CN117820437B - 菊芋肽、其制备方法及其在护肾辅助糖尿病治疗抗氧化产品中的应用 - Google Patents
菊芋肽、其制备方法及其在护肾辅助糖尿病治疗抗氧化产品中的应用 Download PDFInfo
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Abstract
本发明涉及菊芋技术领域,菊芋具有控制血糖、免疫调节和抗肿瘤等多种作用。本发明具体涉及菊芋肽、其制备方法及其在护肾辅助糖尿病治疗抗氧化产品中的应用。该菊芋肽,其选自Pep2,Pep2的氨基酸序列依次如SEQ ID NO.2所示,所述菊芋肽来源于菊芋(Helianthus tuberosus(L.1753))。该菊芋肽于体外具有抗氧化作用和抑菌作用,于体内能够有效降低遗传性高血糖小鼠血糖,并且能够减少糖尿病并发症,尤其对肾脏有明显的保护作用。
Description
技术领域
本发明涉及菊芋技术领域,具体涉及菊芋肽、其制备方法及其在护肾辅助糖尿病治疗抗氧化产品中的应用。
背景技术
菊芋(Helianthus tuberosus)亦称洋姜,为菊科向日葵属宿根生草本植物,又名洋姜、鬼子姜、姜不辣,有通便利胆、消肿去湿、和中益胃等功效,还有治疗糖尿病和风湿病的潜在价值。菊芋地上部分为茎叶和花,地下部分为根和块茎,各部分中均含有菊糖、酚酸和萜类等多种生物活性成分,具有控制血糖、免疫调节和抗肿瘤等多种作用。
菊芋中含有的菊糖具有一定的抗糖尿病效果。Wang等“WANG Z Q,SEUNGHWAN H,SUNYOUB L,et al.Fermentation of purple Jerusalem artichoke extract to improvetheα-glucosidase in hibitory effect in vitro and ameliorate blood glucose indb/db mice[J].Nutrition Research&Practice,2016,10(3):282-287”比较发现,由植物乳杆菌发酵的菊芋(L.plantarum-fermented purple Jerusalem artichoke,LJA)对α-葡萄糖苷酶抑制活性最好,抑制率达49.34%;另外考察了LJA对非胰岛素依赖型糖尿病动物的功效,结果发现口服LJA 400mg/kg组比对照组血糖水平减少42.25%;LJA组小鼠体内的血清胰岛素、高密度脂蛋白和高密度总胆固醇分别增加30%、38.89%和13.52%;糖化血红蛋白和甘油三酯分别减少10.32%和65.27%。
菊芋中还含有丰富的氨基酸、香豆素类和聚乙炔类化合物。Kim等“KIM D,FAN JP,CHUNG H C,et al.Changes in extractability and antioxidant activity ofJerusalem artichoke(Helianthus tuberosus L.)tubers by various highhydrostatic pressure treatments[J].Food Science&Biotechnology,2010,19(5):1365-1371”研究表明菊芋中富含赖氨酸、蛋氨酸、苏氨酸、异亮氨酸、苯丙氨酸、缬氨酸和亮氨酸7种必需氨基酸及天冬氨酸、丝氨酸、谷氨酸、甘氨酸、丙氨酸、胱氨酸、酪氨酸、组氨酸、精氨酸和脯氨酸10种非必需氨基酸。
然而,现有技术并未发现来自于菊芋中的肽类物质具有降血糖作用,为进一步开放菊芋资源,有必要对其来源的肽类物质进一步研究和开发。
发明内容
有鉴于此,本发明公开了降血糖抗氧化的菊芋肽、其制备方法及其应用。该菊芋肽于体外具有抗氧化作用和抑菌作用,于体内能够有效降低遗传性高血糖小鼠血糖,并且能够减少糖尿病并发症,尤其对肾脏有明显的保护作用。
本发明的目的是提供一种降血糖抗氧化的菊芋肽,其选自Pep1、Pep2、Pep3、Pep4或Pep5任一项,Pep1、Pep2、Pep3、Pep4和Pep5的氨基酸序列依次如SEQ ID NO.1~5所示,所述菊芋肽来源于菊芋(Helianthus tuberosus(L.1753))叶片。
前述的Pep1对DPPH自由基清除率的IC50为237.9±20.6μg/ml、对ABTS自由基清除率的IC50为328.1±43.8μg/ml、对羟基自由清除率的IC50为183.5±24.3μg/ml、对超氧负离子清除率的IC50为436.8±50.9μg/ml、对铁离子螯合活性的IC50为169.3±18.5μg/ml、对铜离子螯合活性的IC50为208.7±21.6μg/ml、对脂质过氧化抑制活性的IC50为188.6±19.9μg/ml。
前述的Pep2对DPPH自由基清除率的IC50为562.2±36.4μg/ml、对ABTS自由基清除率的IC50为486.3±43.2μg/ml、对羟基自由清除率的IC50为359.7±29.5μg/ml、对超氧负离子清除率的IC50为836.2±36.8μg/ml、对铁离子螯合活性的IC50为284.8±28.1μg/ml、对铜离子螯合活性的IC50为245.2±24.8μg/ml、对脂质过氧化抑制活性的IC50为235.2±63.5μg/ml。
前述的Pep3对DPPH自由基清除率的IC50为202.3±17.9μg/ml、对ABTS自由基清除率的IC50为286.9±32.0μg/ml、对羟基自由清除率的IC50为179.3±20.7μg/ml、对超氧负离子清除率的IC50为336.9±36.5μg/ml、对铁离子螯合活性的IC50为187.2±20.3μg/ml、对铜离子螯合活性的IC50为193.2±18.4μg/ml、对脂质过氧化抑制活性的IC50为208.3±21.3μg/ml。
前述的Pep4对DPPH自由基清除率的IC50为302.4±29.1μg/ml、对ABTS自由基清除率的IC50为313.5±36.7μg/ml、对羟基自由清除率的IC50为193.2±29.2μg/ml、对超氧负离子清除率的IC50为368.4±30.3μg/ml、对铁离子螯合活性的IC50为197.6±25.4μg/ml、对铜离子螯合活性的IC50为212.3±22.5μg/ml、对脂质过氧化抑制活性的IC50为213.1±29.3μg/ml。
前述的Pep5对DPPH自由基清除率的IC50为216.7±26.5μg/ml、对ABTS自由基清除率的IC50为289.3±37.9μg/ml、对羟基自由清除率的IC50为156.3±22.8μg/ml、对超氧负离子清除率的IC50为305.5±28.7μg/ml、对铁离子螯合活性的IC50为148.6±15.2μg/ml、对铜离子螯合活性的IC50为183.7±16.9μg/ml、对脂质过氧化抑制活性的IC50为157.4±17.2μg/ml。
本发明还提供了菊芋肽的制备方法,包括:
将新鲜菊芋叶片用碱液提取后,榨汁,过滤,滤液用酸液调节pH至6.5,絮凝,离心分离,干燥,得到蛋白成品;
对所述蛋白成品进行木瓜蛋白酶、胃蛋白酶和酰基转移酶I酶解。
前述的方法中,采用所述木瓜蛋白酶酶解的步骤包括:
将所述蛋白成品悬浮于pH 7.0、50mM磷酸盐缓冲液,终浓度为2%(w/v),加入质量体积比为4%的木瓜蛋白酶;水浴65℃反应1h后,在沸水中加热10min,停止反应;冷却后,以10000g离心20min,收集上清液,冷冻干燥。
前述的方法中,采用所述胃蛋白酶酶解的步骤包括:
该冻干品悬浮于pH 1.5、50mM KCl-HCl缓冲液中,终浓度为2%(w/v),加入质量体积比为3%的胃蛋白酶;水浴37℃反应1h后,在沸水中加热10min,停止反应;冷却后,以10000g离心20min,收集上清液,冷冻干燥。
前述的方法中,采用所述酰基转移酶I酶解的步骤包括:
将所述冻干品悬浮于pH 8.0、50mM Tris-HCl缓冲液中,终浓度为2%(w/v),加入质量体积比为6%的酰基转移酶I;水浴57℃,25kHZ超声反应1h后,在沸水中加热10min,停止反应;冷却后,以10000g离心20min,收集上清液,冷冻干燥。
本发明还提供了一种抗氧化的肽组合物,包含Pep1、Pep2、Pep3、Pep4或Pep5中至少一项,Pep1、Pep2、Pep3、Pep4和Pep5的氨基酸序列依次如SEQ ID NO.1~5所示,所述菊芋肽来源于菊芋(Helianthus tuberosus(L.1753))叶片。
本发明还提供了一种抑菌的肽组合物,包含Pep1、Pep2、Pep3、Pep4或Pep5中至少一项,Pep1、Pep2、Pep3、Pep4和Pep5的氨基酸序列依次如SEQ ID NO.1~5所示,所述菊芋肽来源于菊芋(Helianthus tuberosus(L.1753))叶片。
本发明还提供了前述的菊芋肽在制备降低遗传性高血糖药物中的应用。
本发明还提供了前述的菊芋肽在制备减少糖尿病并发症及保护肾脏药物中的应用。
与现有技术相比,本发明至少具有以下有益效果之一:
本发明从新鲜菊芋叶片中提取蛋白,并对其进行多次酶解,并对酶解液进行分离纯化,得到了Pep1~5五种菊芋肽。此五种菊芋肽发现具有明显的抗氧化作用、抗菌作用,并且于体内能够有效降低遗传性高血糖小鼠血糖,并且能够减少糖尿病并发症,尤其对肾脏有明显的保护作用。
附图说明
图1为Pep1(A)、Pep2(B)、Pep3(C)、Pep4(D)的LC-ESI-Q-TOF MS/MS谱图。
图2为Pep5(A)、Pep6(B)、Pep7(C)、Pep8(D)的LC-ESI-Q-TOF MS/MS谱图。
图3为db/db糖尿病实验各组小鼠肾脏组织切片HE染色图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。本发明中未详细单独说明的试剂均为常规试剂,均可从商业途径获得;未详细特别说明的方法均为常规实验方法,可从现有技术中获知。
为了更好地理解本发明而不是限制本发明的范围,在本发明中所用的表示用量、百分比的所有数字、以及其他数值,在所有情况下都应理解为以词语“大约”所修饰。因此,除非特别说明,否则在说明书和所附权利要求书中所列出的数字参数都是近似值,其可能会根据试图获得的理想性质的不同而加以改变。各个数字参数至少应被看作是根据所报告的有效数字和通过常规的四舍五入方法而获得的。
1、材料及提取
以菊芋(Helianthus tuberosus(L.1753))叶片为材料进行蛋白提取。
将新鲜菊芋叶片剪切至1~2cm小段,称取10g加入500mL的1M氢氧化钠溶液提取剂中,充分混匀后,加入至榨汁机榨汁2min,200mm滤布过滤得墨绿色提取液。用1M盐酸调pH至6.5,设定温度下恒温水浴中絮凝20min。冷水冷却至室温,10000rpm离心5min,得到叶蛋白沉淀物,60℃烘箱中烘干,得叶蛋白成品,称重并测定叶蛋白中粗蛋白的含量及叶蛋白得率,计算粗蛋白提取率。其中,根据GB/T5009.5-2010,采用凯氏定氮法测定菊芋叶片中粗蛋白的含量。结果烘干的蛋白成名中蛋白含量为34.5%。
2、酶解
实施例1:
用木瓜蛋白酶、胃蛋白酶和酰基转移酶I酶解上述蛋白成品。
将上述蛋白成品悬浮于50mM磷酸盐缓冲液(pH 7.0),终浓度为2%(w/v),加入质量体积比为4%的木瓜蛋白酶(产品编号76220,Sigma-Aldrich);水浴65℃反应1h后,在沸水中加热10min,停止反应。冷却后,以10000g离心20min,收集上清液,冷冻干燥。
继续将该冻干品悬浮于50mM KCl-HCl缓冲液(pH 1.5)中,终浓度为2%(w/v),加入质量体积比为3%的胃蛋白酶(产品编号1.07185,Sigma-Aldrich);水浴37℃反应1h后,在沸水中加热10min,停止反应。冷却后,以10000g离心20min,收集上清液,冷冻干燥。
继续将该冻干品悬浮于50mM Tris-HCl缓冲液(pH 8.0)中,终浓度为2%(w/v),加入质量体积比为6%的酰基转移酶I(产品编号01818,Sigma-Aldrich);水浴57℃,25kHZ超声反应1h后,在沸水中加热10min,停止反应。冷却后,以10000g离心20min,收集上清液,冷冻干燥。
对比例1:
仅用木瓜蛋白酶酶解上述蛋白成品。
将上述蛋白成品悬浮于50mM磷酸盐缓冲液(pH 7.0),终浓度为2%(w/v),加入质量体积比为15%的木瓜蛋白酶(产品编号76220,Sigma-Aldrich);水浴65℃反应3h后,在沸水中加热10min,停止反应。冷却后,以10000g离心20min,收集上清液,冷冻干燥。
3、酶解品的分离纯化及抗氧化活性检测
将在最优酶解参数下得到的酶解液依次通过分子量为30kDa、10kDa的超滤膜,得到分子量<10kDa的滤液。再将滤液通过装有Millipore超滤膜3KD的超滤杯(型号UFSC20001),将所得滤液进行真空冷冻干燥,得到菊芋肽粗品。
菊芋肽粗品采用Sephadex G-25(源叶,货号S14031)装填的凝胶色谱柱(为2.6×100cm洗脱蒸馏水的流速为1mL/min)进行层析分离,经过分别收集在280nm处具有吸收峰的的组分,然后将其真空冷冻干燥。分别测定各组分的抗氧化活性优选出具有较好抗氧化活性的组分。
采用液相色谱串联三重四级杆串联飞行时间质谱(UPLC-Q-TOF,Waters,Manchester,UK)对优选出的组分进行结构鉴定。其鉴定方法按照“ZHUANG H,TANG N,YUANY.Purification and identification of antioxidant peptides from corn glutenmeal[J].Journal of Functional Foods,2013,5(4):1810-1821.”方法进行测定,并比照NCBI关于Helianthus tuberosus提供的相关参考序列,得到如表1所示的菊芋肽。
结果如表1所示,采用实施例1提供的酶解方法制得了菊芋肽Pep1~5,采用对比例1提供的酶解方法制得了菊芋肽Pep6~8。其质谱图如图1和2所示。
表1菊芋肽及序列
4、菊芋肽抗氧化活性检测
DPPH自由基清除率测定:按照“ZHANG T,LI Y,MIAO M,et al.Purification andcharacterisation of a new antioxidant peptide from chickpea(Cicer arietiumL.)protein hydrolysates[J].Food Chemistry,2011,128(1):28-33.”方法进行测定,计算当DPPH自由基清除率为50%时的菊芋肽浓度IC50-1。
ABTS自由基清除率测定:按照“TIRONI V A,AN M C.Amaranth proteins as asource of antioxidant peptides:Effect of proteolysis[J].Food ResearchInternational,2010,43(1):315-322.”方法进行测定,计算当ABTS自由基清除率为50%时的菊芋肽浓度IC50-2。
羟基清除率测定:按照“WANG J,WANG Y,DANG X,et al.Housefly larvaehydrolysate:orthogonal optimization of hydrolysis,antioxidant activity,aminoacid composition and functional properties[J].BMC Research Notes,2013,6(1):1-10.”方法进行测定,计算当羟基清除率为50%时的菊芋肽浓度IC50-3。
超氧负离子清除率测定:按照“LI Y,JIANG B,ZHANG T,et al.Antioxidant andfree radical-scavenging activities of chickpea protein hydrolysate(CPH)[J].Food Chemistry,2008,106(2):444-450.”方法进行测定,计算当超氧负离子清除率为50%时的菊芋肽浓度IC50-4。
铁离子螯合活性测定:按照“SABEENA FARVIN K H,BARON C P,NIELSEN N S,etal.Antioxidant activity of yoghurt peptides:Part 1-in vitro assays andevaluation inω-3enriched milk[J].Food Chemistry,2010,123(4):1081-1089.”等人的方法进行测定,计算当铁离子螯合活性为50%时的菊芋肽浓度IC50-5。
Cu2+螯合活性测定:按照“ZHU L J,CHEN J,TANG X Y,et al.Reducing,radicalscavenging,and chelation properties of in vitro digests of alcalase-treatedzein hydrolysate[J].Journal of Agricultural and Food Chemistry,2008,56(8):2714-2721.”方法进行测定,计算当Cu2+螯合活性为50%时的菊芋肽浓度IC50-6。
脂质过氧化抑制活性测定:按照“CHANDRASEKARA A,SHAHIDI F.Antioxidantphenolics of millet control lipid peroxidation in human LDL cholesterol andfood systems[J].Journal of the American Oil Chemists'Society,2012,89(2):275-285.”方法进行测定,计算当脂质过氧化抑制活性为50%时的菊芋肽浓度IC50-7。
如表2示出了实施例1制得的菊芋肽Pep1、Pep2、Pep3、Pep4、Pep5分别的IC50-1、IC50-2、IC50-3、IC50-4、IC50-5、IC50-6和IC50-7。表3示出了对比例1制得的菊芋肽Pep6、Pep7、Pep8分别的IC50-1、IC50-2、IC50-3、IC50-4、IC50-5、IC50-6和IC50-7。表2、3中,“-”表示未检出。对比表2和3可知,实施例1制得的菊芋肽Pep1、Pep2、Pep3、Pep4、Pep5能够清除DPPH自由基、ABTS自由基、羟基、超氧负离子,并且还能具有同时螯合铁离子和铜离子的活性,还能抑制脂质过氧化。如表3所示,而对比例1提供的菊芋肽Pep6、Pep7、Pep8不具有同时螯合铁离子和铜离子和抑制脂质过氧化的活性,并且抗氧化作用不如菊芋肽Pep1、Pep2、Pep3、Pep4、Pep5全面,抗氧化能力较弱。
表2(μg/ml)
菊芋肽 | Pep1 | Pep2 | Pep3 | Pep4 | Pep5 |
IC50-1 | 237.9±20.6 | 562.2±36.4 | 202.3±17.9 | 302.4±29.1 | 216.7±26.5 |
IC50-2 | 328.1±43.8 | 486.3±43.2 | 286.9±32.0 | 313.5±36.7 | 289.3±37.9 |
IC50-3 | 183.5±24.3 | 359.7±29.5 | 179.3±20.7 | 193.2±29.2 | 156.3±22.8 |
IC50-4 | 436.8±50.9 | 836.2±36.8 | 336.9±36.5 | 368.4±30.3 | 305.5±28.7 |
IC50-5 | 169.3±18.5 | 284.8±28.1 | 187.2±20.3 | 197.6±25.4 | 148.6±15.2 |
IC50-6 | 208.7±21.6 | 245.2±24.8 | 193.2±18.4 | 212.3±22.5 | 183.7±16.9 |
IC50-7 | 188.6±19.9 | 235.2±63.5 | 208.3±21.3 | 213.1±29.3 | 157.4±17.2 |
表3(μg/ml)
菊芋肽 | Pep6 | Pep7 | Pep8 |
IC50-1 | 1559.2±125.2 | 3638.9±539.8 | 4963.8±832.1 |
IC50-2 | 1824.3±162.1 | 4293.5±835.4 | 7548.2±770.9 |
IC50-3 | 2638.9±256.5 | - | - |
IC50-4 | 4195.5±350.4 | - | - |
IC50-5 | - | - | - |
IC50-6 | - | - | - |
IC50-7 | - | - | - |
5、抗氧化稳定性检测
分别将菊芋肽Pep1、Pep2、Pep3、Pep4、Pep5、Pep6、Pep7、Pep8配制成10mg/mL、5mg/mL、1mg/mL、500μg/mL、200μg/mL、100μg/mL,置于70℃水浴处理2h,后再分别采用上述方法检测其IC50-1、IC50-2、IC50-3、IC50-4、IC50-5、IC50-6和IC50-7。
如表4所示,经70℃水浴处理2h后,实施例1制得的菊芋肽Pep1、Pep2、Pep3、Pep4、Pep5仍然能够清除DPPH自由基、ABTS自由基、羟基、超氧负离子,并且还能具有同时螯合铁离子和铜离子的活性,还能抑制脂质过氧化。表5中,“-”表示未检出。如表5所示,经70℃水浴处理2h后,对比例1提供的菊芋肽Pep6、Pep7、Pep8不具有同时螯合铁离子和铜离子和抑制脂质过氧化的活性,并且抗氧化作用不如菊芋肽Pep1、Pep2、Pep3、Pep4、Pep5全面,抗氧化能力较弱。并且,经70℃水浴处理2h后,Pep6丧失了对羟基、超氧负离子的清除作用,Pep7、Pep8几乎丧失了抗氧化作用。
表4(μg/ml)70℃水浴处理2h
菊芋肽 | Pep1 | Pep2 | Pep3 | Pep4 | Pep5 |
IC50-1 | 535.5±42.8 | 936.2±77.1 | 439.2±30.5 | 516.3±48.6 | 352.5±31.6 |
IC50-2 | 428.1±37.5 | 821.3±64.2 | 332.7±49.1 | 489.7±52.4 | 320.7±40.2 |
IC50-3 | 352.6±31.9 | 562.2±40.5 | 367.8±32.9 | 363.8±34.5 | 193.3±26.4 |
IC50-4 | 802.5±62.4 | 1036.8±92.8 | 527.3±41.6 | 497.3±48.2 | 389.2±36.5 |
IC50-5 | 322.2±40.3 | 497.0±53.7 | 259.6±35.7 | 352.6±32.0 | 186.9±22.7 |
IC50-6 | 343.8±32.1 | 426.3±37.5 | 326.7±31.2 | 423.2±37.2 | 213.5±20.8 |
IC50-7 | 469.5±50.5 | 378.6±43.5 | 292.6±28.6 | 408.7±31.6 | 189.4±23.5 |
表5(μg/ml)70℃水浴处理2h
菊芋肽 | Pep6 | Pep7 | Pep8 |
IC50-1 | 9635.8±263.4 | 18425.7±2671.2 | - |
IC50-2 | 19367.5±649.7 | - | - |
IC50-3 | - | - | - |
IC50-4 | - | - | - |
IC50-5 | - | - | - |
IC50-6 | - | - | - |
IC50-7 | - | - | - |
6、抑菌试验
测试用菌株有沙门氏菌(肠炎沙门氏菌肠炎亚种ATCC14028,上海北诺生物科技有限公司)、变形杆菌(奇异变形杆菌CMCC49027,上海北诺生物科技有限公司)、大肠艾希氏菌(大肠埃希氏菌ATCC35218)、耶尔森菌(Yersinia enterocolitica小肠结肠炎耶尔森菌上海北诺生物科技有限公司)。其中,大肠艾希氏菌用LB培养基培养。沙门氏菌用SS(Salmonella-Shigella)培养基培养。变形杆菌用营养琼脂培养基培养。耶尔森菌用CIN-1培养基培养。
用接种环挑取个测试菌种接种于带玻璃珠的无菌水中,充分分散后注入至各自测试用融化的琼脂培养基黄总,充分冷却后。用镊子将圆片滤纸在含有3mg/mL菊芋肽Pep1、Pep2、Pep3、Pep4、Pep5、Pep6、Pep7或Pep8的水溶液中浸渍1h后取出,置于带菌培养平板中央,盖上盖子,于30℃培养24h后,测量抑菌圈大小。
表6中,“-”表示未检出。表6示出了3mg/mL的菊芋肽Pep1、Pep2、Pep3、Pep4、Pep5、Pep6、Pep7或Pep8对沙门氏菌、变形杆菌、大肠艾希氏菌和耶尔森菌的抑菌圈大小。结果可知,3mg/mL的菊芋肽Pep1、Pep2、Pep3、Pep4、Pep5对沙门氏菌、变形杆菌、大肠艾希氏菌和耶尔森菌均具有抑菌作用,而3mg/mL的菊芋肽Pep6、Pep7或Pep8对沙门氏菌、变形杆菌、大肠艾希氏菌和耶尔森菌均无抑菌作用。
表6抑菌圈大小(mm)
菊芋肽 | 沙门氏菌 | 变形杆菌 | 大肠艾希氏菌 | 耶尔森菌 |
Pep-1 | 5.23±0.52 | 5.72±0.73 | 8.30±0.42 | 4.08±0.34 |
Pep-2 | 5.09±0.49 | 5.58±0.64 | 8.16±0.66 | 4.27±0.41 |
Pep-3 | 4.85±0.37 | 5.47±0.59 | 8.36±0.52 | 4.36±0.32 |
Pep-4 | 4.69±0.35 | 5.32±0.29 | 8.49±0.57 | 4.21±0.26 |
Pep-5 | 5.86±0.67 | 6.14±0.58 | 9.16±0.83 | 4.41±0.39 |
Pep-6 | - | - | - | - |
Pep-7 | - | - | - | - |
Pep-8 | - | - | - | - |
7、α-葡萄糖苷酶抑制活性测定
进一步对本发明提供的菊芋肽的α-葡萄糖苷酶抑制活性测定。
(1)供试品
实验组:用0.01mol/LPBS缓冲液配制含有不同浓度的菊芋肽溶液,Pep1、Pep2、Pep3、Pep4、Pep5、Pep6、Pep7、Pep8配制成10mg/mL、5mg/mL、1mg/mL、500μg/mL、200μg/mL、100μg/mL,作为实验组。
阳性组:取新鲜菊芋块茎、洗净、晾干、切片、60℃烘干、粉碎、过40目筛、菊芋干粉。取200g新鲜菊芋干粉用去离子水(固液比1:20)重复提取两次,提取温度为90℃。合并提取液过滤两次得滤液,减压浓缩至250mL。分次加入一定体积无水乙醇使乙醇终浓度分别为40%,60%,80%,90%并分别在4℃冰箱静置过夜,收集各种醇沉浓度下的沉淀,复溶后离心去除杂质。将各种醇沉浓度下的提取液分别用D101大孔吸附树脂除杂并脱色,将洗脱液分别减压浓缩至200mL,分别加入1/3体积Sevage试剂(三氯甲烷:正丁醇-4:1)搅拌90分钟后置于分液漏斗中萃取30分钟,BCA蛋白浓度测定试剂盒检测蛋白质含量,重复上述操作直至无蛋白质。加水减压浓缩去除有机试剂至165mL,将溶液分别置于截留分子量为1000Da的透析袋中在4℃冰箱中用流动水透析48h,冷冻干燥3天得到菊芋粗多糖。将菊芋粗多糖配置成10mg/mL、5mg/mL、1mg/mL、500μg/mL、200μg/mL、100μg/mL,作为阳性组。
具体如下:向酶标板中加入0.4mL实验组或阳性组的供试品溶液。设置空白组和阴性组,分别加入0.4mL的0.01mol/LPBS缓冲液。分别向实验组、阳性组和阴性组加入0.3U/mL的α-葡萄糖苷酶(G3651,Sigma-Aldrich)溶液0.4mL,振荡均匀后于37℃恒温保存5min实验组、阳性组、空白组和阴性组加入2.5mmol/L PNPG溶液0.4mL,于370C反应30min后加1.6mL0.2mol/LNa2CO3溶液终止反应,以PBS缓冲液为空白对照,于405nm处测定吸光度,每组重复3次,计算8种菊芋肽及菊芋多糖对α-葡萄糖苷酶的抑制率。其中,α-葡萄糖苷酶的抑制率=[1-(OD实验组/阳性-OD空白组)]/(OD阴性组-OD空白组)×100%。
表7α-葡萄糖苷酶的抑制率%
菊芋肽 | 10mg/mL | 5mg/mL | 1mg/mL | 500μg/mL | 200μg/mL | 100μg/mL |
Pep-1 | 49.36±2.37 | 36.57±3.05 | 24.72±1.36 | 18.35±1.15 | 11.12±1.41 | 2.35±0.16 |
Pep-2 | 48.32±2.18 | 34.29±3.26 | 22.49±1.82 | 17.62±1.63 | 10.26±1.23 | 2.09±0.28 |
Pep-3 | 47.59±2.44 | 35.26±2.82 | 25.18±2.15 | 16.49±1.35 | 9.34±0.79 | 2.34±0.31 |
Pep-4 | 50.13±2.59 | 37.29±4.12 | 28.37±2.32 | 19.57±1.68 | 12.36±1.16 | 3.27±0.48 |
Pep-5 | 51.32±2.72 | 39.21±3.05 | 29.53±2.51 | 20.35±1.71 | 13.53±1.25 | 5.08±0.55 |
Pep-6 | - | - | - | - | - | - |
Pep-7 | - | - | - | - | - | - |
Pep-8 | - | - | - | - | - | - |
菊芋多糖 | 26.84±2.68 | 17.36±1.55 | 10.34±0.73 | 5.24±0.19 | - | - |
表7示出了浓度为10mg/mL、5mg/mL、1mg/mL、500μg/mL、200μg/mL、100μg/mL的本发明提供的8种菊芋肽和菊芋多糖分别对α-葡萄糖苷酶的抑制率,其中,“-”表示未检出。由表7可知,Pep1、Pep2、Pep3、Pep4、Pep5和菊芋多糖对α-葡萄糖苷酶具有抑制活性,并且Pep1、Pep2、Pep3、Pep4、Pep5对α-葡萄糖苷酶的抑制活性高于菊芋多糖,并且在浓度低至100μg/mL时仍然检测出具有抑制活性。由此说明,本发明提供的菊芋肽Pep1、Pep2、Pep3、Pep4、Pep5可作为α-葡萄糖苷酶抑制剂,以调节体内糖代谢紊乱的调节剂或作为糖尿病药物。
8、动物实验
(1)遗传性高血糖实验
遗传性高血糖小鼠:KK/Upj-Ay/J小鼠,3月龄,体质量35~45g,雌雄各半,具有高血糖、高胰岛素、葡萄糖耐受不良特征,合格证号00080269,北京华阜康生物科技股份有限公司提供,许可证号SCXK11-00-0006。
取空腹血糖在11.0mmol/L以上的KK/Upj-Ay/J小鼠,雌雄各半,随机分为模型组、实验组和阳性组。模型组每天灌胃蒸馏水1次。阳性组每天灌胃3mg/kg体重的优降糖(天津太平洋制药有限公司)。实验组每天灌胃100mg/kg体重的菊芋肽Pep1、Pep2、Pep3、Pep4、Pep5、Pep6、Pep7或Pep8。连续实验4周,给药12h禁食,末次给药后1.5h从眼眶取血测定血糖。采用血糖测定试剂盒(葡萄糖氧化酶法,北京利德曼生化技术有限公司)检测血糖含量。
表8遗传性高血糖小鼠空腹血糖含量(mM)
由表8可知,给药4周后,与对照组相比,模型组小鼠的体质量显著下降(P<0.01);与模型组相比,各给药组小鼠体质量显著升高(P<0.05),其中复方桦树茸降糖片组小鼠的体质量与桦树茸多糖组及苦荞多糖组相比,虽无显著性差异,但呈升高趋势。说明复方桦树茸降糖片可减轻糖尿病小鼠的消瘦症状,且效果略优于桦树茸多糖和苦荞多糖。
(2)糖尿病并发症及肾脏实验
雄性SPF级m/m正常小鼠(5周龄)和雄性SPF级db/db糖尿病模型小鼠(5周龄)购于常州卡文斯实验动物有限公司,动物许可证编号:2018D046。
将m/m小鼠设置为正常组,每日灌胃等量生理盐水。
db/db糖尿病模型小鼠在适应性饲养7d后测了初始血糖,与正常m/m小鼠相比,均为糖尿病小鼠,符合实验要求。将db/db小鼠随机分为模型组,每日灌胃等量生理盐水;实验组每日灌胃100mg/kg体重的菊芋肽Pep1、Pep2、Pep3、Pep4、Pep5、Pep6、Pep7或Pep8。阳性组,每日灌胃300mg/kg盐酸二甲双胍悬浮液。
小鼠精神状态的观察以及体重和血糖的测定观察小鼠的生理状态,毛发和采食量饮水量和***情况,每周测定小鼠体重,选择小鼠适应性喂养7d后的初始体重,以及灌胃第2、4周后的体重数据进行统计分析。
正常组小鼠生理精神都处于活跃状态,采食量和饮水量正常,***量正常(此处数据在本文中未呈现)。模型组采食量和饮水量明显增多,***量大,到实验中后期出现便秘的情况。行动迟缓呆滞,毛发粗糙,甚至局部掉毛现象,垫料潮湿,须3d更换一次垫料。阳性组和实验组(Pep1~Pep5)生理状态有所改善,毛发顺滑有光泽。
小鼠尾静脉取血,用血糖仪和血糖试纸测定每周血糖值,选择小鼠初始随机血糖,以及灌胃第2、4周后禁食8h的空腹血糖值进行数据分析。结果如表9所示。相对于正常组,模型组在第2、4周空腹血糖含量均显著升高。而相对于模型组,阳性组和实验组(Pep1~Pep5)小鼠空腹血糖含量显著降低,并且分别于第2、4周实验组(Pep1~Pep5)小鼠空腹血糖含量均低于阳性组。由此说明,本发明提供的菊芋肽Pep1~Pep5不仅能够降低db/db糖尿病小鼠空腹血糖含量,而且效果优于盐酸二甲双胍。
表9db/db糖尿病实验各组小鼠空腹血糖含量(mM)
表10示出了db/db糖尿病实验各组小鼠体重数据。结果可知,相对于正常组,模型组在第2、4周小鼠体重均显著升高。而相对于模型组,阳性组和实验组(Pep1~Pep5)小鼠小鼠体重显著降低,并且分别于第2、4周实验组(Pep1~Pep5)小鼠小鼠体重均低于阳性组。由此说明,本发明提供的菊芋肽Pep1~Pep5不仅能够缓解db/db糖尿病小鼠糖尿病引起的肥胖,而且效果优于盐酸二甲双胍。
表10db/db糖尿病实验各组小鼠体重(g)
测定小鼠脏器指数有助于评估菊芋肽对糖尿病小鼠的降血糖活性。实验进行4周后,所有动物用***吸入法麻醉,眼球取血放于2mL离心管中,后脱颈处死实验鼠,30min后于4℃,3000×g离心10min,取上层血清。取其肾脏、肝脏、脾脏和心脏称重,后将脏器、血清液氮速冻储存在-80℃下供后续实验分析使用。脏器指数=小鼠脏器重量/小鼠体重×100%。
由表11可知,正常组小鼠各脏器指标均显著低于模型组,表明糖尿病已经造成db/db小鼠不同程度的脏器病变。阳性组和实验组(Pep1~Pep5)的肾脏指数均显著低于模型组。说明本发明提供的菊芋肽Pep1~Pep5能够保护db/db糖尿病小鼠的脏器,减少糖尿病并发症对小鼠的危害作用。
表11db/db糖尿病实验各组小鼠脏器指数
肾脏丙二醛是脂质过氧化物,糖尿病患者体内自由基过多,氧化反应剧烈,丙二醛含量较高,是肾脏损伤的指标之一。肾功能相关指标的测定治疗4周后,收集24h尿液。以牛血清白蛋白(BSA)作为标准样品,采用Bradford法测定尿蛋白含量。采用ELISA试剂盒(罗氏诊断产品(上海)有限公司)测定血尿素氮、血清肌酐、血尿酸以及肾组织匀浆中丙二醛的含量。各个指标的测定方法严格按照试剂盒说明书操作。
表12db/db糖尿病实验各组小鼠肾脏血清指标
表12示出了db/db糖尿病实验各组小鼠肾脏血清指标。相对于正常组,模型组db/db糖尿病小鼠各项指标均显著升高。而阳性组和实验组(Pep1~Pep5)的肾脏指数均显著低于模型组。说明本发明提供的菊芋肽Pep1~Pep5能够保护db/db糖尿病小鼠的肾脏,减少糖尿病高糖代谢对小鼠肾脏的危害作用。而菊芋肽Pep6~Pep8并未明显降低db/db糖尿病小鼠各项肾脏血清指标,并不具有明显的对小鼠肾脏保护作用。
由图3可知,使用HE进行的组织学分析,正常组正常小鼠血细胞分布均匀,肾小球、肾小囊正常无明显的病理改变,模型组血细胞分布凌乱,肾小囊挤压性萎缩,基底膜增厚,系膜区变宽,这些都是糖尿病肾病的典型病理症状。而采用菊芋肽Pep6~Pep8给药db/db糖尿病小鼠并未取代改善,而采用菊芋肽Pep1~Pep5给药db/db糖尿病小鼠其糖尿病肾病病理症状得到改善,说明本发明提供你的菊芋肽Pep1~Pep5对db/db糖尿病小鼠糖尿病肾病具有改善作用,对肾脏起到了一定程度的保护作用,推测可能是由于其能够显著的降血糖活性,减少ROS,从而降低了胶原蛋白变性导致的基底膜增厚和细胞间基质沉积。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。
Claims (7)
1.降血糖抗氧化的菊芋肽,其序列如如SEQ ID NO.2所示,所述菊芋肽来源于菊芋(Helianthus tuberosus(L.1753))。
2.权利要求1所述的菊芋肽的制备方法,其特征在于,包括:
将新鲜菊芋叶片用碱液提取后,榨汁,过滤,滤液用酸液调节pH至6.5,絮凝,离心分离,干燥,得到蛋白成品;
对所述蛋白成品进行木瓜蛋白酶、胃蛋白酶和酰基转移酶I酶解;
其中,采用所述木瓜蛋白酶酶解的步骤包括:将所述蛋白成品悬浮于pH 7.0、50mM磷酸盐缓冲液,终浓度为2%(w/v),加入质量体积比为4%的木瓜蛋白酶;水浴65℃反应1h后,在沸水中加热10min,停止反应;冷却后,以10000g离心20min,收集上清液,冷冻干燥,得到第一冻干品;
其中,采用所述胃蛋白酶酶解的步骤包括:将所述第一冻干品悬浮于pH 1.5、50mMKCl-HCl缓冲液中,终浓度为2%(w/v),加入质量体积比为3%的胃蛋白酶;水浴37℃反应1h后,在沸水中加热10min,停止反应;冷却后,以10000g离心20min,收集上清液,冷冻干燥,得到第二冻干品;
其中,采用所述酰基转移酶I酶解的步骤包括:将所述第二冻干品悬浮于pH 8.0、50mMTris-HCl缓冲液中,终浓度为2%(w/v),加入质量体积比为6%的酰基转移酶I;水浴57℃,25kHZ超声反应1h后,在沸水中加热10min,停止反应;冷却后,以10000g离心20min,收集上清液,冷冻干燥。
3.一种抗氧化的肽组合物,包含Pep2、Pep3、Pep4或Pep5,Pep2、Pep3、Pep4和Pep5的氨基酸序列依次如SEQ ID NO.2~5所示,所述Pep2、Pep3、Pep4和Pep5来源于菊芋(Helianthus tuberosus)L.1753的叶片。
4.一种抑菌的肽组合物,包含Pep2、Pep3、Pep4或Pep5,Pep2、Pep3、Pep4和Pep5的氨基酸序列依次如SEQ ID NO.2~5所示,所述Pep2、Pep3、Pep4和Pep5来源于菊芋(Helianthustuberosus)L.1753的叶片。
5.权利要求1所述的菊芋肽在制备治疗遗传性高血糖药物中的应用。
6.权利要求1所述的菊芋肽在制备改善糖尿病肾病的病理症状药物中的应用。
7.权利要求1所述的菊芋肽在制备保护肾脏制品中的应用。
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