Formononetin

Manufactured by Yuanye Bio-Technology

Sourced in China

Formononetin is a natural compound derived from various plant species. It functions as a phytoestrogen, exhibiting structural and functional similarities to the human hormone estrogen. This compound is commonly used in laboratory research settings to study estrogenic effects and related biological processes.

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13 protocols using formononetin

Immunohistochemical Analysis of Molecular Markers

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Rabbit anti-ICAM-1, VCAM-1, PECAM-1 and GAPDH polyclonal antibodies; rat anti- DC Marker (33D1); and mouse anti-SRA, αSMA, CD36, Arg1, SRBI, LOX-1, NK Cell Marker, CD4, CD22, Mast Cell Chymase and CD68 monoclonal antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit anti-ABCG1, ABCA1 and Asialo GM1 polyclonal antibodies were purchased from Novus Biologicals (Littleton, CO). Rabbit anti-iNOS polyclonal antibodies were purchased from Proteintech Group, Inc. (Rosemont, IL). Rabbit anti-KLF4 antibody and anti-Lamin A/C monoclonal antibody were purchased from Abcam (Cambridge, MA). Mouse anti-rabbit IgG-R, mouse anti-rabbit IgG-FITC and m-IgGκ BP-FITC antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Formononetin was purchased from Yuanye (Shanghai, China).

Ma C., Xia R., Yang S., Liu L., Zhang J., Feng K., Shang Y., Qu J., Li L., Chen N., Xu S., Zhang W., Mao J., Han J., Chen Y., Yang X., Duan Y, & Fan G. (2020). Formononetin attenuates atherosclerosis via regulating interaction between KLF4 and SRA in apoE-/- mice. Theranostics, 10(3), 1090-1106.

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HPLC Analysis of DBD Water Extract Components

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The qualitative analysis of components in the water extract of DBD was employed by a high-performance liquid chromatography (HPLC) method as previously described (Zhao et al. 2021 (link)). Briefly, the HPLC instrumentation was Waters e2695 and the detector was Waters 2489 UV detector. Quantitative estimation was performed with Empower 3 software programs using the external calibration method. Standard drugs of ferulic acid, calycosin, and formononetin were purchased from Yuanye Biotechnology Company (Shanghai, China).

Li D., Si J., Guo Y., Liu B., Chen X., Qi K., Yang S, & Ji E. (2023). Danggui-Buxue decoction alleviated vascular senescence in mice exposed to chronic intermittent hypoxia through activating the Nrf2/HO-1 pathway. Pharmaceutical Biology, 61(1), 1041-1053.

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Chemical Profiling of NLXT Herbal Extract

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The crude slices of each herb were purchased from Guang Yintang Co., Ltd. (Bozhou, Anhui) and identified by Prof. Yu Nianjun from Anhui university of Chinese medicine, according to the Chinese Pharmacopoeia 2020.
The reference standards of Formononetin, Ferulic acid [110773–201614] and Ginsenoside Re [110704–201223] were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Calycosin [Y14J8Y39707], Calycosin-7-O-β-D-glucoside (CG) [Y27F9H54731], Formononetin [111703–201504], Syringin [111574–201605], Ginsenoside Rb1 [Z20S9X70603], Ginsenoside Rg1 [G22D9Y77957], Notoginsenoside R1 [G27A10Y86799] and HSYA [R25A9F59977] were purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). The structures of these chemical standards are provided in Fig. 2, and the purity of all standard compounds were over 98.0%.Fig. 2

The total ion chromatograms (TIC) of NLXT.

Fig. 2

Acetonitrile and methanol (UPLC-MS grade) were purchased from Oceanpak Corporation (European, Sweden). Formic acid was obtained from Sigma Corporation (USA). Water was purified by a Millipore Milli Q-Plus system (Millipore, Bedford, USA).

Hong L., Shi X., Zhao Y., Zhao G., Jiang H., Liu M., Zhang H., Wu H., Wang L., He L, & Chen W. (2023). Network pharmacology-guided and TCM theory-supported in vitro and in vivo component identification of Naoluoxintong. Heliyon, 9(9), e19369.

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Standardized Analysis of Traditional Chinese Medicine

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Twenty-four authentic standards of adenosine (12), caffeic acid (17b), danshensu (17c), protocatechuic aldehyde (21), trans-5-caffeoylquinic acid (23b), ferulic acid (30), lithospermic acid (45b), salvianolic acid B (48a), baicalin (50a), ononin (50b), salvianolic acid A (61a), salvianolic acid C (70), astragaloside IV (77b), astragaloside III (79b), astragaloside II (83d), farrerol (84b), isoastragaloside II (85b), wogonin (86a), astragaloside I (92b), cyclosieversigenin (97b), dihydrotanshinone I (102a), tanshinone I (112), levistolide A (121) and tanshinone IIA (123) were all purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Five reference standards of calycosin7-O-β-d-glucoside (28b), rosmarinic acid (44), calycosin (62a), formononetin (80) and cryptotanshinone (130) were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). The structures of these reference standards (purity > 98%) are shown in Fig. 2. They were stored at 4 °C before use.
Acetonitrile, formic acid and water of LC-MS grade were purchased from Fisher Scientific Company (USA). Methanol of analytical grade was obtained from Runjie Tech Co. (Shanghai, China).
DZT and its five ingredients, i.e., LC, SM, PA, AM, and HN were provided by Tonghua Baishan Pharmaceutical Co., Ltd. (Jilin, China).

Cheng T., Ye J., Li H., Dong H., Xie N., Mi N., Zhang Z., Zou J., Jin H, & Zhang W. (2019). Hybrid multidimensional data acquisition and data processing strategy for comprehensive characterization of known, unknown and isomeric compounds from the compound Dan Zhi Tablet by UPLC-TWIMS-QTOFMS. RSC Advances, 9(16), 8714-8727.

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Comprehensive HPLC Analysis of Medicinal Metabolites

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Reference compounds of HPLC grade, i.e., salidroside, calycosin-7-O-β-D-glucoside, specnuezhenide, formononetin, and polyphyllin I, were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. with purity ≥98%. Puerarin as an internal standard substance (IS) was obtained from the National Institutes for Food and Drug Control (Beijing, China). Figure 1 lists the chemical structures of these metabolites. Distilled and deionized water was prepared with a water purification system (RODI-220A1, RSJ, China).
HPLC-grade acetonitrile and methanol were obtained from AQA (Cleveland, United States) and Guangzhou Chemical Reagent Factory (Guangzhou, China). The other chemicals and reagents were of analytical grade and were commercially available.

Zhou D., Chen J.R., Yang Z.Q., Xu L, & Huang Y.F. (2023). Establishment of a multicomponent quality control method and the transfer characteristics of five markers from Qidongning Formula to rat tissues by HPLC-QQQ-MS/MS. Frontiers in Pharmacology, 14, 1310266.

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Formononetin Modulation of CD4+ T Cells

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Formononetin (purity ≥ 98.0%) was purchased from Yuanye (Shanghai, China). TNF-α (430904), IFN-γ (430804) and IL-12 (431005) ELISA kits and all flow antibodies were obtained from BioLegend (San Diego, CA, USA). The naive CD4 + T cell isolation (130-104-453) was purchased from Miltenyi Biotec (Auburn CA). anti-IL-4 (504122), anti-IFN-γ (505834), anti-CD28mAb (102116),anti-CD3mAb (100340),anti-CD4-FITC (116004), anti-CD8-PE/Dazzle™ 594 (100762), anti-CD3-APC (100236), anti-CD4-FITC (100510), anti-CD25-Brilliant Violet 421™ (102033), anti-Foxp3-Alexa Fluor 647 (320014) and anti-IL-17a-PE (506903) antibodies were obtained from BioLegend (San Diego, CA). mIL-2 (200-02-10) and hTGF-β1 (100-21) were obtained from PeproTech (USA). p-PI3K (4228 T), PI3K (4249 T), p-Akt (4060 T), Akt (4691 T), Foxp3 (14-5773-80) and β-Actin (8475S) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA) and Invitrogen (Carlsbad, CA, USA).

Lan H., Qiu W., Wu J., Hu Z., Zhang X, & Zhu L. (2024). Formononetin reverses Treg/Th17 imbalance in immune-mediated bone marrow failure mice by regulating the PI3K/Akt signaling pathway. Chinese Medicine, 19, 55.

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UPLC-MS/MS Analysis of Isoflavonoids

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An Ultimate 3000 Ultra High Performance Liquid Chromatography (UPLC) system coupled with LTQ-Orbitrap velos pro mass spectrometer was used (Thermo-Fisher, Waltham, MA, USA), as well as an Acquity UPLC I-Class UPLC system coupled with Xevo TQ-S micro triple quadrupole mass spectrometer (Waters, Milford, MA, USA).
The reference compounds of formononetin, ononin, daidzein, daidzin, genistin, genistein, calycosin-7-O-β-d-glucoside, biochanin A, and chlorogenic acid (purity > 98% for all) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Acetonitrile and formic acid were purchased from Fisher Scientific, San Jose, CA, USA Company (mass spec-grade). Deionized water was prepared by Milli-Q ultrapure water preparation system (Millipore Co., Ltd., Billerica, MA, USA). All other chemicals were purchased from Beijing Chemical Works (Beijing, China, analytical grade).

Luo L., Gao W., Zhang Y., Liu C., Wang G., Wu H, & Gao W. (2020). Integrated Phytochemical Analysis Based on UPLC-MS and Network Pharmacology Approaches to Explore the Quality Control Markers for the Quality Assessment of Trifolium pratense L.. Molecules, 25(17), 3787.

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Extraction and Quantification of Astragalus Compounds

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Radix astragali was purchased from Hunan Sanxiang Chinese Medicine Pieces Co., Ltd. (Changsha, China) BSA and glutaraldehyde (25% w/v aqueous solution) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Acetonitrile was HPLC grade from Tedia Company Inc. (Phoenix, AZ, USA). Ultrapure water (18.2 MΩ·cm resistivity) was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). All of the other chemicals were analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Genistin, calycosin-7-O-β-d-glucoside, ononin and formononetin were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). The purity of each compound was determined to be ≥98% by HPLC analysis.

Liu L., Leng J., Yang X., Liao L., Cen Y., Xiao A, & Ma L. (2016). Rapid Screening and Identification of BSA Bound Ligands from Radix astragali Using BSA Immobilized Magnetic Nanoparticles Coupled with HPLC-MS. Molecules, 21(11), 1471.

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Formononetin Effects in Sprague Dawley Rats

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Formononetin (purity > 98.0%, Yuanye Biotechnology Co., Ltd.) was dissolved in DMSO to prepare a 160 mg/mL stock solution that was stored at 4°C for further use. Sprague Dawley rats (female, 6 weeks old, 200‐220 g) were supplied by Hunan SJA Laboratory Animal Co., Ltd. Animals were bred in a specific pathogen‐free room under a constant temperature of 21‐25°C and 50%‐60% relative humidity with a 12‐hour light/12‐hour dark cycle. All experimental procedures were performed in accordance with the guidelines of the Experimental Research Institute of Guilin Medical University.

Li X., Huang C., Sui C.L., Liang C.M., Qi G.Y., Ren Q.Y., Chen J, & Huang Z.Q. (2019). Formononetin, J1 and J2 have different effects on endothelial cells via EWSAT1‐TRAF6 and its downstream pathway. Journal of Cellular and Molecular Medicine, 24(1), 875-885.

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Comprehensive Chemical Profiling of YGYSG

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The production of YGYSG was undertaken in The First Clinical Medical College at Anhui University of Chinese Medicine. A Milli-Q water purification system (Millipore, Bedford, MA, USA) was employed to acquire deionized water. For mass spectrometry analysis, formic acid of LC-MS grade was purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol were LC-MS grade and obtained from Merck (Darmstadt, Germany). Overall, 27 chemical standards, including chikusetsusaponin IVa, chikusetsusaponin IV, ginsenoside Ro, 25R-inokosterone, β-ecdysone, acacetin, cymaroside, quercetin, gallic acid, oleanolic acid, hyperoside, chlorogenic acid, astragalin, kaempferol, cryptotanshinone, tanshinone II_A, rosmarinic acid, calycosin-7-O-β-D-glucoside, salvianolic acid B, astragaloside IV, formononetin-7-O-β-D-glycoside, calycosin, formononetin, scopoletin, ferulic acid, rutin, and narcissoside, were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China), and their purity levels exceeded 98% as determined through high-performance liquid chromatography (HPLC) analysis. Figure 2 displays the chemical configurations of each reference standard.

Yang F., Zhang K., Dai X, & Jiang W. (2024). Preliminary Exploration of Potential Active Ingredients and Molecular Mechanisms of Yanggan Yishui Granules for Treating Hypertensive Nephropathy Using UPLC-Q-TOF/MS Coupled with Network Pharmacology and Molecular Docking Strategy. Journal of Analytical Methods in Chemistry, 2024, 7967999.

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