Chemicals and reagents
Standards of calycosin-7-O-β-D-glucoside, ononin, calycosin, formononetin and rutin (Internal standard, IS) were supplied by Testing Laboratory for Chinese Medicine of HKUST (Hong Kong, China). The purity of each standard was > 98%, as detected by HPLC-DAD and 13C-NMR analysis. The HPLC grade acetonitrile and formic acid were obtained from Merck (Darmstadt, Germany). Deionized water (18 MW/cm) was supplied with a Direct-Q water purification system (Millipore, Milford, MA). Acquity UPLC C18 column (Waters, Milford, MA). Tris, p-nitrophenol (PNP), p-nitropheny-β-D-glucopyranoside (PNP-D-Glu), α-glucosidase, p-nitrophenyl-α-D-glucopyrano side, bovine serum albumin (BSA) and O-phenylenediamine were purchased from Macklin Biochemical (Shanghai, China). DPPH was gained from TCI Chemical Industry (Shanghai, China). Other materials were obtained from Sigma-Aldrich (St. Louis, MO).
Preparation of herbal decoction
The roots of three-year-old A. memebranaceus var. mongholicus (Astragali Radix; Huangqi; AR) from Shanxi Province  and two-year-old A. sinensis roots (Angelicae Sinensis Radix; Danggui; ASR) from Minxian of Gansu Province  were collected in 2019. The herbs were identified morphologically by Dr. Tina TX Dong. The voucher specimens of AR (Lot: 20190320) and ASR (Lot: 20190412) were recorded in HKUST Shenzhen Research Institute. In preparing DBT, AR and ASR were weighed according to a ratio of 5:1 and then mixed well. The mixture was boiled in 8 volumes of water (v/w) for 2 hours, and the extraction was repeated twice . The extracts were dried by lyophilization and stored at − 80 °C. The chemical analysis of fermented DBT was carried out as described .
Fermentation of DBT extract with L. plantarum
L. plantarum (GDM 1.191) was purchased from Guangdong Microbial Culture Collection Center (ACCC11095; Guangdong, China). The culture was inoculated twice in MRS broth (10 g peptone, 8.0 g lab-lemco’ powder, 4.0 g yeast extract, 20 g glucose, 2.0 g di-potassium hydrogen phosphate, 2.0 g tri-ammonium citrate, 5.0 g sodium acetate with 3 H2O, 0.2 g magnesium sulphate, 0.04 g manganese sulphate with 4 H2O, and 1 mL Tween in 80 liters of water, pH 5.7 ± 0.2; from Hopebio, Qingdao, China) at 37 °C in anaerobic atmosphere (10% H2, 10% CO2, 80% N2) for 24 hours to obtain the strain at end of exponential phase. A stock solution of DBT herbal extract was sterilized with a filter and diluted with MRS medium. The inoculation of L. plantarum was adjusted to a concentration of 1 × 108 CFU per mL, and the fermentation was performed at 37 °C under anaerobic condition, shaking in 100 rpm, until the late stationary phase. The growth of L. plantarum was determined by absorbance at 595 nm.
The stock solutions of calycosin-7-O-β-D-glucoside, ononin, calycosin and formononetin were freshly prepared in methanol at 1 mg/mL. Mixed stock solution (200 µg/mL each) was prepared. Rutin (IS) at 20 µg/mL was diluted from the stock in methanol. The working standard solutions (0.092-200 µg/mL) for analytes were prepared by a serial diluent of mixed stock solution with methanol. The calibration standard solutions (0.023-50 µg/mL) for analytes were prepared by spiking an appropriate amount of working standard solutions into 150 µL blank matrix. The QC concentrations of tested samples were selected in 0.068, 1.84, and 16.6 µg/mL, respectively at low, medium and high levels. The sample after fermentation (200 µL) and IS (50 µL) were shaken with vortex for 30 sec. Then, adding 800 µL methanol to the mixture, vortexed for 2 min and centrifuged at 10,000 rpm for another 10 min. Then, 2 µL supernatant was subjected to UPLC-MS/MS analysis.
UPLC chromatograph coupled with a PerkinElmer QSight®210 MS/MS detector (PerkinElmer, Waltham, MA). The instrument control, analysis and data processing were performed using Simplicity 3Q™ software platform. Sample separation was achieved on an Acquity C18 column (4.6 mm × 50 mm, 1.7 µm) with a constant flow rate of 0.3 mL/min at 30 °C. The mobile phase was composed of water (0.1% formic acid, A) and acetonitrile (C), using a gradient elution of 80%-60% A at 0–4 min, 60%-10% A at 4–6 min, 10%-10% A at 6–7 min, 10%-80% A at 7–8 min, 80%-80% A at 8–10 min. The injected volume was set at 2 µL. The acquired parameters were optimized as follows: drying gas value, 100; nebulizer gas value, 150; electrospray voltage, 5500 V; HSID temperature, 280 °C. The detection was recorded as MRM negative mode. The proposed analytical method was validated and calculated for specificity, linearity, intra-day and inter-day precision, accuracy, extraction recovery, matrix effect and stability, according with the criteria described in the FDA guidelines for bioanalytical samples.
β-glycosidase: The assay for β-glycosidase activity was conducted according to the reported method with minor modifications . Briefly, 100 µL fermented sample was acquired by centrifuging at 10,000 rpm for 10 min. The reaction mixture (1.0 mL) comprised of 1 mM p-nitropheny-β-D-glucopyranoside (PNP-D-Glu), 0.1 M phosphate buffer (pH 6.8) and the sample was incubated at 37 °C for 30 min. The reaction was stopped by adding 500 µL of 0.5 M NaOH centrifuged at 10,000 rpm for 10 min. The amount of PNP released was measured by absorbance at 405 nm in a microplate reader.
α-Glucosidase: The α-glucosidase inhibitory property was performed according to the previous method with modification . The tested sample, diluted 5 times with water, was vortexed at 3,000 rpm for 5 min. The reaction mixture was composed of the tested sample, phosphate buffer (0.1 M, pH 6.8) and α-glucosidase (50 µg/mL). Next, p-nitrophenyl-α-D-glucopyranoside solution (10 mM) was added to the mixture. The incubation was continued for 20 min at 37 °C, and which was stopped by adding 100 mM Na2CO3 solution. Acarbose was used as a positive control at 1 µg/mL. The reaction was measured by monitoring 405 nm. The results were presented as a percentage of α-glucosidase inhibition, calculated according to the following equation: Inhibition (%) = (〖OD〗_(ctrl.)-〖OD〗_sample)/〖OD〗_(ctrl.) × 100%.
α-Amylase: The α-amylase activity was adopted using a modified method . Briefly, the tested sample and α-amylase solution (0.2 U/mL) were incubated at 37 °C for 30 min. Next, 2% soluble starch solution was added to the mixture, and the incubation was continued for another 20 min at 37 °C. HCl (1 M) was added to terminate the enzymatic reaction, followed by iodine reagent (5 mg/mL). Acarbose (200 µg/mL) was used as a positive control. The absorbance was measured at 620 nm, and the percent of inhibition was calculated.
Pancreatic lipase: The pancreatic lipase activity was performed using PNPP as substrate . PNPP was used as a substrate in a solution containing: 40 mg PNPP in isopropanol added to 50 mM Tris-HCl buffer (pH 8.0), 40 mg gum Arabic, 80 mg sodium deoxycholate, and Triton X-100. Orlistat (50 µg/mL) was used as a positive control. Briefly, 20 µL of the tested sample was put into 96-well plates, and lipase enzyme solution (10 mg/mL; porcine pancreatic lipase type II, Sigma-Aldrich) was freshly prepared in 50 mM Tris-HCl buffer (pH 8.0), stirred until fully dissolved and was then added 80 µL to all tests. After 37 °C for 15 min, the substrate solution was added at 37 °C for 25 min. Absorbance was recorded at 405 nm.
DPPH radical-scavenging capacity was estimated as a previous protocol . Vitamin C (100 µg/mL) was used as a positive control. Briefly, 80 µL of each tested sample and 800 µL DPPH (0.5 mmol/L) solubilized in a methanol solution were vortex-mixed and incubated in the dark at 37 °C for 20 min. DPPH radical was determined by measuring the absorbance at 517 nm. Total antioxidant capacity (T-AOC) was measured by biochemical methods following the manufacturer’s instructions (Beijing Solarbio Science and Technology, Beijing, China) .
The lysine-glucose Maillard reaction was determined as recorded previously . Glutamic acid and lysine (both at 1.0 M, 0.2 mL) were mixed with 0.8 mL of tested samples in sodium phosphate buffer (0.1 M, pH 6.8) and 0.5 mL of 0.25 M sodium phosphate buffer at 70 °C for 2 hours. Aminoguanidine (10 mg/mL) was used as a positive sample. The absorbance was measured at 450 nm on a microplate reader. The anti-glycation assay in the BSA-fructose model was performed as described . Fructose (1.5 M, 0.5 mL) was mixed with 0.5 mL tested sample, 2.0 mL sodium phosphate buffer (0.1 M, pH 6.8, with 0.02% sodium benzoate.) at 37 °C for 2 hours. BSA (30 mg/mL, 0.5 mL) was added at 37 °C for 5 days. Aminoguanidine (1 mg/mL) was used as a positive control. The fluorescent advanced glycation end-product (AGE) was monitored (350 nm as the excitation /420 nm as emission) using a fluorescence spectrophotometer. Methylglyoxal (60 mM, 0.5 mL) was mixed with 0.5 mL tested sample and 2.0 mL of sodium phosphate buffer (0.1 M, pH 6.8, with 0.02% sodium benzoate) at 37 °C for 2 hours. BSA (30 mg/mL, 0.5 mL) was added at 37 °C for 5 days. Aminoguanidine (1 mg/mL) was used as a control. The fluorescent AGE was monitored (350 nm as the excitation /420 nm as emission) was measured on the fluorescence spectrophotometer. In arginine-methylglyoxal assay, methylglyoxal (60 mM, 0.5 mL) was mixed with 0.5 mL of tested samples and 2.0 mL sodium phosphate buffer (0.1 M, pH 6.8, with 0.02% sodium benzoate.) at 37 °C, 2 hours. Arginine (60 mM, 0.5 mL) was added to all sets, and the mixtures were incubated at 37 °C for 5 days. Aminoguanidine (1 mg/mL) was used as a positive sample. Then, fluorescent AGE was monitored (350 nm as the excitation /420 nm as emission) was measured.
Methylglyoxal scavenging was conducted by HPLC method according to a previously published method with modification . Methylglyoxal was derivatized with O-phenylenediamine (O-PD) to form 2-methylquinoxaline (2-MQ), highly specific for methylglyoxal. Methylglyoxal and O-PD were dissolved in phosphate buffer (0.1 M, pH 6.8) to 10 mM and 50 mM. Aminoguanidine (1 mg/mL) was used as a control. The mixture of methylglyoxal (50 mM, 0.1 mL) with the sample (0.4 mL) was incubated at 37 °C for 4 hours. Then, O-PD (0.2 mL) was added into all sets. The tubes were kept for 30 min for undergoing derivatization reaction between methylglyoxal and O-PD. Analysis of 2-MQ was performed on a Waters 2695 HPLC platform (Waters Corporation, Milford, MA) and carried out by a Zorbax SB-C18 column (4.6 × 250 mm, 5 µm, Agilent Technologies, Palo Alto, CA). The mobile phase for HPLC system consisted of pure methanol (solvent A) and pure Millipore water (solvent B) with a constant flow rate set at 1.0 mL/min. An injection volume was 10 µL. The linear gradient for elution was: 0–35 min, 5-100% A ; 35–45 min, 100-5%, followed by 5 min to re-equilibrate the system. 2-MQ was detected at 315 nm using a DAD detector having a retention time at 20.09 min. The peak area of 2-MQ in each sample was integrated. The methylglyoxal scavenging was calculated using the percentage (%) calculated from the homologous equation in BSA- fructose model.