Experimental materials: identified as Uralic Glycyrrhiza uralensis Fisch, IsoGlycyrrhiza uralensis Fisch standard (HPLC≥98%, WM256.25, C15H12O4). Hepatocellular carcinoma cells HepG-2, lung cancer cells A549, renal clear carcinoma cells Caki-1, prostate cancer PC-3 cell lines, provided by Mudanjiang Medical College.

Main reagents: Chloro-1-butyl-3-methylimidazole (Beijing Kool Chemical Technology Co., Ltd., China), Bromo-1-butyl-3-methylimidazole (Beijing Kool Chemical Technology Co., Ltd., China), 1-butyl-3-methylimidazole Hydrogen Sulfate (Beijing Kool Chemical Technology Co., Ltd., China), 1-butyl-3-methylimidazole Nitrate (Beijing Kool Chemical Technology Co., Ltd., China), Anhydrous ethanol (Nanjing Xingsha Chemical Co., Ltd., China), methanol (chromatographically pure) (Fisher Chemical, United States), acetonitrile (chromatographically pure) (Fisher Chemical, United States), acetic acid (Tenda Chemical Reagent Factory, Dongli District, Tianjin, China), hydrochloric acid (Tenda Chemical Reagent Factory, Dongli District, Tianjin, China), sodium hydroxide (Tenda Chemical Reagent Factory, Dongli District, Tianjin, China), Agar powder (Beijing Aoboshin Biotechnology Co., Ltd., China), ultrapure water (Hangzhou Wahaha Group Co., Ltd., China), yeast powder (Beijing Aoboshin Biotechnology Co., Ltd., China), RPMI1640 medium (Gibco, United States), fetal calf serum (FCS) (Zheijiang Tianhang Bio-technology Co., Ltd., China), sodium bicarbonate (NaHCO3, AR) (Tianjin Chemical Reagent Sixth Factory Branch Factory, China). Ltd., China), sodium bicarbonate (NaHCO3, AR) (Tianjin Chemical Reagent Six Factory Branch, China), sodium chloride (NaCl, AR) (Tianjin Zhiyuan Chemical Reagent Co., Ltd., China), potassium chloride (KCl, AR) (Tianjin Komeo Chemical Reagent Co., Ltd., China), disodium hydrogen phosphate (Na2HPO4-12H2O, AR) (Tianjin Tianli Chemical Reagent Co., Ltd., China), potassium dihydrogen phosphate (KH2PO4, AR) (Tianjin, China) Dongli Tianda Chemical Reagent Factory, Dongli District, China), Penicillin-Streptomycin Mixed Solution (100X Double Antibody) (Beijing Regen Biotechnology Co., Ltd., China), Tetrazolium Blue Bromide MTT (AR) (Shanghai Yuanmu Biotechnology Co., Ltd., China), PBS Buffer (Shijiazhuang Dingchen Science and Technology Co., Ltd., China), dimethyl sulfoxide DMSO (AR) (Yueyang Xiangmao Pharmaceutical and Chemical Co. Ltd., China), trypsin-EDTA culture medium (Shanghai Yuanmu Biotechnology Co., Ltd., China).

Waters2695 High Performance Liquid Chromatograph (Waters, United States), Mass Spectrometry using LCQ-DecaXP/Ad type (Thermo Electron, United States), AF-08 Miniature High Speed Pulverizer (Wenling City, China, Traditional Chinese Medicine Machinery Manufacturing Co., Ltd.), DHG-9055A Electric Drum Drying Oven (Shanghai Yihang Science and Technology Co., Ltd., China), BCD-206TASJX Intelligent Refrigerator (Qingdao Haier Co., Ltd., China), BSA223S-CW Electronic Analytical Balance (Sartorius, Germany), KQ32000V Ultrasonic Cleaning Machine (Kunshan City Ultrasonic Instrument Co., Ltd., China), ZQZY-C8V Triple Combined Full-Temperature Vibrating Incubator (Shanghai Chuyi Instrument Co., Ltd., China), PB Ltd., PB-10 pH meter (Sartorius, Germany), HiQ SiL C18V column (KYA TECH), ALLEGRA-64R Beckman tabletop centrifuge (Beckman, United States), CKX53 inverted microscope (OLYMPUS), 61M/SG-XSB-102B electron microscope (China Beijing Zhongxi-Yuanda Technology Co. Ltd.), DHP-9052 Constant Temperature Incubator (Shanghai Baidian Instrument Factory, China), YDS-50 Liquid Nitrogen Storage Tank (Changsha Mingjie Instrument Co., Ltd., China), DNM-9602G Enzyme Labeling Instrument (Beijing Plan New Technology Co., Ltd., China), Micropipettes (Gilson, France), ESCOAC2-4S1 Biological Safety Cabinet (ESCO, Singapore), INC153 CO2 Incubator (Memmer, Germany), SX-700 Autoclave (Tomy, Japan), Hematocrit Plate (Shanghai Precision, China), 20∼200uL Pipette Gun (Thermo, United States), 96-well Cell Culture Plate (Corning).

Examination of exclusivity: A sample solution was prepared, and the elution solvent was used as a blank control solution. Following the determination of the standard curve, the sample was injected, and the peak area was measured to assess the method’s exclusivity. Precision test: Five samples of isoliquiritigenin were accurately measured, injected, and the peak area was determined based on the standard curve to evaluate the method’s precision. Repeatability test: Six sample solutions were prepared in parallel, injected, and the peak area was determined under high-performance liquid chromatography (HPLC) conditions to assess the method’s repeatability. Stability test: The same sample solution was injected at 0, 2, 4, 8, 12, and 24 h, and the peak area was measured to evaluate stability over the 0–24 h period. Spiking recovery experiment: Six aliquots of each isoliquiritigenin sample solution were taken, and a control solution of isoliquiritigenin of known quality was added to each. The spiking recoveries were calculated, and the relative standard deviation (RSD) values were determined to assess the method’s accuracy.

A total of 4 g of absolute dry weight licorice powder was weighed and placed in a 100 mL conical flask. Distilled water was added according to the material-liquid ratio, and the mixture was stirred thoroughly to create a solid fermentation medium, which was then sterilized. The effects of different pH values on the yield of isoliquiritigenin from licorice species were examined at pH levels of 3, 4, 5, 6, and 7. Additionally, to assess the impact of varying fermentation durations on isoliquiritigenin yield, incubation times of 2, 4, 6, and 8 days were employed. The influence of different material-liquid ratios on isoliquiritigenin yield was also investigated, with ratios of 1:1, 1:2, 1:3, 1:4, 1:5, and 1:6 being tested. Furthermore, the effect of varying inoculation amounts of Aspergillus niger on isoliquiritigenin yield was analyzed, with inoculation amounts of 1 × 10⁵, 5 × 10⁵, 1 × 10⁶, 2 × 10⁶, 3 × 10⁶, 4 × 10⁶, 5 × 10⁶ used. Following fermentation, 100 mL of a 75% ethanol solution was added to the medium at a 1:25 material-liquid ratio, mixed thoroughly, and extracted using ultrasonic waves in a water bath at 60 °C for 1.5 h. The extraction was performed while hot, and the resulting filtrate was collected and its volume measured.

After the univariate study, the optimal conditions for each variable were determined, with pH, solid-liquid ratio, and Aspergillus niger inoculum as the independent variables, and the yield of isoliquiritigenin fractions as the dependent variable, while keeping the other factors consistent in accordance with the principles of the Response Surface Methodology (RSM) design (Table 1).

Ethanol reflux extraction method: Weigh 4 g of absolute dry licorice in a 250 mL round-bottom flask. Following a 1:25 material-to-liquid ratio, add 100 mL of a 75% ethanol solution, mix thoroughly, and perform reflux extraction at 60 °C in a water bath for 1.5 h. Filter the mixture while hot and collect the filtrate.

Ultrasonic extraction: Weigh 4 g of absolute dry licorice in a 250 mL round-bottom flask. Using the same 1:25 material-to-liquid ratio, add 100 mL of a 75% ethanol solution, mix well, and extract using ultrasonication at 60 °C in a water bath for 1.5 h. Filter the mixture while hot and collect the filtrate.

isoliquiritigenin (200 mg, 0.78 mmol) was dissolved in 10 mL of tetrahydrofuran (THF). To the reaction solution, N,N′-carbonyldiimidazole (506 mg, 3.12 mmol) and two drops of triethylamine were added at room temperature, and the mixture was stirred for 4 h. Following this, a 30% aqueous dimethylamine solution (1.2 mL, 3.9 mmol) was introduced, and the reaction was monitored using thin-layer chromatography (TLC). Upon completion of the reaction, 40 mL of saturated aqueous ammonium chloride solution was added to quench the reaction. The aqueous phase was extracted multiple times with ethyl acetate (20 mL × 3), and the organic phases were combined. Subsequently, the organic phase was washed with saturated saline (5 mL × 2) and dried over anhydrous magnesium sulfate. After filtration and concentration, the yellow solid was obtained via column chromatography (200–300 mesh silica gel, PE/EA = 1:2) (Figure 1A).

Isolation of isoliquiritigenin (200 mg, 0.78 mmol) was achieved by dissolving it in 10 mL of N,N′-dimethylformamide (DMF). A 1.0M solution of lithium bis(trimethylsilyl) sulfamate (LHMDS) in tetrahydrofuran (1.17 mL, 1.17 mmol) was added dropwise to the reaction mixture at −40 °C under nitrogen atmosphere, and the mixture was stirred for 15 min. Subsequently, methyl trifluoromethanesulfonate (0.18 mL, 1.56 mmol) was added dropwise to the reaction solution, with the reaction progress monitored using thin-layer chromatography (TLC). Upon completion, the reaction was quenched by the addition of 40 mL of saturated aqueous ammonium chloride solution. The aqueous phases were extracted several times with ethyl acetate (20 mL × 3), and the organic phases were combined. The combined organic phase was washed with saturated saline (5 mL × 2) and dried over anhydrous magnesium sulfate. The concentrated solution was then filtered and purified by column chromatography (200–300 mesh silica gel, PE/EA = 6:1), yielding a yellow solid (Figure 1B).

isoliquiritigenin (200 mg, 0.78 mmol) was dissolved in 10 mL of THF. A 1.0M lithium bis(trimethylsilyl) diamine (LHMDS) solution in tetrahydrofuran (1.17 mL, 1.17 mmol) was slowly added dropwise to the reaction mixture under nitrogen protection at 0 °C. The reaction was stirred for 15 min, after which acetic anhydride was added dropwise to the reaction mixture (0.15 mL, 1.56 mmol). The progress of the reaction was monitored by TLC, and upon completion, 40 mL of saturated aqueous ammonium chloride solution was added to quench the reaction. The aqueous phase was extracted with ethyl acetate (20 mL × 3), and the organic phases were combined, washed with saturated saline (5 mL × 2), and dried over anhydrous magnesium sulfate. The mixture was then filtered and concentrated, followed by separation on a column (200–300 mesh silica gel, PE/EA = 1:1) to yield a yellow solid. The isoliquiritigenin derivative 3 was successfully prepared, and the synthetic process is depicted in (Figure 1C).

Finally, the derivative yields of the three substances were calculated. Calculation of derivative yields is shown in Equation 1. Yield  % = Actual   yield Theoretical   yield × 100   % (1)

The 1H-NMR spectrum and 13C-NMR spectrum were also determined by NMR using deuterated chloroform as solvent and TMS as internal standard.

Cells were identified through observation and subsequently prepared as a cell suspension. The cell count was determined using a cell counting method, and the suspension was distributed into 96-well culture plates, with each well containing approximately 10⁴ cells in 100 μL. The cells in the 96-well plates were cultured overnight at 37 °C in a 5% CO₂ incubator. After 24 h, the culture solution was aspirated and discarded. The final concentrations of isoliquiritigenin and its derivatives in the 96-well plates were set at 6.25 μmol/L, 12.5 μmol/L, 25 μmol/L, 50 μmol/L, and 100 μmol/L. The blank control group received an equal volume of the culture solution, which consisted of RPMI 1640 medium containing 10% fetal bovine serum. This group was cultured synchronously with the administration groups and the positive control group (37 °C, 5% CO₂ incubator) for 48 h. It underwent the same MTT staining procedure (200 µL MTT solution added to each well for 4 h) and subsequent DMSO dissolution steps. The absorbance value from this group was used as the baseline to correct for interference from medium components, reagents, and operational processes on the experimental results. Each treatment group consisted of six parallel wells, and the cells were incubated at 37 °C in a 5% CO₂ incubator for an additional 48 h. Following this incubation, the supernatant was removed, and 200 μL of fresh culture solution was added to each well. Subsequently, 200 μL of MTT solution was added to each well, and incubation continued for 4 h. After this period, the supernatant was discarded, ensuring minimal liquid residue remained, and 100 μL of DMSO was added and mixed thoroughly by shaking for 10 min. The cell inhibition rate was calculated based on the absorbance values obtained using an enzyme labeling instrument at a wavelength of 490 nm, and is shown in Equation 2. Cell proliferation rate  % = Mean   OD   of   administered   group − Mean   OD   of   blank   control   group Mean   OD   value   of   blank   control   group × 100   % (2)

Caki-1 cells were cultured and suspended in serum-free medium, adjusting the cell concentration to 2 × 10⁶ cells/mL. Under aseptic conditions, the right anterior axillary subcutis and abdomen of the mice were sterilized using 75% ethanol, followed by the inoculation of 0.2 mL of the cell suspension per mouse, ensuring thorough mixing before each aspiration to establish a solid tumor model. Tumor volume was measured every 3 days. The mice were randomly divided into five groups, each containing ten mice, and received intraperitoneal (ip) administration post-inoculation. The experimental groups were administered ISL-bL at high, medium, and low doses of 50 mg/(kg·d), 25 mg/(kg·d), and 12.5 mg/(kg·d), respectively. The positive control group received IL-2 at a dose of 2.5 × 10⁵ IU/(kg·d), while the model control group was given an equal volume of saline. Administration occurred once daily, with 0.2 mL per mouse, starting the day after inoculation. The animal room was well-ventilated, maintained at a temperature of 20 °C–22 °C and a humidity of 45%–50%. The environment was clean, quiet, and followed a natural circadian rhythm. The animals were fed a standard diet, equivalent to 3 g per mouse per day, with water provided continuously. Fresh drinking water was replaced daily, and bedding was changed every 2–3 days.

Tumor tissues of appropriate size were fixed in a 4% paraformaldehyde solution, dehydrated, and embedded in clear paraffin. The samples were then sealed in neutral gel through a series of processes, including dewaxing, gradient ethanol dehydration, differentiation rinsing, and HE staining, after which they were placed under a microscope for observation. Finally, statistical analyses were performed using SPSS 18.0 and GraphPad Prism 6.01 software, with data from each group expressed as x ± s. Sample comparisons were conducted using a one-way ANOVA test, and a P-value of less than 0.05 was considered statistically significant.

The effect of ISL-b on protein expression in Caki-1 cells was determined by Western blot. The protein samples were thawed at room temperature, processed and denatured. Then, gel preparation and electrophoresis were performed, followed by membrane transfer in sequence. The membranes were blocked overnight with 5% milk prepared in TBST, incubated with primary and secondary antibodies successively with washing steps in between. Finally, ECL color development was carried out in the dark, and imaging analysis was performed.

On the first day following the completion of modeling, rat feces were aseptically collected into sterile EP tubes. For both the model group and the IL-2 positive control group, five mice were randomly selected, and their feces were pooled. Two samples were taken from each of the ISL groups categorized by low, medium, and high doses. Each sample consisted of feces from five randomly selected mice, from which 200 mg was extracted. The collected fresh feces were snap-frozen in liquid nitrogen for over 30 s and subsequently stored at −80 °C for future use. DNA extraction, amplification, and library construction were completed within 4 weeks to facilitate subsequent analysis.

The specific sequencing and analysis workflow is as follows: Illumina NovaSeq platform was used for paired-end sequencing (PE250). The sequencing depth was evaluated by rarefaction curves, and the sequencing depth was determined to be reasonable when the curves tended to be flat. Prior to alpha diversity analysis, normalization was performed using a minimum data volume threshold of 59,860. For data standardization, the minimum sample size normalization strategy was adopted, involving random sampling adjustment of effective sequences from all samples. For bioinformatics analysis, QIIME v1.9.1 was used for Tags quality control and alpha diversity index calculation; Uparse v7.0.1001 was used for OTU clustering with 97% identity. Additionally, FLASH v1.2.7 was used for sequence assembly, Vsearch for chimera filtering, and MUSCLE v3.8.31 for constructing phylogenetic trees. The complete workflow was as follows: raw off-machine data were assembled into raw Tags by FLASH, then subjected to QIIME quality filtering to obtain clean Tags. After removing chimeras via Vsearch, effective sequences were obtained, which were subsequently used for OTU clustering, species annotation, and diversity analysis. The quality filtering criteria included truncating consecutive 3 or more bases with a quality score ≤19 and removing Tags with a length less than 75% of the original sequence.

The interactions among these factors were investigated using Response Surface Methodology (RSM) to optimize fermentation pH, solid-liquid ratio, and the inoculum of Aspergillus niger. The experiments were randomized, and were designed to maximize the impact of unexplained variability on extraction efficiency. A total of 17 tests were conducted with 5 replications (specifically the 2nd, 7th, 8th, 12th, and 15th tests) to accurately estimate the pure sum of squares of errors (Table 6).

Based on the data presented, a multiple regression fitting analysis was conducted using Design Expert 8.0.7.1 software. The resulting quadratic equation for the yield of isoglycosides (Y) in relation to each factor variable is as follows:Y% = 1.49–0.016A + 0.054B + 0.20C - 0.095AB + 0.0025AC - 0.062BC - 0.049A² - 0.21B² - 0.33C². The ANOVA results, indicate that the model is statistically significant with a p-value of less than 0.0001. Furthermore, the misfit term F-value of 33.51 suggests that the misfit is not significant (Table 7).

Based on the credibility analysis of the regression equation presented, the model’s adjusted coefficient of determination is R ²_adj = 0.9482, indicating that this model can explain 94.82% of the variation in the response variable. Additionally, the coefficient of determination is R ² = 0.9773 and the predicted coefficient of determination is R ²_pred = 0.8235, with a precision ratio of 18.461. This precision ratio suggests that the model is sufficiently precise, and the goodness of fit results are effective for predicting the yield of isoGlycyrrhiza uralensis Fisch under varying conditions for the yield of isoliquiritigenin. Furthermore, based on the comparison of F-values, it can be concluded that the order of significance under different conditions is as follows: Aspergillus niger inoculum > solid-liquid ratio > pH value (Table 8) (Figures 4A–F).

The optimal combination obtained from the response surface modeling calculation, along with the results analyzed and estimated using prediction software, yielded a pH of 3.694, a solid-liquid ratio of 1:2.155, an Aspergillus niger inoculum concentration of 1,466,745, and a yield of 1.525 mg/g. To enhance practical operational convenience, the extraction process parameters were adjusted to pH 3.694, a solid-liquid ratio of 1:2.155, and an Aspergillus niger inoculum concentration of 1,466,745 for three validation tests. The validation analysis produced a yield of 1.49 ± 0.035 mg/g of isoliquiritigenin, demonstrating that these parameters effectively reflect the impact of screening factors on the yield of isoliquiritigenin.

Cell morphology was observed under an inverted microscope after treatment with ISL and its derivatives on Caki-1 cells at a concentration of 25 μmol/L for 24 h. After 24 h of ISL treatment, most Caki-1 cells appeared to be in good condition, exhibiting adherent growth and well-defined edges; however, a few cells exhibited a rounded morphology. In the presence of the ISL-a compound, the number of rounded cells increased compared to the ISL-treated group, with non-adherent cells becoming evident and sparse growth observed, indicating that most cells were suspended in the culture medium. With the addition of the ISL-c compound, the number of rounded and non-adherent cells further increased, while the growth rate of adherent cells significantly decreased. In the ISL-b treatment group, the morphological changes in Caki-1 cells were more pronounced compared to the ISL group; most cells became rounded, and the transparency and fullness of the cells decreased, with some cells appearing ruptured and a majority exhibiting signs of cell death. These results indicate that all three derivatives had a more pronounced inhibitory effect on Caki-1 cells than ISL, with ISL-b demonstrating the most significant efficacy (Figure 7).