Folates In Vegetables:Determination,Enhancement And Genotypic Variation

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.