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辣木的研究进展许 敏1,2,3，赵三军4，宋 晖2，杨崇仁2,3,*（1.昆明理工大学生命科学与技术学院，云南 昆明 650500；2.云南现代民族药工程技术研究中心，云南 昆明 650101；3.中国科学院昆明植物研究所，植物化学与西部植物资源可持续利用国家重点实验室，云南 昆明 650201；4.云南师范大学生命科学学院，云南 昆明 650500）摘 要：辣木（Moringa oleifera ）为辣木科辣木属多年生热带落叶乔木，广泛种植于亚洲和非洲热带和亚热带地区。
我国广东、台湾、云南等地引种栽培。
辣木因生长快速且具有很高的经济价值，被称为“奇迹之树（miracle tree ）”。
辣木的根、叶和嫩果可食用，种子可榨油，含油量大于30%。
此外，辣木种子含有净水活性很高的蛋白质，这种净水蛋白具有天然、无毒、无副作用、易于降解等特点。
此外，辣木主要含有氨基甲酸酯和酚性成分，具有抗菌、降血压、降糖尿病等生理活性。
本文主要从化学成分和药理研究出发，围绕辣木的安全性和成分检测的研究概况进行综述，为辣木的开发和利用提供依据和参考。
关键词：辣木；化学成分；生物活性；安全性；成分分析Advances in Knowledge of Moringa oleiferaXU Min 1,2,3, ZHAO Sanjun 4, SONG Hui 2, YANG Chongren 2,3,*(1. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China; 2. Center for Drug Discovery & Technology Development of Yunnan Traditional Medicine, Kunming 650101, China; 3. State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China; 4. School of Life Sciences, Yunnan Normal University, Kunming 650500, China)Abstract: Moringa oleifera , belonging to the Moringaceae family, is a biennial deciduous tree that is distributed in tropical and sub-tropical regions of Africa and Asia. The plant has been introduced into China and planted in Guangdong, Yunnan and Taiwan provinces. Moringa oleifera is called as “Miracle Tree” due to its rapid growth. Both its leaves and tender fruits are edible. Moringa oleifera seeds contain more than 30% oil. Moringa oleifera seed proteins are traditionally utilized for water purification owing to advantages such as natural occurrence, no toxic or side effects and easy degradation. Furthermore, Moringa oleifera contains carbamate derivatives and phenolic compounds, which show a diversity of biological activities including antifungal, anti-hypertensive, anti-diabetic effects. This paper reviews the chemical constituents and biological activities of Moringa oleifera with focus on safety evaluation and chemical analysis of Moringa oleifera for the purpose of providing evidence for the development and utilization of Moringa oleifera .Key words: Moringa oleifera ; chemical constituents; biological activities; safety evaluation; constituents analysis DOI:10.7506/spkx1002-6630-201623048中图分类号：R274.9 文献标志码：A 文章编号：1002-6630（2016）23-0291-11引文格式：许敏, 赵三军, 宋晖, 等. 辣木的研究进展[J]. 食品科学, 2016, 37(23): 291-301. DOI:10.7506/spkx1002-6630-201623048. XU Min, ZHAO Sanjun, SONG Hui, et al. Advances in knowledge of Moringa oleifera [J]. Food Science, 2016, 37(23): 291-301. (in Chinese with English abstract) DOI:10.7506/spkx1002-6630-201623048. 收稿日期：2015-06-16基金项目：云南省中青年学术和技术带头人后备人才计划项目（2011CI044）作者简介：许敏（1976—），女，副研究员，博士，主要从事天然药物化学研究。
辣木黄酮和多糖提取方法及其含量影响因素的初步研究的开题报告一、研究背景和意义辣木是一种生长在热带和亚热带地区的常绿乔木，在印度、非洲和南美洲等地广泛种植。
辣木叶和果实富含多种营养成分，如维生素、矿物质、氨基酸、多酚等，具有多种药用价值和保健作用。
其中，辣木黄酮和多糖是辣木中具有重要生物活性的成分之一，具有很高的应用价值。
辣木黄酮是一种黄酮类化合物，具有抗氧化、抗肿瘤、抗炎和保护心血管等多种生物活性。
多糖是一类高分子化合物，具有免疫调节、降血糖、降血脂等多种生物活性。
因此，研究辣木黄酮和多糖的提取方法和含量影响因素，对于充分利用辣木资源，开发辣木营养保健品具有重要的科学意义和实际价值。
二、研究目的和内容本研究旨在探究辣木黄酮和多糖的提取方法和含量影响因素，为深入开发辣木营养保健品提供科学依据和技术支持。
具体研究内容包括：1. 比较不同提取方法对辣木黄酮和多糖的影响，选择最优提取方法。
2. 研究辣木黄酮和多糖的含量与不同时间、温度、pH值和溶剂种类等因素的关系。
3. 分析辣木黄酮和多糖的结构特点及其生物活性，探索其应用前景。
三、研究方法和步骤1. 原料收集：选取辣木叶和果实为研究对象，收集新鲜成熟的辣木叶和果实，洗净晾干备用。
2. 提取方法优化：采用超声波辅助提取、水浴加热提取和微波辅助提取等不同提取方法，比较其对辣木黄酮和多糖提取效果的影响，确定最优提取条件和方法。
3. 影响因素研究：通过单因素实验和正交试验等方法，研究辣木黄酮和多糖的含量与时间、温度、pH值和溶剂种类等因素的关系，筛选出主要影响因素及其相互作用关系。
4. 结构特点和生物活性分析：通过光谱分析、色谱分析等方法，分析辣木黄酮和多糖的结构特点和生物活性，评估其应用前景。
四、研究预期成果1. 确定最优的辣木黄酮和多糖提取方法，提高提取效率和品质。
2. 揭示影响辣木黄酮和多糖含量的主要因素和其相互作用关系，为后续优化工艺提供基础。
3. 分析辣木黄酮和多糖的结构特点和生物活性，为进一步开发辣木营养保健品提供科学依据。
辣木叶黄酮类物质对大豆油的抗氧化作用摘要：以大豆油为对象, 研究了辣木叶黄酮类提取物在不同添加量时对油脂的抗氧化作用。
结果表明, 辣木叶黄酮对大豆油有很强的抗氧化作用, 其在大豆油中的最佳添加量为0.03%。
关键词：辣木叶; 黄酮类化合物; 大豆油; 抗氧化油脂中的不饱和脂肪酸易与空气中的氧发生氧化而酸败变质。
在氧化过程中会产生自由基、氢过氧化物及醛、酮等化合物，使油脂本身的营养成分遭到破坏从而丧失营养价值。
其中一些酸败产物是公认的致癌物，危及人体健康。
防止油脂氧化酸败的方法很多，目前工业上广泛使用的是合成抗氧化剂，如BHT、TBHQ等，虽然效果较好，但其安全性一直受到质疑[1]。
于是高效、安全、无毒的天然抗氧化剂的开发利用日益受到人们的重视。
辣木提取物作为一种天然抗氧化剂，具有安全、高效等显著优势。
辣木（Moringa oleifera）为辣木科（Moringaceae）辣木属（Moringa Adans）植物，起源于印度北部喜马拉雅山南麓地区[2]，又称鼓槌树（Drumsticktree），一直以来被西方科学家誉为上帝赐予人类的珍贵礼物，人们把这种礼物称作“奇迹之树（Miracle tree）”。
辣木中起抗氧化性作用的主要功能性成分为黄酮及多酚类物质。
早期国内外关于辣木的研究多集中在食用和药用、育种和栽培技术、作为植物饲料[3]及饮水净化[4]等方面。
试验将对辣木叶提取物黄酮类物质在油脂中的抗氧化作用进行研究，以期为辣木资源的进一步综合利用提供理论依据。
1 材料与方法1.1 材料与仪器辣木干叶，德宏芒市天佑辣木岛云南天佑科技开发有限公司；大豆油，金龙鱼；芦丁；硅胶（200-300目），青岛海洋化工厂；其他试剂均为分析纯。
KQ-5200B超声波清洗机：巩义市予华仪器有限责任公司；GL-20L高速离心机：上海安亭科学仪器厂；RE-2000B旋转蒸发器：上海亚荣生化仪器厂；LGL-18C真空冷冻干燥机：北京四环科学仪器厂有限公司；DHG-9076A恒温箱：上海精宏实验设备有限公司；722N紫外可见分光光度计：上海精科实业有限公司；HD-A色谱工作站：上海青浦泸西仪器厂。
基于新资源食品辣木的糖尿病防治研究进展该文探讨辣木营养治疗糖尿病的可行性与有效性。
通过查阅中国知网和万方数据库等数据库辣木防治糖尿的相关文献，发现辣木营养治疗对于降低糖尿病病人血糖水平，改善临床症状，可有效减少并发症，开展糖尿病的辣木营养治疗具有可行性与有效性。
标签：辣木；糖尿病；营养治疗糖尿病、恶性肿瘤以及心脑血管疾病已经成为严重危害我国人民健康的三大慢性非传染性疾病，伴随着国人生活方式的不断变化特别是膳食结构的改变，劳动和体力活动的减少，不远的将来我国糖尿病患病人数还有一个较长时期的增长过程。
糖尿病是由于各种原因导致胰岛素分泌减少、需求增加或机体细胞对胰岛素不敏感而导致的以糖代谢紊乱为主代谢紊乱综合征，临床上以持续高血糖为主要特征的、“三多一少”（多饮、多尿、多食以及体重减少）典型症状为主的慢性全身性代谢性疾病。
长期高血糖常导致各种脏器尤其是脑、心、肾及眼底等损害。
临床上主要有1型（胰岛素依赖型）和2型（非胰岛素依赖型）两种类型，其中2型糖尿病最多见，占糖尿病总数的90%左右，且发病年龄也呈现低龄化趋势。
而我国糖尿病患者人数多，增速快，已严重危害国民身心健康，造成巨大经济负担，如何防治糖尿病也显得越发重要。
2012年国家卫生部公布辣木叶为国家新资源食品，辣木研究方兴未艾，众多的辣木研究者对辣木的适应性种植、生产加工、保健食品开发等领域做了许多有益研究，下面主要对辣木在糖尿病及相关并發症的防治方面的重要文献进行回顾性研究。
1 辣木的生物学特性辣木又称奇树、鼓槌树，为单科单属植物，归为辣木亚目、辣木科、辣木属，根据树形和根分为纤细型、粗壮型以及块根型三型。
是一种神奇的热带、亚热带的速生、多功能植物，在亚洲、非洲的亚热带和热带地区均有大片种植。
辣木喜温，辣木适宜生长温度18～32 ℃；辣木耐旱，年降水量800～1 800 mm。
辣木对土壤质地和酸碱度要求不严，能在PH4-9的壤土或沙壤土良好生长。
毕业论文辣木黄酮和多糖提取方法及其含量影响因素的初步研究摘要为了稳定和提高辣木产品的有效成分含量，本研究比较了辣木有效成分黄酮和多糖的提取和测定方法，找到了辣木黄酮和多糖提取方法和条件的最佳优化组合。
并应用乙醇回流法和苯酚-硫酸法探讨了叶龄、器官、产地、采收期、管理水平、朝向等与有效成分含量的关系。
研究结果如下：辣木总黄酮的最佳提取条件为：用70%的乙醇作为提取溶剂，乙醇用量为20倍，提取温度为80℃，提取3次，每次90min。
在此提取条件下，辣木叶总黄酮量为6.59％。
辣木多糖的最佳提取条件为：以15倍的溶剂用量，在90℃水浴条件下，提取3次，每次120min，在此提取条件下，辣木叶多糖量为25.51％。
辣木叶片总黄酮和多糖含量均以45d的壮龄叶含量最高，总黄酮含量可达6.11％，多糖含量可达21.97％；幼龄叶和老龄叶中的总黄酮和多糖含量都比较少。
辣木不同器官的总黄酮含量为花柄中最多，根中最少，其变化围为0.53%-4.47%；不同器官的多糖含量为根中最多.叶柄中最少.其变化围为8.16%～33.61％。
不同采收期的辣木有效成分含量均在11月采收时含量最高，其中总黄酮含量为叶5.69％、叶柄3.13％、茎2.01％；多糖含量为叶28.85%、叶柄9.71％、茎12.24％。
不同管理水平的辣木有效成分含量为：叶在中等管理水平下有效成分含量最高，叶柄和茎中有效成分含量依次为低>中>高。
不同朝向的辣木有效成分含量为各器官在向阳和背阳区之间均没有显著性差异。
关键词：辣木；总黄酮；多糖；提取方法；发育毕业设计（论文）原创性声明和使用授权说明原创性声明本人重承诺：所呈交的毕业设计（论文），是我个人在指导教师的指导下进行的研究工作及取得的成果。
尽我所知，除文中特别加以标注和致的地方外，不包含其他人或组织已经发表或公布过的研究成果，也不包含我为获得及其它教育机构的学位或学历而使用过的材料。
对本研究提供过帮助和做出过贡献的个人或集体，均已在文中作了明确的说明并表示了意。
智者论道智库时代 ·249·糖尿病是一组严重威胁人类健康的代谢性疾病，由胰岛素分泌绝对或相对不足引起，已成为继心血管、肿瘤、艾滋病之后第四大易致人死亡的疾病，是与遗传、自身免疫和环境因素有关的多因素综合征[1,2]。
随着生活水平的提高，生活模式的改变及社会老龄化，无论是发达国家还是发展中国家，糖尿病的发病率日趋增加。
根据统计，全世界糖尿病的患者数已超过4.15亿，中国的糖尿病患者已超1.39亿，中国已经成为糖尿病第一大国[3]。
因此，防治糖尿病已是一个迫不及待的科学任务。
目前，胰岛素及各种口服降血糖药物使用可以有效控制糖尿病病情，但糖尿病的根治迄今为止仍是个难题。
近年的研究发现，辣木具有降血糖、降血脂、抗肿瘤、抗氧化、抗炎、免疫调节和保护肝肾功能等生物活性[4,5]，具有多种保健功能。
因此开发药食两用的道地药材—辣木，探索其降糖作用和机制，针对糖尿病进行有效治疗，预防并发症的发生，提高患者生存质量，具有重要的现实意义。
现就近年来对辣木降血糖作用研究进展情况进行综述。
一、辣木的生物学特性辣木（Moringa）属于辣木科辣木属植物，因其有辛辣味的根而得名，主要生长在热带、亚热带，是一种多功能植物。
原产于印度，现广植于各热带地区，我国主要栽种在广东、台湾、广西、海南、福建、云南、四川以及重庆等地，亦有野生的，如产崖县牛尾山大抱杠，生于杂林中。
辣木的根、叶和嫩果均可食用，种子可榨油，含油30％[6]。
辣木的根、茎、叶、花、枝、种子和树皮都含有丰富的营养成分及药用活性，被称为“奇迹之树”，可作为蔬菜、保健食品，在营养保健 、医疗药品、功能原料、水净化、牲畜饲料等方面得到利用，有着极大发展潜力和应用价值。
二、辣木的主要成分（一）辣木籽辣木籽含有33.1%原油，37.8%蛋白质，16.3%碳水化合物及5.2%的灰分。
Monica Premi 等人从辣木籽提取物中鉴定出黄酮类及酚苷类等12 种化合物[7]。
辣木叶有效成分的提取、分离纯化及其活性研究辣木(Moringa oleifera Lam.)是一种辣木科辣木属植物,普遍分布在亚洲、非洲的热带和亚热带地区,在我国主要分布于云南、广东、福建、台湾等地。
辣木含有丰富的营养成分和许多活性物质,具有多种生理活性,在医药和保健品行业具有广泛的用途及开发前景,但目前国内对辣木研究较少且多为单一品种粗提物的研究。
本论文对比研究不同品种辣木叶有效成分及其生物活性,有助于充分利用该植物资源,为辣木叶保健品或药物的进一步研究和开发利用提供理论基础。
以非洲辣木叶为原料,采用响应面分析法对辣木叶总黄酮的超声辅助提取工艺条件进行优化,确定最佳提取条件为:超声功率300 W,乙醇浓度70%(v/v),料液比1:27(m/v),提取时间46 min,提取温度50°C,在此条件下,总黄酮得率为48.93±0.44 mg RT/g,氧自由基吸收能力(ORAC)值为2747.17±301.51μmol TE/g。
采用聚酰胺层析柱对辣木叶总粗黄酮进行纯化,黄酮含量和ORAC值分别为纯化前的3.01和2.16倍,且均具有较强的DPPH和ABTS自由基清除能力。
采用超声提取法和加热回流法对非洲辣木、印度改良种辣木(pkm1)及pkm1改良种辣木总黄酮和总多糖进行提取,总黄酮得率分别为21.30 g/100g干基(DW)、22.29g/100g DW、20.78 g/100g DW,总多糖得率分别为6.18 g/100g DW、7.71 g/100g DW、6.71 g/100g DW。
使用不同标准曲线对两大类物质进行含量测定,黄酮含量分别为21.91%、35.95%、16.40%,多糖含量分别为34.80%、43.30%及35.32%。
对比研究了三个品种辣木叶黄酮和多糖粗提物的抗氧化活性、糖尿病关键酶抑制活性和抗补体活性。
结果表明:三种辣木叶中黄酮的抗氧化活性及糖尿病关键酶抑制活性强于多糖,而抗补体活性弱于多糖。
辣木籽中总黄酮的提取工艺研究华帅'T雄"陈海丰2师晶晶'1.中国科学院西双版纳热带植物园，云南西双版纳666303；2.云南中医学院，云南昆明650500【摘要】目的：优选辣木籽中总黄酮的提取工艺「方法：以浸膏中总黄酮的转移率为评价指标，利用正交试验匚（34）优选辣木籽总黄酮提取工艺。
结果：通过正交试验选出辣木籽中总黄酮的最佳提取工艺条件为：微波提取2次，乙醇浓度70%,溶剂量10倍，时间3min。
此条件下浸膏中总黄酮的转移率为93.89%。
结论：该提取工艺总黄酮转移率高，工艺稳定。
【关键词】辣木籽；总黄酮；微波提取；正交试验【中图分类号】R284【文献标志码】A【文章编号】1007-8517（2019）1-0045-05辣木（Morin ga oleifera）又名洋椿树、鼓槌树，为辣木科（Morin gaceae）辣木属（Morin ga Adans.）植物，起源于印度西北部的喜马拉雅山南麓，是辣木属中可以食用且利用和研究最多、最有商业价值的品种。
我国于20世纪60年代初开始在台湾、海南、广东、云南南部等广大热带、亚热带地区引种辣木。
辣木为药食两用的植物，其树叶、花、果荚不仅含有钙、钾和氨基酸等营养物质，且在印度和非洲国家常用于退热、消炎、降压、强心和抗菌等⑴。
目前，国内外学者主要集中于辣木营养学方面的研究辣木的药用保健功能日益受到人们的重视⑷。
辣木籽作为纯天然绿色食品，营养物质含量非常丰富，食用辣木籽可预防疾病、延缓衰老、改善睡眠、增强免疫力和记忆力⑸。
经初步分析,辣木籽中含有黄酮类化合物⑷。
黄酮类化合物的生理活性比较广泛，具有清除自由基、抗氧化、抗癌、防癌、治疗心血管病、降血糖等作用。
近年来对辣木的开发已成热点，但从辣木籽中提取黄酮类化合物的研究鲜有报道力。
本文以辣木籽总黄酮转移率为指标，采用正交试验方法分别考察微波提取法、回流提取法、超声提取法中的各种影响因素，旨在提高辣木籽中总黄酮的提取率,并通过对比产生最佳的提取工艺，为辣木籽的进一步开发利用提供了理论依据。
辣木的有效成分、保健功能和开发利用研究进展作者：孙丹管俊岭许玫李涛陈文品来源：《热带农业科学》2016年第03期摘要辣木（Moringa oleifera）是辣木科辣木属的热带落叶木本蔬菜及油料作物，有着丰富的营养价值和独特的生物活性，极具开发潜力。
从辣木有效成分辣木油、蛋白质、总黄酮等的提取，抗氧化及保护肝脏、降血糖、降血脂、抗菌消炎等保健功能和开发利用等方面进行综述，为辣木资源的研究利用提供参考。
关键词辣木；有效成分；保健功能；开发利用分类号 S58 Doi：10.12008/j.issn.1009-2196.2016.03.007Abstract Moringa oleifera belongs to Moringaceae Moringa Adans'hot tropical deciduous woody vegetables and oil crops， having a high nutritional value and unique biological activity， with great development potential. This article reviewed the extraction of moringa oil， protein， total flavonoids et al； and the function of anti-oxidation and protecting the liver， lowering blood sugar and blood fat， anti-bacterial and anti-inflammatory and other health functions，besides， the development and utilization were also reviewed， hoping to provide a reference for the comprehensive development and utilization of Moringa oleifera resources.Keywords Moringa oleifera ； active ingredients ； health function ； exploitation辣木（Moringa oleifera）属于辣木科（Moringaceae）辣木属（Moringa Adans.）的热带落叶木本蔬菜及油料植物，又名鼓槌树、洋椿树等，商品名奇树，树龄约为20 a[1-2]。
Subcritical ethanol extraction of flavonoids from Moringa oleifera leaf and evaluation of antioxidantactivityYongqiang Wang a ,Yujie Gao b ,c ,Hui Ding d ,⇑,Shejiang Liu d ,Xu Han d ,Jianzhou Gui e ,Dan Liu eaSchool of Chemical Engineering and Technology,Tianjin University,Tianjin 300072,China bTianjin Academy of Environmental Sciences,Tianjin 300191,China cTianjin United Environmental Engineering Design Company Limited,Tianjin 300191,China dSchool of Environmental Science and Engineering,Tianjin University,Tianjin 300072,China eSchool of Environmental and Chemical Engineering,Tianjin Polytechnic University,Tianjin 300387,Chinaa r t i c l e i n f o Article history:Received 27November 2015Received in revised form 24August 2016Accepted 8September 2016Available online 13September 2016Keywords:Response surface methodology FlavonoidsMoringa oleifera leafSubcritical ethanol extraction Antioxidant propertya b s t r a c tA large-scale process to extract flavonoids from Moringa oleifera leaf by subcritical ethanol was developed and HPLC–MS analysis was conducted to qualitatively identify the compounds in the extracts.To opti-mize the effects of process parameters on the yield of flavonoids,a Box-Behnken design combined with response surface methodology was conducted in the present work.The results indicated that the highest extraction yield of flavonoids by subcritical ethanol extraction could reach 2.60%using 70%ethanol at 126.6°C for 2.05h extraction.Under the optimized conditions,flavonoids yield was substantially improved by 26.7%compared with the traditional ethanol reflux method while the extraction time was only 2h,and obvious energy saving was observed.FRAP and DPPH Åassays showed that the extracts had strong antioxidant and free radical scavenging activities.Ó2016Elsevier Ltd.All rights reserved.1.IntroductionMoringa oleifera (M.oleifera )is a widely growing tree in India,and also being cultivated in Niger,Haiti,Mexico,China,etc (Morton,1991).Recent years,M.oleifera has attracted great attention among researchers because of the potential use in many fields.Almost every part of the tree can be eaten,and more impor-tant thing is that all parts of the tree have great potential as medicines (Abdulkarim,Long,Lai,Muhammad,&Ghazali,2005).Therefore,many research workers paid great attention to the medicinal and nutritional uses of M.oleifera (Anwar,Latif,Ashraf,&Gilani,2007).Meanwhile,environmental uses of M.oleifera seed such as treatment of waste water also aroused scholarly interest (Kansal &Kumari,2014).M.oleifera leaf contains lots of nutrients which can be absorbed into human body,such as vitamins,minerals,and fatty acids (Moyo,Masika,Hugo,&Muchenje,2011).Additionally,the leaf has been certified to contain various compounds like flavonoids,phenolics,and carotenoids which can be used as antioxidant (Alhakmani,Kumar,&Khan,2013;Vongsak,Sithisarn,&Gritsanapan,2014).Flavonoids are widely distributed in plants and have many roles and functions.The wide distribution compared with other bioactive compounds makes the animals and humans ingest signif-icant amounts of flavonoids in their diet (Manach,Scalbert,Morand,Remesy,&Jimenez,2004).In human bodies,oxidation is an essential process to product energy,but sometimes the produc-tion of oxygen-derived free radicals is uncontrolled and damaging to human cells.Antioxidants such as flavonoids have ability to scavenge these free radicals and reduce the risk of death from coro-nary heart disease (Hertog,Feskens,Hollman,Katan,&Kromhout,1993).Therefore,it is essential to develop and utilize antioxidants to protect human body from free radicals (Singh &Rajini,2004).Many new extraction technologies have been developed to separate flavonoids from plants (Dai &Mumper,2010),such as ultrasound-assisted extraction (UAE)(Albu,Joyce,Paniwnyk,Lorimer,&Mason,2004;Zhang,Yang,Li,&Wang,2008),subcriti-cal water extraction (SWE)(Jo et al.,2013;Matshediso,Cukrowska,&Chimuka,2015),supercritical fluid extraction (SFE)(Maran,Manikandan,Priya,&Gurumoorthi,2015),microwave-assisted extraction (MAE)(Rostagno,Palma,&Barroso,2007).Subcritical extraction does not require an alternative energy source such as microwave and ultrasound.In addition,subcritical water extrac-tion (SWE)is a new and ‘green’method to extract bioactive com-pounds from plants,and the dielectric constant of subcritical water is different under different conditions.But SWE needs high temperature to reach the subcritical condition which may destroy the bioactive compounds (Dai &Mumper,2010),and extraction/10.1016/j.foodchem.2016.09.0580308-8146/Ó2016Elsevier Ltd.All rights reserved.⇑Corresponding author.E-mail address:dinghui@ (H.Ding).efficiency of subcritical water still needs more investigation.Etha-nol is a widely used solvent for bioactive compounds extraction and relatively safe for human(Shi et al.,2005),and supercritical or subcritical temperature of ethanol is much lower than water. However,few studies have been done on subcritical ethanol extraction offlavonoids from M.oleifera leaf.There are many factors influencing the subcritical extraction process,and the purpose of the present study was tofind an optimal condition to extractflavonoids by large-scale subcritical ethanol extraction and investigate antioxidant ability of the extracts.Response surface methodology(RSM)was used to opti-mize process conditions.The antioxidant property of the extracts was investigated by FRAP and DPPHÅassay.2.Experimental procedures2.1.MaterialsMoringa oleifera leaf was obtained from Guangxi province, China.Rutin and oligomeric proantho cyanidins(OPC)standards were purchased from J&K Scientific Co.(Beijing,China),and the purity was P98%and99%respectively according to the manufac-turer.2,4,6-tri(2-pyridyl)-1,3,5-triazine(TPTZ)and2,2-diphenyl-1-picrylhydrazyl radical(DPPHÅ)were purchased from J&K Scientific Co.(Beijing,China).Iron(III)chloride hexahydrate(FeCl3Á6H2O) and ferrous sulfate(FeSO4)were obtained from Bodi Chemical Reagents Co.(Tianjin,China).All other solvents and chemicals were obtained from Jiangtian Chemical Reagents Co.(Tianjin, China)and were analytical grade.2.2.HPLC–MS conditions for identification offlavonoidsBefore the HPLC–MS analysis,a purification process was con-ducted by the following procedure to enrich the antioxidant com-pounds.A column(30mmÂ500mm)was packed with150g D101macroporous adsorbent resin,and washed with300mL ethanol,5%HCl,2%NaOH respectively.After each wash,water was used to eliminate the residual ethanol,HCl or NaOH.An extract tank containing600mL extracts wasfixed on the top of the column to prepare the purification process.When the purifica-tion was performed,a pump was used to supply the extracts to the tank and keep the volume of extractsfixed in the tank,and theflow rate of extracts was 1.5mL/min.The purification process was lasted for8h,and then the purified extracts were analyzed by the HPLC–MS analysis.The compounds were separated on a C18column (150mmÂ4.60mm)operated at25°C with the elution solvents A(0.1%formic acid in water)and B(0.1%formic acid in acetoni-trile).In addition,aflow-rate of 1.5mL/min for the following gradient:10–30%B in20min and30%B in20–40min was performed to conduct the HPLC separation.To identify the separated compounds,an electrospray ion mass spectrometer(ESI-MS)was used under positive ion mode and scanned from m/z100to1000.Detailed conditions were as follows, needle voltage at3.5kV,capillary temperature at350°C,nitrogen as the drying gas at12L/min and350°C and nebulizer pressure at 40psi.2.3.Reflux extraction offlavonoidsA round-bottomflask and an attached reflux condenser were used to conduct this experiment.10.0g dried powder of M.oleifera leaf was extracted with200mL of90%ethanol for3h at a controlled temperature,and heating capacity wasfixed at500W to maintain the temperature.Then the extracts werefiltered,the solvent was evaporated at50°C under vacuum,and the residue was extracted again under the same conditions.The sample was extracted for3times in total.This whole experiment was con-ducted for3times for accuracy.Finally the products were analyzed by UV–vis spectrophotometry.2.4.Subcritical extraction offlavonoids60.0g dried powder of M.oleifera leaf was dissolved by1.5L ethanol.Then the mixture was introduced into a gas-cooled fast (GCF)reactor(shown in Fig.S1)to extractflavonoids,and a vac-uum pump was used to evacuate the air in the reactor.Heating capacity wasfixed at2000W to heat up the mixture at the begin-ning,and then turned to200W to maintain the temperature.And the pressure was approximately equaled to the saturated vapor pressure of over-heated ethanol solution due to the high vacuum at the beginning.The extracts werefiltered,and the solvent was evaporated at50°C under vacuum.UV–vis spectrophotometry was used to analyze the products.Three factors were considered to be the most important in this extraction process.Therefore we conducted a set of single factor analysis.Flavonoids were extracted with different concentrations of ethanol(55%,70%,85%and100%) for a given time ranging from1to 2.5h,while the extraction temperature ranged from110to140°C.2.5.UV–vis spectrophotometry analysisA standard solution(60l g/mL)of rutin was prepared.The solvent used in this process was ethanol–water(60:40,v/v).And then1,2,3,4and5mL rutin solutions were removed infive volumetricflasks(10mL)respectively.Next,we added2mL of AlCl3(0.1mol/L)solution and3mL of CH3COONa(1mol/L)solu-tion,waiting for5min,followed by adding ethanol–water(60:40, v/v)solvent to the scale.The sample solution without coloration was used as a reference.Determination wavelength of420nm was used to analyze the samples.Results were used to draw the rutin standard curve.1mL of the extracts were removed and diluted to10mL in a volumetricflask(10mL)by ethanol–water(60:40,v/v)solvent. Then1mL of this solution was colorated and analyzed,using the method stated above.The extraction yield Y1(mg RE/g)which meant milligrams of rutin equivalent from1g M.oleifera leaf was calculated as the following Eq.(1),where C(l g/mL)was theflavo-noid concentration calculated by rutin standard curve,V(mL)was the volume of the extracts,M(g)was the mass of M.oleifera leaf used in extraction process.And the extraction yield Y(%)could be calculated as the following Eq.(2).For simplicity,we used Y as the extraction yield in the following text.Y1¼ð100ÂCÂVÂ10À3Þ=Mð1ÞY¼Y1Â10À3Â100%ð2Þ2.6.Experimental design and statistical analysisIn the present study,we used Design Expert Version8.0soft-ware as a design and analysis tool to conduct experiments.Regres-sion coefficients,significance of the process variables,conformity of the experimental data to models and optimal response variables can be obtained by using this software.Response variable was predicted by a quadratic model shown as the following Eq.(3)Y¼AþX3i¼1B i X iþX3i¼1C ii X2iþX2i¼1X3j¼iþ1C ij X i X jð3ÞY.Wang et al./Food Chemistry218(2017)152–158153where Y was the predicted dependent variable,X i were the indepen-dent variables,A was the constant coefficient,B i were the linear regression coefficients,C ij were the interaction effect terms,and C ii were the quadratic effect terms,respectively.A three-factor RSM was conducted in this study to investigate the relationship between the response variables and process variables,and optimize the extraction process conditions. Concentration of ethanol(X1:55%–85%),extraction temperature (X2:120–140°C)and extraction time(X3:1.5–2.5h)were chosen as independent or process variables,while response variable was the extraction yield(Y)offlavonoids.After optimization,each vari-able was coded at three levels ofÀ1,0,+1as shown in Table S1.The accuracy of the model was investigated by the regression analysis(R2).And F-test was conducted to analyze the significance of the model terms.The response surface plots and contour plots were used in combination to show how the process variables affect the response variables.2.7.Antioxidant assayThe total antioxidant activity of the extracts and standards was determined by ferric reducing antioxidant power(FRAP)assay,and FeSO4solution was used as a standard to compare with the extracts.The antioxidant activity was also determined by 2,2-diphenyl-1-picrylhydrazyl radical(DPPHÅ)assay to investigate the free radical scavenging activity of the extracts,and the oligomeric proantho cyanidins(OPC)was used as a standard to compare with the extracts.2.7.1.FRAP assayFRAP reagent included50mmol/L acetate buffer which con-tained20.4g C2H3NaO2and80mL C2H4O2per liter;10mmol/L TPTZ(2,4,6-tripyridyl-s-triazine)HCl solution was used as the solvent;20reagent was obtained by mixing100mLsolution,and10mL FeCl3Á6H2O solution(0.1mL different amounts of FeSO4added into6mL FRAP reagent,and thenfor30min.Determination wavelengthmonitor the absorbance of samples.2.7.2.DPPHÅassay60l M DPPHÅwas dissolved in3mLdifferent amounts of extracts or OPCments which contained0.5mL of99%extract were also conducted.The517nm(Cervato et al.,2000).Theradical(IR)was calculated by Eq.(4)(Guirado,del Mar Rebolloso-Fuentes,&where A0was the absorbance of thewas the absorbance in the presence ofIR¼ðA0ÀA sÞ=A0Â100%When the IR is50%,theIC50.Therefore,the value of IC50wasconcentration and the inhibition rate(2010).3.Results and discussion3.1.Standard curve of rutin and results ofThe rutin standard curve was drawntion of rutin standard solution versusbency,as shown in Fig.S2.The where A was the absorbance of the sample,C(l g/mL)was the concentration of rutin.And R2of this equation was0.9997.A¼33:6574ÂCÀ0:45632ð5ÞThe extraction yield was calculated by Eq.(1),and the volume of extracts was about100mL in the reflux extractions while 10.67g M.oleifera leaf was used.And the absorbances of the three experiments were0.666,0.667and0.664.According to the Eqs.(5) and(1),the average extraction yield which was20.6mg RE/g (2.06%)could be obtained.3.2.Single factor analysisThere are many factors affecting the extraction yield,among which the ethanol concentration,extraction time,and extraction temperature are the main factors.Single factor analysis was performed with one factor changed and the others kept unvaried.The extraction by different ethanol concentrations(55%,70%, 85%,100%)was investigated,while the other conditions were kept the same(extraction temperature was140°C,and extraction time was2h).And the extraction by extraction time of1,1.5,2and2.5h was investigated,while the other conditions were kept the same (extraction temperature was130°C,and ethanol concentration was100%).Finally,the extraction by extraction temperature of 110,120,130and140°C was investigated,while the other condi-tions were kept the same(extraction time was2h,and ethanol concentration was100%).The detail experimental conditions and results were shown in Table.S2.As shown in Fig.1,the extraction efficiency was the highest when the extraction temperature was130°C.When the tempera-ture was higher,the extraction efficiency decreased.It could speculate that some of the heat-sensitive components in M.oleiferaFig.1.Effects of extraction temperature,extraction time and ethanol concentration on yield offlavonoids.154Y.Wang et al./Food Chemistry218(2017)152–1582.Three-dimensional(3D)response surface and contour plot curve illustrating combined effects of(a)extraction time and ethanol concentration(b)extraction time extraction temperature(c)extraction temperature and ethanol concentration on extraction yield.As we can see in Table1,F value of the model was26.23while the P value was only0.0004,which showed a high significance of the model.For a good accuracy of a model,R2must be more than 75%(Chauhan&Gupta,2004).Thus the model stated above with a relatively high coefficient of determination value(R2=0.9752) illustrated that almost all extraction data could be explained by this model.Therefore,using this model to predict the influence of the process variables on the extraction yield was reasonable and reliable.The quadratic variable X22was statistically very signif-icant because the P value was lower than0.0001;two-variable interaction X1X2,linear variable X2,quadratic variables X12and X32 had significant influences(P<0.01)on the extraction yield offlavo-noids;linear variable X3and two-variable interaction X1X3had influences(P<0.05)on the extraction process,whereas the linear variable X1and two-variable interaction X2X3had no significant influence(P>0.1)on the extraction yield offlavonoids.The linearand quadratic coefficients of each process variable indicated that extraction temperature had morethan extraction time,whilethan ethanol concentration.3.4.Response surface analysisThe effects of the processtions on the extraction yield cansurface plots and their contourthe process variables areplots is elliptical(Muralidhar,2001).The effects of extraction timeextraction yield were shown in Fig.ture was set at130°C.Fig.2(a)between extraction time andimpact on the yield offlavonoids,significant influence than ethanolyield increased as the extractionapproximately 2.1h,and thenincreased from about2.1–2.5h,at a certain value(70%).It was notthe ethanol concentration affectedextraction time,but there wasciency while ethanol concentrationAs shown in Fig.2(b),in whichboth extraction time andimpact on extraction efficiency,cant impact.When extractionextraction yield obviouslyethanol concentration had noaddition,the interactionextraction temperature had avonoids.All the results were inin the ANOVA.3.5.Optimization of extraction andAccording to the analysis ofdition was obtained:ethanolperature,126.6°C;extractioncondition,the estimated value2.61%.The extracts obtained frompurified by D101macroporouschapter 2.2,and analyzed byHPLC–MS was shown in Fig.S3shown in Fig.S4.In Fig.S4,two303)were obviously observedquercetin glycosides respectively.glycosides in the Moringa oleifera leaf extracts are quercetin and Table2Identifiedflavonoid compounds of Moringa oleifera leaf extracts.Peak t R(min)MS(m/z)MS fragmention(m/z)Identitiesa13.4595.2303Quercetin-diRb18.8465.1303Quercetin-Gc21.0449.1287Kaempferol-Gd21.6507.1303Quercetin-G-Ace23.4507.1303Quercetin-G-Acf24.4491.1287Kaempferol-G-Acg27.1533.1303Quercetin-Xyl/Api-S h27.8517.1287Kaempferol-Xyl/Api-S diR:dirhamnosyl,G:Glucosyl/Galactosyl moiety,Ac:Acetyl,Xyl:Xylosyl,Api: Apiosyl,S:Succinoyl.Fig.3.Total antioxidant activity of(a)extracts and(b)FeSO4solutions.156Y.Wang et al./Food Chemistry218(2017)152–158reliable,and efficient.Extraction yield of subcritical extraction increased about26.70%compared with the traditional ethanol reflux method.Most importantly,this subcritical extraction pro-cess only needed4h(including heating time)to reach the optimal yield which was2.60%while the traditional method needed nearly 11h to reach a yield of2.06%.And we also observed the power consumption of traditional method totaled 4.6kWÁh while2.4kWÁh in the subcritical ethanol extraction process.3.6.Antioxidant activity3.6.1.FRAP assayAs shown in Fig.3,FeSO4solution was used as a standard to evaluate the total antioxidant activity of extracts.Thefigure showed a linear correlation between the concentration of FeSO4 solution or extracts and the absorbance,and the concentration of (IR%)and IC50to characterize the free radical scavenging activity of extracts.The value of IR increased with the concentration added. For extracts from M.oleifera leaf,the IC50value was0.7440mg/L, while the IC50value of OPC was0.0195mg/L.Therefore,the free radical scavenging activity of extracts from1mg M.oleifera leaf was approximately equivalent to that of0.026mg OPC.4.ConclusionsIn the present study,RSM in combination with three-factor and three-level BBD was successfully applied to study and optimize the process variables for the subcritical ethanol extraction offlavo-noids from M.oleifera leaf.According to the HPLC–MS analysis, quercetin and kaempferol glycosides were found in the extracts. The experiment results showed that,extraction time and extrac-tion temperature had significant effects on the extraction yield. Analysis of variance(ANOVA)showed a high coefficient of deter-mination value(R2>0.95).Therefore,the mathematical model developed by Box-Behnken design can be used to predict the extraction efficiency offlavonoids.Under the optimal conditions (extraction temperature:126.6°C,extraction time:2.05h,ethanol concentration:70%),the experimental result was2.60%and shown to be in agreement with the predicted one.And the subcritical extraction used only4h to reach the optimal result,while the traditional ethanol reflux method needed nearly half a day to obtain a yield of2.06%.The traditional method spent twice as much energy as subcritical extraction did.Subcritical ethanol extraction will have great potential use in industry.In addition, antioxidant assays showed that the extracts had strong antioxidant ability,and extracts from1mg M.oleifera leaf approximately equaled0.95–1.35mmol FeSO4or0.026mg OPC. AcknowledgementThis work wasfinancially supported by the National Natural Science Foundation of China(Grant No.21376166).Appendix A.Supplementary dataSupplementary data associated with this article can be found,in the online version,at /10.1016/j.foodchem.2016.09.058.ReferencesAbdulkarim,S.M.,Long,K.,Lai,O.M.,Muhammad,S.K.S.,&Ghazali,H.M.(2005).Some physico-chemical properties of Moringa oleifera seed oil extracted using solvent and aqueous enzymatic methods.Food Chemistry,93(2),253–263. Albu,S.,Joyce,E.,Paniwnyk,L.,Lorimer,J.P.,&Mason,T.J.(2004).Potential for the use of ultrasound in the extraction of antioxidants from Rosmarinus officinalis for the food and pharmaceutical industry.Ultrasonics Sonochemistry,11(3–4), 261–265.Alhakmani,F.,Kumar,S.,&Khan,S.A.(2013).Estimation of total phenolic content, in-vitro antioxidant and anti-inflammatory activity offlowers of Moringa n Pacific Journal of Tropical Biomedicine,3(8),623–627.discussion 626–627.Anwar,F.,Latif,S.,Ashraf,M.,&Gilani,A.H.(2007).Moringa oleifera:A food plant with multiple medicinal uses.Phytotherapy Research,21(1),17–25.Arabshahi-Delouee,S.,&Urooj,A.(2007).Antioxidant properties of various solvent extracts of mulberry(Morus indica L.)leaf.Food Chemistry,102(4),1233–1240. Benzie,I.F.,&Strain,J.J.(1996).The ferric reducing ability of plasma(FRAP)as a measure of‘‘antioxidant power”:The FRAP assay.Analytical Biochemistry,239(1),70–76.Cervato,G.,Carabelli,M.,Gervasio,S.,Cittera,A.,Cazzola,R.,&Cestaro,B.(2000).Antioxidant properties of oregano(Origanum vulgare)leaf extracts.Journal of Food Biochemistry,24(6),453–465.Chauhan,B.,&Gupta,R.(2004).Application of statistical experimental design for optimization of alkaline protease production from Bacillus sp.RGR-14.Process Biochemistry,39(12),2115–2122.Dai,J.,&Mumper,R.J.(2010).Plant phenolics:Extraction,analysis and their antioxidant and anticancer properties.Molecules,15(10),7313–7352.Fig.4.Scavenging activity of(a)extracts and(b)OPC on DPPH free radical.Y.Wang et al./Food Chemistry218(2017)152–158157Guil-Guerrero,J.L.,Martínez-Guirado, C.,del Mar Rebolloso-Fuentes,M.,& Carrique-Pérez,A.(2006).Nutrient composition and antioxidant activity of10 pepper(Capsicum annuum)varieties.European Food Research and Technology, 224(1),1–9.Hertog,M.G.,Feskens,E.J.,Hollman,P.C.,Katan,M.B.,&Kromhout,D.(1993).Dietary antioxidantflavonoids and risk of coronary heart disease:The Zutphen Elderly ncet(London,England),342(8878),1007–1011.Jo,E.-K.,Heo,D.-J.,Kim,J.-H.,Lee,Y.-H.,Ju,Y.-C.,&Lee,S.-C.(2013).The effects of subcritical water treatment on antioxidant activity of golden oyster mushroom.Food and Bioprocess Technology,6(9),2555–2561.Kansal,S.K.,&Kumari,A.(2014).Potential of M.oleifera for the treatment of water and wastewater.Chemical Reviews,114(9),4993–5010.Manach,C.,Scalbert,A.,Morand,C.,Remesy,C.,&Jimenez,L.(2004).Polyphenols: Food sources and bioavailability.American Journal of Clinical Nutrition,79(5), 727–747.Maran,J.P.,Manikandan,S.,Priya,B.,&Gurumoorthi,P.(2015).Box-Behnken design based multi-response analysis and optimization of supercritical carbon dioxide extraction of bioactiveflavonoid compounds from tea(Camellia sinensis L.)leaf.Journal of Food Science and Technology-Mysore,52(1),92–104. Matshediso,P.G.,Cukrowska,E.,&Chimuka,L.(2015).Development of pressurised hot water extraction(PHWE)for essential compounds from Moringa oleifera leaf extracts.Food Chemistry,172,423–427.Morton,J.(1991).The horseradish tree,Moringa pterygosperma(Moringaceae)—A boon to Arid Lands?Economic Botany,45(3),318–333.Moyo,B.,Masika,P.J.,Hugo,A.,&Muchenje,V.(2011).Nutritional characterization of Moringa(Moringa oleifera Lam.)leaf.African Journal of Biotechnology,10(60), 12925–12933.Muralidhar,R.V.,Chirumamila,R.R.,Marchant,R.,&Nigam,P.(2001).A response surface approach for the comparison of lipase production by Candida cylindracea using two different carbon sources.Biochemical Engineering Journal,9(1),17–23. Rostagno,M.A.,Palma,M.,&Barroso,C.G.(2007).Microwave assisted extraction of soy isoflavones.Analytica Chimica Acta,588(2),274–282.Shi,J.,Nawaz,H.,Pohorly,J.,Mittal,G.,Kakuda,Y.,&Jiang,Y.(2005).Extraction of polyphenolics from plant material for functional foods—Engineering and technology.Food Reviews International,21(1),139–166.Singh,N.,&Rajini,P.S.(2004).Free radical scavenging activity of an aqueous extract of potato peel.Food Chemistry,85(4),611–616.Song,H.,Zhang,Q.,Zhang,Z.,&Wang,J.(2010).In vitro antioxidant activity of polysaccharides extracted from Bryopsis plumosa.Carbohydrate Polymers,80(4), 1057–1061.Vongsak,B.,Sithisarn,P.,&Gritsanapan,W.(2014).Simultaneous HPLC quantitative analysis of active compounds in leaves of Moringa oleifera Lam..Journal of Chromatographic Science,52(7),641–645.Zhang,H.-F.,Yang,T.-S.,Li,Z.-Z.,&Wang,Y.(2008).Simultaneous extraction of epimedin A,B,C and icariin from Herba Epimedii by ultrasonic technique.Ultrasonics Sonochemistry,15(4),376–385.158Y.Wang et al./Food Chemistry218(2017)152–158。
