CN117229380A - 一种青刺果肽复合物的制备及其应用 - Google Patents
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Abstract
本发明公开一种青刺果肽复合物的制备及其应用,属于保健食品及药品领域。本发明通过对青刺果肽干粉经葡聚糖凝胶G‑25层析柱进行分离,再经RP‑HPLC进行进一步分离纯化,得到包含11个青刺果肽的青刺果肽复合物,该复合物对高血糖导致的糖代谢异常、糖化血红蛋白升高、胰岛素抵抗以及胰腺损伤具有良好的疗效,可应用于制备降糖产品,毒副作用小。
Description
技术领域
本发明涉及一种青刺果肽复合物的制备及其应用,属于保健食品及药品领域。
背景技术
II型糖尿病是一种病因尚未完全明确,发病机制与遗传、免疫功能异常、环境因素、饮食习惯、体力活动量过少、胰岛素抵抗等多种因素有关的多病因疾病。长期高血糖可引发机体慢性并发症,如视网膜病变、糖尿病肾病、脑出血、糖尿病足等疾病,甚至危及生命。目前,糖尿病的血糖控制主要服用化学类的双胍类、磺脲类、格列奈类、格列酮类药物和抑制剂,容易引起低血糖、体重增加、皮肤过敏、食欲减退、胃肠道不适等不良反应以及血脂异常、血细胞比容增加等副作用。因此,寻找天然、安全的降糖物质,并在食品和医药中的应用成为人们关注的热点。
青刺果(Prinsepia utilis Royle)别名苦果子、为蔷薇科扁核木属灌木,主要产于云南西部至南部和贵州西部。青刺果在当地作为植物食物或药草用于治疗咳嗽、牙痛、跌打损伤、风湿痹痛、高血压及动脉硬化症,对心血管疾病有辅助疗效。但是将青刺果肽用于干预糖尿病保健食品和制备药物未见报道。青刺果粕是青刺果榨油后的废弃物,含有大量的蛋白质。研究已证实,天然降血糖活性肽对机体安全且可以有效调控血糖、减少和缓解糖尿病引发的组织器官损伤。因此,从青刺果粕中寻找新的、安全的降血糖肽,应用于保健食品和医药制备有重要意义。
发明内容
为了解决现有药物在治疗II型糖尿病中的不足,本发明提供了一种青刺果肽复合物的制备方法,该肽复合物可用于II型糖尿病人群保健产品的开发。所述青刺果肽复合物包含11个青刺果肽,包括LVLP(Leu-Val-Leu-Pro)、LYTP(Leu-Tyr-Thr-Pro)、ALLP(Ala-Leu-Leu-Pro)、VLLP(Val-Leu-Leu-Pro)、LALP(Leu-Ala-Leu-Pro)、FVSP(Phe-Val-Ser-Pro)、LGGL(Leu-Gly-Gly-Leu)、LGLG(Leu-Gly-Leu-Gly)、HLGLP(His-Leu-Gly-Leu-Pro)、APAG(Ala-Pro-Ala-Gly)、YAYF(Tyr-Ala-Tyr-Phe),它们的氨基酸序列如SEQ ID NO:1所示。所述青刺果肽复合物具有显著的体外α-淀粉酶抑制活性;对高血糖导致的糖代谢异常、糖化血红蛋白升高、胰岛素抵抗以及胰腺损伤具有良好的疗效,可应用于降糖保健食品或制备降糖药物。
本发明的另一目的是提供一种青刺果肽复合物的制备,具体步骤如下所述:
(1)通过碱溶酸沉法提取,得到青刺果粗蛋白;
(2)将步骤(1)中得到的青刺果粗蛋白用模拟胃液进行溶解,然后用HCI将pH调节至3.0得到青刺果粗蛋白溶液,在青刺果粗蛋白溶液中青刺果粗蛋白的质量百分比浓度为3.3%;
(3)在青刺果粗蛋白溶液中加入2000U/mL胃蛋白酶混匀,在37℃、静态条件下孵育4小时,用NaOH将pH调节至7.0得到混合物,在混合物中加入胰蛋白酶和胆汁盐,其中胰蛋白酶的加入量为混合物质量的8%,胆汁盐在混合物中的浓度为68.5mmol/L;在37℃下孵育3.5小时模拟肠消化过程,然后调节pH值至中性,煮沸灭酶,冷却,离心,收集上清液即为青刺果蛋白消化液;
(4)将步骤(3)中得到的青刺果蛋白消化液用截留分子量为5kDa的超滤膜进行超滤,收集分子量小于5kDa的超滤液,冻干得到青刺果肽干粉;
(5)将步骤(4)中得到的青刺果肽干粉用超纯水配置成溶液,经葡聚糖凝胶G-25层析柱进行分离,得到4个不同的组分;
(6)将步骤(5)中得到的4个不同的组分别进行α-淀粉酶抑制活性检测;
(7)将步骤(6)中检测出α-淀粉酶抑制活性最高的组分用RP-HPLC进行进一步分离纯化,得到包含11个青刺果肽的青刺果肽复合物。
优选的,步骤(2)中所述模拟胃液的成分包括:3.5mmol/L的KH2PO4、10mmol/L的CaCl2·2H2O、3.6mmol/L的MgCl2·6H2O、14mmol/L的KCl和20mmol/L的NaCl。
优选的,步骤(3)中煮沸时间为10min,离心条件为10000rpm/min离心10min。
优选的,步骤(5)中青刺果肽干粉与超纯水的比例为:每毫升超纯水加入20mg青刺果肽干粉。
本发明的有益效果
(1)本发明提供了一种可用于制备降糖产品的青刺果多肽复合物,与传统药物相比本发明所述青刺果多肽复合物具有毒副作用小的特点。
(2)青刺果粕是青刺果榨油后的废弃物,本发明利用青刺果粕制备得到青刺果肽,实现了资源的最大化利用。
(3)本发明所得到的青刺果多肽复合物稳定性比青刺果肽干粉好,易于保存。
附图说明
图1为实施例1中青刺果肽复合物经葡聚糖凝胶G-25柱层析分离的结果;
图2为实施例2中正常小鼠胰腺组织病理切片;
图3为实施例2中模型组小鼠胰腺组织病理切片;
图4为实施例2中二甲双胍干预下II型糖尿病小鼠胰腺组织病理切片;
图5为实施例2中低剂量青刺果肽复合物干预下II型糖尿病小鼠胰腺组织病理切片;
图6实施例2中高剂量青刺果肽复合物干预下II型糖尿病小鼠胰腺组织病理切片;
图7为实施例2中不同组小鼠口服葡萄糖耐量的影响;
图8为实施例2中不同组小鼠胰岛素抵抗的影响;
图9为实施例2中不同组小鼠糖化血红蛋白的影响。
具体实施方式
下面结合具体实施例对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
1.本实施例所述青刺果复合肽的制备方法,具体包括以下步骤:
(1)将100g青刺果粕经粉碎、过80-100目筛(例如80目、90目、100目,本实施例中选择90目),得到青刺果粕粉。
(2)向步骤(1)获得的青刺果粕粉中加入超纯水,青刺果粕粉与超纯水的质量比为1:20,然后用1.0mol/LNaOH调节pH至8.8,在30℃下超声搅拌提取2.5小时,接着以8000r/min离心15分钟,收集上清液,用1.0mol/LHCl将上清液调pH至4.2,酸沉淀40分钟后,以8000r/min离心15分钟,收集沉淀,然后将沉淀重新溶解于超纯水中,其中沉淀与超纯水的质量比为1:2,调节pH至7.0(此处调节pH可用1.0mol/LNaOH或1.0mol/LHCl),冷冻干燥即可得到青刺果粗蛋白。
(3)将步骤(2)得到的青刺果粗蛋白用含有3.5mmol/LKH2PO4、10mmol/LCaCl2·2H2O、3.6mmol/LMgCl2·6H2O、14mmol/LKCl和20mmol/LNaCl的模拟胃液进行溶解,以1mol/LHCI将pH调节至3.0,在青刺果粗蛋白溶液中青刺果粗蛋白的质量百分比浓度为3.3%,接着加入2000U/mL胃蛋白酶混匀,在37℃、静态条件下孵育4小时,然后用1mol/LNaOH将pH调节至7.0,随后在混合物中加入胰蛋白酶和胆汁盐,其中胰蛋白酶的加入量为混合物质量的8%,胆汁盐在混合物中的浓度为68.5mmol/L,在37℃下孵育3.5小时模拟肠消化过程,接下来将混合物pH值调至中性(此处调节pH可用1.0mol/LNaOH或1.0mol/LHCl),然后煮沸10min灭酶,冷却,以10000rpm/min离心10min,收集上清液即为青刺果蛋白消化液。
(4)将步骤(3)得到青刺果蛋白消化液用截留分子量为5kDa的超滤膜进行超滤,收集分子量小于5k Da的超滤液,将超滤液冻干得到青刺果肽干粉。
(5)将步骤(4)得到的青刺果肽干粉用超纯水配置成青刺果肽溶液,其中每毫升超纯水加入20mg青刺果肽干粉,接着将青刺果肽溶液经葡聚糖凝胶G-25层析柱进行分离,如图1所示,将青刺果肽溶液上样葡聚糖凝胶G-25层析柱后,以1.0mL/min的流速进行洗脱,用自动收集器每隔2min收集1管,每管收集2mL,共收集30管,接着在220nm处测定各管的吸光度值,将得到4个峰,第一个峰为收集的1-9管的吸光度值,第二个峰为收集的10-14管的吸光度值,第三个峰为收集的15-20管的吸光度值,第四个峰为收集的21-30管的吸光度值,分别记为PT1,PT2,PT3和PT4。
(6)α-淀粉酶抑制率测定:分别将0.2mL步骤(5)中得到样品PT1,PT2,PT3和PT4和0.2mL的α-淀粉酶(700U/g)置于1.5mL离心管中混匀,在37℃水浴孵育10min。加入0.2mL1%的可溶性淀粉,在37℃水浴孵育3min后加入0.2mLDNS试剂,煮沸10min,迅速冷却,加入5mL超纯水,在540nm波长下测定其吸光值记录为样品,其他条件不变的情况下将磷酸盐缓冲液代替DNS测得的吸光值记录为对照,其他条件不变的情况下将超纯水代替样品测得的吸光值记录为空白,按照下面公式计算α-淀粉酶抑制率:
抑制率=[1-(样品组-对照组)/空白组]×100%
青剌果肽溶液经过葡聚糖凝胶G-25层析柱分离得到四个峰的α-淀粉酶抑制率如表1所示,在相同肽浓度(5mg/mL)下,PT2组分的α-淀粉酶抑制率为68.89±1.02%,显著高于其它组分(P<0.05),因此,选择PT2组分进行进一步的分离纯化。
表1青刺果肽经G-25分离出4个洗脱峰的α-淀粉酶抑制活性
洗脱峰组分 | α-淀粉酶抑制率(%) |
PT1 | 53.12±0.62c |
PT2 | 68.89±1.02d |
PT3 | 41.65±0.21b |
PT4 | 35.26±0.32a |
(7)将PT2用RP-HPLC进行进一步分离纯化,所用色谱柱为Agilent C18色谱柱,规格为250mm×4.6mm,5μm,检测波长为220nm,柱温为30℃,流动相A为水和三氟乙酸,水和三氟乙酸的体积比为1000∶1,流动相B为乙腈和三氟乙酸,乙腈和三氟乙酸的体积比为1000∶1,采用0-30min,B相10-45%的洗脱程序,以0.5mL/min的流速梯度洗脱色谱柱,分离纯化得到青剌果肽混合物。
(8)将步骤(7)中分离纯化得到的青剌果肽混合物用LC-MS/MS进行序列鉴定,所用色谱柱为Agilent Eclipse Plus C18色谱柱,规格为2.1mm×150mm,3.5μm,其中A相为水和甲酸,水和甲酸体积比为1000∶1,B相为乙腈和甲酸,乙腈和甲酸体积比为1000∶1,流速0.2mL/min。梯度洗脱:B相5%,0-5min;B相5%-20%,5-10min;B相20%-40%,10-25min;B相40%-75%,25-35min;B相5%,35-40min。柱温30℃,进样量为5μL,检测波长220nm,质谱条件:分辨率为Full MS 35000,dd-MS217500,质谱检测在正离子模式下进行,质量范围为100-2000m/z,碰撞能量为10eV,20eV,30eV,使用PeaksStudio软件鉴定氨基酸序列,将所得的液质图据质谱的片段信息通过De NovoTM软件自动识别肽序列和分子量,选择平均局部置信度>85%的肽序列,最终得到包含11个青剌果肽的青刺果肽复合物,它们的氨基酸序列分别为:LVLP(Leu-Val-Leu-Pro)、LYTP(Leu-Tyr-Thr-Pro)、ALLP(Ala-Leu-Leu-Pro)、VLLP(Val-Leu-Leu-Pro)、LALP(Leu-Ala-Leu-Pro)、FVSP(Phe-Val-Ser-Pro)、LGGL(Leu-Gly-Gly-Leu)、LGLG(Leu-Gly-Leu-Gly)、HLGLP(His-Leu-Gly-Leu-Pro)、APAG(Ala-Pro-Ala-Gly)和YAYF(Tyr-Ala-Tyr-Phe)。
实施例2
本实施例通过将实施例1中的青刺果肽复合物用于动物实验进一步证明该青刺果多肽复合物对II型糖尿病症状的改善作用,具体实验如下:
1.小鼠II型糖尿病的诱导与干预
将40只小鼠随机分为5组,每组8只,即每组有8个重复,5组包括正常组(Control)、模型组(Model)、二甲双胍组(Positive)、青刺果肽复合物高剂量组(PMPH)和青刺果肽复合物低剂量组(PMPL),正常组小鼠饲喂普通饲料,喂养方式为自由喂养,二甲双胍组用二甲双胍进行灌胃,其灌胃剂量为每只小鼠每1千克体重灌200mg,青刺果肽复合物高剂量组用青刺果肽复合物灌胃,其灌胃剂量为每只小鼠每1千克体重灌200mg,青刺果肽复合物低剂量组用青刺果肽复合物灌胃,其灌胃剂量为每只小鼠每1千克体重灌100mg,模型组饲喂高糖高脂饲料,喂养方式为自由喂养,其含有20%蔗糖,含10%猪油和2.5%胆固醇,按照上述喂养方法,5个组饲养周期为1个月,1个月后禁食禁水过夜,除正常组以外,各组小鼠腹腔注射3次链脲佐菌素溶液,注射量剂量为40mg/kg,注射间隔为24h,诱导II糖尿病小鼠模型,之后每天记录动物的体重、摄食量(正常小鼠喂食普通饲料,诱导Ⅱ型糖尿病小鼠喂食高糖高脂饲料,喂养方式为自由喂养)和饮水量,期间二甲双胍组、青刺果肽复合物高剂量组和青刺果肽复合物低剂量组用相应药物干预,持续4周。
2.动物实验指标
小鼠胰腺组织病理学检测
4周后,小鼠禁食过夜,小鼠颈椎脱位后即刻取出胰腺,放入4%的多聚甲醛组织固定液中固定24小时,用生理盐水漂洗三次,再依次用30%、50%、70%、85%、95%的乙醇和无水乙醇进行脱水处理,最后经液氮速冻后转移至-80℃冰箱存放,将组织放在熔化的石蜡和二甲苯的等量混合液浸渍1小时,再移入石蜡液中浸渍4小时左右,将包埋的组织块切成0.2μm厚的切片,用二甲苯浸泡脱蜡,接着将切片进行H&E染色,通过倒置显微镜观察小鼠胰腺组织病理学改变,糖尿病可增加胰腺的炎症反应,导致调节糖代谢的重要器官胰腺的进一步损害,如图2-图6所示,正常组小鼠的胰腺组织结构清晰,有大量胰岛细胞,细胞形态规则,边界清晰,呈椭圆形或圆形,细胞质丰富,模型组小鼠胰腺中胰岛细胞明显减少,体积变小,边界模糊,细胞簇排列不规则,且胰腺有大量脂肪滴,胰腺细胞收缩。灌胃青刺果肽复合物和二甲双胍的小鼠胰岛也出现了一些脂肪滴,但病变程度较模型组轻,胰岛细胞恢复到正常胰岛细胞大小,排列整齐,细胞数量有所增加,边界清晰,即给青刺果肽复合物和二甲双胍后减轻了胰腺的损伤。
口服葡萄糖耐量试验(OGTT)
在持续喂养4周的第3周,对所有禁食过夜的小鼠口服葡萄糖水溶液,服用量为每只小鼠每1千克体重口服2g葡萄糖水溶液,分别于0、30、60和120min采集尾静脉血,用血糖仪监测血糖,计算OGTT值,口服葡萄糖耐量(OGTT)是反映机体的胰岛β细胞功能和葡萄糖负荷后的血糖调节功能的重要指标,在正常情况下,机体的血糖在2h内可以恢复到空腹血糖水平,如图7所示,模型组小鼠显示明显的糖耐量升高,各处理组的血糖值在120min时均显著低于模型组,结果表明,青刺果肽复合物和二甲双胍的干预可改善II型糖尿病小鼠的糖代谢紊乱。
胰岛素抵抗(HOMA-IR)的稳态模型评估
4周后,每组取8只小鼠,以0.3%戊巴比妥钠腹腔注射麻醉,摘小鼠除眼球后,取血样放入1.5mL离心管中,血样在室温下放置20min,然后以3000rpm/min的转速,在4℃下离心15min,收集上层血清,胰岛素通过ELISA试剂盒测定;胰岛素抵抗是糖尿病的关键特征之一,通过计算HOMA-IR水平可以评估胰岛素抵抗程度。如图8所示模型组组的HOMA-IR水平明显高于其他组;在改善胰岛素抵抗方面,各处理组的HOMA-IR值显著低于模型组;结果表明,青刺果肽复合物可以下调糖尿病小鼠的HOMA-IR值。
糖化血红蛋白(HbA1c)分析
4周后,每组取8只小鼠,以0.3%戊巴比妥钠腹腔注射麻醉,摘除小鼠一侧眼球,用抗凝采血管收集小鼠血液,在室温下静置20min,于3000rpm/min、4℃下离心10min,收集上层血浆,用商用试剂盒检测糖化血红蛋白(HbA1c),如图9所示,模型组组血浆HbA1c水平显著高于正常组,通过二甲双胍和青刺果肽复合物干预后都可以降低HbA1c水平,其中青刺果肽复合物高、低剂量组与二甲双胍差异不显著。
本实施例中,数据进行平均值和标准差计算。用SPSS软件对数据进行单因素方差分析,Duncan's法进行多重比较分析,P<0.05为显著性水平。采用Origin9.0软件绘图。
Claims (6)
1.一种青刺果肽复合物,其特征在于:所述青刺果肽复合物由11个青刺果肽组成,包括LVLP、LYTP、ALLP、VLLP、LALP、FVSP、LGGL、LGLG、HLGLP、APAG、YAYF,如SEQ ID NO:1所示。
2.权利要求1所述青刺果肽复合物的制备方法,其特征在于,具体包括以下步骤:
(1)通过碱溶酸沉法提取,得到青刺果粗蛋白;
(2)将步骤(1)中得到的青刺果粗蛋白用模拟胃液进行溶解,然后用HCI将pH调节至3.0得到青刺果粗蛋白溶液,在青刺果粗蛋白溶液中青刺果粗蛋白的质量百分比浓度为3.3%;
(3)在青刺果粗蛋白溶液中加入2000U/mL胃蛋白酶混匀,在37℃、静态条件下孵育4小时,用NaOH将pH调节至7.0得到混合物,在混合物中加入胰蛋白酶和胆汁盐,其中胰蛋白酶的加入量为混合物质量的8%,胆汁盐在混合物中的浓度为68.5mmol/L;在37℃下孵育3.5小时模拟肠消化过程,然后调节pH值至中性,煮沸灭酶,冷却,离心,收集上清液即为青刺果蛋白消化液;
(4)将步骤(3)中得到的青刺果蛋白消化液用截留分子量为5kDa的超滤膜进行超滤,收集分子量小于5k Da的超滤液,冻干得到青刺果肽干粉;
(5)将步骤(4)中得到的青刺果肽干粉用超纯水配置成溶液,经葡聚糖凝胶G-25层析柱进行分离,得到4个不同的组分;
(6)将步骤(5)中得到的4个不同的组分别进行α-淀粉酶抑制活性检测;
(7)将步骤(6)中检测出α-淀粉酶抑制活性最高的组分用RP-HPLC进行进一步分离纯化,得到包含11个青刺果肽的青刺果肽复合物。
3.根据权利要求2所述青刺果肽复合物的制备方法,其特征在于:步骤(2)中所述模拟胃液的成分包括:3.5mmol/L的KH2PO4、10mmol/L的CaCl2·2H2O、3.6mmol/L的MgCl2·6H2O、14mmol/L的KCl和20mmol/L的NaCl。
4.根据权利要求2所述青刺果肽复合物的制备方法,其特征在于:步骤(3)中煮沸时间为10min,离心条件为10000rpm/min离心10min。
5.根据权利要求2所述青刺果肽复合物的制备方法,其特征在于:步骤(5)中青刺果肽干粉与超纯水的比例为:每毫升超纯水加入20mg青刺果肽干粉。
6.权利要求1所述青刺果肽复合物在制备降糖产品中的应用。
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