CN117447624A - 一种鸡软骨提取物、其制备方法及应用 - Google Patents
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Abstract
本发明涉及软骨提取技术领域,具体涉及一种鸡胸软骨提取物、其制备方法及应用,该一种鸡胸软骨提取物,以饲养40天到60天的鸡胸软骨为原料,所得到的软骨提取物获得硫酸软骨素A/C值更高。
Description
技术领域
本发明涉及软骨提取技术领域,具体涉及一种鸡胸软骨提取物、其制备方法及应用。
背景技术
软骨是一种坚韧而有弹性的结缔组织,主要存在于关节和支撑骨骼的部位。
软骨提取物是以优质动物软骨为原料,采用特殊酶解工艺制备得到,通常具有许多生物活性成分,如胶原蛋白、硫酸软骨素钠和其他天然生物活性物质。因此,软骨提取物被广泛应用于保健食品或功能性食品行业。
为了提高软骨提取物的利用价值,也有很多关于软骨提取物相关的研究,例如,专利公开号为CN114045322A的专利申请公开的一种采用鸡胸软骨提取鸡软骨提取物的方法及其制品,在提取II型胶原蛋白时,以鸡胸软骨为原料通过脱脂,粉磨,酸解,酶解,电泳透析,二次粉磨,杀菌,制得II型胶原蛋白,再将II型胶原蛋白通过载体膜的制备,二次电泳,真空封存制得制品。该发明制得的鸡软骨提取物为水解分子量约为几千的小分子多肽易被吸收,制得的敷料膜制品能为细胞提供所需的胶原蛋白多肽,促进细胞的增值分化,以达到伤口修复的效果。又如专利公开号为CN112544778A的专利申请公开的一种纯度高口感好的高品质软骨提取物的制备方法及制备的软骨提取物的应用,选用一种酶就能达到多种酶复合使用的功效,既提升了产品品质,又简化了生产工序,使得产品品质和生产效率得到双效提升,可控制杂质以及有效降低产品中的钠含量,对于软骨提取物的色泽、口感及应用领域都有显著提升。
目前,软骨提取物的相关研究主要集中在提取、分离纯化方法上,对原料特性、品质本身的研究还较少。
发明内容
本发明从原料出发,对原料特性进行深入研究,获得硫酸软骨素A/C值更高的软骨提取物。
本发明将软骨提取物的核心集中在A/C值上,根据文章“The MolecularEvolution of Cartilage”的研究,人类在1岁以后,体内硫酸软骨素A含量逐年降低,至45岁后保持在相对较低水平;人体内硫酸软骨素C含量随年龄增加几乎没有变化。由此可见,随着年龄增加,选择特定品质的鸡胸软骨作为原料,获得更高硫酸软骨素A含量的软骨提取物,弥补人体中硫酸软骨素A含量下降问题是必要的。
因此,本发明旨在提供A/C值更高的软骨提取物,以期满足人体对硫酸软骨素A的需求。
为此,本发明的第一方面,提供一种鸡胸软骨提取物,以饲养40天到60天的鸡胸软骨为原料。尤其是45天到60天。
进一步优选地,以圈养的40天到60天,尤其是45天到60天的鸡胸软骨为原料。该生长周期内的鸡胸软骨中,硫酸软骨素A/C比值最高达4.1。
本发明的第二方面,提供一种鸡胸软骨提取物,以圈养鸡的鸡胸软骨为原料。
进一步优选地,以圈养的40天到60天,尤其是45天到60天的鸡胸软骨为原料。该生长周期内的鸡胸软骨中,硫酸软骨素A/C比值最高达4.1。
本发明的第三方面,提供一种鸡胸软骨提取物的制备方法,以饲养40天到60天的鸡胸软骨为原料。优选地,以饲养45天到60天的鸡胸软骨为原料。
进一步优选地,以圈养的40天到60天,尤其是45天到60天的鸡胸软骨为原料。
上述制备方法,具体包括如下的步骤:
S1. 取新鲜鸡胸骨,进行预处理后,用清水冲洗干净;
S2. 将冲洗干净的新鲜鸡胸骨加水煮沸4小时以上,再冷却至50℃至55℃,调节pH至6-7;
S3. 向步骤S2的鸡胸骨中加入蛋白酶,充分酶解,酶解结束后,升温灭酶,过滤得到澄清滤液,经纳滤去除灰分,浓缩后进行真空冷冻干燥得到鸡胸软骨提取物。
进一步优选,所述蛋白酶为木瓜蛋白酶、菠萝蛋白酶、碱性蛋白酶、角蛋白酶、中性蛋白酶中的一种或几种。
进一步优选,所述蛋白酶的添加量为1%(w/v)20万酶活力单位的,酶解时间为4-7小时。
进一步优选地,预处理包括去掉残余的肌肉、脂肪和带血红骨的操作。
本发明的第四方面,提供所述鸡胸软骨提取物的应用,用于关节炎、骨质疏松等骨骼相关疾病的治疗和预防。
附图说明
图1为圈养和散养鸡在不同生长周期下软骨中硫酸软骨素钠含量变化趋势曲线图;
图2为圈养和散养鸡在不同生长周期下软骨中硫酸软骨素A/C比变化趋势曲线图;
图3为圈养和散养鸡在不同生长周期下软骨中胶原蛋白变化趋势曲线图;
图4为不同处理组大鼠机械痛值柱形图;
图5为不同处理组大鼠血液炎症因子测定结果柱形图;
图6为各组小鼠关节组织HE染色图。
具体实施方式
下面通过具体实施例,对本发明的技术方案作进一步的具体说明。应当理解,本发明的实施并不局限于下面的实施例,对本发明所做的任何形式上的变通和/或改变都将落入本发明保护范围。所涉及的参数范围如无特别说明均包括端点值。
在本发明中,若非特指,所有的份、百分比均为重量单位,所采用的设备和原料等均可从市场购得或是本领域常用的。下述实施例中的方法,如无特别说明,均为本领域的常规方法。
实施例1 鸡胸软骨提取物制备
1.1 制备鸡胸软骨提取物的原料筛选
分别取散养和圈养30天、45天、60天、90天、120天、150天的白羽鸡,屠宰后取新鲜鸡胸骨,去掉残余的肌肉、脂肪和带血红骨,用清水冲洗干净,打碎后,进行低温冷冻干燥。
圈养是指厂房内进行笼养或者平地养殖,鸡的活动范围小,定期自动投喂饲料。
散养是指户外(大多数是指林下)养殖,鸡的活动范围大,除饲料外还可自行捕捉昆虫、果等。
1.2 鸡胸软骨提取物的制备
将1.1所获得的鸡胸骨置于烧杯内,加水没过软骨,在水浴锅内煮沸4小时以上,再将温度冷却至55℃,调节pH至6-7,加入1%(w/v)20万酶活力单位的木瓜蛋白酶,充分酶解6小时,酶解结束后,升温至80℃灭酶,过滤得到澄清滤液,经纳滤去除灰分,浓缩后进行真空冷冻干燥得到鸡胸软骨提取物。
1.3 鸡胸软骨提取物中指标成分测定方法
硫酸软骨素钠及硫酸软骨素A、C含量测定采用《中国药典》(2020年版)硫酸软骨素钠项下含量测定方法测定;胶原蛋白含量胶原蛋白检测采用GB/T 9695.23羟脯氨酸测定方法,换算系数为8,即胶原蛋白含量(%)=羟脯氨酸测含量(%)×8。结果见表1。
表1的结果显示:在以鸡胸软骨为原料制备鸡胸软骨提取物过程中,鸡的生长时间、饲养方式不同,所制备的软骨提取物中的硫酸软骨素钠的含量和其同分异构体的比例不同:(1)散养鸡与圈养鸡在同一生长周期内鸡胸软骨中硫酸软骨素钠的含量相当,但圈养鸡胸软骨胶原蛋白含量更高且硫酸软骨素A/C比更高;(2)同一饲养方式下,随着鸡的生长,鸡胸软骨中硫酸软骨素钠的同分异构体硫酸软骨素A含量先升高后降低,硫酸软骨素C含量几乎没变化,在饲养45天左右时,不同饲养方式的鸡体内硫酸软骨素A/C值均达到最高,圈养鸡硫酸软骨素A/C值达最高4.1,散养鸡硫酸软骨素A/C值达最高3.6。
实施例2 鸡胸软骨提取物的功效研究
2.1试验造模与分组
雄性SPF 级 Wistar 大鼠,体质量180~220 g,每组8只,分别为正常对照组(A)、OA模型组(B)、鸡胸软骨提取物低剂量组(C)、鸡胸软骨提取物高剂量组(D)。A组大鼠膝关节腔内单次注射50微升生理盐水。C、D组于造模后分别以鸡胸软骨提取物(低剂量45mg/kg/天、高剂量180mg/kg/天)灌胃,A、B组每日灌胃同体积的生理盐水,每日 1 次,连续给药5周。其中,鸡胸软骨提取物是以圈养方式、生长时间45天鸡来源的白羽鸡胸软骨为原料制备所得的鸡胸软骨提取物。试验期间每周测量足底机械痛。试验结束后心脏采血分析血清中IL-6、TNF-α含量,同时取各组大鼠右后肢膝关节以4%多聚甲醛固定,脱钙后进行病理学检查(HE染色)。
2.2实验检测指标
2.2.1机械痛指标检测
采用法国bioseb Bio-EVF5型电子机械测痛仪,在造模前(第0 d) 、造模后第7、14、21、28 、35d 时测定机械痛阈值,观察各组大鼠机械痛的差异。
2.2.2血液指标检查
实验结束后心脏采血,用ELISA试剂盒检测血清中IL-6、TNF-α含量,观察各组大鼠血液炎症因子的变化。
2.2.3病理检测
2.3统计学处理
所有数据用SPSS软件(版本号)进行ANOVA检验。四组大鼠机械痛值及血液相关因子测量结果以均数±标准差(x±s)表示,组间整体比较均采用单因素方差分析,组间两两比较采用 LSD-t 检验,P<0.05为差异有统计学意义。
2.4鸡胸软骨提取物的功效研究结果
2.4.1各组大鼠机械痛结果
参见图4和如下表3。
结果显示,在造模后的第1周至第5周,与正常对照组比较,OA模型组大鼠机械痛值均显著下降(P<0.05)。第1周至第3周,鸡胸软骨提取物高、低剂量组与OA模型组比较,大鼠机械痛值无明显差异。第4周时,鸡胸软骨提取物高、低剂量组与OA模型组比较,机械痛值显著升高(P<0.05)。与第0周时比较,鸡胸软骨提取物高、低剂量组大鼠在造模后第1周时,机械痛值均下降,并在第2、3、4、5周机械痛值总体呈上升趋势,鸡胸软骨提取物高剂量组在第5周时机械痛值接近于正常对照组,但仍存在显著性差异(P<0.05)。
2.4.2各组大鼠血液炎症因子测定结果
结果参见图5和如下表4。
与正常对照组比较,OA 模型组血液中的IL-6、TNF-α含量显著上升(p<0.05)。与OA模型组比较,鸡胸软骨提取物高、低剂量组IL-6、TNF-α含量显著下降(p<0.05),说明鸡胸软骨提取物具有较好的抗炎效果。
2.4.3各组大鼠病理结果
对各处理组小鼠关节组织进行HE染色,结果参见图6。
根据图6显示:
正常对照组(A):关节结构正常,关节面被覆透明软骨、表面光滑。关节腔正常,滑膜面光滑,关节囊及周围纤维组织未见异常。关节面下的骨组织结构正常。软骨细胞及基质染色正常,潮线完整、清晰可见。
OA模型组(B):骨关节表面被覆的透明软骨变薄,局部软骨细胞变性、坏死,软骨细胞数量减少;关节软骨表面可见增生的纤维组织,关节腔变窄,局部关节面粘连。滑膜组织充血、增厚,局部滑膜细胞变性、坏死、消失,滑膜细胞周围成纤维细胞增生。局部骨关节下方骨组织受累,骨小梁断裂。软骨细胞及基质染色浅淡,潮线完整性被破坏,Mankin’s评分较正常对照组显著升高(p<0.05)。
低剂量组(C):骨关节表面被覆的透明软骨变薄,局部软骨细胞增生,软骨细胞数量增多;关节软骨表面可见增生的纤维组织。滑膜组织充血、增厚,滑膜细胞周围成纤维细胞增生。软骨细胞及软骨基质染色减少,潮线完整性被破坏。Mankin’s评分较OA模型组显著降低(p<0.05)。
高剂量组(D):局部骨关节表面被覆的透明软骨变薄、表面不光滑,大部分样品软骨细胞增生,软骨细胞数量增多。局部滑膜组织充血、增厚。骨关节下方骨组织正常。软骨细胞及软骨基质染色减少,潮线的完整性被破坏。Mankin’s评分较OA模型组显著降低(p<0.05)。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种鸡胸软骨提取物,其特征在于,以饲养40天到60天的鸡的软骨为原料。
2.根据权利要求1所述的鸡胸软骨提取物,其特征在于,以圈养的40天到60天鸡的软骨为原料。
3.根据权利要求1所述的鸡胸软骨提取物,其特征在于,以圈养的45天到60天鸡的软骨为原料。
4.一种鸡胸软骨提取物,其特征在于,以圈养的鸡的软骨为原料。
5.根据权利要求4所述的鸡胸软骨提取物,其特征在于,以圈养的40天到60天鸡的软骨为原料。
6.根据权利要求4所述的鸡胸软骨提取物,其特征在于,以圈养的45天到60天鸡的软骨为原料。
7.一种鸡胸软骨提取物的制备方法,其特征在于,以圈养的40天到60天的鸡的软骨为原料。
8.根据权利要求7所述的一种鸡胸软骨提取物的制备方法,其特征在于,以圈养的45天到60天的鸡的软骨为原料。
9.根据权利要求8所述的制备方法,其特征在于,具体包括如下的步骤:
S1. 取新鲜鸡胸骨,进行预处理后,用清水冲洗干净;
S2. 将冲洗干净的新鲜鸡胸骨加水煮沸4小时以上,再冷却至50℃至55℃,调节pH至6-7;
S3. 向步骤S2的鸡胸骨中加入蛋白酶,充分酶解,酶解结束后,升温灭酶,过滤得到澄清滤液,经纳滤去除灰分,浓缩后进行真空冷冻干燥得到鸡胸软骨提取物。
10.如权利要求1-8任一项所述的鸡胸软骨提取物的应用,用于治疗和预防关节炎、骨质疏松等骨骼相关疾病的药物、保健食品或功能性食品中。
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