CN112794931A - 一种具有抗氧化及抗炎活性的豆腐柴叶rg-i果胶及其制备方法和应用 - Google Patents
一种具有抗氧化及抗炎活性的豆腐柴叶rg-i果胶及其制备方法和应用 Download PDFInfo
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- CN112794931A CN112794931A CN202110122216.5A CN202110122216A CN112794931A CN 112794931 A CN112794931 A CN 112794931A CN 202110122216 A CN202110122216 A CN 202110122216A CN 112794931 A CN112794931 A CN 112794931A
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- pectin
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- premna microphylla
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Abstract
本发明公开了一种具有抗氧化及抗炎活性的豆腐柴叶RG‑I果胶及其制备方法和应用,属于功能食品及食品技术领域,该提取方法包括以下步骤:豆腐柴鲜叶清洗、去杂、干燥、粉碎后,超声辅助碱法提取,提取液过滤,取滤液浓缩、醇沉、节能热泵干燥得RG‑I果胶粗品,复溶、纯化、干燥得豆腐柴叶天然RG‑I果胶成品。本制备方法具有流程简单、提取率高、活性保护好、无有害加工助剂残留等优点,适宜规模化生产;本制备方法生产的豆腐柴叶RG‑I果胶无细胞毒性,具有极强的抗氧化能力和较好的抗慢性炎性反应活性,显著降低肠道有害微生物诱导的促炎性因子水平,可作为功能性食品原料应用于健康食品和功能食品。
Description
技术领域
本发明涉及功能食品及食品技术领域,具体涉及到一种具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶及其制备方法和应用。
背景技术
豆腐柴(Premna microphylla Turcz)又名观音草或腐蜱,是马鞭草科多年生落叶灌木,广泛分布于我国华东、华南、华中、四川、贵州等省区,含有大量对人体健康有益的天然食用果胶成分,是一种药食兼用的植物,现为重庆市地方特色食物资源。其根、茎和叶入药,有清热解毒、排毒养颜、祛风止痛、收敛止血、强筋健骨、祛风湿等功效,主治毒蛇咬伤,无名肿毒,创伤出血等。豆腐柴叶富含果胶、蛋白质、粗纤维、粗脂肪、维生素和矿质元素,自古以来在民间就被用来制作凝胶状的传统食品“神仙豆腐”,味道鲜美,风味独特。
果胶属于多糖类化合物,通常为白色或淡黄色粉末,无臭,味微甜,稍带酸味,不溶于乙醇、甲醇等有机溶剂,溶于热水,微溶于冷水,大量存在于植物的果实、块茎、块根内,在植物中一部分呈溶解状态存在于细胞液中,一部分充塞在植物细胞间质。果胶由于具有优良的凝胶性和乳化稳定性,使其成为食品工业中一种重要的添加剂,现已被广泛应用于果酱、果冻、糖果、乳酸及果汁饮料等食品中。近年来,作为可溶性膳食纤维,果胶也被发现具有抗氧化、抗腹泻、抗癌、治疗糖尿病等功效,具有很多潜在的生物特性。尤其是鼠李糖半乳糖醛酸聚糖I型(RG-I)果胶由于其丰富的侧链和独特的单糖组成特点,表现出比聚半乳糖醛酸聚糖型(HG)果胶更显著的生物活性和独特的加工特性,作为新型的功能性食品原料、药物、保健品及化妆品活性添加物,具有广阔的市场前景。目前商品果胶主要是提取自柑橘皮、柠檬皮、苹果皮、柚子皮中的HG型果胶,主要作为增稠剂、稳定剂使用,远远不能满足市场需求,因此亟待开发其他果胶原料,以弥补不足。豆腐柴叶中果胶含量可达30~40%,在目前已知蔬果类植物中首屈一指,是优质的果胶原料来源。
目前常用的果胶提取方法有热酸法、草酸铵法、醇氨法、混合酸提取法、纤维素酶法等。但上述技术主要存在以下问题:(1)酸性环境下提取的果胶粘度较高,溶解速率较慢,溶解时间较长;(2)热酸法可能引起果胶的水解和降解,导致产率降低,所使用的无机试剂易对环境造成危害,酸性萃取液易腐蚀设备等;(3)草酸铵法所用试剂草酸铵具有毒性,所提取的果胶会有部分草酸铵残留而难以清除;(4)利用纤维素酶等酶法提取果胶的成本较高;(5)利用上述方法提取的果胶主要为HG型果胶(高甲氧基果胶(HMP)和低甲氧基果胶(LMP)),主要用于食品增稠、凝胶,体内外抗氧化及抗炎作用的报道较少。同时,豆腐柴果胶现有提取手段主要包括热酸法、草酸铵法、混合酸提取法,现有技术手段获取的豆腐柴果胶也均为HG型果胶,且未见确切的生物活性报道。
发明内容
针对上述的不足或缺陷,本发明的目的是提供一种具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶及其制备方法和应用,可有效解决现有果胶提取技术无法获取豆腐柴叶中RG-I果胶的问题。
为达上述目的,本发明采取如下方案:
本发明提供一种具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶的制备方法,包括以下步骤:
步骤(1):豆腐柴叶原料预处理,得到豆腐柴叶粉末;
步骤(2):将步骤(1)所得的豆腐柴叶粉末利用超声辅助碱法提取,得到提取液;然后将提取液过滤、离心、真空减压浓缩得到浓缩液;再向浓缩液中加入乙醇混匀,得到沉淀产物,过滤沉淀并干燥,得到RG-I果胶粗品;其中,碱法提取时料液质量体积比为1:10~50,乙醇与浓缩液体积比为2~10:1;
步骤(3):将步骤(2)所得的RG-I果胶粗品复溶于水,然后过滤去除沉淀,得到RG-I果胶粗品滤液,再经DEAE-纤维素层析柱纯化,收集组分后经真空减压浓缩,膜分离,干燥,得到豆腐柴叶RG-I果胶成品。
进一步地,步骤(1)中豆腐柴叶原料预处理过程为:采摘成熟新鲜的豆腐柴叶,去杂、清洗、低温干燥、粉碎后过40~80目筛,取筛下物。
进一步地,步骤(1)中低温干燥为热泵干燥或真空冷冻干燥;热泵干燥的参数为:温度为80℃以下;真空冷冻干燥的参数为:升华干燥阶段温度-50~-10℃,解析干燥阶段0~50℃,真空度为1~20Pa。
进一步地,步骤(2)中超声辅助碱法提取的参数为:超声波功率为400~600W,pH为8~12,温度为50~95℃,提取时间为30~150分钟。
进一步地,步骤(2)中离心的转速为4000~8000r/min,时间为10~30分钟。
进一步地,步骤(2)中干燥为热泵干燥或真空冷冻干燥;热泵干燥的参数为:温度为60℃以下;真空冷冻干燥的参数为:升华干燥阶段温度-50~-10℃,解析干燥阶段温度40-60℃,真空度1~10Pa。
进一步地,步骤(3)中DEAE-纤维素层析柱纯化的参数为:洗脱液为0.1~1mol/L的NaCl溶液,RG-I果胶粗品滤液的浓度为0.5~1mol/L,采用苯酚-硫酸法监测多糖浓度。
进一步地,步骤(3)中收集的组分为通过苯酚-硫酸法监测多糖浓度结果中多糖浓度最高的组分。
进一步地,步骤(3)中膜分离的具体过程为:使用截留分子量为2kDa的膜分离设备纯化,得到截留部分,再经40℃-60℃热泵干燥或真空冷冻干燥;真空冷冻干燥参数:升华干燥温度-40℃~-10℃,解析干燥温度50-70℃,真空度1~10Pa。
进一步地,步骤(2)和步骤(3)中真空减压浓缩的参数为:真空度为-0.095~-0.01MPa,温度为50~70℃,浓缩至原溶液体积的1/4~1/2。
本发明还提供上述制备方法制得的具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶。
本发明还提供上述豆腐柴叶RG-I果胶在制备功能性食品中的应用,如可降低肠道有害微生物等功能性食品。
本发明具有以下优点:
1、本发明提供一种具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶的制备方法,在碱处理条件下通过控制超声辅助碱提法的功率、温度、处理时间和pH参数,能够充分结合超声空化作用和碱性pH,通过破坏纤维素和半纤维素的交联聚合物网络结构而促进果胶的释放,且碱性条件容易破坏果胶的酯化羧基,诱导HG结构域的降解,剩余一些中性糖附着在RG-I骨架上,从而显示出高比例的RG-I果胶比例,达到减少HG果胶的提取、增加RG-I果胶溶出且不破坏其活性结构的效果;且该制备方法具有流程简单、提取率高、活性保护好、无有害加工助剂残留等优点,适宜规模化生产;
2、本发明提供的具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶的制备方法的提取率显著增加,多糖含量更高,蛋白质等杂质含量低;
3、本发明提供的豆腐柴叶RG-I果胶在扫描电镜下观察可知,该果胶颗粒度更小,具有粗糙、松散的表面结构,表现出具有优良的水溶性;
4、本发明提供的豆腐柴叶RG-I果胶具有更为丰富的单糖组成、更多中性糖侧链,且具有更高的细胞内自由基清除能力,可降低细胞炎性反应,显著降低肠道有害微生物胞外脂多糖(LPS)诱导的肿瘤坏死因子(TNF-α)、白介素-6(LP-6)、白介素-1β(IL-1β)等促炎性因子水平,具有良好的生物活性,可用于制备功能性食品,具有实际的应用价值。
附图说明
图1为本发明实施例1和对比例1提取得到的果胶表面微观结构图。
具体实施方式
下面通过具体实施例对本发明作进一步说明,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。
实施例1
本实施例1提供了一种具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶及其制备方法,具体包括以下步骤:
(1)原料预处理:
将摘于5月下旬的豆腐柴鲜叶去杂、清洗、干燥、粉碎后过60目筛;
(2)豆腐柴叶果胶的提取:
用0.5mol/L的NaOH溶液调整pH为10,90℃温度下浸提60min,然后在60℃、500W的温度和超声条件下继续浸提60min。所得提取液用滤布过滤,除去滤渣,以5000r/min的转速离心15min,浓缩醇沉,洗涤沉淀,于45℃下热泵干燥,得到RG-I果胶粗品,其中,提取时料液比为1:20,醇沉时乙醇与浓缩液体积比为3:1;
(3)豆腐柴叶果胶的纯化:
将RG-I果胶粗品复溶过滤,调整浓度为0.5mol/L,采用DEAE-纤维素层析柱法纯化,用溶度0.3~0.5M的NaCl溶液进行洗脱,使用苯酚-硫酸法监测多糖浓度,收集多糖浓度最高组分的果胶溶液,滤膜过滤(滤膜孔径小于0.5μm),真空减压浓缩至原体积1/4,45℃热泵干燥得到豆腐柴叶RG-I果胶成品;其中,真空减压浓缩的真空度为-0.05MPa,温度为60℃,浓缩至原体积1/4。
实施例2
本实施例2提供了一种具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶及其制备方法,具体包括以下步骤:
(1)原料预处理:
将摘于5月下旬的豆腐柴鲜叶去杂、清洗、干燥、粉碎后过60目筛。
(2)豆腐柴叶果胶的提取:
用0.5mol/L的NaOH溶液调整pH为8.5,90℃温度下浸提60min,然后在60℃、500W的温度和超声条件下继续浸提60min。所得提取液用纱布过滤,除去滤渣,以5000r/min的转速离心15min,浓缩醇沉,洗涤沉淀,冷冻干燥得到RG-I果胶粗品,冷冻干燥参数为:升华干燥温度-30℃,解析干燥温度50℃,真空度5Pa。其中,提取时料液比为1:20,醇沉时乙醇与浓缩液体积比为3:1;
(3)豆腐柴叶果胶的纯化:
将RG-I果胶粗品复溶过滤,调整浓度为0.5mol/L,采用DEAE-纤维素层析柱法纯化,用溶度0.1~0.5M的NaCl溶液进行洗脱,使用苯酚-硫酸法监测多糖浓度。收集多糖浓度最高组分的果胶溶液,滤膜过滤(滤膜孔径小于0.5μm),真空减压浓缩至原体积1/4,真空冷冻干燥得到豆腐柴叶RG-I果胶成品,冷冻干燥参数为:升华干燥温度-20℃,解析干燥温度60℃,真空度5Pa;其中,真空减压浓缩的真空度为-0.06MPa,温度为60℃,浓缩至原体积1/4。
实施例3
本实施例3提供了一种具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶及其制备方法,具体包括以下步骤:
(1)原料预处理:
将摘于5月下旬的豆腐柴鲜叶去杂、清洗、干燥、粉碎后过60目筛。
(2)豆腐柴叶果胶的提取:
用0.5mol/L的NaOH溶液调整pH为11.5,90℃温度下浸提60min,然后在60℃、500W的温度和超声条件下继续浸提60min。所得提取液用纱布过滤,除去滤渣,以5000r/min的转速离心15min,浓缩醇沉,洗涤沉淀,冷冻干燥得到RG-I果胶粗品,冷冻干燥参数为:升华干燥温度-40℃,解析干燥温度55℃,真空度5Pa。其中,提取时料液比为1:20,醇沉时乙醇与浓缩液体积比为3:1;
(3)豆腐柴叶果胶的纯化:
将RG-I果胶粗品复溶过滤,调整浓度为0.5mol/L,采用DEAE-纤维素层析柱法纯化,用溶度0.1~0.5M的NaCl溶液进行洗脱,使用苯酚-硫酸法监测多糖浓度。收集多糖浓度最高组分的果胶溶液,滤膜过滤(滤膜孔径小于0.5μm),真空减压浓缩至原体积1/4,真空冷冻干燥得到豆腐柴叶RG-I果胶成品,冷冻干燥参数为:升华干燥温度-25℃,解析干燥温度65℃,真空度5Pa;其中,真空减压浓缩的真空度为-0.05MPa,温度为60℃,浓缩至原体积1/4。
对比例1
本对比例1提供了一种豆腐柴叶果胶的制备方法,具体包括以下步骤:
(1)原料预处理:
豆腐柴鲜叶摘自5月下旬,鲜叶去杂、清洗、干燥、粉碎后过60目筛;
(2)豆腐柴叶果胶的提取:
用0.5mol/L的HCl溶液调整pH为4,80℃温度下浸提45min,然后在60℃、500W超声条件下浸提90min。所得提取液用滤布过滤,除去滤渣,以4000r/min的转速离心12min,浓缩醇沉,洗涤沉淀,真空冷冻干燥得RG-I果胶粗品,冷冻干燥参数为:升华干燥温度-25℃,解析干燥温度50℃,真空度5Pa;其中,提取时料液比为1:20,醇沉时乙醇与浓缩液体积比为3:1;
(3)豆腐柴叶果胶的纯化:
将RG-I果胶粗品复溶过滤,调整浓度为0.5mol/L,采用DEAE-纤维素层析柱法纯化,用溶度0.1~0.5M的NaCl溶液进行洗脱,使用苯酚-硫酸法监测多糖浓度。收集多糖浓度最高组分的果胶溶液,滤膜过滤(滤膜孔径小于0.5μm),真空减压浓缩至原体积1/4,真空冷冻干燥得到豆腐柴叶RG-I果胶成品,冷冻干燥参数为:升华干燥温度-25℃,解析干燥温度65℃,真空度5Pa;其中,真空减压浓缩的真空度为-0.08MPa,温度为60℃,浓缩至原体积1/4。
实验例
为了考察本发明制得的豆腐柴叶RG-I果胶的性质,本实验例将实施例1-3和对比例1所得的豆腐柴叶果胶通过高效液相(HPLC)和高效凝胶色谱(HPGPC)分别测定果胶的单糖组成和分子量;采用自由基清除实验和细胞实验测定其抗氧化及抗炎活性,细胞实验测定其抗氧化及抗炎活性时分为正常组、模型组、阳性对照组和处理组。模型组为细菌胞外脂多糖LPS刺激后的巨噬细胞,100mg/L乙酰半胱氨酸处理组为阳性对照组,50-100mg/L豆腐柴叶RG-I果胶干预为处理组,测定氧化应激水平(ROS)和丙二醛(MDA)水平,以及细胞抗氧化酶类过氧化氢酶CAT、超氧化物歧化酶SOD、谷胱甘肽过氧化物酶GPx的活性,促炎性因子肿瘤坏死因子TNF-α、白介素-6(IL-6)、白介素-1β(IL-1β)水平;通过扫描电镜(SEM)观察实施例1和对比例1所得的豆腐柴叶果胶的微观结构。
1.单糖组成的测定方法
采用1-苯基-3-甲基-5-11吡唑碄酮(PMP)柱前衍生化反相高效液相色谱法(RP-HPLC)分析果胶多糖的单糖组成。称取10mg左右多糖样品于20mL的钳口瓶中,加入5mL的2mol/L的TFA,充氮气封管,于110℃烘箱中水解6h。冷却后取1mL溶液加入1mL甲醇,70℃水浴用氮气吹干,重复加甲醇多次以去除TFA;加入1mL 0.3mol/L的NaOH溶液充分溶解残渣,制备多糖水解液。分别取400μL的混合单糖标准液或多糖水解液于5mL的具塞试管中,加入400μL的PMP甲醇溶液,漩涡混匀,于70℃水浴中反应2h后冷却至室温。加入400μL的0.3mol/L的盐酸中和至pH中性,加入1200μL水和氯仿,振荡混匀去除氯仿相,重复2次。将水相用0.45μm微孔膜过滤后用HPLC进样分析。色谱条件:色谱柱C18柱,250mm×4.6mm,粒度5μm;流动相A:100mM磷酸钠缓冲液(pH6.6);流动相B:乙腈;检测波长:250nm;柱温:30℃;流速1mL/min;进样量5μL。
2.分子量的测定方法
采用高效凝胶渗透色谱法(HPGPC)测定样品的分子量。取适量多糖用超纯水(0.02%叠氮化钠)配制成约2-3mg/mL的溶液,用0.22μm微孔膜过滤后进行色谱分析。色谱条件:色谱柱Shodex OH-pak SB-806和803串联;流动相:0.02M的磷酸二氢钠缓冲液(pH6);柱温:40℃;流速:1mL/min;进样量:500μL;Mw测试范围:200-109Daltons。通过将测量值与葡聚糖标准曲线比较来确定分子量。
3.微观结构观察方法
采用扫描电镜SEM观察果胶微观形态。取适量样品粉末喷金后观察果胶微观形态,加速电压15kV。
4.抗氧化活性测定方法
4.1羟自由基清除能力测定
将1mL不同浓度的果胶溶液(0.25、0.5、1、2和4mg/mL)依次加入1mL FeSO4溶液(1.5mM)、0.7mL H2O2溶液(3%v/v)和0.3mL水杨酸溶液(20mM),充分摇匀。在37℃下孵育30min后,在734nm处测定吸光度值。以同浓度的VC作为阳性对照。计算羟基自由基清除能力,计算公式如下:
羟基自由基清除率(%)=[1-(A1-A2)/A0]×100%
其中,A0为不加样品的吸光度值;A1为含样品的吸光度值;A2为背景溶液吸光度值。
4.2细胞毒性、细胞内ROS和MDA水平测定
4.2.1细胞培养及处理
人角质层细胞HaCaT、小鼠巨噬细胞RAW 264.7细胞用含10%FBS的DMEM培养基,于5%CO2、37℃、饱和湿度条件下的恒温培养箱培养,待细胞生长至对数生长期进行传代,2~3d传代1次,取第3代细胞用于实验。
试验分为四组:正常组、模型组、阳性对照组和处理组。模型组为LPS(1μg/mL)刺激后的巨噬细胞,100mg/L乙酰半胱氨酸处理组为阳性对照组,50-100mg/L豆腐柴叶RG-I果胶干预为处理组。乙酰半胱氨酸和果胶预处理2h后加入LPS刺激。
4.2.2细胞毒性
HaCaT细胞以1×105细胞/mL的密度接种至细胞板中,于5%CO2、37℃、饱和湿度条件下的恒温培养箱培养24小时。待细胞贴壁生长后,随机分组进行处理,将培养基替换为DMEM培养基与本实施例所得果胶溶液(溶解于培养基),继续培养24小时。CCK8试剂盒测定细胞存活率。
4.2.3ROS和MDA水平测定
细胞培养及处理方法如4.2.1所示。使用DCFH-DA荧光染色法测定细胞内产生的ROS。将巨噬细胞以1×105细胞/mL的密度接种至细胞板中,于5%CO2、37℃、饱和湿度条件下的恒温培养箱培养24小时。待细胞贴壁生长后,随机分组进行处理,将培养基替换为DMEM培养基与本实施例所得果胶溶液(溶解于培养基),继续培养24小时。在处理结束时,添加100μL的20μM DCFH-DA代替培养基,并在黑暗中室温下孵育30min。然后用PBS洗涤细胞,在荧光显微镜下观察细胞。使用酶标仪在485nm激发波长和528nm发射波长下测量荧光强度。通过硫代巴比妥酸法测定上清液中MDA。
4.2.4细胞内抗氧化酶活性测定
细胞培养及处理方法如4.2.1所示。通过NBT显色法测定上清液中的SOD。通过过氧化氢显色法测定上清液中的过氧化氢酶的酶活力。通过有机过氧化物试剂(Cum-OOH)特异检测谷胱甘肽过氧化物酶的活力。将巨噬细胞以1×105细胞/mL的密度接种至细胞板中,于5%CO2、37℃、饱和湿度条件下的恒温培养箱培养24小时。待细胞贴壁生长后,随机分组进行处理,将培养基替换为DMEM培养基与本实施例所得果胶溶液(溶解于培养基),继续培养24小时。使用RIPA缓冲液制备细胞裂解液,测定超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、丙二醛(MDA)和谷胱甘肽过氧化物酶(GPx)。
5.抗炎活性测定方法
细胞培养及处理方法如4.2.1所示。用酶联免疫法测定促炎性因子TNF-α、IL-1β和IL-6的水平。将细胞以1×105细胞/mL的密度,每孔100μL接种至细胞板中,于5%CO2、37℃、饱和湿度条件下的恒温培养箱培养24小时。待细胞贴壁生长后,随机分组进行处理,将培养基替换为DMEM培养基与本实施例所得果胶溶液(溶解于培养基),继续培养24小时。以10000rpm离心5min取上清。通过酶联免疫法,用酶标仪在450nm下测定TNF-α、IL-1β和IL-6的值。
6.实施例1-3和对比例1中豆腐柴叶果胶测试结果
1)分子量、单糖组成与微观结构特点
实施例1-3和对比例1中豆腐柴叶果胶的分子量、单糖组成、溶解性及果胶类型见表1,由表1可知,本发明实施例1-3制备得到的豆腐柴叶RG-I果胶测试结果分别为平均分子量280、272和290kDa,单糖组成包括葡萄糖37.6%、37.2%和36.8%,半乳糖醛酸27.4%、30.1%、和28.3%,半乳糖13.2%、12.7%和12.3%,鼠李糖6.3%、5.8%和7.1%等,半乳糖醛酸/鼠李糖比值4.3、5.8和4.0,水中溶解度7.9、7.5和8.1,根据国际粮农组织和欧盟标准,为含中性糖侧链较多的RG-I型果胶;而对比例1中豆腐柴叶果胶平均分子量为213kDa,单糖组成包括:半乳糖醛酸67.2%、葡萄糖16.12%、半乳糖2.42%、鼠李糖1.0%等,半乳糖醛酸占比大于65%,半乳糖醛酸/鼠李糖比例67.2,水中溶解度为6.5g/100g按照国际粮农组织标准,为侧链较少、主链较光滑的HG型果胶。
扫描电镜SEM观察结果如图1所示,由图1可知,本发明制得的豆腐柴叶RG-I果胶表面微观结构具有多孔、粗糙、分散的特点,而对比例1所得豆腐柴叶HG型果胶表面微观结构较平坦、致密。
表1实施例1-3和对比例1中豆腐柴叶果胶的分子量、单糖组成、溶解性及果胶类型
实施例1-3和对比例1中豆腐柴叶果胶及各对照组细胞存活率、羟自由基清除效果、细胞内ROS、MDA水平及CAT、SOD、GPx活性测试结果见表2,由表2可知,实施例1中500mg/L豆腐柴叶RG-I果胶处理48h,HaCaT细胞存活率98.2%,与正常组无显著差异;豆腐柴叶RG-I果胶清除羟自由基达到50%的有效浓度(IC50)为1.18μg/mL。模型组巨噬细胞氧化应激水平为正常组的3.88倍,阳性对照(100μg/mL NAC处理)组降低到正常组的1.31倍,100μg/mL本实施例1中RG-I果胶可使ROS降低到正常组的1.62倍,仅为模型组的42%;正常组脂质过氧化指标MDA水平为2.88nmol/mg,模型组为9.32nmol/mg,阳性对照组约8.01nmol/mg,而本实施例1RG-I果胶可使MDA水平降低到3.64nmol/mg,仅为模型组的39%。正常组过氧化氢酶CAT、超氧化物歧化酶SOD、谷胱甘肽过氧化物酶GPx水平为38.3、118.7、121.6U/mg,模型组下降为26.4、72.8、58.1U/mg,阳性对照组提高至于正常组相当的水平,本实施例1中RG-I果胶干预组可使CAT、SOD活力提高到模型组的1.54倍和2.3倍,正常组的1.06倍和1.42倍,将GPx活力提高到模型组的1.86倍、正常组的89%。
实施例2中500μg/mL豆腐柴叶RG-I果胶处理48h,HaCaT细胞存活率97.8%,与正常组无显著差异;豆腐柴叶RG-I果胶清除羟自由基达到50%的有效浓度(IC50)为1.32μg/mL。模型组巨噬细胞氧化应激水平为正常组的3.88倍,阳性对照(100μg/mL NAC处理)组降低到正常组的1.31倍,100μg/mL本实施例RG-I果胶可使ROS降低到正常组的1.45倍,仅为模型组的37%;正常组脂质过氧化指标MDA水平为2.88nmol/mg,模型组为9.32nmol/mg,阳性对照组约8.01nmol/mg,而本实施例RG-I果胶可使MDA水平降低到4.12nmol/mg,仅为模型组的44%。正常组过氧化氢酶CAT、超氧化物歧化酶SOD、谷胱甘肽过氧化物酶GPx水平为38.3、118.7、121.6U/mg,模型组下降为26.4、72.8、58.1U/mg,阳性对照组提高至于正常组相当的水平,本实施例RG-I果胶干预组可使CAT、SOD活力提高到模型组的1.47倍和2.4倍,正常组的1.01倍和1.48倍,将GPx活力提高到模型组的1.90倍、正常组的91%。
实施例3中500μg/mL豆腐柴叶RG-I果胶处理48h,HaCaT细胞存活率101.2%,与正常组无显著差异;豆腐柴叶RG-I果胶清除羟自由基达到50%的有效浓度(IC50)为1.01μg/mL。模型组巨噬细胞氧化应激水平为正常组的3.88倍,阳性对照(100μg/mL NAC处理)组降低到正常组的1.31倍,100μg/mL本实施例RG-I果胶可使ROS降低到正常组的1.35倍,仅为模型组的35%;正常组脂质过氧化指标MDA水平为2.88nmol/mg,模型组为9.32nmol/mg,阳性对照组约8.01nmol/mg,而本实施例RG-I果胶可使MDA水平降低到3.42nmol/mg,仅为模型组的37%。正常组过氧化氢酶CAT、超氧化物歧化酶SOD、谷胱甘肽过氧化物酶GPx水平为38.3、118.7、121.6U/mg,模型组下降为26.4、72.8、58.1U/mg,阳性对照组提高至于正常组相当的水平,本实施例RG-I果胶干预组可使CAT、SOD活力提高到模型组的1.56倍和2.4倍,正常组的1.08倍和1.49倍,将GPx活力提高到模型组的1.89倍、正常组的90%。
对比例1中500μg/mL果胶处理24h,HaCaT细胞存活率98.7%,与正常组无显著差异;果胶清除羟自由基达到50%的有效浓度(IC50)为4.3μg/mL。模型组巨噬细胞氧化应激水平为正常组的3.88倍,阳性对照(100μg/mL NAC处理)组降低到正常组的1.31倍,100μg/mL RG-I果胶可使ROS降低到正常组的3.66倍,为模型组的94%;正常组脂质过氧化指标MDA水平为2.88nmol/mg,模型组为9.32nmol/mg,阳性对照组约8.01nmol/mg,而RG-I果胶可使MDA水平降低到8.78nmol/mg,为模型组的94%。正常组过氧化氢酶CAT、超氧化物歧化酶SOD、谷胱甘肽过氧化物酶GPx水平为38.3、118.7、121.6U/mg,模型组下降为26.4、72.8、58.1U/mg,阳性对照组提高至于正常组相当的水平,RG-I果胶干预组可使CAT、SOD活力提高到模型组的1.05倍和1.1倍,仅为正常组的72%和68%,GPx活力仅为模型组的95%、正常组的45%。
上述结果显示,本实施例1-3制备得到的豆腐柴叶RG-I果胶无细胞毒性,具有显著的细胞内外抗氧化活性,并能显著提高细胞内源性抗氧化酶系的活力;而对比例1制备的果胶无细胞毒性,100μg/mL本实施例制备的果胶对正常巨噬细胞和模型组巨噬细胞中ROS、MDA水平没有显著降低作用;对细胞内抗氧化酶活力提高作用不明显。
表2实施例1-3和对比例1中豆腐柴叶果胶及各对照组细胞存活率、羟自由基清除效果、细胞内ROS、MDA水平及CAT、SOD、GPx活性
实施例1-3和对比例1中豆腐柴叶果胶及各对照组TNF-α、IL-1β、IL-6水平测试结果见表3,由表3可知,实施例1中正常组促TNF-α、IL-1β、IL-6水平为9.88ng/mL、13.47pg/mL和0.59ng/mL,模型组升高为37.5ng/mL、36.19pg/mL和5.63ng/mL,分别为正常组的3.8倍、2.7倍和9.54倍;阳性对照组降低为27.56ng/mL、27.49pg/mL和4.03ng/mL,为模型组的73.5%、76.0%和71.5%;本实施例干预组为22.46ng/mL、30.48ng/mL和3.58ng/mL,与阳性对照相当。
实施例2中正常组促TNF-α、IL-1β、IL-6水平为9.88ng/mL、13.47pg/mL和0.59ng/mL,模型组升高为37.5ng/mL、36.19pg/mL和5.63ng/mL,分别为正常组的3.8倍、2.7倍和9.54倍;阳性对照组降低为27.56ng/mL、27.49pg/mL和4.03ng/mL,为模型组的73.5%、76.0%和71.5%;本实施例干预组为24.13ng/mL、29.98ng/mL和3.42ng/mL,与阳性对照组相当。
实施例3中正常组促TNF-α、IL-1β、IL-6水平为9.88ng/mL、13.47pg/mL和0.59ng/mL,模型组升高为37.5ng/mL、36.19pg/mL和5.63ng/mL,分别为正常组的3.8倍、2.7倍和9.54倍;阳性对照组降低为27.56ng/mL、27.49pg/mL和4.03ng/mL,为模型组的73.5%、76.0%和71.5%;本实施例干预组为21.18ng/mL、29.67ng/mL和3.38ng/mL,与阳性对照相当。
对比例1中正常组促TNF-α、IL-1β、IL-6水平为9.88ng/mL、13.47pg/mL和0.59ng/mL,模型组升高为37.5ng/mL、36.19pg/mL和5.63ng/mL,分别为正常组的3.8倍、2.7倍和9.54倍;阳性对照组降低为27.56ng/mL、27.49pg/mL和4.03ng/mL,为模型组的73.5%、76.0%和71.5%;干预组为35.73ng/mL、35.22ng/mL和5.29ng/mL,与模型组无明显差异。
上述结果表明,对比例1中所得果胶对模型组巨噬细胞、正常细胞中的促炎性因子TNF-α、IL-1β和IL-6水平没有显著影响,抗炎活性不明显。而本实施例1-3制备得到的豆腐柴叶RG-I果胶能显著降低细胞中促炎性因子水平,具有显著的抗炎活性,可作为功能性食品原料,具有实际的应用价值。
表3实施例1-3和对比例1中及各对照组TNF-α、IL-1β、IL-6水平
样品 | TNF-α(ng/mL) | IL-1β(pg/mL) | IL-6(ng/mL) |
正常组 | 9.88 | 13.47 | 0.59 |
模型组 | 37.57 | 36.19 | 5.63 |
阳性对照组(NAC,100μg/mL) | 27.56 | 27.49 | 4.03 |
实施例1(200μg/mL) | 22.46 | 30.48 | 3.58 |
实施例2(200μg/mL) | 24.13 | 29.98 | 3.42 |
实施例3(200μg/mL) | 21.18 | 29.67 | 3.38 |
对比例1(200μg/mL) | 35.73 | 35.22 | 5.29 |
综上所述,本发明制备的天然豆腐柴叶RG-I型果胶是一种有效的具有抗氧化及抗炎作用的天然多糖。它无细胞毒性且能够有效清除DPPH自由基、ABTS阳离子自由基、羟基自由基,同时激活多种细胞抗氧化酶类,清除ROS,具有较高的抗氧化能力,能显著抑制LPS刺激的巨噬细胞的氧化应激和炎症反应,降低炎性细胞因子TNF-α、IL-1β和IL-6水平,具有极强的抗氧化能力和较好的抗慢性炎性反应活性,显著降低肠道有害微生物诱导的促炎性因子水平,可作为功能性食品原料应用于健康食品和功能食品。
以上内容仅仅是对本发明结构所作的举例和说明,所属本领域的技术人员不经创造性劳动即对所描述的具体实施例做的修改或补充或采用类似的方式替代仍属本专利的保护范围。
Claims (10)
1.一种具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶的制备方法,其特征在于,包括以下步骤:
步骤(1):豆腐柴叶原料预处理,得到豆腐柴叶粉末;
步骤(2):将步骤(1)所得的豆腐柴叶粉末利用超声辅助碱法提取,得到提取液;然后将提取液过滤、离心、真空减压浓缩得到浓缩液;再向浓缩液中加入乙醇混匀,得到沉淀产物,过滤沉淀并干燥,得到RG-I果胶粗品;其中,碱法提取时料液质量体积比为1:10~50,乙醇与浓缩液体积比为2~10:1;
步骤(3):将步骤(2)所得的RG-I果胶粗品复溶于水,然后过滤去除沉淀,得到RG-I果胶粗品滤液,再经DEAE-纤维素层析柱纯化,收集组分后经真空减压浓缩,膜分离,干燥,得到豆腐柴叶RG-I果胶成品。
2.如权利要求1所述的具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶的制备方法,其特征在于,所述步骤(1)中豆腐柴叶原料预处理过程为:采摘成熟新鲜的豆腐柴叶,去杂、清洗、低温干燥、粉碎后过40~80目筛,取筛下物。
3.如权利要求2所述的具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶的制备方法,其特征在于,所述步骤(1)中低温干燥为热泵干燥或真空冷冻干燥;热泵干燥的参数为:温度为80℃以下;真空冷冻干燥的参数为:升华干燥阶段温度-50~-10℃,解析干燥阶段0~50℃,真空度为1~20Pa。
4.如权利要求1所述的具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶的制备方法,其特征在于,所述步骤(2)中超声辅助碱法提取的参数为:超声波功率为400~600W,pH为8~12,温度为50~95℃,提取时间为30~150分钟;离心的转速为4000~8000r/min,时间为10~30分钟;干燥为热泵干燥或真空冷冻干燥;热泵干燥的参数为:温度为60℃以下;真空冷冻干燥的参数为:升华干燥阶段温度-50~-10℃,解析干燥阶段温度40-60℃,真空度1~10Pa。
5.如权利要求1所述的具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶的制备方法,其特征在于,所述步骤(3)中DEAE-纤维素层析柱纯化的参数为:洗脱液为0.1~1M的NaCl溶液,RG-I果胶粗品滤液的浓度为0.5~1mol/L,采用苯酚-硫酸法监测多糖浓度。
6.如权利要求1所述的具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶的制备方法,其特征在于,所述步骤(3)中膜分离的具体过程为:使用截留分子量为2kDa的膜分离设备纯化,得到截留部分,再经40℃-60℃热泵干燥或真空冷冻干燥;真空冷冻干燥参数:升华干燥温度-40℃~-10℃,解析干燥温度50-70℃,真空度1~10Pa。
7.如权利要求1所述的具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶的制备方法,其特征在于,所述步骤(3)中收集的组分为通过苯酚-硫酸法监测多糖浓度结果中多糖浓度最高的组分。
8.如权利要求1所述的具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶的制备方法,其特征在于,所述步骤(2)和步骤(3)中真空减压浓缩的参数为:真空度为-0.095~-0.01MPa,温度为50~70℃,浓缩至原溶液体积的1/4~1/2。
9.权利要求1-8任一项所述的制备方法制得的具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶。
10.权利要求9所述的具有抗氧化及抗炎活性的豆腐柴叶RG-I果胶在制备功能性食品中的应用。
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