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6 protocols using glycitein

1

Extraction and Purification of Soybean Compounds

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The stachyose (pure>80%) was extracted from soybean residue [22 (link)]. Soybean isoflavones (pure>95%) includes 50% of daidzin, 6% of daidzein, 26% of glycitin, 3% of glycitein, 10% of genistin and 2% of genistein for animal experiments and the analytical standards of daidzein, glycitein and genistein (pure>98%) were bought from Shanghai Yuanye Biotechnology co., Ltd. (Shanghai, China). Assay kits of total cholesterol (TC), total triglyceride (TG), high-densitylipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), glutamic-oxal(o)acetic transaminase (GOT) and glutamicpyruvic transaminase (GPT) were purchased from HuiliBiotechnology Co. Ltd. (Changchun, China). Additionally, enzyme linked immunosorbent assay ELISA kits of interleukin-1 (IL-1), tumor necrosis factor (TNF-α), nuclear factor kappa-B (NF-κB), acetyl CoA carboxylase (ACC), fatty acid synthetase (FAS), glucose, insulin, leptin and lipopolysaccharide (LPS) were purchased from Jiancheng Bioengineering Institute (Nanjing, China). All other reagents and chemicals were analytical grade or higher.
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2

Comprehensive Analysis of Fructus Puerariae

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A total of 13 batches of FP were collected from Zhejiang (S1), Guangdong (S2 and S3), Guangxi (S4, S5 and S6), Hebei (S8), Hunan (S9, S10, S11 and S12) and Sichuan (S13) provinces of China. Besides, the control herbs (S7) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Reference standards (purity >98%) including rutin, daidzin, daidzein, and quercetin were obtained from the National Institutes for Food and Drug Control (Beijing, China). Glycitin, genistin, tectoridin, glycitein, genistein, tectorigenin, and biochanin A with the purities over 98% were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Nicotiflorin and ononin with the purities over 98% were bought from Chengdu Alfa Biotechnology Co., Ltd. (Chengdu, China). Meanwhile, formononetin and puerarin were bought from Chengdu Must Bio-Technology Co., Ltd.(Chengdu, China). HPLC grade acetonitrile (ACN) and methanol (MeOH) were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid of HPLC grade was provided by Kermel Chemical Reagent Co., Ltd. (Tianjin, China) and pure water was obtained from Wahaha (Hangzhou, China). All the other reagents were of analytical grade.
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3

Quantification of Soy Isoflavones and SCFAs

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Sodium chloride (NaCl) and foline-phenol (C6H6O) were purchased from Tianjin Fengchuan Chemical Reagent Co., Ltd. Potassium dihydrogen phosphate (KH2PO4) and 3,5-dinitrosalicylic acid (DNS) were purchased from Sinopharm Chemical Reagent Co., Ltd. Disodium hydrogen phosphate (Na2HPO4) was purchased from Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd. SIs (H05M9R54983), composed of daidzin (57.98%), glycitin (23.09%), genistin (6.92%), daidzein (2.12%), glycitein (0.74%), and genistein (0.4%), were purchased from Shanghai Yuanye Biotechnology Co., Ltd.
Reference standards, including acetic acid (≥ 99.5%), propionic acid (≥ 99.0%), isobutyric acid (≥ 99.0%), butyric acid (≥ 99.0%), isovaleric acid (≥ 99.0%), valeric acid (≥ 98%), caproic acid (≥ 99.5%), and isocaproic acid (≥ 98%), were purchased from Sigma-Aldrich (Merck, Darmstadt, Germany).
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4

Extraction and Characterization of Bioactive Compounds

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Chemical reagents like ethanol, methanol, acetonitrile, and formic acid were all of chromatographic grade from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Using a Milli-Q Plus instrument from Merk KGaA, 18.2 MΩ.cm water was made (Darmstadt, Germany1). Folin–Ciocalteu and aluminum nitrate were supplied by Solarbio Science & Technology Co., Ltd. (Beijing, China). Chemical standards including chlorogenic acid, ferulic acid, vitexin, rutin, gallic acid, myricitrin, morin, glycitein, wogonin, kaempferol-3-O-sophoroside, liquiritigenin, and butin were obtained from Yuanye Biological Technology Co., Ltd. (Shanghai, China), with >98% purity. The extract was concentrated on Bond Elut PPL 6 cc, 500 mg C18 solid phase extraction (SPE) cartridge that was purchased from Agilent Technology Co., Ltd. (Beijing, China). High-glucose (4.5 g/L) Dulbecco’s modified eagle’s medium (DMEM), which contains 10% (v/v) heat-inactivated fetal bovine serum, 100 µg/mL of streptomycin, and 100 U/mL of penicillin, was obtained from Gibco Laboratories (Carlsbad, CA, USA). Escherichia coli 0111: B4 LPS was a Sigma product (St. Louis, MO, USA).
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5

Soybean Fermentation with R. oligosporus

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The high purity (≥97%) standards, including ascorbic acid, vanillic acid, syringic acid, epicatechin, daidzin, glycitin, ferulic acid, genistin, daidzein, glycitein, quercetin, and genistein were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Other reagents of analytical grade. Soybeans (Liaodou, yellow seed coat, mainly planted in northern China, weigh 21.6 g of one hundred seeds) were obtained from a local market. R. oligosporus RT-3 isolated, screened and identified from Tempeh in our laboratory was used for soybean fermentation (11 (link), 12 ). The strain was cultivated on potato dextrose agar at 30°C for 3 days. Then, spores produced were scraped in water (1 × 107 spores/mL).
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6

Fermentation of Soybean Isoflavones

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Daidzein, glycitein, genistein, daidzin, glycitin, and genistin were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China) with a purity ≥98%. Methanol (HPLC grade) was purchased from Thermo Fisher Scientific (Shanghai, China). All other chemicals and reagents were analytical grade, and reverse-osmosis Milli-Q water was used.
The fresh soybeans were purchased from the local supermarket. Bacillus subtilis natto ATCC 65301 used for fermentation was purchased from American Type Culture Collection (ATCC) and maintained in 20% glycerol at −80 °C. Its cultural conditions and activation methods are applied according to the report of Li [21 (link)].
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