摘要
叶酸是一种重要的水溶性B族维生素,常以多种形式存在,其英文名称除folic acid外,也被称为folate.叶酸生理功能包括参与DNA和RNA的合成;参与氨基酸代谢,在同型半胱氨酸与蛋氨酸之间的相互转化中发挥作用,因此,叶酸与人体重要的生化过程密切相关。叶酸缺乏会引起多种疾病,包括阿尔茨海默病、心血管疾病、骨质疏松症、乳腺癌和结肠癌、抑郁症、痴呆、唇腭裂、听力受损症状。此外,研究还发现,叶酸对孕妇尤其重要,孕妇膳食中添加叶酸可以预防新生儿神经管缺陷。由于人类自身无法合成叶酸,因此,需要从膳食中摄取叶酸。美国食品和营养委员会推荐成年人叶酸摄取量为400μg/天,怀孕期妇女为600μg/天,哺乳期妇女为500μg/天。但调查发现大量人群叶酸摄取不足,特别是孕妇。在人体叶酸营养强化策略中,食物中天然存在的叶酸的营养强化效果明显优于药物和强化食品中人工合成的叶酸。绿叶蔬菜作为天然叶酸的主要来源,其叶酸常以多谷氨酸叶酸的形式存在。然而,目前对蔬菜中的叶酸生物强化研究相对较少。
蔬菜是中国居民摄取叶酸的主要来源,用高效液相色谱方法检测蔬菜中叶酸类物质具有重要意义。本研究建立了高效液相色谱方法检测蔬菜中多谷氨酸叶酸的检测方法,具有良好的灵敏度,应用在中国市场上出售的不同种类蔬菜中叶酸类物质的检测中发现,蔬菜中的豆芽、菠菜和小白菜的叶酸含量最高。因此,本研究通过进一步研究不同品种豆类种子发芽时间对豆类叶酸及其形式的分析,以期获得最佳的发芽时间。除此外,也通过对不同品种菠菜和小白菜叶酸总量和形态的分析,研究了基因型对两种叶菜中叶酸的影响,以揭示蔬菜叶酸的遗传多样性。主要结果如下：
(1)不同形式叶酸的稳定性,及低浓度是植物样品中叶酸检测的难点。因此,建立一个稳定可靠的叶酸及不同形式叶酸的分析检测技术对于蔬菜叶酸强化领域研究尤为重要。本研究通过建立快速、简便、灵敏的高效液相色谱方法应用于植物样品中叶酸的测定。该方法采用磷酸缓冲液煮沸法、添加老鼠血清(RS)轭合酶法、固相萃取柱纯化法(SPE)提取和纯化植物中的叶酸,通过Zorbax SB C-18色谱柱分离,使用高效液相色谱(HPLC)-紫外-荧光检测器的串联法对蔬菜中的叶酸总量及不同形式叶酸(四氢叶酸、5-甲基四氢叶酸、5-甲酰基四氢叶酸、10-甲酰叶酸和叶酸)进行定性和定量测定,结果表明,叶酸在范围内,峰面积与进样量呈良好线性关系,叶酸标准物质回收率高,试验重复性好。应用该方法对三种叶菜(菠菜、小白菜和生菜)的不同形态叶酸检测发现,5-甲基四氢叶酸是三种叶菜叶酸的主要形式。因此,本研究建立的叶酸检测方法是一种快速、简便、灵敏的特点,适用于植物不同形式叶酸的检测。
(2)叶酸缺乏会增加人体患慢性疾病的风险,这些疾病包括婴幼儿神经管缺陷,成年人巨幼细胞性贫血,心血管疾病和某些癌症。中国是已知的神经管畸形高发区。通过足量食用新鲜的绿叶蔬菜可以减少中国人群叶酸缺乏问题,但目前对中国居民经常食用的蔬菜中总叶酸及其不同形式叶酸含量的研究较少。本研究采用高效液相色谱法对中国市场上出售的不同种类蔬菜(绿叶蔬菜、水果蔬菜、根类蔬菜)叶酸分析检测,根据结果估算以蔬菜为主要叶酸来源的中国居民膳食摄入叶酸量。结果表明,不同种类蔬菜可食部分的总叶酸含量在14.78-145.54μg/100g之间,平均含量为61.99μg/100g。其中以小白菜和菠菜的叶酸含量最高(>140μg/100g)。绿叶蔬菜、水果蔬菜、根类蔬菜的总叶酸含量分别为17.22-145.54μg/100g、18.14-86.04g/100g、14.78-75.81μg/100g。以上结果说明,中国居民常食用的不同类型蔬菜之间叶酸含量差异很大,总体来说,叶菜类蔬菜的叶酸含量高于水果和根茎类蔬菜。
(3)叶酸缺乏和叶酸摄入量不足所导致的人体健康问题及社会经济负担问题日益受到关注。本试验研究不同发芽时间对不同品种豆类(黄豆、绿豆)种子的叶酸、维生素C、总酚和总抗氧化能力含量的影响,以期豆芽营养富集的最佳发芽时间。结果表明,发芽第4天,黄豆芽和绿豆芽叶酸含量达到最高值分别为815.2μg/100g鲜重,675.4μg/100g鲜重。相比未发芽对照,分别提高了3.5和3.9倍；超过4天之后,黄豆芽和绿豆芽的叶酸含量随之下降。5-甲基四氢叶酸是豆芽中叶酸的主要形式,并在种子发芽第4天含量最高。维生素C在未发芽对照中未检测到,发芽的第4天,黄豆芽和绿豆芽的维生素C含量达到最大值,分别达到了29mg/100g鲜重和27.7mg/100g鲜重。发芽4天的黄豆芽和绿豆芽的总叶酸、5-甲基四氢叶酸、维生素C、多酚和抗氧化能力也达到最大值。因此,本研究认为豆类发芽最适宜时间为4天。
(4)叶酸生物强化为包括发展中国家在内的世界各国解决人体叶酸缺乏提供了一种成本低廉、有效的途径,该方法主要通过育种手段提高植物可食用部分叶酸含量来实现。高叶酸的种质可用于常规育种或直接作为品种利用。在蔬菜中,菠菜中叶酸含量相对较高,然而,对于不同品种菠菜中总叶酸及不同形式叶酸含量分析检测的报道较少。本研究在人工气候室统一种植条件下,种植供试67个菠菜品种(来自于美国农业部和亚洲蔬菜研究发展中心种质资源)收获后采用高效液相色谱法检测供试菠菜品种的总叶酸及不同形式叶酸含量。结果表明,菠菜总叶酸含量范围为54.1-173.2gg/100g鲜重,不同基因型之间相差约3倍,并筛选出4个高叶酸品种(PI499372,NSL6095,PI261787和TOT7337-B),其总叶酸含量均超过150gg/100g鲜重。不同形式叶酸(四氢叶酸,5-甲基-四氢叶酸,5-甲酰-四氢叶酸和叶酸)存在显著的基因型差异是造成总叶酸含量变化的原因。菠菜叶酸的主要形式是5-甲基四氢叶酸占总叶酸量的52%以上。菠菜总叶酸及不同形式叶酸含量的基因型差异为高叶酸育种研究提供了可能。
(5)叶酸是生物体必需的微量营养素。然而叶酸摄入不足造成的人体叶酸缺乏的问题相当普遍。利用育种生物强化途径是解决人体叶酸缺乏具有较好前景。种质资源遗传多样性的那些样品,在遗传育种中可作为亲本被优先利用。在本研究采用高效液相色谱法对不同品种小白菜叶酸总量及其不同形式叶酸含量分析检测,研究小白菜叶酸的遗传多样性。结果表明,小白菜叶酸总量范围在52.7-166.9μg/100g鲜重之间。小白菜中不同形式叶酸(四氢叶酸,5-甲基-四氢叶酸,5-甲酰-四氢叶酸和叶酸)存在显著的基因型差异造成了叶酸总量的变化。小白菜叶酸主要形式是5-甲基-四氢叶酸含量,其占总叶酸含量的54.6%,不同基因型之间相差4.5倍。对叶酸合成代谢途径中11个关键基因的表达水平研究发现,叶酸含量的变异受转录后水平的调控,故叶酸代谢基因表达的转录后调控可能是造成小白菜种质资源叶酸积累量差异的主要原因,这也是利用小白菜叶酸含量的基因型差异进行叶酸生物强化育种的遗传基础。
Folate, or folic acid, is one of the most nutritionally significant water soluble B vitamin that exists in many different forms. Its presence is necessary for synthesis of DNA and RNA and methylation of homocysteine to methionine. The importance of folate in the diet of pregnant women for the prevention of neural tube defects (spina bifida and anencephaly) in babies is well-known. Folate deficiency has also been implicated in a wide variety of disorders including Alzheimer's disease, cardiovascular diseases, osteoporosis, breast and colon cancers, depression, dementia, cleft lip/palate and hearing loss. Humans cannot synthesise folate by themselves and therefore depend on an adequate supply through food intake. The US Food and Nutrition Board have nominated a recommended dietary allowance (RDA) for folate of400μg for adults,600μg during pregnancy and500μg during lactation per day. However, the recommended daily intake of folate is not reached by many people, especially women in fertile ages. Natural sources of folate are reportedly more effective than supplementation or fortification with folic acid. Green leafy vegetables are excellent sources of folates, which occur naturally in many forms or vitamers. However, little effort has been taken to folate biofortification in vegetables.
In this series of experiment, we develop and validate a rapid, simple and sensitive Liquid Chromatographic (LC) method suitable for analysis of most common monoglutamate folates in vegetables. Using this validated method, further we analyze most commonly consumed important vegetables that contribute to folate intake in Chinese population, and we found that legume sprouts, spinach and pakchoi are the richest sources of folate. Therefore, further we optimization of the germination process of different cultivars of legume seeds to maximize content and composition of folate. Further we investigate natural variation in the folate content and composition of spinach genotypes grown under controlled environmental conditions to screen for natural diversity. We also investigate natural genetic diversity of folate in pakchoi genotypes grown under controlled environmental conditions to screen for natural diversity for breeding program. The salient findings are as follows:
(1) Folate analysis is challenging because of large number of structural analogs, their stability and low level in natural samples. A reliable, relatively simple and rapid method will enable to assess the biofortification interventions of folate content and composition. The prime concern of this study was to develop and validate a rapid, simple and sensitive liquid chromatography (LC) method suitable for analysis of most common monoglutamate folates in plant samples. The method involved extraction of folates from the plant sample by heat treatment, deconjugation of folate polyglutamates to monoglutamates by incubation with rat serum (RS) conjugase and purification of food extracts by solid-phase extraction (SPE) and LC-UV/FL detection with Zorbax SB C18column for separation of main monoglutamate folates from plant samples. It was possible to separate and determine five folate derivatives: tetrahydrofolate,5-methyltetrahydrofolate,5-formyltetrahydrofolate,10-formylfolic acid and folic acid with fluorescence and ultraviolet detection. Validation of the method included linearity tests, the addition of standard folates for the determination of recovery and repeatability tests. The method developed was applied to analysis of spinach, pakchoi and lettuce;5-methyltetrahydrofolate was found to be the main folate form in all these vegetables. Our validate method offers a rapid, simple and sensitive approach to determine the level of main monoglutamate folates in plant samples.
(2) Folate deficiency increases the risk of chronic diseases, including neural tube defects (NTDs) in infants, megaloblastic anemia, cardiovascular disease, and some cancers in adults. China is the most NTDs prevalent area in the world. Folate deficiency in China can be reduced by proper supply of fresh leafy green vegetables but little is known about the folate content and vitamers in the vegetables commonly consumed by Chinese population. The purposes of this study were first to analyze most commonly consumed important vegetables that contribute to folate intake in the Chinese population and second to estimate the significance of selected vegetables as a source of dietary folate intake. Folate content and vitamers forms in vegetables were analyzed using a valid liquid chromatography (LC) method. Mono-enzyme treatment was used for leafy green and some fruit vegetables, and di-enzyme treatment for some root vegetables. Total folate content in commonly consumed vegetables ranged from14.78to145.54μg/100g in edible portion with an average61.99μg/100g. The highest folate content (>140μg/100g) were found in pakchoi and spinach. Total folate content in leafy vegetables, fruit vegetables, and root vegetables were in the range of17.22to145.54μg/100g,18.14to86.04μg/100g, and14.78to75.81p.g/100g, respectively. The considerable variations in folate content were found in different types of vegetables commonly consumed by Chinese population. Leafy vegetables are a better source of folate than fruit and root vegetables commonly consumed by Chinese population.
(3) Folate deficiency associated with low dietary intake is a well-documented public health problem, resulting in serious health and socioeconomic burdens. Therefore, optimization of the germination process of different cultivars of legume seeds in relation to the content and composition of folate, vitamin C, and total phenolics and total antioxidant capacity was carried out to maximize the health-promoting properties. The content and composition of folate, vitamin C, and total phenolic and total antioxidant capacities varied between species, among cultivars, and with germination time. During germination, total folate content was maximum at815.2μg/100g fresh weight in soybean sprout and at675.4μg/100g fresh weight in mungbean sprout on the fourth day, which were equivalent to, respectively,3.5-and3.9-fold increases in the seed's content, and total folate content strongly decreased thereafter.5-CH3-H4folate was the most abundant folate species in legume sprouts and reached a maximum on the fourth day. Vitamin C was not detected in raw seeds, and its content increased sharply in soybean and mungbean sprouts and reached a maximum at the fourth day of germination (29and27.7mg/100g fresh weight, respectively). Germination of soybean and mungbean for4days provided the largest amount of total folate as well as the more stable species5-CH3-H4folate and also brought about large amounts of vitamin C and total phenolics and substantial antioxidant capacities.
(4) Breeding to increase folate levels in edible parts of plants, termed folate biofortification, is an economical approach to fight against folate deficiency in humans, especially in the developing world. Germplasm with elevated folates are a useful genetic source for both breeding and direct use. Spinach is one of the well-know vegetables that contain a relatively high amount of folate. Currently, little is known about how much folate, and their composition varies in different spinach accessions. The aim of this study was to investigate natural variation in the folate content and composition of spinach genotypes grown under controlled environmental conditions. The folate content and composition in67spinach accessions were collected from the United States Department of Agriculture (USDA) and Asian Vegetable Research and Development Center (AVRDC) germplasm collections according to their origin, grown under control conditions to screen for natural diversity. Folates were extracted by a monoenzyme treatment and analyzed by a validated liquid chromatography (LC) method. The total folate content ranged from54.1to173.2μg/100g of fresh weight, with3.2-fold variation, and was accession-dependent. Four spinach accessions (PI499372, NSL6095, PI261787, and TOT7337-B) have been identified as enriched folate content over150μg/100g of fresh weight. The folate forms found were H4-folate,5-CH3-H4folate, and5-HCO-H4folate, and10-CHO-folic acid also varied among different accessions and was responsible for variation in the total folate content. The major folate vitamer was represented by5-CH3-H4folate, which on average accounted for up to52%of the total folate pool. The large variation in the total folate content and composition in diverse spinach accessions demonstrates the great genetic potential of diverse genotypes to be exploited by plant breeders.
(5) Folates are essential micronutrient required by most living organisms. Folate deficiency in human remains prevalent due to inadequate dietary intake. Biofortification through breeding is a potential strategy to reduce widespread human folate deficiency. Genetically diverse germplasm can potentially be used as parents in breeding programs. Therefore, we investigated natural genetic diversity of folate in pakchoi genotypes grown under controlled environmental conditions. Folates were extracted by a monoenzyme treatment and analyzed by a validated liquid chromatography method. The total folate content ranged from52.7to166.9μg/100g of fresh weight, with3.2-fold variation. The foiate forms found were H4folate,5-CH3-H4folate, and5-HCO-H4folate, and10-CHO-folic acid also varied among different accessions and was responsible for variation in the total folate content. The major folate vitamer was represented by5-CH3-H4folate, with4.5-fold variation, which on average accounted for up to54.6%of the total folate pool. A general correlation between total folate, pteridine and pABA was observed. Analysis of the transcription of11genes involved in folate metabolism suggested that variation in folate concentrations may be controlled primarily at the post-transcriptional level. Transcriptional regulation of folate metabolism gene expression might contribute to the different levels of folate accumulation in pakchoi germplasm. The diverse genotypic variation in pakchoi germplasm demonstrates the great genetic potential to integrate breeding programs for folate biofortification.
引文
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