Formononetin

The 50% chlormequat aqueous solution was obtained from Sichuan Guoguang Agrochemical Co., Ltd. (Chengdu, China). The standard compound of chlormequat chloride (98.0%) was purchased from Agro-Environmental Protection Institute (Tianjin, China). Thirteen standard compounds (with purities all over 98.0% confirmed by HPLC-UV analysis), including astragaloside IV, astragaloside I, astragaloside II, astragaloside III, isoastragaloside II, isoastragaloside IV, cycloastragenol, methylnissolin-3-O-glucoside, astraisoflavan-7-O-β-d-glucoside, formononetin, ononin, calycosin, and calycosin-7-glucoside, were provided by Chengdu Must Bio-technology Co. Ltd. (Chengdu, China). HPLC-grade acetonitrile and methanol were sourced from Fisher Scientific (Fair Lawn, NJ, USA). Other reagents and chemicals were purchased from Sinopharm Chemical Regent Beijing Co., Ltd. (Beijing, China). Ultrapure water intended for HPLC-MS/MS analysis was obtained from a Milli-Q system (Merck Millipore, Billerica, MA, USA).

Formononetin (For, A0232), isorhamnetin (Iso, A0514), kaempferol (Kae, A0129), calycosin (Cal, A0190), and quercetin (Que, A0083) (purity ≥ 98%) were provided by Chengdu Must Biotechnology Co., Ltd. (Chengdu, China), which were mixed with dimethyl sulfoxide (DMSO, 0.1%, v/v; Sigma, St. Louis, MO, United States). In the meantime, the HEK293-derived recombinant human TGF-β1 (100-21) was purchased from PeproTech (New Jersey, United States). The anti-α-SMA (14395-1-AP) and anti-GAPDH (6004-1-lg) antibodies, together with the HRP-labeled AffiniPure goat anti-mouse IgG antibody (H + L) (SA00001-1), were provided by Proteintech (Wuhan, China). The HRP-labeled goat anti-rabbit IgG antibody (H + L) (BS13278) was provided by Bioworld Technology (St. Paul, MN, United States). The Alexa fluor 568-conjugated goat anti-rabbit IgG (H + L) antibody was purchased from Abcam (Cambridge, MA, United States). The enhanced BCA protein assay kit (P0010), RIPA lysis buffer (P0013) and DAPI staining solution (C1005) were provided by Beyotime Institute of Biotechnology (Shanghai, China).

Hydroxysafflor yellow A (batch no. MUST-13121913), paeoniflorin (batch no. MUST-13113009), calycosin-7-O-β-D-glucoside (batch no. MUST-14031314), calycosin (batch no. MUST-13081501), and formononetin (batch no. MUST-14030710) were purchased from Chengdu Must Bio-technology Co. Ltd. (Sichuan, China). Ononin (batch no. 130507) was obtained from Chengdu Pufei De Biotech Co. Ltd. (Sichuan, China). The purity of all the above chemical reference substances were determined higher than 98 %. Rabbit anti-rat neuron specific nuclear protein (NeuN) and mouse anti-rat glial fibrillary acidic protein (GFAP) were bought from Millipore Co. Ltd (USA). Rabbit anti-rat CD34 was purchased from Abcam (UK).

Methanol, acetonitrile, and formic acid were of LC–MS grade, and 2-propanol was high-performance liquid chromatography (HPLC) grade. These chemicals were all purchased from Thermo Fisher Scientific Inc (Waltham, Massachusetts, United States). The ferulic acid (MW: 194.19, LOT: MUST-17010908, purity ≥98% HPLC)，caffeic acid (MW: 180.15, LOT: MUST-17010908, purity ≥98% HPLC), calycosin (MW: 284.26, LOT: MUST-14070923, purity ≥98% HPLC), and formononetin (MW: 268.26, LOT: MUST-14091205, purity ≥98% HPLC) were provided by Chengdu Must Bio-Technology Co. Ltd., Ligustilide (MW: 284.26, LOT: wkq-00246, purity ≥98% HPLC LOT: wkq-00246) was obtained from Sichuan Weikeqi Biological Technology Co., Ltd. Rat SOD, MDA, TNF-α, IL-1β, IL-18, IL-6 and insulin ELISA Kits and ROS kit were all purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.
Danggui (the root of A. sinensis (Oliv.) Diels, Place of Origin: Gansu Province, Batch number: 259200103) and Huangqi (the root of Astragalus mongholicus Bge, Place of Origin: Inner Mongolia, Batch number: 190919001) were purchased from Beijing Tong Ren Tang Nanjing Pharmacy (Han Zhong Road, Nanjing, China). After purchase, the herbs were maintained in closed, lucifugal, and dry conditions at 23 ± 2°C.

The 50% chlormequat aqueous solution was obtained from Sichuan Guoguang Agrochemical Co., Ltd. (Chengdu, China). The standard compound of chlormequat chloride (98.0%) was purchased from Agro-Environmental Protection Institute (Tianjin, China). Thirteen standard compounds (with purities all over 98.0% confirmed by HPLC-UV analysis), including astragaloside IV, astragaloside I, astragaloside II, astragaloside III, isoastragaloside II, isoastragaloside IV, cycloastragenol, methylnissolin-3-O-glucoside, astraisoflavan-7-O-β-d-glucoside, formononetin, ononin, calycosin, and calycosin-7-glucoside, were provided by Chengdu Must Bio-technology Co. Ltd. (Chengdu, China). HPLC-grade acetonitrile and methanol were sourced from Fisher Scientific (Fair Lawn, NJ, USA). Other reagents and chemicals were purchased from Sinopharm Chemical Regent Beijing Co., Ltd. (Beijing, China). Ultrapure water intended for HPLC-MS/MS analysis was obtained from a Milli-Q system (Merck Millipore, Billerica, MA, USA).