Isoflavone Yield Research Articles

The Influence of the Parameters of the Subcritical Water Extraction Process on the Efficiency of Isoflavones Elicitation from Soy Meal

Topicality. Incidental products of soy processing (okara, meal, millcake) contain a large amount of protein, and, additionally, other valuable biologically active substances. Among them, isoflavones are of particular researchers’ interest. Subcritical water extraction is a promising method for elicitation biologically active substances from plant raw material. However, such a method of extracting isoflavones, and its rational parameters for soy meal, remain insufficiently investigated. Aim and methods. The aim of this work is to study the influence of parameters (temperature: 120...160°С, duration of extraction: 5...15 min, hydromodule: 1:15...25) of the extracting process of soy meal with subcritical water on the isoflavones content in the dry material of the extract. The empirical research method is applied, using the second-order orthogonal compositional outline for a fractional factor experiment. The samples of soy meal extract are obtained using the experimental setup, based on the high-pressure reactor “RHP-2-500”. The total isoflavones content in the dry extract material is defined in terms of gallic acid with absorption spectrophotometry method. Results. Based on the obtained results, the quadratic interpolation model is constructed. This model links the total isoflavones content in the dry extract material with the extraction parameters. The analysis of the constructed model makes it possible to establish rational measures of extraction parameters. Conclusions and discussion. In this study, the intensive growth of soflavones content in the dry extract with highing the temperature process, and a relatively small influence of the process duration on this indicator, were defined. The influence of the hydromodule is ambiguous. The lowest yield of isoflavones is noted at hydromodule ≈1:21,41. Its increase or decrease lead to the growth of isoflavones content in the extract. In this paper, for the first time, the existence of rational measures of parameters in the process of extracting isoflavones with subcritical water from soy meal is studied and established. The obtained results can be used in the elaboration of the technology for producing isoflavones enriched soy protein concentrate.

Cyclodextrin-Assisted Extraction Method as a Green Alternative to Increase the Isoflavone Yield from Trifolium pratensis L. Extract.

Trifolium pratense L. is receiving increasing attention due to the isoflavones it contains, which have been studied for their benefits to human health. A common problem with isoflavone aglycones is a rather low water solubility and limited pharmaceutical applications. The use of excipients, such as cyclodextrins in the production of isoflavone rich extracts, could become one of the new strategies for the extraction of target compounds. The aim of this study was to evaluate an eco-friendly method using the effects of α-, β- and γ-cyclodextrins for isoflavone solubilization in plant extracts in comparison to a standard extract without excipients. Extractions of red clover were prepared using ultrasound-assisted combined with thermal hydrolysis and heat reflux. It was determined that cyclodextrins significantly increased the isoflavones aglycone yields. By increasing cyclodextrins in the extraction media from 1 to 5%, the daidzin concentration increased on average by 1.06 (α-cyclodextrins), 1.4 (β-cyclodextrins) and 1.25 (γ-cyclodextrins) times. Genistein concentration increased using α- and γ-cyclodextrins (1.28 and 1.12 times, α- and γ-cyclodextrins, respectively), but decreased using β-cyclodextrins. The results showed that the cyclodextrin-assisted extraction enhanced the yields of isoflavones from red clover, which suggests using cyclodextrins as a green alternative and a cost-effective method to increase its pharmaceutical application.

Isolation of individual isoflavone species from soybean by solvent extraction followed by the combination of macroporous resin and aluminium oxide separation.

Growing interest in the health benefits of soy isoflavones has led to research in the isolation of individual isoflavone species for further application. Herein, we develop a new strategy to isolate daidzein, genistein, daidzin and genistin in soybean. We investigated the impact of solvents used and the extraction time on the extracted isoflavone contents from soybean. A 30-min extraction with 65% aqueous methanol gave a total isoflavone yield of 345mg/100g soybean, the highest value among tested conditions. Further, we proposed a two-stage adsorption/desorption chromatography comprising macroporous resin and aluminium oxide to isolate isoflavone. First, HP-20 resin was used to separate the glucosidic and aglyconic forms of isoflavone, then individual species of isoflavone could be isolated using aluminium oxide by specific retention of 5-hydroxy isoflavone. This process achieved overall high recovery (82-97%) and purity (92-95%) of the four isoflavones, which confirms a high separating efficiency for isoflavones from soybean.

Cytosine Methylation of Isoflavone Synthase Gene in the Genic Region Positively Regulates Its Expression and Isoflavone Biosynthesis in Soybean Seeds.

Plants, being sessile organisms, have evolved several dynamic mechanisms of gene regulation. Epigenetic modification especially cytosine methylation and demethylation actively regulates the expression of genes. To understand the role of cytosine methylation during isoflavonoid biosynthesis and accumulation, we performed cytosine methylation analysis in the coding region of two isoforms IFS1 and IFS2 gene, in two contrasting soybean genotypes differing in total isoflavone content (NRC37: high isoflavone; and NRC7: low isoflavone). The results indicated increased 5-mC in both the isoforms in NRC37 (∼20.51% in IFS2 and ∼85% in IFS1) compared with NRC7 (∼7.8% in IFS2 and ∼2.5% in IFS1) genotype, which signifies the positive role of 5-mC in the coding region of the gene leading to enhanced expression. In addition, temporal expression profiling [35 days after flowering (DAF), 45, 55, and 65 DAF] of both the isoforms showed increasing trend of accumulation in both the genotypes with maximum in NRC37 at 65 DAF. To further establish a correlation between methylation and expression of transcripts, we quantified the different isoforms of isoflavone in both the genotypes across all the stages. Therefore, the finding of this study would certainly increase our understanding of epigenetic regulation of isoflavone biosynthetic pathway mediated by the cytosine methylation that would assist molecular breeders to get high-performing soybean genotypes with better isoflavone yield.

Isoflavone production in hairy root cultures and plantlets of Trifolium pratense.

The aim of this study was to develop a Trifolium pratense hairy root (HR) production protocol and select HR lines with high isoflavone yield following elicitor treatments. We obtained 13 independent HR lines, producing approximately three times more isoflavonoids than seedlings (3.3mg/g dry weight) and in which 27 isoflavonoids were detected. Each HR line had its own isoflavonoid profile. These lines produced as major components daidzein, genistein, formononetin and biochanin A. Sucrose, salicylic acid (SA), yeast extract (YE) and flagellin 22 (flg22) were tested as elicitors. Using SA 140mg/L, allowed the maximum isoflavonoid production in plantlets (11.9mg/g dry weight) but reduced root growth, possibly as a result of its toxicity. The highest isoflavone production in HR (27.9mg/g dry weight) was obtained using sucrose 60g/L, for 3.5days. This work reports the high production of various isoflavonoids with T. pratense elicited HR cultures.

Regulation of Isoflavone Biosynthesis by miRNAs in Two Contrasting Soybean Genotypes at Different Seed Developmental Stages.

Owing to the presence of nutritionally important, health-promoting bioactive compounds, especially isoflavones, soybean has acquired the status of a functional food. miRNAs are tiny riboregulator of gene expression by either decreasing and/or increasing the expression of their corresponding target genes. Despite several works on identification and functional characterization of plant miRNAs, the role of miRNAs in the regulation of isoflavones metabolism is still a virgin field. In the present study, we identified a total of 31 new miRNAs along with their 245 putative target genes from soybean seed-specific ESTs using computational approach. The Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that miRNA putatively regulates metabolism and genetic information processing. Out of that, a total of 5 miRNAs (Gma-miRNA12, Gma-miRNA24, Gma-miRNA26, Gma-miRNA28, and Gma-miRNA29) were predicted and validated for their probable role during isoflavone biosynthesis. We also validated their five target genes using RA-PCR, which is as good as 5'RLM-RACE. Temporal regulation [35 days after flowering, 45, 55, and 65 DAF] of miRNAs and their targets showed differential expression schema. Differential expression of Gma-miR26 and Gma-miRNA28 along with their corresponding target genes (Glyma.10G197900 and Glyma.09G127200) showed a direct relationship with the total isoflavone content. Therefore, understanding the miRNA-based genetic regulation of isoflavone pathway would assist in selection and manipulation to get high-performing soybean genotypes with better isoflavone yield.

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%).

Acetone-butanol-ethanol fermentation and isoflavone extraction using kudzu roots

The economics of Acetone-butanol-ethanol (ABE) fermentation is greatly affected by raw materials, and the use of readily available starchy materials from marginal farming lands could be a viable option for reducing costs. Kudzu, a rapidly growing perennial leguminous vine, has been planted on marginal farming land and widely distributed in Asia and America. This study investigated ABE fermentation by C. acetobutylicum ATCC 824 using kudzu roots and isoflavone extraction from kudzu fermentation residue (KFR). The kudzu roots could be used as a sole substrate for ABE fermentation without nutritional supplements. Batch culture containing 140 g kudzu/L produced 17.99 ± 1.08 g/L solvent (ABE), including 11.20 ± 0.79 g/L butanol, 5.54 ± 0.20 g/L acetone, and 1.15 ± 0.09 g/L ethanol, with a productivity of 0.19 g/(L/h) and a yield of 0.33 g solvent/g sugar after 96 h of fermentation. Isoflavone yield extracted from KFR was 1.90/100 g KFR, approximately 48% higher compared with that extracted from raw kudzu. A kinetic analysis of the extraction process showed that both the isoflavone yield and the extraction rate obtained from KFR were higher than the corresponding values obtained from raw kudzu. These results indicate that kudzu may provide a new potential raw material for ABE production and the process of ABE fermentation integrated with isoflavone extraction may provide a new way to reduce fermentable substrate costs.

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.

Ionic liquid based ultrasonic assisted extraction of isoflavones from Iris tectorum Maxim and subsequently separation and purification by high-speed counter-current chromatography

We developed an ionic liquid based ultrasonic assisted extraction (ILUAE) method for the extraction of the three isoflavones, namely tectoridin, iristectorin B and iristectorin A from Iris tectorum Maxim of the Iridaceae family. Three kinds of 1-alkyl-3-methylimidazolium ionic liquids with different alkyl chain and anion were investigated. The results indicated that ionic liquids (ILs) showed remarkable effects on the extraction yield of isoflavones. In addition, the ILUAE, including several ultrasonic parameters, such as the concentration, extraction time and solvent to solid ratio have been optimized. Under these optimal conditions (e.g., with 30 min extraction time and the solvent to solid ratio of 30 ml/g), this approach gained the highest extraction yields of tectoridin (37.45 mg/g), iristectorin B (2.88 mg/g) and iristectorin A (5.28 mg/g). Meanwhile, tectoridin, iristectorin B and iristectorin A in the ILUAE extract were separated and purified successfully through the high-speed counter-current chromatography (HSCCC) with a two-phase solvent system consisting of n-butanol–water (1:1, v/v). The additional advantage of this approach is that 60.21 mg tectoridin, 4.33 mg iristectorin B and 8.24 mg iristectorin A with more than 95.0% purities have been obtained from 400 mg ILUAE extract of I. tectorum within 5 h and one-step elution under the most optimized conditions (e.g., a flow rate of 2.0 ml/min, 900 rpm and the wavelengh of 280 nm). The obtained fractions were successfully analyzed by HPLC and identified by 1H-NMR and 13C-NMR.

Continuous microwave-assisted isoflavone extraction system: Design and performance evaluation

The purpose of this research was to design, test, and optimize a continuous microwave extraction method using temperature and residence time during and after microwave exposure as optimizing parameters for extraction of major isoflavones (genistin, genistein, daidzin, and daidzein) from soy flour. The extraction yield of four isoflavones at different heating temperatures (55 and 73 °C) and extraction times (0, 4, 8, 12, and 16 min) were investigated and compared with yields provided by a conventional solvent extraction method. The microwave prototype consisted of multiple, commercially available, batch-type, house-hold microwave units placed on top of each other in series to impart a continuous operation. The optimum parameters for microwave-assisted extraction of isoflavones were 73 °C for 8 min using a 3:1 ethanol-to soy flour ratio. At these parameters, the total yield of isoflavones extracted doubled, while the amount of oil extracted was 12%. Continuous microwave-assisted solvent extraction is a viable method for extraction of soybean isoflavones at relatively short residence times and high throughput.

Extraction yield of isoflavones from soybean cake as affected by solvent and supercritical carbon dioxide

Soybean cake has been shown to be a rich source of isoflavone and can be produced during processing of soybean oil as byproduct. The objectives of this study were to compare the extraction yield of isoflavone from soybean cake by solvent and supercritical carbon dioxide, and study the conversion of isoflavone glucosides to the biologically active aglycone by employing β-glucosidase. Results showed that with supercritical carbon dioxide extraction, a maximum yield of malonylglucoside and glucoside was generated at 60 °C and 350 bar, while a high level of acetylglucoside and aglycone was produced at 80 °C and 350 bar. Supercritical carbon dioxide extraction resulted in a lower yield of total isoflavone than solvent extraction, but the former was more applicable to extraction of acetylglucoside and aglycone, and the latter to malonylglucoside and glucoside. A peak level of aglycone was attained from conversion of isoflavone glucoside by β-glucosidase at 55 °C, pH 5, concentration 50 U/ml and incubation time of 2 h.

Irrigation Level Affects Isoflavone Concentrations of Early Maturing Soya Bean Cultivars

AbstractField experiments were conducted in 2003/2004 in Québec to determine the effects of irrigation levels (none, low and high) and cultivars (AC Orford, AC Proteina and Golden) on soya bean [Glycine max (L.) Merr.] isoflavone concentrations and yields. Seed yield, yield components, and oil and crude protein (CP) concentrations were concurrently determined. Response to irrigation was greater in 2003, which was substantially warmer and drier than in 2004. In both years, most responses were observed with the lower of the two irrigation levels evaluated, which increased total isoflavones concentration by an average of 45 % compared with a non‐irrigated control. Cultivars, however, responded differently to irrigation. In 2003, response of AC Proteina was greater than that of AC Orford, while Golden did not respond. In 2004, some responses were observed with AC Proteina and Golden but none with AC Orford. Overall, in both years, AC Proteina had the greatest isoflavone concentrations and AC Orford the lowest. Responses of seed yield and yield components depended on the year and were also greater in 2003. Both irrigation treatments generally increased seed yield and yield components compared with a non‐irrigated control; the response was greater with the higher irrigation level. Irrigation had no effect on oil and CP concentrations. Finally, isoflavone yield response to irrigation was again greater in 2003, and depended on the cultivar. Results thus demonstrate that specific soil moisture levels will maximize soya bean isoflavone concentrations, excess irrigation sometimes negating any potential benefits.

Preparative Isolation of Isoflavones from Defatted Soy Flakes

Defatted soy flakes (DSF) were used as the starting material for the preparative isolation of isoflavones in this study. First, the crude DSF extracts were prepared using an extractor with solvent refluxing, operated under optimal extraction conditions (using 500 mL 80% ethanol aqueous for 3 h per 100 g DSF). The extraction yield was about 10% and the purity of isoflavones was about 2.0–2.5 wt%. Before the isolation operations, the extracts were dissolved in deionized water. The isolation procedures included the method of liquid–liquid extraction and the method of column chromatography. For the method of liquid–liquid extraction using a mixed solvent of 10% n‐butanol and 90% ethyl acetate operated under the optimal extraction conditions, the purity and yield of isoflavones were ∼35% and ∼70%. For the method of column chromatography, XAD‐7HP and XAD‐4 adsorbents with different polarities were used as the packing materials. For the XAD‐4 column, a part of non‐polar impurities was efficiently separated with the majority of isoflavones by a proper step gradient elution, which resulted in an efficient isolation: the purity and yield of isoflavones were ∼58% and ∼89%. In comparison, the method of column chromatography using XAD‐4 adsorbents achieved both the highest purity and yield, and was found to be the best isolation method in the current isolation stage.

Seeding date, row spacing, and weed effects on soybean isoflavone concentrations and other seed characteristics

Soybean [Glycine max (L.) Merr.] seeds contain isoflavones that may have positive impacts on human health. Field experiments were conducted in 2003/2004 in Québec, Canada to determine the effects of seeding date (late May and mid-June), row spacing (20-, 40- and 60-cm) and weeds (presence or absence) on soybean isoflavone concentrations and isoflavone yield. Total and individual isoflavone concentrations were determined by HPLC. Seed yield, and oil and crude protein (CP) concentrations were concurrently determined. Year, seeding date, and weeds affected total and individual isoflavone concentrations, while row spacing had no effect. Total isoflavone concentration was 84% greater in 2003 than 2004. Seeding in mid-June increased isoflavone concentration by 38%, compared with seed ing in May. The presence of weeds increased total isoflavone concentration by 9%. Year, row spacing, and weeds significantly affected seed yields. Seed yields were greatest in 2004, at 20- or 40-cm row spacing, and in the absence of weeds. Seeding date affected CP and oil concentrations. Greater CP concentration was observed with earlier seeding, the reverse was observed for oil. Weeds also affected CP and oil concentrations: higher CP and oil concentrations were observed in weedy and weed-free plots, respectively. Total isoflavone yield was affected by all factors evaluated. Isoflavone yield was greater in 2003 than 2004, with mid-June rather than late May seeding, when seeded at row spacing of 20- or 40- than 60-cm, and without weeds. Finally, negative correlations were observed between isoflavone concentrations and CP concentration and seed yield. It thus seems that certain agronomic practices may need to be tailored specifically to isoflavone production if concentrations in soybean are to be maximized. The negative correlations observed between isoflavone concentrations and other important seed characteristics warrant further research. Key words: Soybean, isoflavone, daidzein, genistein, glycitein, protein, oil, seed yield, weeds, row spacing, seeding date

An improved method for determination of isoflavones in soybean powder by liquid chromatography

Several extraction and separation methods were evaluated and then developed for determination of isoflavone in soybean powder. Results showed that the highest yield of isoflavones was achieved by using defatted soybean powder as raw material, followed by shaking extraction for 2h with a mixture of acetone and 0.1 M hydrochloric acid as solvent. A mobile phase of acetonitrile (A) and water (B) with the following gradient elution was developed: 5% A in B initially, then increased to 10% A in 5 min, 25% A in 15 min, 32% A in 22 min and returned to 5% A in 27 min. Twelve isoflavones in soybean powder were resolved within 20 min using a Vydac 201TP54 column flow rate 20 mL min−1 and detection at 262 nm. The amounts of various isoflavones in soybean powder were also determined.

Isoflavonoid production by callus cultures of Maackia amurensis

Callus cultures were established from the different parts of Maackia amurensis plants and analyzed for isoflavonoids. The isoflavones daidzein, retuzin, genistein and formononetin and the pterocarpans maakiain and medicarpin were found to be produced by these cultures. The content of isoflavones and pterocarpans was essentially the same in cultures derived from leaf petioles, inflorescences and apical meristems of the plant. The maximal yield of isoflavones and pterocarpans in calluses was 20.8 mg/g cell dry wt., approximately four times higher than the content of the heartwood of M. amurensis plants. Unlike wild-growing plants, none of the cell cultures had the ability to accumulate stilbenes.