CN106509885A - 一种铁皮石斛、三七、红景天口服液及其制备方法 - Google Patents
一种铁皮石斛、三七、红景天口服液及其制备方法 Download PDFInfo
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- CN106509885A CN106509885A CN201610887890.1A CN201610887890A CN106509885A CN 106509885 A CN106509885 A CN 106509885A CN 201610887890 A CN201610887890 A CN 201610887890A CN 106509885 A CN106509885 A CN 106509885A
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- dendrobium candidum
- protocorm
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
本发明涉及一种口服液,其采用铁皮石斛、尤其是铁皮石斛原球茎鲜萃液,复配三七、红景天,三者功效协同互补,强化了该口服液的功效,能够有效用于增强人体免疫力。此外,本发明还提供了所述口服液的制备方法,包括鲜萃、提取浓缩、复配等步骤,使得石斛多糖、石斛碱等活性物质的质量可控,口服液品质稳定、成本低。
Description
技术领域
本发明属于中药保健品技术领域,具体而言,涉及一种由铁皮石斛、三七、红景天为主要原料制备而成的口服液,以及该口服液的制备方法。
背景技术
世界卫生组织调查显示,全世界亚健康人群比例高达75%。亚健康状态已经成为严重影响人们工作、生活和健康的重大隐患。专家分析认为,85%以上的疾病和亚健康状态都与人体免疫力下降有关,同时认为有目的地适当服用具有调节免疫机能功效的保健品,对消除或避免亚健康状态十分有益。
铁皮石斛(学名:Dendrobium officinale)主要含有石斛多糖、石斛碱等活性成分,性味甘淡微咸,寒,归胃、肾、肺经。益胃生津,滋阴清热。用于阴伤津亏,口干烦渴,食少干呕,病后虚热,目暗不明。铁皮石斛名列“九大仙草”之首,与其它品种如霍山石斛、金钗石斛等都属于传统名贵中草药,在中国及东南亚等地区备受青睐。富硒铁皮石斛中含有丰富的有机硒,硒是人体必需的微量元素,参与合成人体内多种含硒酶和含硒蛋白;能提高人体免疫力,促进淋巴细胞的增殖及抗体和免疫球蛋白的合成;硒对多种癌症具有明显的抑制和防护作用;并且硒与维生素E、大蒜素、亚油酸、锗、锌等营养素具有协同抗氧化的功效,增加抗氧化活性。
三七(学名:Panax pseudo-ginseng)性温,味甘微苦,入肝、胃、大肠经,具有止血、散瘀、定痛的功效,同时还具有治疗糖尿病和心血管疾病的作用。
红景天(学名:Rhodiola rosea L.),能够补气清肺,益智养心,是一味作用广泛的中药,亦有很强的美容效果,可作护肤品,也可食用。
市场上含有铁皮石斛的功能性食品种类繁多,例如,中国专利CN 104435749公开了一种铁皮石斛复方制剂及其制备方法和应用,其采用铁皮石斛与枸杞子、西洋参、灵芝等搭配,用于增强免疫力、缓解体力疲劳。然而,将铁皮石斛与三七、红景天配伍的复方制剂尚未见报道。
另一方面,现有铁皮石斛复方制剂在制备过程中,常常直接采用市售铁皮石斛作为原料,但因种源质量、栽培方式、栽培基质、采收时间的不同,造成铁皮石斛品质不一,其石斛多糖、石斛碱等活性成分含量存在差异,进而使得铁皮石斛复方制剂的质量难以控制;其次,在复方制剂制备过程中多采用干燥粉碎、热水煎煮(例如上述文献中提到的复方制剂)以及醇提取等手段对铁皮石斛进行处理,造成石斛多糖、石斛碱等有效成分的流失,保健效果无法保证;此外,现有铁皮石斛复方制剂添加的原料较多,导致生产成本居高不下。
发明内容
针对现有技术中存在的上述问题,本发明的目的是提供一种原料精简、工艺简单、活性成分稳定可控、功效提升的功能性保健食品及其制备方法。
因此,本发明提供了一种由铁皮石斛、三七、红景天作为主要原料制备而成的口服液,其采用铁皮石斛原球茎、尤其是铁皮石斛原球茎鲜萃液,复配三七、红景天,三者功效协同互补,强化了口服液功效,能够有效用于增强人体免疫力。
另一方面,本发明还提供了所述口服液的制备方法,使得石斛多糖、石斛碱等活性物质的质量可控,口服液品质稳定、成本低。
具体而言,本发明的技术方案通过如下段落进行描述:
[1]一种由铁皮石斛、三七和红景天作为主要原料制备而成的口服液。
[2]如段落[1]所述的口服液,其特征在于,以重量份计,制备所述口服液的原料包含:铁皮石斛1~10份、三七1~10份、红景天1~10份、辅料1.5~3.5份。
[3]如段落[1]或[2]所述的口服液,其特征在于,所述铁皮石斛为富硒铁皮石斛。
[4]如段落[2]所述的口服液,其特征在于,所述辅料包含赤藓糖醇和柠檬酸,优选地,以重量份计,制备所述口服液的辅料包含赤藓糖醇1~2份、柠檬酸0.5~1.5份。
[5]如段落[1]-[4]中任一项所述的口服液的制备方法,所述方法包括:
鲜萃:将铁皮石斛原球茎经洗净、除杂后,进行打浆,过滤,分别得到铁皮石斛原球茎鲜萃液和滤渣;
提取浓缩:将三七与红景天混合、粉碎,再加入铁皮石斛原球茎滤渣,混合,进行水提,提取液经过滤、浓缩,得到提取浓缩液;
复配:将所述铁皮石斛原球茎鲜萃液与所述提取浓缩液混合,再加入辅料,经搅拌混合、灭菌、离心、灌装、再灭菌,得所述口服液。
[6]如段落[5]所述的方法,其特征在于,所述鲜萃步骤如下:
洗净除杂:采用10倍重量的水对铁皮石斛原球茎漂洗30~60min,重复3次,沥干表面水分;
萃取:将洗净除杂后的铁皮石斛原球茎与2倍重量的水一起打浆,经200目过滤,分别得到铁皮石斛原球茎鲜萃液和滤渣。
[7]如段落[5]所述的方法,其特征在于,所述提取浓缩步骤如下:
将三七与红景天混合,粉碎至80目,再加入鲜萃步骤中得到的滤渣进行混合,获得混合物;
向所述混合物中加入10倍重量的水,煮沸提取1~3h,经200目过滤,得滤液,重复提取3次;
合并滤液,将滤液减压浓缩至相对密度1~1.5,得到提取浓缩液。
[8]如段落[5]所述的方法,其特征在于,所述复配步骤如下:
将鲜萃步骤中得到的铁皮石斛原球茎鲜萃液、提取浓缩步骤中得到的提取浓缩液、赤藓糖醇和柠檬酸混合,搅拌溶解,得到口服液原液;
口服液原液灭菌后,静置5~10h,然后以5000r/min离心30min,收集上清液,灌装、再灭菌,得所述口服液。
[9]如段落[5]-[8]中任一项所述的方法,其特征在于,所述方法还包括铁皮石斛原球茎的培育步骤:
播种:无菌条件下,将铁皮石斛种子播撒在萌发培养基表面,于25±1℃、1000~2000Lux光照10~16h/d条件下,培养50~80d,使种胚萌发,其中,所述萌发培养基如下:1/4~1MS培养基、蔗糖10~40g/L、琼脂3~10g/L,pH 7.0,121℃灭菌20min;
诱导增殖:无菌条件下,将铁皮石斛种胚转接于原球茎诱导增殖培养基,于25±1℃、1000~2000Lux光照10~16h/d条件下,培养60~90d,进行原球茎的诱导增殖,其中,所述诱导增殖培养基如下:1/2~2MS MS培养基、NAA 1~5mg/L、6-BA 1~5mg/L、蔗糖20~50g/L、琼脂3~15g/L,pH 7.0,121℃灭菌20min。
[10]如段落[9]所述的方法,其特征在于,所述铁皮石斛原球茎的培育步骤为富硒铁皮石斛原球茎的培育步骤,包括:
播种:无菌条件下,将铁皮石斛种子播撒在萌发培养基表面,于25±1℃,1000~2000Lux光照10~16h/d条件下,培养50~80d,使种胚萌发,其中,所述萌发培养基如下:1/4~1MS培养基、蔗糖10~40g/L、亚硒酸钠3~10g/L、琼脂3~10g/L,pH 7.0,121℃灭菌20min;
诱导增殖:无菌条件下,将铁皮石斛种胚转接于原球茎诱导增殖培养基,于25±1℃,1000~2000Lux光照10~16h/d条件下,培养60~90d,进行富硒原球茎的诱导增殖,其中,所述诱导增殖培养基如下:1/2~2MS MS培养基、NAA 1~5mg/L、6-BA 1~5mg/L、蔗糖20~50g/L、亚硒酸钠3~10g/L、琼脂3~15g/L,pH 7.0,121℃灭菌20min。
本发明的有益效果为:本发明以铁皮石斛、三七、红景天为主要原料,铁皮石斛可滋阴生津、补五脏虚劳,为君;三七、红景天益气活血,通脉平喘,为臣,辅助铁皮石斛滋阴润五脏,补气活血、缓解疲劳,提升机体运动能力,并强化对心血管的保护作用。此外,在优选的实施方式中,本发明采用了通过富硒培养基培养获得的富硒铁皮石斛原球茎作为铁皮石斛原料,其含有丰富的活性硒元素,参与合成人体内多种含硒酶和含硒蛋白,能提高人体免疫。本发明利用各个组分的功效特点,达到增强免疫、缓解疲劳、抗缺氧、延缓衰老以及及改善心血管功能等多重功效,非常适合运动休闲人群和亚健康人群的养生与保健。
在选材方面,本发明采用人工组培条件下培养的铁皮石斛原球茎,通过无菌、可控的培养条件,能有效控制石斛多糖、石斛碱等活性物质指标,具有安全可靠、品质可控的优点,有效避免了当前人工大棚、露天培育的铁皮石斛品质不一、卫生条件不达标、易出现发霉、染菌等微生物污染等问题。
在工艺方面,本发明应用的“鲜萃”技术最大程度保留了多糖、石斛碱等有效成分,大大提升了其利用价值;其次,省去了铁皮石斛的干燥步骤,避免因干燥而导致多糖、石斛碱等有效成分营养成分的流失(为避免焦化,往往采用中低温、长时间干燥,这样多糖及石斛碱会通过细胞的代谢作用而消耗);再次,萃取液色泽翠绿、气味清香、口感微甜,鲜萃工艺可使产品色泽、气味和口感更佳,极好地保留了铁皮石斛鲜品的特色。再结合提取、复配、灌装等工艺,制成方便服用的口服液,非常适合现代人快节奏的生活。
本发明的口服液从组方配伍、原料的选择、加工工艺、产品剂型等方面较之市场同类产品均有明显优势,有着良好的市场前景。
附图说明
图1是铁皮石斛原球茎的培养流程图。
图2是铁皮石斛原球茎的照片。
图3是铁皮石斛原球茎的鲜萃流程图。
图4是提取浓缩流程图。
图5是作为具体实施方式之一的铁皮石斛、三七、红景天口服液工艺流程图。
图6示出了铁皮石斛原球茎多糖含量测定标准曲线。
图7示出了铁皮石斛原球茎生物碱含量测定标准曲线。
具体实施方式
在本发明中,采用以下测定方法对铁皮石斛中的有效成分含量进行测定。
1.石斛多糖测定方法:
(1)试剂
D-无水葡萄糖购自中国药品生物制品检定所;蒽酮(AR)购自国药集团;其它试剂均为分析纯;铁皮石斛原球茎采用本文所述的培养方法进行培养。
(2)仪器
TU-1800spc型紫外分光光度计(北京普析通用仪器有限责任公司);KQ-500E型超声波清洗器(昆山市超声仪器有限公司);RE-52型旋转蒸发仪(上海亚荣生化仪器厂)。
(3)分析步骤
①最大波长的测量:精密吸取葡萄糖对照品溶液0.2mL于10mL量瓶中,加蒸馏水补足至1.0mL,加入0.2%的蒽酮-硫酸试剂4.0mL,混匀,置于沸水浴中,加热20min,取出迅速浸入冰水浴中10min。另取蒸馏水1.0mL,加入4.0mL 0.2%蒽酮-硫酸试剂同上操作,作为参比。在400~700nm波长处扫描,测定吸光度。确定吸光度的最大测定波长为572nm。
②标准曲线绘制:分别精密吸取葡萄糖对照品溶液0.1mL、0.2mL、0.3mL、0.4mL、0.5mL于10mL量瓶中,在572nm波长处测定吸光度。以吸光度A为纵坐标,葡萄糖浓度C为横坐标,作对照曲线,得回归方程:
A=0.0367C+0.0412
r=0.9998,线性范围4.36~21.8μg/mL。
③样品处理:将铁皮石斛样品置于索氏提取器中,加入石油醚回流5h,脱去叶绿素等脂类,得到铁皮石斛试样。称取干燥的铁皮石斛试样,采用水提醇沉法,用10倍重量的水提取,水浴温度90℃,提取4次,每次2.5h。提取液浓缩后,加入无水乙醇使其浓度达到80%,低温静置过夜,离心得沉淀。沉淀用Sevag法反复去除蛋白5次,再依次用无水乙醇、丙酮反复洗涤,减压干燥得黑色石斛多糖粉末。
④测定:精密称取105℃下干燥至恒重的D-无水葡萄糖对照品0.0109g,置于50mL量瓶中,加蒸馏水溶解,摇匀,定容,作为对照品溶液。精密称取0.2000g蒽酮,加浓硫酸溶解,摇匀,冷却至室温,于100mL棕色容量瓶定容,得0.2%蒽酮-硫酸试剂。精密称取经提取纯化的石斛多糖粉末0.0508g,用蒸馏水定容于50mL量瓶,作为供试品溶液,在572nm波长处测定吸光度。
⑤结果计算:
试样中多糖的含量按下面公式计算:
式中:X:试样中多糖的含量(g/100g);
m1:试样的质量(g);
m2:通过线性回归方程算得的待测液中多糖的质量(mg);
v1:待测液定容后的体积(mL);
v2:测定用的溶液体积(mL);
x:试样的含水量(g/100g)。
计算结果保留三位有效数字。
⑥精密度
在重复性条件下获得的两次独立测定结果的绝对差值不得超过算术平均值的10%。
2.石斛碱测定方法:
(1)试剂
pH 4.5缓冲溶液:0.2M邻苯二甲酸氢钾(pH 4.0),用0.2M氢氧化纳调至pH 4.5;0.04%溴甲酚绿溶液:称取溴甲酚绿40.0mg,加100mL pH 4.5缓冲溶液溶解,滤过即得;0.01N氢氧化钠、无水乙醇溶液、氯仿、无水乙醇(以上均为国产分析纯试剂)。
(2)仪器
721型分光光度计,上海第三分析仪器厂。
(3)分析步骤
①标准曲线绘制:精密称取石斛碱1.00mg,置100ml容量瓶中,加氯仿至刻度。精密量取1.0mL、2.0mL、3.0mL、4.0mL、5.0mL分别置于分液漏斗中,用氯仿准确稀释至10.0mL加入pH 4.5缓冲液5.0ml和0.04%溴甲酚绿溶液1.0mL,剧烈振摇3min,静置30min,经氯仿浸泡处理并干燥后的药棉滤过,取滤液6.0mL加0.01N氢氧化钠无水乙醇液1.0mL摇匀。以氯仿10.0mL同样操作,做空白对照。于波长620nm处分别测得吸光度。以吸光度A为纵坐标,石斛碱浓度C为横坐标,作对照曲线,得回归方程:
A=0.0438C+0.0391
r=0.9998,线性范围3.26~20.16μg/mL。
②测定:精密称取石斛样品(60目)0.500g,用适量氨水浸湿密封塞放置30min,精密加入氯仿1000mL称重,置水溶回流加热2h,冷后称重;补充氯仿达到原重量,过滤,精密吸取续滤液1.0mL,置10mL容量瓶中,加氯仿至刻度摇匀,从中吸取2.0mL置25mL容量瓶中,加氯仿稀释至刻度,摇匀,为样品试液。
精密吸取样品试液10.0ml,同法加入缓冲溶液及溴甲酚绿试液,显色后,测定OD620mm,并以石斛碱标准溶液(25mg/ml)2.0mL置25mL容量瓶中,加氯仿稀释至刻度,再准确吸取10mL同法显色,测吸光度。
③计算:按标准品对照法计算总生物碱含量(以石斛碱计算)计算公式如下:
式中:X:试样中生物碱的含量(g/100g);
m1:试样的质量(g);
m2:通过线性回归方程算得的待测液中生物碱的质量(mg);
v1:待测液定容后的体积(mL);
v2:测定用的溶液体积(mL);
x:试样的含水量(g/100g)。
计算结果保留三位有效数字。
⑥精密度
在重复性条件下获得的两次独立测定结果的绝对差值不得超过算术平均值的10%。
样品准备:
样品1:100g铁皮石斛原球茎;
样品2:将100g铁皮石斛原球茎采用本法明的鲜萃方法获得的鲜萃液;
样品3:将100g铁皮石斛原球茎在60±5℃下干燥、粉碎至60目,加入5倍量的水煎煮2小时,获得提取液。
结果:测量结果如下表所示:
石斛多糖 | 石斛碱 | |
样品1 | 25.46% | 0.035% |
样品2 | 25.09% | 0.032% |
样品3 | 22.57% | 0.027% |
由上表可以看出,本发明的鲜萃工艺很好地保留了石斛多糖、石斛碱等活性成分。
实施例
下面通过实施例对本发明作进一步阐述,这些实施例将帮助更好地理解本发明,但本发明并不仅仅局限于下述实施例。
实施例1
1.富硒铁皮石斛原球茎的培养:
(1)无菌条件下,将铁皮石斛种子均匀播种在萌发培养基(1/2MS培养基、蔗糖30g/L、亚硒酸钠5g/L、琼脂5g/L,pH 7.0,121℃灭菌20min)表面,于25±1℃,1600Lux光照12h/d条件下,培养60d,使种胚萌发;
(2)无菌条件下,将铁皮石斛种胚转接于原球茎诱导增殖培养基(1/2MS培养基、NAA3mg/L、6-BA2mg/L、蔗糖40g/L、亚硒酸钠6g/L、琼脂6g/L,pH 7.0,121℃灭菌20min),于25±1℃,1800Lux光照14h/d条件下,培养80d,获得富硒铁皮石斛原球茎。
2.富硒铁皮石斛原球茎的鲜萃:
(1)洗净除杂:采用10倍重量的水对富硒铁皮石斛原球茎漂洗50min,重复3次,沥干表面水分;
(2)萃取:将洗净除杂后的富硒铁皮石斛原球茎3份与纯化水6份用打浆机进行打浆,经200目尼龙纱布过滤,分别获得富硒铁皮石斛原球茎鲜萃液和滤渣。
3.提取浓缩:
(1)以重量份计,将三七5份与红景天5份混合,粉碎至80目,再加入上述滤渣进行混合,得到混合物;
(2)向上述混合物中加入10倍重量的纯化水混合,煮沸提取2h,经200目尼龙纱布过滤得滤液,重复提取3次;
(3)合并滤液,将滤液减压浓缩至相对密度1.2,得到提取浓缩液。
4.复配:
(1)将鲜萃步骤中得到的铁皮石斛原球茎鲜萃液、提取浓缩步骤中得到的提取浓缩液与赤藓糖醇1份、柠檬酸0.2份混合、搅拌溶解,得到口服液原液;
(2)口服液原液经灭菌后,静置8h,然后在5000r/min转速下离心30min,收集上清液,灌装、再灭菌,即得本发明的口服液。
实施例2
1.富硒铁皮铁皮石斛原球茎的培养:
(1)筛选优质铁皮石斛作为种源;无菌条件下,将铁皮石斛种子均匀洒落在萌发培养基(1/4MS培养基、蔗糖10g/L、亚硒酸钠3g/L、琼脂3g/L,pH 7.0,121℃灭菌20min)表面,于25±1℃,1000Lux光照10h/d条件下,培养50d,使种胚萌发;
(2)无菌条件下,将铁皮石斛种胚转接于原球茎诱导增殖培养基(MS培养基、NAA1mg/L、6-BA1mg/L、蔗糖20g/L、亚硒酸钠4g/L、琼脂3g/L,pH 7.0,121℃灭菌20min),于25±1℃,1000Lux光照10h/d条件下,培养50d,获得富硒铁皮石斛原球茎。
2.富硒铁皮铁皮石斛原球茎的鲜萃:
(1)洗净除杂:采用10倍重量的水对富硒铁皮石斛原球茎漂洗30min,重复3次,沥干表面水分;
(2)萃取:将洗净除杂后的富硒铁皮石斛原球茎1份与纯化水2份一起用打浆机进行打浆,经200目尼龙纱布过滤,分别得到铁皮石斛原球茎鲜萃液和滤渣。
3.提取浓缩:
(1)以重量份计,将三七1份与红景天1份混合,粉碎至80目,再加入上述鲜萃步骤中得到的滤渣进行混合,获得混合物;
(2)向所述混合物中加入10倍重量的水,煮沸提取1h,经200目尼龙纱布过滤,得滤液,重复提取3次;
(3)合并滤液,将滤液减压浓缩至相对密度为1,得到提取浓缩液。
4.复配:
(1)将鲜萃步骤中得到的铁皮石斛原球茎鲜萃液、提取浓缩步骤中得到的提取浓缩液与赤藓糖醇0.5份、柠檬酸0.1份混合、搅拌溶解,得到口服液原液;
(2)口服液原液经灭菌后,静置5h,然后以5000r/min离心30min,收集上清液,灌装、再灭菌,即得本发明的口服液。
实施例3
1.富硒铁皮石斛原球茎的培养:
(1)无菌条件下,将铁皮石斛种子均匀洒落在萌发培养基(MS培养基、蔗糖40g/L、亚硒酸钠9g/L、琼脂10g/L,pH 7.0,121℃灭菌20min)表面,于25±1℃,2000Lux光照16h/d条件下,培养80d,使种胚萌发;
(2)无菌条件下,将铁皮石斛种胚转接于原球茎诱导增殖培养基(2MS培养基、NAA5mg/L、6-BA5mg/L、蔗糖50g/L、亚硒酸钠10g/L、琼脂15g/L,pH 7.0,121℃灭菌20min),于25±1℃,2000Lux光照16h/d条件下,培养90d,获得富硒铁皮石斛原球茎。
2.富硒铁皮石斛原球茎的鲜萃:
(1)洗净除杂:采用10倍重量的水对富硒铁皮石斛原球茎漂洗60min,重复3次,沥干表面水分;
(2)萃取:将洗净除杂后的富硒铁皮石斛原球茎5份与纯化水10份一起用打浆机进行打浆,经200目尼龙纱布过滤,分别得到铁皮石斛原球茎鲜萃液和滤渣。
3.提取浓缩:
(1)以重量份计,将三七10份与红景天10份混合,粉碎至80目,再加入鲜萃步骤中得到的滤渣进行混合,获得混合物;
(2)向所述混合物中加入10倍重量的水,煮沸提取3h,经200目尼龙纱布过滤,得滤液,重复提取3次;
(3)合并滤液,将滤液减压浓缩至相对密度1.5,得到提取浓缩液。
4.复配:
(1)以重量份计,将鲜萃步骤中得到的铁皮石斛原球茎鲜萃液、提取浓缩步骤中得到的提取浓缩液与赤藓糖醇2份、柠檬酸0.5份混合、搅拌溶解,得到口服液原液;
(2)口服液原液经灭菌后,静置10h,然后以5000r/min离心30min,收集上清液,灌装、再灭菌,即得本发明的口服液。
对比实施例1
1.富硒铁皮石斛原球茎的培养:
(1)无菌条件下,将铁皮石斛种子均匀播种在萌发培养基(1/2MS培养基、蔗糖30g/L、亚硒酸钠5g/L、琼脂5g/L,pH7.0,121℃灭菌20min)表面,于25±1℃,1600Lux光照12h/d条件下,培养60d,使种胚萌发;
(2)无菌条件下,将铁皮石斛种胚转接于原球茎诱导增殖培养基(1/2MS培养基、NAA3mg/L、6-BA2mg/L、蔗糖40g/L、亚硒酸钠6g/L、琼脂6g/L,pH7.0,121℃灭菌20min),于25±1℃,1800Lux光照14h/d条件下,培养80d,获得富硒铁皮石斛原球茎。
2.富硒铁皮石斛原球茎的鲜萃:
(1)洗净除杂:采用10倍重量的水对富硒铁皮石斛原球茎漂洗50min,重复3次,沥干表面水分;
(2)萃取:将洗净除杂后的铁皮石斛原球茎3份与2倍重量的水一起用打浆机进行打浆,经200目尼龙纱布过滤,分别得到铁皮石斛原球茎鲜萃液和滤渣。
3.提取浓缩:
(1)以重量份计,将上述滤渣3份与纯化水20份混合,煮沸提取2h,经200目尼龙纱布过滤,得滤液,重复提取3次;
(2)合并滤液,将滤液减压浓缩至相对密度1.2,得到提取浓缩液。
4.复配:
(1)以重量份计,将所述富硒铁皮石斛原球茎鲜萃液、提取浓缩步骤中得到的提取浓缩液与赤藓糖醇1份、柠檬酸0.2份混合、搅拌溶解,获得口服液原液;
(2)口服液原经液灭菌后,静置8h,然后以5000r/min离心30min,收集上清液,灌装、再灭菌,即得铁皮石斛口服液。
对比实施例2
1.提取浓缩:
(1)将三七粉碎至80目,以重量份计,将三七粉5份与纯化水50份混合,煮沸提取3h,经200目尼龙纱布过滤,得滤液,重复提取3次;
(2)合并滤液,将滤液减压浓缩至相对密度1.5,得到提取浓缩液。
2.复配:
(1)以重量份计,将所述提取浓缩液与赤藓糖醇2份、柠檬酸0.5份混合、搅拌溶解,获得口服液原液;
(2)口服液原液经灭菌后,静置10h,然后以5000r/min离心30min,收集上清液,灌装、灭菌,即得三七口服液。
对比实施例3
1.提取浓缩:
(1)将红景天粉碎至80目,以重量份计,将红景天粉5份与纯化水50份混合,煮沸提取3h,经200目尼龙纱布过滤,得滤液,重复提取3次;
(2)合并滤液,将滤液减压浓缩至相对密度1.5,得到提取浓缩液。
2.复配:
(1)以重量份计,将所述提取浓缩液与赤藓糖醇2份、柠檬酸0.5份混合、搅拌溶解,获得口服液原液;
(2)口服液原液经灭菌后,静置10h,然后以5000r/min离心30min,收集上清液,灌装、再灭菌,即得红景天口服液。
试验实施例1:药效试验
经动物实验证实本品具有增强免疫力和缓解体力疲劳的功能。
一、增强免疫力功能试验
以氢化可的松模型小鼠为试验对象,通过口腔给予不同剂量的本发明的口服液1个月,结果显示,本发明的口服液具有升高小鼠外周血白细胞数,增加模型小鼠胸腺、脾脏指数,增强巨噬细胞的吞噬功能,促进模型鼠细胞免疫功能的功效。因此认为,本发明的口服液具有增强免疫力的功能。
1材料和方法
1.1材料
试验样品:实施例1制备的本发明的口服液,规格为25mL/瓶;对比实施例1-3制备的口服液作为对照组;实验动物:清洁级ICR小鼠180只,雌雄各半,体重18~22g,由广东省医学实验动物中心提供;主要试剂:印度墨汁,绵羊红细胞(SRBC)。
1.2方法
(1)对小鼠白细胞数和胸腺、脾脏指数的影响
如表1所示,取健康小鼠80只,雌雄各半,按体重随机分为8组。正常组、模型组小鼠每天灌胃给予生理盐水0.01mL/g,对照组和给药组按表1剂量分别灌胃给予试验样品,剂量分别为0.4mL/kg体重(低剂量组)、1.2mL/kg体重(中剂量组)与4.0mL/kg体重(高剂量组),1次/天,连续30天。
除正常组外,其它各组于实验第18天开始皮下注射氢化可的松(40mg/kg)5次,隔天注射1次。末次给药1.5h后,小鼠眼眶采血,测定白细胞数,并用颈椎脱臼法处死动物,剖取胸腺、脾脏并称重量,计算脏器指数,再用t检验进行统计处理。
(2)对小鼠巨噬细胞吞噬功能的影响
小鼠分组、给药及造模同上述方法(1)。末次给药1h后,各组小鼠尾静脉注射印度墨汁(用生理盐水稀释4倍)0.01mL/g,于注射后2min、10min时分别眼眶取血20μL,溶于2mL0.1%(wt)碳酸钠水溶液中,摇匀,测定OD600nm,计算吞噬指数a:
吞噬指数a=(logA1-logA2)÷(t1-t2)。
(3)对绵羊红细胞(SRBC)诱导小鼠足跖肿胀度的影响
取健康小鼠80只,雌雄各半,按体重随机分为8组,见表3。正常组、模型组小鼠每天灌胃生理盐水0.01mL/g,对照组和给药组分别按表3以试验样品0.4mL/kg体重、1.2mL/kg体重与4.0mL/kg体重的剂量灌胃给药,1次/天,连续30天。30天后,除正常组外,腹腔注射2%(wt)SRBC,每只小鼠注射0.2mL(约1×108个SRBC)。免疫4天后,测量左后足跖部厚度或肿胀度,然后在测量部位皮下注射20%(V/V)SRBC,每只鼠20μL(约1×108个SRBC),注射后24h测定左后足跖部厚度或肿胀度,同一部位测定三次,取平均值。
2结果
2.1对小鼠白细胞数及胸腺、脾脏指数的影响
结果如表1所示,与模型组相比,实施例1的口服液1.3mL/kg、4.1mL/kg能有效抵制氢化可的松所引起的外周血白细胞数的急剧下降,减轻氢化可的松应用的副作用,并能有效抑制氢化可的松引起的免疫器官分泌淋巴细胞的减少;与正常组相比,实施例1的口服液1.3mL/kg、4.1mL/kg能够较明显地提升外周血白细胞的效应,促进免疫器官的增殖(P<0.05),且作用优于各对照组。为平均值±标准差。
表1试验样品对小鼠白细胞数和胸腺、脾脏指数的影响(n=10,)
注:与正常组比较,*:P<0.05;**:P<0.01。
2.2对小鼠碳廓清指数的影响
结果如表2所示,与模型组相比,实施例1的口服液1.3mL/kg、4.1mL/kg能有效抑制氢化可的松加入后引起的小鼠碳粒廓清速度减慢;与正常组相比,实施例1的口服液1.3mL/kg、4.1mL/kg能明显提高吞噬速率a,增加小鼠碳粒廓清能力,且作用优于各对照组。
表2试验样品对小鼠碳粒廓清速度的影响(n=10,)
注:与正常组比较,与模型组比较,*:P<0.05;**:P<0.01。
2.3对绵羊红细胞(SRBC)诱导小鼠足跖肿胀度的影响
结果如表3所示,模型组的足跖肿胀度与正常组相比,有显著的统计学差异(P<0.01)。实施例1的口服液1.3mL/kg、4.1mL/kg能显著减少SRBC致敏引起的足跖肿胀程度,有效促进细胞免疫。与正常组相比,实施例1的口服液1.3mL/kg、4.1mL/kg可以有效促进小鼠迟发型变态反应,促进细胞免疫功能(P<0.05,P<0.01),且作用优于各对照组。
表3试验样品对绵羊红细胞诱导小鼠足跖肿胀度的影响(n=10,)
注:与正常组比较,*:P<0.05,**:P<0.01;***:P<0.01。
3结论
上述试验实施例证明本发明的富硒铁皮石斛、三七、红景天口服液具有升高氢化可的松模型小鼠外周血白细胞数,增加模型鼠胸腺、脾脏指数,增强巨噬细胞的吞噬功能,促进模型鼠细胞免疫功能。表明该制剂具有免疫增强作用,可增强机体的特异性和非特异性免疫功能。
Claims (10)
1.一种由铁皮石斛、三七和红景天作为主要原料制备而成的口服液。
2.如权利要求1所述的口服液,其特征在于,以重量份计,制备所述口服液的原料包含:铁皮石斛1~10份、三七1~10份、红景天1~10份、辅料1.5~3.5份。
3.如权利要求1或2所述的口服液,其特征在于,所述铁皮石斛为富硒铁皮石斛。
4.如权利要求2所述的口服液,其特征在于,所述辅料包含赤藓糖醇和柠檬酸,优选地,以重量份计,制备所述口服液的辅料包含赤藓糖醇1~2份、柠檬酸0.5~1.5份。
5.权利要求1-4中任一项所述的口服液的制备方法,所述方法包括:
鲜萃:将铁皮石斛原球茎经洗净、除杂后,进行打浆,过滤,分别得到铁皮石斛原球茎鲜萃液和滤渣;
提取浓缩:将三七与红景天混合、粉碎,再加入铁皮石斛原球茎滤渣,混合,进行水提,提取液经过滤、浓缩,得到提取浓缩液;
复配:将所述铁皮石斛原球茎鲜萃液与所述提取浓缩液混合,再加入辅料,经搅拌混合、灭菌、离心、灌装、再灭菌,得所述口服液。
6.如权利要求5所述的方法,其特征在于,所述鲜萃步骤如下:
(1)洗净除杂:采用10倍重量的水对铁皮石斛原球茎漂洗30~60min,重复3次,沥干表面水分;
(2)萃取:将洗净除杂后的铁皮石斛原球茎与2倍重量的水一起打浆,经200目过滤,分别得到铁皮石斛原球茎鲜萃液和滤渣。
7.如权利要求5所述的方法,其特征在于,所述提取浓缩步骤如下:
(1)将三七与红景天混合,粉碎至80目,再加入鲜萃步骤中得到的滤渣进行混合,获得混合物;
(2)向所述混合物中加入10倍重量的水,煮沸提取1~3h,经200目过滤,得滤液,重复提取3次;
(3)合并滤液,将滤液减压浓缩至相对密度1~1.5,得到提取浓缩液。
8.如权利要求5所述的方法,其特征在于,所述复配步骤如下:
(1)将鲜萃步骤中得到的铁皮石斛原球茎鲜萃液、提取浓缩步骤中得到的提取浓缩液、赤藓糖醇和柠檬酸混合,搅拌溶解,得到口服液原液;
(2)口服液原液灭菌后,静置5~10h,然后以5000r/min离心30min,收集上清液,灌装、再灭菌,得所述口服液。
9.如权利要求5-8中任一项所述的方法,其特征在于,所述方法还包括铁皮石斛原球茎的培育步骤:
(1)播种:无菌条件下,将铁皮石斛种子播撒在萌发培养基表面,于25±1℃、1000~2000Lux光照10~16h/d条件下,培养50~80d,使种胚萌发,其中,所述萌发培养基如下:1/4~1MS培养基、蔗糖10~40g/L、琼脂3~10g/L,pH 7.0,121℃灭菌20min;
(2)诱导增殖:无菌条件下,将铁皮石斛种胚转接于原球茎诱导增殖培养基,于25±1℃、1000~2000Lux光照10~16h/d条件下,培养60~90d,进行原球茎的诱导增殖,其中,所述诱导增殖培养基如下:1/2~2MS MS培养基、NAA 1~5mg/L、6-BA 1~5mg/L、蔗糖20~50g/L、琼脂3~15g/L,pH 7.0,121℃灭菌20min。
10.如权利要求9所述的方法,其特征在于,所述铁皮石斛原球茎的培育步骤为富硒铁皮石斛原球茎的培育步骤,包括:
(1)播种:无菌条件下,将铁皮石斛种子播撒在萌发培养基表面,于25±1℃,1000~2000Lux光照10~16h/d条件下,培养50~80d,使种胚萌发,其中,所述萌发培养基如下:1/4~1MS培养基、蔗糖10~40g/L、亚硒酸钠3~10g/L、琼脂3~10g/L,pH 7.0,121℃灭菌20min;
(2)诱导增殖:无菌条件下,将铁皮石斛种胚转接于原球茎诱导增殖培养基,于25±1℃,1000~2000Lux光照10~16h/d条件下,培养60~90d,进行富硒原球茎的诱导增殖,其中,所述诱导增殖培养基如下:1/2~2MS MS培养基、NAA 1~5mg/L、6-BA 1~5mg/L、蔗糖20~50g/L、亚硒酸钠3~10g/L、琼脂3~15g/L,pH 7.0,121℃灭菌20min。
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CN105770816A (zh) * | 2016-04-10 | 2016-07-20 | 佛山市朗达信息科技有限公司 | 一种调理脾胃的养胃组合物 |
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