技术人员可以按照本发明公开的组合物的配方和设计的产品类型,分别精确称取各配料和相关辅料的规定量,充分混合均匀,制备成压片糖、固体饮料、异形片或胶囊等食品或药学可以接受的食用剂型或形式,用于预防或治疗饮酒、情志或其他各种原因引起的肝损伤等。
本发明所述“枸杞提取物”为市场购买的枸杞,按照中国药典枸杞项下标准检验合格后,按照30︰1即30份枸杞得1份枸杞提取物;具体的制备方法是65%医用乙醇,按照中国药典“渗滤法”操作,得枸杞渗滤液,常规方法回收乙醇后,减压浓缩,真空干燥,分装即双铝包装,即得。
与现有技术相比,本发明的优点在于:(1)本发明提供一种牡丹花瓣超细破壁微粉的制备方法,并且研究了细粉和微粉在细胞破壁率及生物活性方面的不同,结果表明微粉在治疗肝损伤方面的作用显著提高。(2)本发明公开了牡丹花瓣超细破壁微粉的主要技术参数和营养成分及其含量。(3)本发明提出以牡丹花瓣超细破壁微粉为主料的配方和制备成食品或药品但不限于的共识并可供方便应用的产品。(4)本发明证实以牡丹花瓣超细破壁微粉为主配料的组方具有防治肝损伤的食疗作用。
附图说明
图1是细粉显微特征图(A:薄壁细胞;B:导管;C:腺毛;D:花粉粒);
图2是细粉的显微计数图;
图3是微粉的显微计数图;
图4是对照品和样品HPLC图;1-没食子酸;2-羟基芍药苷;3-芍药苷;4-野漆树苷:5-紫云英苷;6-芹菜素-7-O-β-D-葡萄糖苷;上-微粉水溶液;下-对照品溶液。
具体实施方式
1、细粉和微粉的通用制备方法:
(1)牡丹花瓣细粉的制备:将新鲜牡丹花瓣粉碎,冷冻干燥后,过80目筛,得牡丹花瓣细粉(简称“细粉”);
(2)牡丹花瓣超细破壁微粉的制备:取步骤(1)得到的牡丹花瓣细粉,使用超微破壁粉碎机进一步粉碎后,过300目筛,即得所述牡丹花瓣超细破壁微粉(简称“微粉”)。
步骤(1)、(2)中粉碎时的温度不高于10℃,优选在-15~10℃条件下粉碎。
以下技术方案中使用的细粉、微粉的粉碎温度为0~10℃。
2、细粉的主要技术参数和营养成分及其含量
(1)细粉细胞计数以及其标准曲线的绘制:精密称取0.1020,0.2914,0.5122,0.7132, 0.8876,1.1349g的细粉,分别置于25mL容量瓶中,加水合氯醛,超声处理10min,使细粉分散均匀,定容至刻度;用移液枪精密吸取细粉溶液30μL,装片应无气泡,分别置显微镜下(40×50倍)观察;细粉的显微特征包括薄壁细胞、导管、腺毛、花粉粒(细粉的显微特征见图1);其薄壁细胞显微镜下易于观察,适用作为检测指标对破壁率的测定,即以细粉中完整的薄壁细胞为计数为指标进行计数(以1个视野为1个计数单位,每个浓度的溶液取8个计数单位的平均值),结果见表1;以细粉的称样量为横坐标,每个计数中完整的薄壁细胞平均数为纵坐标,绘制标准曲线,得回归曲线方程 Y=4.3393x+1.3339,R2=0.9746,说明细粉中的薄壁细胞在0.1020~1.1349g范围内线性关系良好,可用来作为显微特征计数。
表1不同称样量中完整薄壁细胞的个数(n=8)
(2)细粉的细胞检测计数方法学考察
精密度实验:精密称取0.7132g细粉,同方法制片,分别观察6个视野,计算6 个视野相对标准偏差(RSD)为9.80%,结果表明本方法精密度较好。
稳定性实验:同方法制片,分别于0、3、6、9、12h于显微镜(40×50倍)下观察,各时间点取三个视野平均值,计算五个时间点相对标准偏差(RSD)为9.35%,结果表明细粉混悬液在12h内较为稳定。
重复性实验:取同一批细粉6份,室温放置,同法制片,置于显微镜(40×50倍) 下观察,计算6份相对标准偏差(RSD)为7.90%,结果表明本方法重复性较好。
微粉细胞破壁率:分别取微粉与细粉,精密称取,同法制片,于显微镜下观察,分别观察25个视野(代表性视野如图2、3所示),按下列公式计算细粉和微粉的显微特征个数。牡丹花瓣粉显微特征(薄壁细胞)个数计算的相关参数(n=25),结果见表2。
经计算可得微粉的破壁率为100%。
式中:X为每个盖玻片下的薄壁细胞特征数;V为牡丹花瓣粉混悬液总体积(mL);V′为盖玻片下混悬液体积(mL);W为牡丹花瓣粉称取量(mg)。再根据细粉和微粉的显微特征个数,按下列公式计算细胞破壁率:Y=(A-B)/A×100%,Y为细胞破壁率; A、B为细粉显微特征个数;C、D为微粉的显微特征个数。
表2细粉和微粉的显微特征即薄壁细胞个数计算的相关参数(n=25)
(3)微粉中主要营养成分及其含量
常规理化指标测定:蛋白质测定方法按照GB5009.5-2016[11]第一法;水分测定方法按照GB5009.3-2016[12]第一法;脂肪测定方法按照GB5009.6-2016[13]第一法;总灰分测定方法按照GB5009.4-2016[14]第一法;镉、钛、铜、锑、硒、猛、铁测定方法按照 GB5009.268-2016[15]第一法。维生素C测定方法按照GB5009.86-2016[16]。
营养成分测定结果:如表3所示,蛋白质,水分,总灰分含量相对较高,脂肪含量偏低,维生素C含量最高。维生素C是公认的抗氧化和抗坏血酸的活性物质,是人体不可或缺的一类维生素;所以,牡丹花瓣超细破壁微粉是一种对人体健康十分有益的营养物质,其作为药食同源配料极具开发和利用价值。
表3营养成分测定结果
矿物元素测定结果:从表4中得出,常量和微量元素符合国家相关标准的规范。从微量元素角度看,牡丹花瓣粉主要富含人体必需元素如钛、锑、硒、锰。
表4矿物元素测定结果
主要化学成分及其含量:采用HPLC方法,色谱条件:Inno C18-AQ(250×4.6mm, 5μm);流动相为乙腈(A)-0.1%乙酸(B),梯度条件为0-2.5min:90-87%B,2.5-6min, 87-86%B,6-14min,86-85%B,14-15min,85-77%B,15-17min,77-77%B,17-23min, 77-69%B,23-25min,69-69%B,25-40min,69-53%B。流速为1mL/min;柱温为35℃;没食子酸,羟基芍药苷,芍药苷,野漆树苷,紫云英苷,芹菜素-7-O-β-D-葡萄糖苷在 250nm波长下均有较大吸收峰。因此,选择250nm作为检测波长;进样量10μL。检测证实,微粉中主要可检出没食子酸、羟基芍药苷、芍药苷、野漆树苷、紫云英苷、芹菜素-7-O-β-D-葡萄糖苷等6种化学成分,其含量依次为紫云英苷>没食子酸>芹菜素 -7-O-β-D-葡萄糖苷>野漆树苷>羟基芍药苷>芍药苷;色谱实验结果如图4所示。
3、以微粉为主料的配方及其制品的制备方法
(1)基础配方
牡丹花瓣微粉1-8份,L-半胱氨酸0.1-1.0份、L-丙氨酸0.1-1.0份,二氢杨梅素0.1-0.8 份,枸杞提取物0.1-3份(枸杞提取物:市购枸杞,按照中国药典枸杞项下标准检验合格后,按照30:1即30份枸杞得1份枸杞提取物。具体的制备方法是65%医用乙醇,按照中国药典“渗滤法”操作,得枸杞渗滤液,常规方法回收乙醇后,减压浓缩,真空干燥,分装即双铝包装,即得);必要时,依据食品或药品外形特征及应用依从性可添加适量食药工业生产允许的辅料,如赋型剂娇味剂等(注:“份”表述为质量份即“克”或公斤)。
(2)制备方法
按照基础配方和设计的产品类型,分别精确称取各配料和相关辅料的规定量,充分混合均匀,制备成压片糖、固体饮料、异形片或胶囊等食品或药学可以接受的食用剂型或形式,用于预防或治疗饮酒、情志或其他各种原因引起的肝损伤等。
具体制备例
实施例1:取牡丹花瓣微粉2000克,L-半胱氨酸200克、L-丙氨酸200克,二氢杨梅素500克,枸杞提取物500克;上述配料,充分混合均匀,用适量无水乙醇湿法制粒,压片(规格:0.5g/片),60℃以下烘干,选片,包薄膜衣,包装,即得。
实施例2:取牡丹花瓣微粉1500克,L-半胱氨酸300克、L-丙氨酸300克,二氢杨梅素1200克,枸杞提取物900克;上述配料,充分混合均匀,用适量无水乙醇湿法制粒,压片(规格:0.5g/片),60℃以下烘干,选片,包薄膜衣,包装,即得。
实施例3:取牡丹花瓣微粉3000克,L-半胱氨酸375克、L-丙氨酸375克,二氢杨梅素300克,枸杞提取物1125克;上述配料,充分混合均匀,用适量无水乙醇湿法制粒,压片(规格:0.5g/片),60℃以下烘干,选片,包薄膜衣,包装,即得。
实施例4:取牡丹花瓣微粉1000克,L-半胱氨酸500克、L-丙氨酸300克,二氢杨梅素500克,枸杞提取物500克;上述配料,充分混合均匀,用适量无水乙醇湿法制粒,压片(规格:0.5g/片),60℃以下烘干,选片,包薄膜衣,包装,即得。
实施例5:取牡丹花瓣微粉2000克,L-半胱氨酸200克、L-丙氨酸200克,二氢杨梅素500克,枸杞提取物500克;上述配料,充分混合均匀,用适量95%乙醇湿法制粒,60℃以下烘干,整粒,包装(每包2克),即得。
实施例6:取牡丹花瓣微粉3000克,L-半胱氨酸37.5克、L-丙氨酸37.5克,二氢杨梅素37.5克,枸杞提取物37.5克;上述配料,充分混合均匀,用适量85%乙醇湿法制粒,60℃以下烘干,整粒,包装(每包2克),即得。
实施例7:取L-半胱氨酸200克、L-丙氨酸200克,二氢杨梅素500克,枸杞提取物500克;上述配料,充分混合均匀,用适量无水乙醇湿法制粒,压片(规格:0.5g/ 片),60℃以下烘干,选片,包薄膜衣,包装,即得。
实施例8:取牡丹花瓣细粉(步骤(1)过80目筛后得到的)2000克,L-半胱氨酸 200克、L-丙氨酸200克,二氢杨梅素500克,枸杞提取物500克;上述配料,充分混合均匀,用适量无水乙醇湿法制粒,压片(规格:0.5g/片),60℃以下烘干,选片,包薄膜衣,包装,即得。
配方的应用实施例
实施例9:相关产品对急性酒精性肝损伤具有显著预防作用
(1)方法:将C57BL/J小鼠110只适应喂养1周,随机分为正常对照组10只和造模组100只。除正常对照组外其余100只随机分为10组,分别为模型对照组、联苯双酯阳性药对照组、实施例1、实施例2、实施例3、实施例4、实施例5、实施例6、实施例7和实施例8组,每组按15mL/kg灌胃,一日1次,连续2天;建立乙醇急性肝损伤模型,同时给予对应的药物处理。给药方式和剂量为:联苯双酯150mg/kg、实施例1、实施例2、实施例3、实施例4、实施例5、实施例6、实施例7和实施例8组灌胃给予试物的剂量相同即均为200mg/kg,每天灌胃1次;正常对照组和模型对照组灌胃等体积生理盐水。
(2)观察指标:包括肝脏指数(%)、脾脏指数(%);血清中AST和ALT,肝组织匀浆中抗氧化指标MDA和SOD的含量;具体操作按试剂盒说明书操作步骤进行检测。
(3)结果:由表5说明,实施例1-6的样品,采用200mg/kg剂量同时灌胃给予乙醇造模小鼠,具有显著预防和治疗乙醇所致的急性肝和脾脏损伤的作用;同时,缺牡丹花瓣超微粉(实施例7组)基本无效(P>0.05),而实施例8牡丹花瓣细粉组亦具有明显防治作用,但不及牡丹花瓣超微粉的作用强(P<0.05)。
注:##P<0.01有显著性差异(与正常组比较);*P<0.05,**P<0.01,有显著性差异(与模型组比较)。
注:#P<0.05有显著性差异,##P<0.01,有显著性差异(与正常组比较);*P<0.05,**P<0.01,有显著性差异(与模型组比较)。
实施例10:配方制备的产品对CC14诱导小鼠慢性肝损伤具有显著预防作用
(1)方法:将C57BL/J小鼠110只适应喂养1周,随机分为正常对照组10只和造模组100只。除正常对照组外其余100只随机分为10组,分别为模型对照组、联苯双酯阳性药对照组、实施例1、实施例2、实施例3、实施例4、实施例5、实施例6、实施例7和实施例8组,每组按10mL/kg腹部皮下注射20%CC14橄榄油溶液,一周2次,造模6周;建立CC14慢性肝损伤模型,同时给予对应的药物处理。给药方式和剂量为:联苯双酯150mg/kg、实施例1、实施例2、实施例3、实施例4、实施例5、实施例6、实施例7和实施例8组灌胃给予试物的剂量相同即均为200mg/kg,每天灌胃1次;正常对照组和模型对照组灌胃等体积生理盐水。
(2)观察指标:同实施例9。
(3)结果:由表7说明,实施例1-6的样品,采用200mg/kg剂量同时灌胃给予CC14造模小鼠,具有显著预防和治疗CC14所致的慢性肝和脾脏损伤的作用;同时,实施例 7组基本无效(P>0.05),实施例8组亦具有明显防治作用,但不及牡丹花瓣超微粉的作用强(P<0.05)。
注:##P<0.01有显著性差异(与正常组比较);*P<0.05,**P<0.01,有显著性差异(与模型组比较)。
注:#P<0.05,##P<0.01,有显著性差异(与正常组比较);*P<0.05,**P<0.01,有显著性差异(与模型组比较)。
实施例11:对卡介苗加脂多糖诱发性小鼠免疫性肝损伤具有保护作用
(1)方法:将C57BL/J小鼠110只适应喂养1周,随机分为正常对照组10只和造模组100只。除正常对照组外其余100只随机分为10组,分别为模型对照组、联苯双酯阳性药对照组、实施例1、实施例2、实施例3、实施例4、实施例5、实施例6、实施例7和实施例8组,每组于灌胃给药前4小时尾静脉注射卡介苗2.5mg/0.2mL/只,每天按照规定的剂量灌胃给药,每天1次,连续12天,最后一次给药3h后按10μg/ 只/0.2mL尾静脉注射脂多糖造成免疫性肝损伤小鼠模型,同时给予对应的药物处理;给药方式和剂量为:联苯双酯150mg/kg、实施例1、实施例2、实施例3、实施例4、实施例5、实施例6、实施例7和实施例8组灌胃给予试物的剂量相同即均为200mg/kg,每天灌胃1次;正常对照组和模型对照组灌胃等体积生理盐水。
(2)观察指标:同实施例9。
(3)结果:由表9说明,实施例1-6的样品,采用200mg/kg剂量同时灌胃给予卡介苗+脂多糖造模小鼠,具有显著预防和治疗卡介苗+脂多糖所致的慢性肝和脾脏损伤的作用;实施例7组基本无效(P>0.05)实施例8组亦具有明显防治作用,但不及牡丹花瓣超微粉的作用强(P<0.05)。
&卡介苗加脂多糖(下表同)。注:##P<0.01有显著性差异(与正常组比较);*P<0.05,**P<0.01,有显著性差异(与模型组比较)。
注:##P<0.01,有显著性差异(与正常组比较);*P<0.05,**P<0.01,有显著性差异(与模型组比较)。
参考文献
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Claims (9)
1.一种牡丹花瓣超细破壁微粉,其特征在于所述牡丹花瓣超细破壁微粉的制备方法包括如下步骤:
(1)牡丹花瓣细粉的制备:将新鲜牡丹花瓣粉碎,冷冻干燥后,过80目筛,得牡丹花瓣细粉;
(2)牡丹花瓣超细破壁微粉的制备:取步骤(1)得到的牡丹花瓣细粉,使用超微破壁粉碎机进一步粉碎后,过300目筛,即得所述牡丹花瓣超细破壁微粉。
2.权利要求1所述的牡丹花瓣超细破壁微粉,其特征在于步骤(1)、(2)中粉碎时的温度不高于10℃,优选在-15~10℃条件下粉碎。
3.一种牡丹花瓣超细破壁微粉的制备方法,其特征在于包括权利要求1-2任一项所述的步骤。
4.权利要求1-2任一项所述的牡丹花瓣超细破壁微粉在制备防治肝损伤的功能食品中的应用。
5.权利要求4所述的应用,其特征在于所述功能性食品的形式优选功能饮料、咀嚼片、压片糖、固体饮料、异形片或胶囊等食品或药学可以接受的食用剂型或形式。
6.一种治疗肝损伤的组合物,其特征在于该组合物以上述牡丹花瓣微粉作为有效成分。
7.权利要求6所述的组合物,其特征在于该组合物优选包含牡丹花瓣超细破壁微粉1-8份,L-半胱氨酸0.1-1.0份、L-丙氨酸0.1-1.0份,二氢杨梅素0.1-0.8份,枸杞提取物0.1-3份。
8.权利要求7所述的组合物,其特征在于优选牡丹花瓣超细破壁微粉3-5份,L-半胱氨酸0.2-0.8份、L-丙氨酸0.2-0.8份,二氢杨梅素0.2-0.6份,枸杞提取物0.2-2.0份。
9.权利要求6-8任一项所述的组合物在预防和/或治疗肝损伤中的应用。
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