Total Isoflavone Concentration Research Articles

Effect of Origin, Seed Coat Color, and Maturity Group on Seed Isoflavones in Diverse Soybean Germplasm.

Soybeans are grown worldwide owing to their protein, oil, and beneficial bioactive compounds. Genetic and environmental factors influence soybean seed isoflavones. In the present study, we profiled the seed isoflavones in world diverse soybean germplasm grown in two locations over two years in China. Significant differences (p < 0.001) were observed between the accessions, accession origins, seed coat colors, and maturity groups for individual and total isoflavone (TIF) content. TIF content of the soybean accessions ranged from 677.25 μg g-1 to 5823.29 μg g-1, representing an 8-fold difference. USA soybean accessions showed the highest mean TIF content (3263.07 μg g-1), followed by Japan (2521.26 μg g-1). Soybean with black seed coat showed the highest (3236.08 μg g-1) TIF concentration. Furthermore, isoflavone levels were significantly higher in late-maturity groups. Correlation analysis revealed significant positive associations between individual and TIF content. Malonyldaidzin and malonylgenistin showed higher correlations with TIF content (r = 0.92 and r = 0.94, respectively). The soybean accessions identified as having high and stable TIF content can be utilized in the food and pharmaceutical industries and breeding programs to develop soybean varieties with enhanced isoflavone content.

Effects of Light Orientation and Mechanical Damage to Leaves on Isoflavone Accumulation in Soybean Seeds

Soybean is largely cultivated worldwide providing high amounts of proteins and oil for food and feed, and isoflavones for nutraceutical uses. The increasing interest in agroforestry practices for improving carbon sequestration and mitigating climate changes suggests the need to assess soybean response to variations of light availability and direction. A two-year pot trial was carried out at Legnaro (NE Italy) in order to mimic the response of the soybean var. Sponsor to contrasting light orientation (east or west) by artificial shading, associated or not with mechanical leaf damage, in terms of protein accumulation, total cotyledon isoflavone concentration (TCIC) and isoflavone profile. Here, we demonstrate that a different intensity of the isoflavone metabolism exists in response to lighting conditions, with higher TCIC and slightly increased seed crude proteins in plants lighted from the east (morning time) and after mechanical leaf damage. The isoflavone profile was not changed, but low temperatures and high rainfall during seed filling (1st year) were associated with increased accumulation of medium-high molecular weight (MW) forms (i.e., glycosyls and malonyls), while high temperatures and low rainfall (2nd year) with increased accumulation of medium-low MW forms (i.e., glycosyls and aglycones). It is concluded that within agroforestry systems, there is possibly a large scope for maximizing isoflavone accumulation by selecting the harvesting area in the neighboring of the east side of the tree alleys, with further improvements if a mild shoot stripping is applied before flowering.

Determination of Isoflavones in Nuts, Dried Fruits and Vegetables by High Performance Liquid Chromatography

In this study, the amounts of daidzin, daidzein and genistein of isoflavone components were determined in nuts, dried fruits and vegetables by high performance liquid chromatography (HPLC). Firstly, the extraction conditions like the rate of the aqueous and organic phase, the total volume of the extraction solution, time and rotation per minute of centrifugation were optimizaed. After extraction conditions were specified, the parameters of HPLC device have investigated. The isoflavone components were extracted by a mixture of 0.1% acetic acid and methanol (20:80 v/v), at the room temperature. The chromatographic separation was conducted on C18 with wavelength of 260 nm at 30 oC. The diode array detector was used. The analytical parameters given as the dedection and quantification limits, linearity, precision and accuracy were determined. The relative standard deviation of method was determinated ≤3 % (n=21) and the calibration curse was worked in the range 1-100 mg L-1. The total isoflavone concentration in real samples from Sivas centrum was determined in the range 9.6 ˗ 469.1 μg g-1.

Seed isoflavone profiling of 1168 soybean accessions from major growing ecoregions in China.

Soybean (Glycine max L. Merrill) isoflavones are secondary metabolites of great interest because of their beneficial impact on human health. We profiled the seed isoflavone composition of 1168 soybean accessions collected from diverse ecoregions of China in three locations over two years. We observed significant differences in isoflavone content among the accessions, accession types, years of growth and ecoregions of origin. Total isoflavone (TIF) concentration of the soybean accessions ranged from 745μgg-1 to 5253.98μgg-1, which represents a 7-fold difference. The highest mean TIF concentration (2689.27μgg-1) was observed in the Huang Huai Hai Valley Region (HR) accessions, followed by accessions from the Southern Region (SR) and Northern Region (NR) with TIF concentration of 2518.91μgg-1 and 1942.78μgg-1, respectively. Thirty-five accessions were identified as elite soybean resources based on their higher TIF concentration (4024.74μgg-1 to 5253.98μgg-1). Pairwise correlation analysis showed significant positive correlations between individual isoflavones and TIF concentrations. Malonyldaidzin and malonylgenistin showed the highest correlations with TIF concentration (r=0.90 and r=0.92, respectively), whereas acetyldaidzin showed the lowest correlation. The main isoflavone components had significant negative correlations with latitude and longitude, indicating that the geographical origin of the accessions influenced their seed isoflavone composition. Based on principal component analysis, glycosides and malonylglycosides of isoflavones were the major discriminative components for the soybean accessions. The present study demonstrated the geographical distribution of soybean seed isoflavone concentrations across the main ecoregion of China. The identified soybean accessions with both high and low TIF concentrations, which are desirable materials for industrial uses and could also be used as parents to breed soybean lines with improved isoflavone quantity and composition in the seeds.

Optimization of isoflavones extraction from soybeans using full factorial design

This study focuses on the optimization of isoflavones extraction from soybeans cultivated in Quangnam province, Vietnam, using aqueous ethanol solvent extraction. The total isoflavone concentration was determined with six isoflavone standards including daidzin, glycitin, genistin, daidzein, glycitein, and genistein by Reversed Phase HPLC method. The effects of temperature, extraction time, and ratio of solvent to material on the extracted isoflavone content were investigated for use in the experimental planning method with full element 23 and optimization using the Box-Wilson model. The results of the optimization showed that the optimal conditions of isoflavones extraction from soybeans using ethanol 80% was in temperature of 72.5°C, extraction time of 67.5 min, and solvent to dry soybean ratio of 26.5/1 (ml/g). The maximum amount of isoflavone content obtained from this extraction condition was 1,932.44 µg/g dry matter. Practical applications The extraction of isoflavones from soybeans involves the interaction between various factors including solvent concentration, temperature, extraction time, and ratio of solvent to material. There is a need to study the combined effect of these factors to optimize the extraction conditions within a minimum number of experiments. Hence, this work applies the full factorial design to study the effects of several factors on one or more responses to achieve the optimum extraction condition. This research work is important to obtain the most efficient extraction conditions for isoflavones production without consuming too much efforts on the experimentation. The results attain from this work can contribute to the extension of efficient isoflavones extraction in industrial scale.

LC-ESI-MS based characterisation of isoflavones in soybean (Glycine max (L.) Merr.) from India.

A total of twenty-one soybean varieties were screened for their morphological characteristics followed by isoflavone content analysis by HPLC. The total isoflavone (TI) content was found within a wide range of 140.9-1048.6μg/g of soy in different varieties. The highest isoflavone content was found in MAUS-2 followed by DS_2613 and lowest in Karunae (140.9μg/g of soy). Various isoflavone forms were identified by LC-ESI+ MS. Significant differences in the isoflavone content were observed for all the aglycones and their glucoside conjugates as well as total daidzein, total genistein, total glycitein, and TI. A positive correlation between TI content and growth stages was found during the progression of seed development. An increase of 5.4-fold and 5.3-fold of TI concentration was observed for JS 335 and MAUS-2 respectively, from early to green mature (R5-R8) stage of bean development.

Genotype, environment, and genotype by environment interaction for seed isoflavone concentration in soybean grown in soybean cyst nematode infested and non-Infested environments

Soybean (Glycine max [L.] Merr.) is a major isoflavone producing legume. Isoflavones and their derivatives function as phytoalexins that may function in protecting plants against pathogens. They are also known as phytoestrogens in the human diet, which have putative positive human health effects such as reduced risk of breast and prostate cancers, cardiovascular disease, and high blood cholesterol levels. Therefore, increasing isoflavone concentration could be a desirable target for developing added value for food-grade soybean cultivars. Soybean cyst nematode (SCN; Heterodera glycines) is the most yield-limiting pathogen for soybean world-wide. The objectives of this study were to: 1) determine genotype (G) and genotype-by-environment (GE) interaction variation for seed isoflavone concentration and, 2) determine the effects that SCN resistance may have on seed isoflavone concentration in SCN infested and non-infested environments. A population of 109 recombinant inbred lines (RILs), derived from a cross between DH4202 and RCAT1004, along with 11 commercial check cultivars were grown in two SCN infested and two non-infested fields in southern Ontario, Canada, in 2015 and 2016. The genotypes were categorized into resistant, moderately resistant, and susceptible lines based on SCN female indices calculated from SCN infested environments. Significant G, environment (E), and GE interaction effects were observed for total isoflavone concentration as well as for yield. Higher isoflavone concentrations were observed in non-infested environments when compared with SCN infested environments for all three levels of resistance. Within SCN infested environments, resistant genotypes had significantly higher isoflavone concentrations than susceptible genotypes, indicating a potential role of isoflavones in plant defense from SCN in resistant genotypes. SCN infested environments were found to be more reliable for genotype evaluation of isoflavone concentration than non-infested environments due to similar relative performance of genotypes in all SCN infested environments. A strong relationship between the level of SCN resistance and relative yield among the genotypes was observed in SCN environments. When evaluating genotypes, soybean breeders should consider the negative impact SCN has on seed isoflavone concentration, the positive relationship between SCN resistance and isoflavone concentration in SCN environments, as well as the greater stability in isoflavone concentration across genotypes within SCN environments compared to the non-infested ones.

Variation for Concentrations of Various Phytoestrogens and Agronomic Traits Among a Broad Range of Red Clover (Trifolium pratense) Cultivars and Accessions

Agronomic characteristics and phytoestrogen concentrations were measured on 17 cultivars and 47 accessions of red clover (Trifolium pratense). These accessions included a range of currently recommended cultivars—from Australia and overseas—and germplasm accessed from genetic resource collections. All lines were grown in the field at Hamilton Vic in 2000 and 2001. Significant genetic variation was detected for key agronomic parameters such as growth habit, leaf shape and markings, leaf area, herbage yield, flowering time, and prolificacy. Significant variation in the concentration of the four main phytoestrogens was found; total isoflavone concentration ranged from 0.14–1.45% DM. Maximum concentrations of daidzein, genistein, formononetin, and biochanin were 0.06, 0.08, 0.86, and 0.91% DM respectively. Multivariate analysis showed that the accessions grouped into 10 distinct clusters that had between 1 and 10 members. Several accessions were superior to existing cultivars—notably Mediterranean accessions with regard to cool season vigour—and valuable for breeding programs to develop high yielding cultivars with either high (for possible medicinal purposes) or low (for grazing) phytoestrogen concentrations.

Heterosis and combining ability estimates in isoflavone content using different parental soybean accessions: wild soybean, a valuable germplasm for soybean breeding.

Isoflavone, a group of secondary metabolites in soybean, is beneficial to human health. Improving isoflavone content in soybean seeds has become one of the most important breeding objectives. However, the narrow genetic base of soybean cultivars hampered crop improvement. Wild soybean is an extraordinarily important gene pool for soybean breeding. In order to select an optimal germplasm for breeding programs to increase isoflavone concentration, 36 F1 soybean progenies from different parental accessions (cultivars, wild, Semi-wild and Interspecific) with various total isoflavone (TIF) concentration (High, Middle, Low) were analyzed for their isoflavone content. Results showed that male parents, except for Cultivars, showed positive GCA effects. In particular, wild soybean had higher positive GCA effects for TIF concentration. Both MP and BP heterosis value declined in the hybrid in which male parents were wild soybean, semi-wild soybean, interspecific offspring and cultivar in turn. In general, combining ability and heterosis in hybrids which had relative higher TIF concentration level parents showed better performance than those which had lower TIF concentration level parents. These results indicated characteristics of isoflavone content were mainly governed by additive type of gene action, and wild relatives could be utilized for breeding of soybean cultivars with this trait. A promising combination was found as the best potential hybrid for isoflavone content improvement.

Isoflavone Content of Soybean Cultivars from Maturity Group 0 to VI Grown in Northern and Southern China.

Soybean isoflavone content has long been considered to be a desirable trait to target in selection programs for their contribution to human health and plant defense systems. The objective of this study was to determine isoflavone concentrations of various soybean cultivars from maturity groups 0 to VI grown in various environments and to analyze their relationship to other important seed characters. Forty soybean cultivars were grown in replicated trials at Wuhan and Beijing of China in 2009/2010 and their individual and total isoflavone concentrations were determined by HPLC. Their yield and quality traits were also concurrently analyzed. The isoflavone components had abundant genetic variation in soybean seed, with a range of coefficient variation from 45.01% to 69.61%. Moreover, individual and total isoflavone concentrations were significantly affected by cultivar, maturity group, site and year. Total isoflavone concentration ranged from 551.15 to 7584.07 μg g−1, and averaged 2972.64 μg g−1 across environments and cultivars. There was a similar trend regarding the isoflavone contents, in which a lower isoflavone concentration was generally presented in early rather than late maturing soybean cultivars. In spite of significant cultivar × year × site interactions, cultivars with consistently high or low isoflavone concentrations across environments were identified, indicating that a genetic factor plays the most important role for isoflavone accumulation. The total isoflavone concentration had significant positive correlations with plant height, effective branches, pods per plant, seeds per plant, linoleic acid and linolenic acid, while significant negative correlations with oleic acid and oil content, indicating that isoflavone concentration can be predicted as being associated with other desirable seed characteristics.

Effects of High-Temperature Stress on Soybean Isoflavone Concentration and Expression of Key Genes Involved in Isoflavone Synthesis

Isoflavones have been reported to have putative health-beneficial properties, which has led to increased interest and demand for soybeans and soy-based products. This study was conducted to determine the effects of high-temperature stress on isoflavone concentration and expression of four key genes involved in isoflavone synthesis (i.e., CHS7, CHS8, IFS1, and IFS2) in both soybean pods and seeds during their late reproductive stage (i.e., R5-R8). Isoflavone concentrations were quantified using high-performance liquid chromatography (HPLC), and gene expression was studied using quantitative real-time (qRT)-PCR. High-temperature stress [33/25 °C (day/night temperatures)] imposed at the late reproductive stage (R5-R8) reduced total isoflavone concentration by 46-86 and 20-73% in seeds and pods, respectively, the reduction depending on the stage of maturity. At stage R5, the reduction in total isoflavone concentration was greater in seeds than in pods, whereas at subsequent stages, the reverse was observed. High-temperature stress had a large impact on the expression of CHS7, CHS8, IFS1, and IFS2 in both seeds and pods. In seeds, temperature stress reduced the expression of one gene at the R5 stage (CHS8), two genes at the R6 stage (CHS7 and IFS1), and all four genes at the R7 stage, the reduction ranging between 35 and 97%. In pods, high-temperature stress affected the expression of two genes at the R6 stage (CHS7 and IFS2) and all four genes at the R7 stage. Unlike in seeds, at the R6 stage, high temperature increased the expression of CHS7 and IFS2 by 72 and 736%, respectively, whereas at R7 stage the expression of all genes was reduced by an average of 97%. The present study reveals that high-temperature stress initiated at the R5 stage and maintained until maturation (i.e., R8 stage) has a rapid and sustained negative effect on isoflavone concentration in both seeds and pods. High temperature also affects gene expression; however, there was no clear correlation between isoflavone concentration and gene expression.

Analysis of Isoflavone, Phenolic, Soyasapogenol, and Tocopherol Compounds in Soybean [Glycine max (L.) Merrill] Germplasms of Different Seed Weights and Origins

This study investigated the functional compounds, including isoflavones, phenolics, soyasapogenols, and tocopherols, that were detected in 204 soybean [ Glycine max (L.) Merrill] germplasms. The soybean samples were divided into three groups according to origin: America, China, and Korea. The soybean samples were also classified into three groups on the basis of 100-seed weight: small (<13 g), medium (13-24 g), and large (>24 g). Among the soybean germplasms, CSRV121 (Bosukkong) had the highest level of isoflavone content (4778.1 μg g(-1)), whereas CS01316 had the lowest isoflavone content (682.4 μg g(-1)). Of the soybeans from the three different countries of origin, those from Korea showed the highest average concentration of total isoflavones (2252.6 μg g(-1)). The small seeds had the highest average total isoflavone concentration (2520.0 μg g(-1)) of the three different seed sizes. Among the 204 soybean germplasms, CS01405 had the highest content of total phenolics (5219.6 μg g(-1)), and CSRV017 (Hwangkeumkong) had the lowest phenolic content (654.6 μg g(-1)). The mean concentrations of total phenolic compounds were 2729.1 μg g(-1) in American soybean seeds, 1680.4 μg g(-1) in Chinese soybean seeds, and 1977.6 μg g(-1) in Korean soybean seeds. Of the soybean seeds from the three different countries of origin, American soybean seeds had the highest average concentration of total phenolic compounds, and Korean varieties showed the second highest value. Small soybean seeds had the highest average content of total phenolic compounds (2241.7 μg g(-1)), whereas medium-sized (1926.8 μg g(-1)) and large (1949.9 μg g(-1)) soybeans had lower concentrations of phenolic compounds. In whole soybean germplasms, the level of total soyasapogenols was higher in CS01173 (1802.3 μg g(-1)) and CS01346 (1736.8 μg g(-1)) than in the other types of soybeans. The mean concentrations of total soyasapogenol were 1234.0 μg g(-1) in American, 1294.5 μg g(-1) in Chinese, and 1241.5 μg g(-1) in Korean soybean varieties. Chinese soybean varieties showed the highest mean concentration of total soyasapogenol, and Korean soybean seeds showed the second highest level. The medium-seed group had the highest soyasapogenol content (1269.3 μg g(-1)) of the seeds that were grouped by size. A larger amount of soyasapogenol B than soyasapogenol A was detected. In whole soybeans, CS01202 showed the highest level of total tocopherols (330.5 μg g(-1)), whereas CSRV056 (Pungsannamulkong) had the lowest content (153.3 μg g(-1)). Chinese soybeans had the highest average concentration of total tocopherols (255.1 μg g(-1)). By comparison, the medium-sized Chinese soybean group had the highest (256.1 μg g(-1)) average total tocopherol content.

Variation of isoflavones production in red clover as related to environment, growth stage and year

Trifolium pratense cv. Minshan is a dominant red clover variety for animal feeding and pharmaceutical industry in Alpine Pasture Region of Gansu province, northwest China. In order to achieve the best isoflavone production, a 3-year trial was conducted at three locations to study variations of hay yield, total isoflavone concentration and isoflavone yield at different growth stages under different environmental conditions. Predominant isoflavone compounds and distribution of isoflavones in leaves, stems and inflorescences were also investigated. The results indicated that there were large variations on hay yield and isoflavone concentration among different sites, growth stages, age and parts of the plant. Minshan red clover harvested at the flower bud stage had the highest isoflavone yield (86.30 kg ha−1. The temperate climate region, Guanghe, provided the best growth conditions among three sites for isoflavone production (58.93 kg ha−1). The highest isoflavone production (53.42 kg ha−1) appeared at the second year of crop. In Minshan red clover, isoflavones are mainly distributed in leaves (1.12%). The predominant isoflavone compounds were formononetin (0.27%) and biochanin A (0.25%).

Soybean isoflavone patterns in main stem and branches as affected by water and nitrogen supply

Several isoflavones, such as genistein, daidzein, and their glucosyl and malonyl forms were detected by HPLC–UV in cotyledons, together with glycitein forms in the hypocotyl of two 1-maturity class contrasting soybean varieties (Ales with high isoflavone and low protein concentrations vs. Nikir, showing opposite behaviour). Cultivation involved drip irrigation and rainfed conditions, factorially combined with two levels of nitrogen supply (0 and 100kgha−1 at R1–R3 stages) at Legnaro (NE Italy) for two years (2006–2007). Yield components and hypocotyl and cotyledon isoflavone concentrations were measured in stems and branches separately.Ales was confirmed as having a higher total cotyledon isoflavone concentration (TCIC) than Nikir, with higher fractions of total daidzein in cotyledons and total glycitein in hypocotyl. In 2007, a 40% decline in total hypocotyl isoflavone concentration (THIC) was observed compared with 2006, due to a higher daily range temperature regime during seed filling.Irrigation increased seed yield and protein, but its effect on TCIC was negligible. Irrigation reduced THIC (∼8%) in the drier year (2007), perhaps due to greater hypocotyl weight (dilution effect) and lower fungal disease attack. TCIC and THIC were generally increased by the unusual nitrogen fertilisation practice (TCIC in year 2: 1.85 vs. 1.58mgg−1), especially in Nikir.The most important finding was a 24.4% cotyledon (1.54 vs. 1.96mgg−1) and 4.2% hypocotyl (14.9 vs. 14.3mgg−1) greater isoflavone concentration in seeds of branches compared with those of the main stem, and this may have a practical application in sowing density.It is currently concluded that nitrogen fertilisation improves the nutraceutical properties of soybean, although its effect depends on the activity of the isoflavone biosynthetic pathway and the concurrent extent of seed bulging.

Effects of High Temperature Stress at Different Development Stages on Soybean Isoflavone and Tocopherol Concentrations

Soybean contains a range of compounds with putative health benefits including isoflavones and tocopherols. A study was conducted to determine the effects on these compounds of high temperature stress imposed at specific development stages [i.e., none, pre-emergence, vegetative, early reproductive (R1-4), late-reproductive (R5-8), or all stages]. Two cultivars (AC Proteina and OAC Champion) were grown in growth chambers set at contrasting temperatures [i.e., stress conditions of 33/25 °C (day/night temperature) and control conditions of 23/15 °C] in order to generate these treatments. Isoflavone and tocopherol concentrations in mature seeds were determined using high-performance liquid chromatography. In both cultivars isoflavone response was greatest when stress occurred during the R5-8 stages and during all development stages, these treatments reducing total isoflavone concentration by an average of 85% compared to the control. Stress imposed at other stages also affected isoflavone concentration although the response was smaller. For example, stress during the vegetative stages reduced total isoflavones by 33% in OAC Champion. Stress imposed pre-emergence had an opposite effect increasing daidzein concentration by 24% in AC Proteina. Tocopherol concentrations were affected the most when stress was imposed during all stages of development, followed by stress restricted to stages R5-8; response to stress during other stages was limited. The specific response of tocopherols differed, α-tocopherol being increased by high temperature by as much as 752%, the reverse being observed for δ-tocopherol and γ-tocopherol. The present study demonstrates that while isoflavone and tocopherol concentrations in soybeans are affected the most by stress occurring during seed formation, concentrations can also be affected by stress occurring at other stages including stages as early as pre-emergence.

Effects of Pseudomonas aureofaciens 63-28 on Defense Responses in Soybean Plants Infected by Rhizoctonia solani

The objective of this work was to investigate the ability of the plant growth-promoting rhizobacterium Pseudomonas aureofaciens 63-28 to induce plant defense systems, including defense-related enzyme levels and expression of defense-related isoenzymes, and isoflavone production, leading to improved resistance to the phytopathogen Rhizoctonia solani AG-4 in soybean seedlings. Seven-dayold soybean seedlings were inoculated with P. aureofaciens 63-28, R. solani AG-4, or P. aureofaciens 63-28 plus R. solani AG-4 (P+R), or not inoculated (control). After 7 days of incubation, roots treated with R. solani AG-4 had obvious damping-off symptoms, but P+R-treated soybean plants had less disease development, indicating suppression of R. solani AG-4 in soybean seedlings. Superoxide dismutase (SOD) and catalase (CAT) activities of R. solani AG-4-treated roots increased by 24.6% and 54.0%, respectively, compared with control roots. Ascorbate peroxidase (APX) and phenylalanine ammonia lyase (PAL) activities of R. solani AG-4-treated roots were increased by 75.1% and 23.6%, respectively. Polyphenol oxidase (PPO) activity in soybean roots challenged with P. aureofaciens 63-28 and P+R increased by 25.0% and 11.6%, respectively. Mn-SOD (S1 band on gel) and Fe-SOD (S2) were strongly induced in P+R-treated roots, whereas one CAT (C1) and one APX (A3) were strongly induced in R. solani AG-4- treated roots. The total isoflavone concentration in P+Rtreated shoots was 27.2% greater than the control treatment. The isoflavone yield of R. solani AG-4-treated shoots was 60.9% less than the control.

Fast analysis of isoflavones by high-performance liquid chromatography using a column packed with fused-core particles

The recent development of fused-core technology in HPLC columns is enabling faster and highly efficient separations. This technology was evaluated for the development of an fast analysis method for the most relevant soy isoflavones. A step-by-step strategy was used to optimize temperature (25–50 °C), flow rate (1.2–2.7 mL/min), mobile phase composition and equilibration time (1–5 min). Optimized conditions provided a method for the separation of all isoflavones in less than 5.8 min and total analysis time (sample-to-sample) of 11.5 min. Evaluation of chromatographic performance revealed excellent reproducibility, resolution, selectivity, peak symmetry and low limits of detection and quantification levels. The use of a fused-core column allows highly efficient, sensitive, accurate and reproducible determination of isoflavones with an outstanding sample throughout and resolution. The developed method was validated with different soy samples with a total isoflavone concentration ranging from 1941.53 to 2460.84 μg g −1 with the predominant isoflavones being isoflavone glucosides and malonyl derivatives.

Intraspecific variability for soybean cotyledon isoflavones in different cropping and soil conditions

Cotyledon isoflavones, such as genistein, daidzein and their glucosyl and malonyl forms, were detected by HPLC–UV in 18 0- and 1-maturity class soybean varieties, cultivated under organic and conventional farming (3 years) and various soils (2 years) at Legnaro, NE Italy. Among varieties, the overall coefficient of variation for total cotyledon isoflavone concentration (TCIC) was 22%, and cluster analysis revealed three variety groups. Group L had 1.22 mg g −1 TCIC (low) and 40.3% proteins; group H, mainly including black-hilum varieties, 2.10 mg g −1 (high) and 39.2%, and group I 1.77 mg g −1 (intermediate) and 39.4%. Isoflavone profiles were similar in groups L and H (55% genistein and 45% daidzein aglycone equivalents), whereas group I had increased genistein (61%), which is recognised to have lower intestinal bioavailability (nutraceutics). Under organic management, a 2% protein increase (all groups) was associated with a 5–15% variety-dependent decrease – in groups H and I – and stable TCIC in group L. Isoflavone accumulation was positively affected by rainfall and limited daily range temperature regimes during pod filling, and hail damage (year 2005). In the Nikir variety (maturity class 1), grown in four lysimeter soils, TCIC was negatively correlated with soil C/N ratio, partially explaining the worse TCIC in organic cultivation. Based on this relationship, TCIC followed the order clay-loam > silty-loam >organic > sandy, with a 64% difference between extremes.

Seasonal changes in temperature and precipitation influence isoflavone concentration in short-season soybean

Future soybean [ Glycine max (L.) Merr.] cultivars with elevated or reduced concentrations of isoflavones may be required to satisfy the food market. Climate influences isoflavone concentration making their prediction problematic. Our research examined the associations between precipitation and temperature during a growing season with the isoflavone concentration in the harvested seed. We grew 14 cultivars across 12 years, at one location and calculated climate parameters for readily identifiable growth stage intervals (GSI). We correlated the climate parameters within each GSI with seed daidzein, genistein and total isoflavone (TIF) concentrations. We found that cumulative precipitation from the mid-vegetative to the flowering period was correlated with daidzein and TIF concentration. Mean daily average T during early vegetative development was positively correlated with daidzein and TIF concentration and this was largely a result of higher minimum daily T. Accumulated heat stress T greater than 27 °C during seed development was significantly correlated with a reduction in genistein and TIF, but not daidzein concentration. Accumulated cold stress T's less than 20, 15 and 17 °C during seedling emergence were significantly correlated with higher seed daidzein, genistein and TIF concentrations, respectively in all short-season cultivars tested. Cold stress T during seedling emergence could be used to predict seasonal changes in isoflavone concentrations.

The effects of phytoestrogenic isoflavones on the formation and disposition of paracetamol sulfate in the isolated perfused rat liver.

This study examines the potential for the phytoestrogenic isoflavones, a type of complementary medicine, to be involved in pharmacokinetic interactions in the liver. Rat livers were isolated and perfused to steady state, in single-pass mode, with either 5 microM paracetamol (n = 6), or 5 microM paracetamol with a 50:50 molar mixture of genistein and biochanin A or daidzein and formononetin, at a total isoflavone concentration of 1 and 10 microM (n = 6 for each mixture at each concentration). At 1 microM, neither isoflavone mixture had any effect, while at 10 microM both mixtures decreased the clearance of paracetamol and the formation clearance to paracetamol sulfate. Genistein and biochanin A (10 microM) also increased the biliary extraction of hepatically-generated paracetamol sulfate. Additional livers were perfused with an infusion of 5 microM (14)C-paracetamol in the absence (n = 4), or presence, of a 10 microM genistein and biochanin A mixture (n = 4). Analysis of washout perfusate and bile samples (up to 30 min after stopping the infusion) revealed that the isoflavones reduced the first-order rate constant for paracetamol sulfate transport into perfusate, but not for transport into bile. The results indicate that isoflavones can reduce the formation of paracetamol sulfate and that its enhanced excretion into bile arises from the inhibition of sinusoidal efflux transport.