Astragaloside IV

AS-IV (batch number: 140913) was obtained from Weikeqi Biotechnology (Sichuan, China). Betloc tablets (batch number: 2010038) were purchased from AstraZeneca (Wuxi, China). Cell Counting Kit-8 (CCK-8, CAS Number: ZP328-3) was purchased from Zomanbio (Beijing, China).

The immobilization of P. canescens spores (Figure 2b) in CAG beads was prepared according to the method described by Yao et al. (2014). AMHRCs (Figure 2d) were initiated by culturing a high-productive hairy root line (AMHRL VI) (Figure 2c) under the optimal conditions as reported by Jiao et al. (2015). AG standards including AG I, AG II, IAG II and AG IV were purchased from Weikeqi Biological Technology Co. Ltd. (Sichuan province, China). MiniBEST Plant RNA Extraction Kit, PrimeScript™ RT reagent Kit and SYBR Premix Ex Taq™ II Kit were provided by TaKaRa (Dalian, China). Other reagents of either analytical or optical grades were purchased from Beijing Chemical Reagents Co. (Beijing, China). Ultrapure water was prepared from a Milli-Q system at 18.3 M resistance (Millipore, Bedford, MA).

Tetraethtylamine (TEA, no. 134473), 4-aminopyridine (4-AP, no. 275875), BaCl2 (no. 342920), glyburide (Gli, no. Y0001511), ethylene glycol-bis (2-aminoethylether)-N, N, N′, N′-tetraacetic acid (EGTA, no. E3889), and 9, 11-dideoxy-9α, 11α-methanoepoxy prostaglandin F2α (U46619, no. D8174) were purchased from Sigma. Astragaloside IV (no. wkq16042601) and ferulic acid (no. wkq17022303) were provided by Weikeqi Biological Technology Co., Ltd. Formononetin (no. DST201025-011) and ligustalide (no. DST200610-007) were procured from Lemeitian medicine.

Astragaloside and isoavonoid standards, including astragaloside I (AG I), astragaloside II (AG II), isoastragaloside II (IAG

II), astragaloside III (AG III), astragaloside IV (AG IV), cycloastragenol (CY), calycosin-7-O-b-D-glucoside (CAG), ononin

(ON), astraisoavan-7-O-b-D-glucoside (ASG), calycosin (CA) and

formononetin (FO), were purchased from Weikeqi Biological

Technology Co. Ltd. (Sichuan province, China). Other reagents

of either analytical or optical grade were obtained from Beijing

Chemical Reagents Co. (Beijing, China).

All chemicals and reagents are of analytical grade unless there are specific descriptions. The Astragalus membranaceu roots were purchased from Beijing Tong Ren Tang Co., Ltd (China). The crude astragaloside IV (ASI), standards of ASI, cycloastragenol-6-O-β-D-glucoside (CMG), and cycloastragenol (CA) were purchased from Sichuan Weikeqi Biotechnology Co., Ltd (China). The crude CMG and cycloastragenol-3-O-β-D-xyloside (CMX) were prepared from ASI, as reported previously (Cheng et al. 2020).

The TWMM was provided by Beijing HanDian Pharmaceutical Co., Ltd. (Beijing, China). Chlorogenic acid, linarin, luteolin, jaspolyside, tolbutamide, stachydrine, magnoflorine, calycosin-7-O-β-d-glucoside, stepharanine, jatrorrhizine, epiberberine, columbamine, ononin, berberine, palmatine, berberrubine, calycosin, formononetin, 2α-hydroxyoleanic acid, hydroxytyrosol, salidroside, luteolin-7-O-glucoside, verbascoside, specnuezhenide, oleuropein, physcion, oleonuezhenide, astragaloside IV, astragaloside II, isoastragaloside II, astragaloside I, luteolin, formononetin, and tolbutamide (purity ≥ 98%) were purchased from Shanghai Yuanye Pharmaceutical Technology Co., Ltd and Sichuan Weikeqi Pharmaceutical Technology Co., Ltd. Acetonitrile and formic acid both in HPLC grade were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Ultrapure water was prepared using a Milli-Q water purification system (Millipore, Bredford, MA, USA). Other reagents were all analytical grade.

AG standards including AG I, AG II, IAG II, AG III, AG IV and CY were purchased from Weikeqi Biological Technology Co., Ltd. (Sichuan province, China). UV-A, UV-B and UV-C lamps (40 W) were bought from Beijing Institute of Electric Light Source (Beijing, China).

The crude extracts of astragaloside IV (ASI) (80 % purity), the standards of ASI and cycloastragenol (CA) were purchased from Sichuan Weikeqi Biotechnology Co., Ltd (China). The commercial cellulase (Model: Cellic CTec3) was purchased from Novozymes.

JuB was supplied by the Nanjing Spring & Autumn Biological Engineering Co. (Jiangsu, China). The internal standards (ISs), palmatine hydrochloride (IS1), daidzin (IS2) and astragaloside IV (IS3) were obtained from Chengdu Weikeqi Biological Technology Co. (Chengdu, China).

Mature seeds of A. membranaceus were generously provided by Heilongjiang University of Chinese Medicine. AG standards including AG I, AG II, IAG II, AG III, AG IV and CY were purchased from Weikeqi Biological Technology Co. Ltd. (Sichuan province, China). Other reagents either of analytical or optical grades were obtained from Beijing Chemical Reagents Co. (Beijing, China).

SK injection samples were manufactured by Xi'an Shi Ji Sheng Kang Pharmaceutical industry (Xi'an, China). The samples were diluted by four times to inject. The reference standards aloe-emodin, rhein, emodin, chrysophanol, physcion and astragaloside IV were purchased from Chengdu Weikeqi Bio-technology (Sichuan, China). Gallic acid, chlorogenic acid, caffeic acid, ferulic acid, rosmarinic acid, ethyl ferulate, protocatechuic acid, protocatechu aldehyde, catechin, hydroxysafflor yellow A

The standards of compounds A1–A35 were purchased from Chengdu Must Biotechnology. (Sichuan, China) and Sichuan WEIKEQI Biotechnology (Sichuan, China), and these compounds were all determined with the purities >98% by HPLC or GC method. Ten batches of DHQYCs were obtained from Jilin Long Xin Pharmaceutical.

sucrose and 1 g/L casein hydrolyzate but without NH4NO3, at 25 ± 1 °C in the dark. AG standards including astragaloside I (AG I), astragaloside II (AG II), isoastragaloside II (IAG II), astragaloside III (AG III), astragaloside IV (AG IV) and cycloastragenol (CY) were purchased from Weikeqi Biological Technology Co., Ltd. (Sichuan province, China). Elicitors (MJ, SA and ASA) were obtained from Sigma–Aldrich Co.

Glycyrrhetinic acid was purchased from Shanghai Yuanye Biological Technology Co. Astragaloside I, astragaloside II, astragaloside IV, calycosin‑7‑O‑β‑d‑glucoside, ononin, formononetin and liquiritin were obtained from Sichuan Weikeqi Biotechnology Co. The purity of each standard substance was larger than 98%. The roots of Astragalus propinquus Schischkin were collected in Longxi County, Gansu Province (lot: 160901) and authenticated by Prof. Chen Jianwei in the field. The raw materials of AP

3′-Hydroxypuerarin, puerarin, 3′-methoxypuerarin, mirificin, daidzin and daidzein were obtained from Chengdu Purechem-standard Co. Ginsenoside Rg1, ginsenoside Rh1, ginsenoside Rd, astragaloside IV and 20(S)-ginsenoside Rg3 were purchased from Chendu Herbpurify Co. Astragalin was obtained from Sichuan Weikeqi Biotechnology Co. Linoleic acid and palmitic acid were purchased from Shanghai Ryon Biological Technology Co. HPLC-grade acetonitrile was obtained from Merck Co.

Astragaloside IV (chemical structure in Fig. 1, purity 98%; 3-O-β-D-xylopyranosyl-6-O-β-D-glucopyranosyl-cycloastragenol) was purchased from Sichuan Weikeqi Biotech Co., Ltd. (Chengdu, Sichuan, China).