CN108849771A - 牡丹花蕊在延长线虫寿命中的应用 - Google Patents
牡丹花蕊在延长线虫寿命中的应用 Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
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Abstract
本发明公开了牡丹花蕊在延长线虫寿命中的应用,所述牡丹花蕊水提液能够延长线虫的寿命,本发明通过体内试验鉴定了牡丹花蕊的抗衰老功能,提供了牡丹花蕊在延长线虫寿命中的应用,以及其应用的具体方法,该方法步骤简单,效果优势。
Description
技术领域
本发明涉及牡丹花蕊在延长线虫寿命中的应用,属于牡丹花蕊新应用技术领域。
背景技术
根据第六次全国人口普查结果显示,中国65岁以上人口已经占全国的8.87%,已进入人口老龄化社会。随着社会人口的老龄化日趋加重,老年性疾病如糖尿病、冠心病等明显增加(满都拉等,2017),而衰老是导致这些疾病增加的重要因素。衰老是生命体随年龄的增长必须经历的过程(于周龙,2016),受基因、环境和饮食等影响,是人类一直研究而未得到解决的重大问题。目前已研发出各种中草药和西药来缓解衰老(刘仁俊,2009;王福海,2013;张海平,2012),但由于取材不便、成本过高、合成复杂等问题导致效果不显著或应用不广泛。因此,寻找一种取材方便、成本较低、应用广泛的日常饮品来有效缓解衰老显得尤为必要。
牡丹(Paeonia suffruticosa Andr.)是中国传统名花,其花大色艳,雍容华贵,有“花中之王”的美誉(卢宗元,2014)。牡丹花蕊是牡丹精华之所在,是牡丹生长最集中的部位,更是牡丹数千年来得以生存的根本。牡丹花蕊中含有丰富的氨基酸、蛋白质、多糖、总黄酮、矿物元素等多种生物活性成分(刘俊民等,2015),具有较高的营养保健功能,有良好的开发前景。因此,开展牡丹花蕊功能鉴定研究十分必要。目前对牡丹花蕊的功能鉴定研究仅在抗氧化方面有部分报道。如陈庆敏等在《牡丹雄蕊的抗氧化活性及酚类成分分析》一文中通过测定牡丹花蕊提取物DPPH自由基清除能力证明了花蕊的抗氧化功能。傅茂润等在《牡丹雄蕊的营养成分和抗氧化能力研究》一文中利用DPPH自由基清除能力同样说明了牡丹花蕊具有较强的抗氧化能力,且乙醇提取物的能力最强。李朝苹等在《牡丹花蕊水提取液抗氧化功能研究》也发现牡丹花蕊水提液具有抗氧化功能。这些研究均认为牡丹花蕊具有抗氧化功能,然而均是通过体外研究得到的结果,此外,它们所采用的实验材料并不是目前市场上普遍认可的单瓣型牡丹品种‘凤丹’的雄蕊,所得结果不具有一定的代表性,其在体内是否真正具有抗氧化功能还值得探究。而关于牡丹花蕊的抗衰老功能鉴定方面的研究更是未见报道。因此,开展牡丹花蕊的抗衰老功能鉴定和应用研究具有十分重要的现实意义。
发明内容
发明目的:针对上述技术问题,本发明提供了牡丹花蕊在延长线虫寿命中的应用,为牡丹花蕊的进一步开发利用与推广提供参考理论依据。
技术方案:为了达到上述发明目的,本发明所采用的技术方案如下:
牡丹花蕊在延长线虫寿命中的应用。
优选:
所述线虫为秀丽隐杆线虫。
所述牡丹花蕊的品种为“凤丹”。
所述的应用包括以下步骤:
(1)取牡丹花初开第一天、第二天的花蕊,烘干至恒重,筛去花粉,去掉杂质,然后加入去离子水,配成牡丹花蕊水提液母液,无菌过滤;
(2)将上述过滤后的牡丹花蕊水提液母液加入到NGM空白培养基中,制成终浓度范围为10-4000mg/mL牡丹花蕊水提液的NGM空白培养板;
(3)挑取E.coli OP50单克隆过夜摇菌,涂布在上述NGM空白培养板上培养,制成NGM食物培养板;
(4)将同步化的L4时期线虫挑虫上板,20℃恒温培养,定期将线虫转移到新的NGM食物培养板上。
步骤(1)中牡丹花蕊水提液母液的浓度为15-25mg/mL。
步骤(3)中,挑取E.coli OP50单克隆过夜摇菌培养,按照1:1000比例转摇至OD600在0.4-0.6之间,将菌液均匀涂布在含有牡丹花蕊水提液的NGM空白培养板上,37℃过夜培养制成NGM食物培养。
技术效果:相对于现有技术,本发明通过体内试验鉴定了牡丹花蕊的抗衰老功能,提供了牡丹花蕊在延长线虫寿命中的应用,以及其应用的具体方法,该方法步骤简单,效果优势。
附图说明
图1为不同浓度牡丹花蕊水提液(PSWE)作用下线虫的生存曲线。
图2不同浓度牡丹花蕊水提液(PSWE)对线虫存活率、体长及头摆动影响。
图3不同浓度牡丹花蕊水提液(PSWE)对线虫体内相对荧光强度的影响。
具体实施方式
下面通过具体实施例对本发明所述的技术方案给予进一步详细的说明,但有必要指出以下实施例只用于对发明内容的描述,并不构成对本发明保护范围的限制。
实施例1牡丹花蕊的抗衰老功能鉴定
(1)仪器与试剂:YW-042-L电热鼓风干燥箱(上海元沃仪器设备有限公司)、T-500型电子天平(常熟双杰测试仪器厂)、赛多利斯电子天平(赛多利斯科学仪器有限公司)、SHZ-Ⅲ循环水式多用真空泵(上海贤德实验仪器有限公司)、RE-52AA旋转蒸发器(上海亚荣生化仪器厂)、HP 300GS-C型智能人工气候箱(武汉瑞华仪器设备有限责任公司)、CX31RTSF光学显微镜(日本东京奥林巴斯公司)、SZX2-ILLK荧光显微镜(日本东京奥林巴斯公司)、SW-CJ-2D型双人单面净化工作台(苏州净化设备有限公司)、美的微波炉(广东美的厨房电器制造有限公司)。
氯化钙(CaCl2)、硫酸镁(MgSO4)、磷酸氢二钾(K2HPO4)、磷酸二氢钾(KH2PO4)、氢氧化钠(NaOH)、次氯酸钠(NaClO)、磷酸氢二钠(Na2HPO4)、氯化钠(NaCl)均购自国药集团化学试剂有限公司,胆固醇和琼脂购自索莱宝生物科技有限公司,蛋白胨和酵母购自上海金畔生物科技有限公司。
(2)材料选择:以单瓣型牡丹品种‘凤丹’为试材,取其花蕊作为研究对象开展工作,其花期较早,花瓣白色,田间栽植于扬州大学园艺与植物保护学院芍药种质资源圃中(32°30′N,119°25′E)。
(3)牡丹花蕊水提液母液配制:取‘凤丹’初开第一天、第二天的花蕊经过简单处理烘干至恒重,筛去花粉,去掉杂质,然后加入去离子水,经过合并滤液,减压浓缩等步骤配成20mg/mL牡丹花蕊水提液母液,使用针式滤器无菌过滤,置于4℃冰箱待用。
(4)实验储备液配制
①1mol/L CaCl2:取CaCl2粉末110.9g,加入蒸馏水,定容至1L,121℃,20min灭菌待用。
②1mol/L MgSO4:取MgSO4粉末120.3g,加入蒸馏水,定容至1L,121℃,20min灭菌待用。
③5mg/mL胆固醇:取0.25g胆固醇溶解于50mL无水乙醇中,使用针式滤器无菌过滤,待用。
④1mol/L PBS缓冲液(pH=6.0):量取132mL的K2HPO4(1mol/L)加到868mL的KH2PO4(1mol/L)溶液中,121℃,20min灭菌待用。
⑤5mol/L NaOH:取6g NaOH溶解于24mL蒸馏水待用。
⑥10%NaClO:取3ml NaClO溶液加入27mL蒸馏水待用。
⑦M9溶液:分别取Na2HPO4、KH2PO4和NaCl 6g、3g和5g,加入去离子水定容至1L,121℃,20min灭菌待用。
⑧S-buffer溶液:分别取K2HPO4、KH2PO4和NaCl 1.47g、5.93g和5.85g,加入去离子水定容至1L,121℃,20min灭菌待用。
⑨Bleach溶液(裂解线虫):分别取5mol/L NaOH和10%NaClO 1mL和2mL,加入去离子水定容至10mL,需现配现用。
(5)LB培养基配制
①液体培养基:分别称取蛋白胨、酵母提取物和NaCl 10g、5g和10g,加入去离子水定容至1L,121℃,20min灭菌后4℃冰箱备用。
②固体培养基:分别称取蛋白胨、酵母提取物、NaCl和琼脂10g、5g、10g和15g,加入去离子水定容至1L,121℃,20min灭菌后倒平板,待凝固后4℃冰箱备用。
(6)NGM空白培养基配制:分别取NaCl、琼脂粉和蛋白胨3g、17g和2.5g,加入去离子水定容至1L,高压灭菌后,等溶液温度降到60℃左右,在超净工作台中加入以下试剂:1mLCaCl2(1mol/L)、1mL MgSO4(1mol/L)、25mL(1mol/L,pH=6.0)PBS缓冲液和1mL胆固醇(5mg/mL),混和均匀后,倒入直径为90mm或者60mm的培养皿中,吹风晾干30min,紫外照射5min,平板晾干后4℃冰箱备用。
(7)牡丹花蕊水提液的NGM空白培养基的配制:用20mg/mL牡丹花蕊水提液母液配制10、50、250、1000、2000、4000mg/L的NGM空白培养基。先将氯化钠和琼脂粉、蛋白胨加入锥形瓶,高温灭菌后,等溶液温度降到60℃左右加入CaCl2(1mol/L)、MgSO4(1mol/L)、PBS缓冲液(1mol/L,pH=6.0)和胆固醇溶液(5mg/mL),最后加入再加入已计算好量的牡丹花蕊水提液。例如,终浓度为10mg/mL的NGM空白培养基,即100mL的NGM空白培养基中所需牡丹花蕊水提液母液50μL,后倒板,冷却,封板。标记好后4℃冰箱备用。
(8)秀丽隐杆线虫的培养
实验中使用的秀丽隐杆线虫为N2野生型线虫,线虫食物大肠杆菌OP50(E.coliOP50)是一种尿嘧啶缺陷型菌株。
①E.coli OP50的培养:取出储存于-80℃的OP50,用接种环蘸取少量菌液在LB固体培养基上,交叉划线。37℃过夜培养,再蘸取过夜培养的菌液加入200mL LB液体培养基中,37℃过夜培养至OD600在0.4-0.6之间,放入4℃冰箱备用。
②涂布:从4℃冰箱中取出OP50菌液,在超净台上吸取适量的菌液于NGM空白培养基以及牡丹花蕊水提液的NGM空白培养基中央,用涂布棒涂抹均与,涂抹完毕后在超净台内待其干透,封板后置于37℃的生化培养箱中过夜培养。待细菌长出薄薄的一层时,然后进行紫外灭菌,置于4℃冰箱备用。
③线虫的一般培养:实验室常用雌雄同体的秀丽隐杆线虫,保证无菌条件下用挑虫针将线虫挑取至含有OP50的NGM培养基上,置于20℃生化培养箱培养。
④线虫的同期化培养:用1.5mL M9溶液冲洗含有大量虫卵与孕期线虫的培养板,将收集的液体置于2mL EP管中,涡旋30s,然后离心弃上清(4000r/min,1min),继续加入1mLM9溶液,涡旋30s后离心弃上清(4000r/min,1min)。加入1mL Bleach溶液,涡旋1min,静置5min后离心弃上清(4000r/min,1min),再加入1mL S-buffer溶液,涡旋30s后离心弃上清(4000r/min,1min),重复上一步操作两次,最后将虫卵混合液用移液枪打在含OP50的NGM边缘20℃恒温培养,大约48h之后成长为L4期线虫,此阶段的线虫可以进行实验。本实验中同步化均采用此法。
(9)秀丽线虫寿命的测定:挑取同期化的L4期线虫到各组(每个浓度)培养板中,每组三个培养板,每个板大约40条,此时记为寿命试验的第0天,20℃恒温培养,为确保药液的浓度,每两天将线虫转移到新的NGM培养板上。每天观察统计线虫死亡、存活及剔除条数,直至最后一条线虫死亡为止。线虫死亡标志为用铂丝轻触虫体无任何反应。各实验组(每个浓度)最后一条线虫死亡的时间为该组线虫的最长寿命,实验重复3次。
(10)秀丽线虫的安全性评估
同期化的L4期线虫给药24h后进行以下指标测定。
①存活率测定:统计各组(每个浓度)线虫的存活率。实验重复3次。
②身体长度测定:24h后将各组(每个浓度)线虫用M9洗下,用移液枪吸取100μL虫液放置在双凹载玻片上,酒精灯杀死线虫,用Nikon Eclipse 80i测量,通过显微镜尺和NISElements测体长,实验重复3次。
③头摆动能力测定:取10条(每个浓度)放置在空白NGM培养基上(不含OP50),恢复运动2min后记录1min内线虫头部摆动的次数。一次头部摆动是指线虫的头部从一个方向摆向另一方向后又摆回来,实验重复3次。
④肠道自发荧光测定:载玻片预热后,滴加1滴2%的琼脂液,垂直盖上另一片载玻片,压平琼脂,排除气泡。待凝固冷却后,移除上层载玻片,滴加1滴浓度为5μm左旋咪唑于琼脂中央,将给药24h的线虫各组(每个浓度)挑10只置于左旋咪唑液滴中,盖上盖玻片。用荧光显微镜观察并拍照。用Image J软件分析线虫体内的荧光强度,实验重复3次。
(11)秀丽隐杆线虫寿命和安全性分析
①寿命分析:图1为不同浓度牡丹花蕊水提液(PSWE)作用下线虫的生存曲线。与对照相比,10-4000mg/L牡丹花蕊水提液都能延长线虫的寿命,其中浓度为1000mg/L最为明显;表1为牡丹花蕊水提液(PSWE)对秀丽隐杆线虫抗衰老的影响。与对照相比,10-4000mg/L牡丹花蕊水提液都能延长线虫的平均寿命和最长寿命,其中1000mg/L的牡丹花蕊水提液效果最好,平均寿命延长了2.31天,延长百分比为16.40%;最长寿命延长了3.67天,延长百分比达到了17.46%。因此,牡丹花蕊具有抗衰老作用。
表1
注:同一列不同小写字母表示差异性显著,P<0.05。
②安全性分析:图2为不同浓度牡丹花蕊水提液(PSWE)对线虫存活率、体长及头摆动影响。与对照相比,10-4000mg/L牡丹花蕊水提液对线虫的存活率(图2A),身体长度(图2B),头部摆动(图2C)没有显著影响;图3为不同浓度牡丹花蕊水提液(PSWE)对线虫体内相对荧光强度的影响。与对照相比,10-4000mg/L牡丹花蕊水提液对幼虫体内相对荧光强度没有显著影响。10-4000mg/L牡丹花蕊水提液不会对幼虫产生致死性伤害且不会影响幼虫的生长,也不会影响幼虫主要靶器官正常的生理状态,所以牡丹花蕊没有毒性,是比较安全的。。
尽管本发明已通过上述实施例作了详细介绍,但应当认识到上述的描述不应该被认为是本发明的限制,在本领域技术人员阅读了上述内容后,对于本发明的多种修改和替代都将是显而易见的。因此,本发明的保护范围应由所附的权利要求来限定。
Claims (6)
1.牡丹花蕊在延长线虫寿命中的应用。
2.根据权利要求1所述的应用,其特征在于,所述线虫为秀丽隐杆线虫。
3.根据权利要求1所述的应用,其特征在于,所述牡丹花蕊的品种为“凤丹”。
4.根据权利要求1所述的应用,其特征在于,包括以下步骤:
(1)取牡丹花初开第一天、第二天的花蕊,烘干至恒重,筛去花粉,去掉杂质,然后加入去离子水,配成牡丹花蕊水提液母液,无菌过滤;
(2)将上述过滤后的牡丹花蕊水提液母液加入到NGM空白培养基中,制成终浓度范围为10-4000mg/mL牡丹花蕊水提液的NGM空白培养板;
(3)挑取E.coli OP50单克隆过夜摇菌,涂布在上述NGM空白培养板上培养,制成NGM食物培养板;
(4)将同步化的L4时期线虫挑虫上板,20℃恒温培养,定期将线虫转移到新的NGM食物培养板上。
5.根据权利要求4所述的应用,其特征在于,步骤(1)中牡丹花蕊水提液母液的浓度为15-25mg/mL。
6.根据权利要求4所述的应用,其特征在于,步骤(3)中,挑取E.coli OP50单克隆过夜摇菌培养,按照1:1000比例转摇至OD600在0.4-0.6之间,将菌液均匀涂布在含有牡丹花蕊水提液的NGM空白培养板上,37℃过夜培养制成NGM食物培养。
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