CN112322681B - 一种中药五谷虫活性肽及其制备方法和用途 - Google Patents
一种中药五谷虫活性肽及其制备方法和用途 Download PDFInfo
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Abstract
本发明属于医药技术领域,具体涉及一种中药五谷虫活性肽;还涉及该中药五谷虫活性肽的制备方法和用途;该中药五谷虫活性肽,可按以下方法制备得到:取干燥中药五谷虫脱脂药渣;加入5~8倍质量的浓度为1~2wt%的NaCl,室温搅拌4~5h,离心取上清液;调节pH,析出沉淀,透析,离心取沉淀冷冻干燥得中药五谷虫蛋白,配制底物浓度为7~13%的溶液,按照中药五谷虫蛋白质量的0.5~1.5wt%加入胃蛋白酶,35~40℃条件下提取,加热煮沸,冷却后过滤,冷冻干燥得产物;该方法提高了中药五谷虫的资源利用,降低原料成本,又减少了中药五谷虫活性肽的制备过程中的脱脂步骤,且中药五谷虫活性肽的得率显著提高。
Description
技术领域
本发明属于医药技术领域;具体涉及一种中药五谷虫活性肽;还涉及该中药五谷虫活性肽的制备方法和用途。
背景技术
五谷虫为蝇科或丽蝇科昆虫,据《本草求源》、《本草便读》中记载,五谷虫性寒,经干燥研末后,外敷,能治疗臁烂、唇疔。中医认为五谷虫性味咸寒、无毒,具有清热消滞、解毒消炎、去腐生肌等功效,可治疗臁疮等感染性外科疾病。目前五谷虫在美国和欧洲允许在临床上使用,主要将活体五谷虫用于清创治疗,但其使用并不方便、患者接受程度低、医疗用活体五谷虫饲养与保存难度大,最终很难被广泛应用。近些年来,研究者对中药五谷虫活性物质基础进行研究,制备成常规药物制剂,改变活体给药方式,提高患者接受程度。有研究者发现,中药五谷虫中肽类成分抗菌与促创面愈合活性显著,但其制备过程复杂、获得难度大、获得量少,很难应用于产业化生产过程。能够寻找一种中药五谷虫活性肽的制备方法显得十分重要。
经检索发现,公开号为CN102675409A,专利名称为《可保持五谷虫分泌排泄物活性的收集液》。该专利通过刺激活体五谷虫,使其分泌活性肽等物质,并进行收集,由于五谷虫自身分泌肽类成分量很少,应用该专利的方法很难获得大量活性肽类成分,无法达到产业化要求。结合公开号为CN108392496A,专利名称为《一种中药五谷虫脂肪酸提取工艺》,该专利将五谷虫中脂肪酸类成分进行提取,最终得到五谷虫脂肪酸和脱脂药渣,其中中药五谷虫脱脂药渣没有得到很好利用,造成资源的浪费。公开号为CN10394863,专利名称为《蟾饲五谷虫抗菌肽的医药用途》,该专利中虽然涉及五谷虫抗菌肽的提取方法,但该方法需要依赖离心机,甚至需要使用高速离心机进行15000rpm的离心操作,该离心机设备投入资金大,且高速离心的方法单次处理量少,不易应用于大批量样品的处理,此外,该方法中使用正己烷萃取,正己烷有麻醉和刺激作用,对操作者而言,长期接触可致周围神经炎,且还涉及萃取剂残留问题以及后续除杂等问题。因此,需要研发一种中药五谷虫活性肽提取工艺来解决中药五谷虫生产问题非常必要。
发明内容
本发明针对现有技术的不足,提供一种中药五谷虫活性肽的制备方法和用途。所述方法直接利用中药五谷虫脱脂药渣对中药五谷虫活性肽进行提取,即提高了中药五谷虫的资源利用,降低原料成本,又减少了中药五谷虫活性肽的制备过程中的脱脂步骤。且使用该方法获得的中药五谷虫活性肽,其得率高于公开号为CN108392496A专利名称为《一种中药五谷虫脂肪酸提取工艺》中制备得到中药五谷虫肽类成分的得率。
本发明涉及的一种中药五谷虫活性肽的制备方法,主要包括以下制备步骤:
(1)盐提法:取干燥中药五谷虫脱脂药渣;加入5~8倍质量的浓度为1~2wt%的NaCl,室温搅拌4~5h,离心取上清液;本发明中使用的中药五谷虫脱脂药渣的来源是经过脱脂处理的蝇科、丽蝇科的干燥幼虫,但使用其它近缘昆虫的干燥幼虫也可以实现本发明的技术效果。
(2)重复上述过程(1),合并2次上清液;
(3)取步骤(2)所得的上清液,调节pH6~7,析出沉淀,离心,取沉淀;
(4)对沉淀进行透析,离心,取沉淀进行冷冻干燥,得中药五谷虫蛋白,得率不低于65.0%(得到蛋白与脱脂药渣的质量比);
(5)酶解法:取中药五谷虫蛋白,配制底物浓度质量体积比为7~13%的溶液,按照中药五谷虫蛋白质量的0.5~1.5wt%加入胃蛋白酶,35~40℃条件下提取2~4h,加热煮沸,冷却后过滤,取滤液,离心,取上清液进行冷冻干燥,得中药五谷虫活性肽,最终中药五谷虫活性肽与中药五谷虫蛋白的质量比不低于60.0%。
对于上文所述的制备方法,优选的,所述干燥中药五谷虫脱脂药渣粉碎后过60~80目筛。
对于上文所述的制备方法,优选的,步骤(3)的pH6~7是加入8~12%HCl或8~12%磷酸进行调节。
对于上文所述的制备方法,优选的,步骤(4)的透析时间为20~26h。
对于上文所述的制备方法,优选的,步骤(4)按照中药五谷虫蛋白质量的1~1.5wt%加入胃蛋白酶。
对于上文所述的制备方法,优选的,所述的离心条件为,在4000~5000r/min离心10~20min。
本发明的再一方面,公开了所述方法制备的中药五谷虫活性肽产品,所述产品中的中药五谷虫活性肽与中药五谷虫蛋白的质量比不低于60.0%。
本发明的另一方面,公开了上文所述方法制备的中药五谷虫活性肽在制备促创面愈合药物或保健品中的应用。
对于上文所述的应用,优选的,以五谷虫活性肽为主要活性成分(由于五谷虫活性肽活性高,也可以做为唯一活性成分),用于制备治疗糖尿病溃疡或褥疮的药物或保健品中的应用。
本发明的优点在于采用了脱脂五谷虫药渣为原料,提高了中药五谷虫资源的利用,发现具有促创面愈合作用的中五谷虫活性肽,并且制备方法简单、收率较高、促创面愈合效果明显,为工业化生产以及相关药物制剂的开发提供可能。
具体实施方式
下面结合实例,对本发明的具体实施方式作进一步的详细说明。
试验仪器:pH计(PHS-3C上海佑科仪器仪表有限公司)、高速万能粉碎机、电子分析天平、台式高速冷冻离心机、冷冻干燥机。
本发明中使用的中药五谷虫脱脂药渣的来源是经过脱脂处理的蝇科、丽蝇科的干燥幼虫,但使用其它近缘昆虫的干燥幼虫也可以实现本发明的技术效果。
实施例1
一种中药五谷虫活性肽的制备方法,包括以下制备步骤:
(1)盐提法:取适量干燥中药五谷虫脱脂药渣,粉碎过80目筛。取100g中药五谷虫脱脂药渣,加入500ml 1%NaCl,室温搅拌4h,4000r/min离心10min,取上清液。重复上述过程,合并2次上清液。取上清液,加入10%HCl调节至pH6~7,可见析出沉淀,4000r/min离心10min。应用透析方法对沉淀进行透析,透析24h后,4000r/min离心10min,取沉淀进行冷冻干燥,得蛋白类成分。依照药典中蛋白含量测定要求,应用凯氏定氮法,最终测得蛋白质的量为72.2g(占脱脂药渣的72.2%)。
(2)酶解法:取中药五谷虫蛋白50g,在37℃、pH2.0、底物浓度10%(蛋白质量与溶液体积比)的条件下,加入胃蛋白酶0.5g(批号:101644990,Sigma公司),搅拌3h后,加热煮沸5min,冷却后过滤,取滤液,4000r/min离心10min,取上清液进行冷冻干燥,得中药五谷虫活性肽44.8g(占得到蛋白的89.6%)。
实施例2
一种中药五谷虫活性肽的制备方法,包括以下制备步骤:
(1)盐提法:取适量干燥中药五谷虫脱脂药渣,粉碎过60目筛。取200g中药五谷虫脱脂药渣,加入1L 2%NaCl,室温搅拌4h,4500r/min离心20min,取上清液。重复上述过程,合并2次上清液。取上清液,加入10%磷酸调节至pH6~7,可见析出沉淀,4500r/min离心20min。应用透析方法对沉淀进行透析,透析24h后,4500r/min离心20min,取沉淀进行冷冻干燥,得中药五谷虫蛋白140.9g(占脱脂药渣的70.5%)。
(2)酶解法:取中药五谷虫蛋白100g,在37℃、pH2.0、底物浓度8%(蛋白质量与溶液体积比)的条件下,加入胃蛋白酶1.5g(批号:101644990,Sigma公司),搅拌4h后,加热煮沸5min,冷却后过滤,取滤液,4000r/min离心10min,取上清液进行冷冻干燥,得中药五谷虫活性肽62.1g(占得到蛋白的62.1%)。
实施例3
一种中药五谷虫活性肽的制备方法,包括以下制备步骤:
(1)盐提法:取适量干燥中药五谷虫脱脂药渣,粉碎过80目筛。取500g中药五谷虫脱脂药渣,加入2.5L 1%NaCl,室温搅拌4h,5000r/min离心10min,取上清液。重复上述过程,合并2次上清液。取上清液,加入10%HCl调节至pH6~7,可见析出沉淀,5000r/min离心10min。应用透析方法对沉淀进行透析,透析24h后,5000r/min离心10min,取沉淀进行冷冻干燥,得中药五谷虫蛋白330.6g(占脱脂药渣的66.1%)。
(2)酶解法:取中药五谷虫蛋白300g,在37℃、pH2.0、底物浓度12%(蛋白质量与溶液体积比)的条件下,加入胃蛋白酶3g(批号:101644990,Sigma公司),搅拌3h后,加热煮沸5min,冷却后过滤,取滤液,4000r/min离心10min,取上清液进行冷冻干燥,得中药五谷虫活性肽187.5g(占得到蛋白的62.5%)。
试验例1:中药五谷虫活性肽制备工艺研究
试验方法:以中药五谷虫脱脂药渣为原料,以中药五谷虫活性肽得率为考察指标,通过底物浓度(蛋白质量与酶解反应溶液体积比)、加酶量、酶解时间3因素3水平正交试验确定中药五谷虫活性肽的最佳制备工艺条件,正交试验因素水平表见表1,正交试验结果见表2。
表1正交试验因素水平表
水平 | A底物浓度(%) | B加酶量(%) | C提取时间(h) |
1 | 8 | 0.5 | 2 |
2 | 10 | 1.0 | 3 |
3 | 12 | 1.5 | 4 |
表2正交试验设计及结果
试验结果:从表2结果的极差分析可知,对中药五谷虫活性肽得率的影响因素大小依次为:提取时间>加酶量>底物浓度,各个因素中K值大小可知,其最优化工艺为A2B2C3。因此中药五谷虫的最佳萃取工艺为:底物浓度10%、加酶量1.0%、酶解时间4h。
试验例2:药效学试验
1.促创面愈合试验
试验材料:雄性昆明小鼠
试验方法:采用乙醚对小鼠进行麻醉,小鼠背部剃毛,面积约3cm×2cm,75%乙醇消毒。轻提小鼠皮肤,放置在两个圆形磁碟之间(直径12mm,厚5mm),形成一个5mm的“皮肤桥”。施压周期:(16h挤压+8h释放)2个周期。造模完成后去除磁碟,见创面局部皮缘红肿溃烂,即造模成功。将本专利所述中药五谷虫活性肽涂抹于压疮创面,每日1次。第3、7、10天,分别取创伤小鼠,麻醉后,应用涤纶投影薄膜覆盖于创面,描记创面轮廓,并将其复印于坐标纸上,计算创面面积,结果见表3。
表3:促压疮创面愈合试验结果
试验结果:从表3可以看出实施例1、2、3活性肽组在第3、7、10天时,小鼠创面愈合率均高于空白组,第10天的创面愈合率的效果与阳性组接近,证明本发明获得的中药五谷虫活性肽对小鼠压疮创面具有促愈合作用。
2.促HaCaT细胞增殖试验
试验材料:按照试验例1的方法制备中药五谷虫活性肽、HaCaT细胞系(购于上海宇翔生物科技有限公司,产品编号:2019H06235)、高糖DMEM培养液、PBS、CCK-8、超净工作台、CO2培养箱。
试验方法:
2.1HaCaT培养:HaCaT细胞在5%CO2,37℃恒温条件下,采用含有10%胎牛血清的高糖DMEM培养液进行培养。细胞融合约瓶底70%~80%时,弃去培养液,用PBS洗涤2~3次后加入适量胰酶进行消化。
2.2CCK-8实验:收集HaCaT细胞,计数并按每孔5×103个细胞浓度接种至96孔板中,孵育过夜。弃去96孔板内培养液,向孔中加入100μl含有不同浓度中药五谷从活性肽的培养基,在5%CO2、37℃的条件下培养24h,去除培养液,向每孔中加入100μl含10%CCK-8溶液,继续孵化2h,使用酶标仪在450nm波长下测量吸光度。
2.3统计学方法:使用SPSS 20.0软件进行统计,计量资料以均数±标准差(x±s)表示,各组间计量资料的比较采用单因素方差分析,P<0.05为差异有统计学意义,结果见表4。
表4:不同浓度中药五谷虫活性肽对HaCaT细胞增殖的影响
注:与空白对照组比较*p<0.05
试验结果:从表4可以看出,经方差分析,中、高浓度中药五谷虫活性肽组吸光度值与空白对照组进行比较,具有显著性差异(P<0.05)。因此可知中、高浓度中药五谷虫活性肽促HaCaT细胞增殖作用具有显著性。
总之,根据以上药效学试验结果,证明该发明涉及的中药五谷虫活性肽,具有促创面愈合作用。
以上所揭露的仅为本发明的最佳实施例,不能以此来限定本发明的权利范围,本领域技术人员可以实现上述实施例的全部或部分流程,并依本发明权利要求所作的等同变化,仍属于本发明所涵盖范围。
Claims (7)
1.一种中药五谷虫活性肽的制备方法,其特征在于,包括以下制备步骤:
(1)盐提法:取干燥中药五谷虫脱脂药渣;加入5~8倍质量的浓度为1~2wt%的NaCl,室温搅拌4~5h,离心取上清液;
(2)重复上述过程(1),合并所得上清液;
(3)取步骤(2)所得的上清液,调节pH6~7,析出沉淀,离心,取沉淀;
(4)对沉淀进行透析,离心,取沉淀进行冷冻干燥,得中药五谷虫蛋白,得率不低于65.0%;
(5)酶解法:取中药五谷虫蛋白,配制底物浓度质量体积比为7~13%的溶液,按照中药五谷虫蛋白质量的0.5~1.5wt%加入胃蛋白酶,35~40℃条件下提取2~4h,加热煮沸,冷却后过滤,取滤液,离心,取上清液进行冷冻干燥,得中药五谷虫活性肽产品;
所述的中药五谷虫脱脂药渣的来源是经过脱脂处理的蝇科、丽蝇科的干燥幼虫;
所述的离心条件为,在4000~5000r/min离心10~20min。
2.根据权利要求1所述的制备方法,其特征在于,所述干燥中药五谷虫脱脂药渣粉碎后过60~80目筛。
3.根据权利要求1所述的制备方法,其特征在于,步骤(3)的pH6~7是加入8~12%HCl或8~12%磷酸进行调节。
4.根据权利要求1所述的制备方法,其特征在于,步骤(4)的透析时间为20~26h。
5.根据权利要求1所述的制备方法,其特征在于,步骤(4)按照中药五谷虫蛋白质量的1~1.5wt%加入胃蛋白酶。
6.如权利要求1所述方法制备的中药五谷虫活性肽产品,所述产品中的中药五谷虫活性肽与中药五谷虫蛋白的质量比不低于60.0%。
7.如权利要求1所述方法制备的中药五谷虫活性肽在制备促创面愈合药物中的应用。
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