Total Triterpenoid Extraction from Inonotus Obliquus Using Ionic Liquids and Separation of Potential Lactate Dehydrogenase Inhibitors via Ultrafiltration High-Speed Countercurrent Chromatography

Extracts of the fungus Inonotus obliquus exhibit cytotoxic properties against different cancers; hence, this fungal species has been extensively studied. This study aimed to extract total triterpenoids from Inonotus obliquus using ionic liquids (ILs) and separate potential lactate dehydrogenase (LDH) inhibitors via ultrafiltration (UF)-high-speed countercurrent chromatography (HSCCC). Total triterpenoids from Inonotus obliquus were extracted by performing a single-factor experiment and employing a central composite design via ultrasonic-assisted extraction (UAE) and heat-assisted extraction (HAE). The extract was composed of 1-butyl-3-methylimidazolium bromide as the IL and methanol as the dispersant. Ultrafiltration-liquid chromatography (UF-LC) was used to rapidly scan the LDH inhibitors and betulin and lanosterol were identified as potential inhibitors. To obtain these target compounds, betulin and lanosterol with the purities of 95.9% and 97.8% were isolated from HSCCC within 120 min. Their structures were identified using several techniques, among which IL-HAE was fast and effective. This study reports the extraction of triterpenoids from Inonotus obliquus by IL for the first time. Collectively, the findings demonstrate that UF-LC is an effective tool for screening potential LDH inhibitors from crude extracts of I. obliquus and may help to identify bioactive substances against myocardial infarction, whereas high-purity compounds can be separated via UF-HSCCC.


Introduction
The fungus Inonotus obliquus is mainly distributed in northeastern China, northern Russia, and other cold regions [1,2]. The sclerotium of its fruiting body is nodular (sterile block), sessile, 25-40 cm in diameter, dark gray in appearance, irregularly furrow-marked with a deep crack, and hard and brittle when dry [3]. This fungus can be used as food and medicine for preventing and treating malignant tumors, diabetes, liver diseases, vascular diseases, and acquired immune deficiency syndrome [4,5]. Therefore, I. obliquus has attracted considerable attention from researchers owing to its potential anti-cancer properties. For instance, Baek et al. evaluated its cytotoxic activity against four human lung adenocarcinoma cell lines, each with different p53 statuses. They found that the triterpenoids from I. obliquus have potential therapeutic effects against lung cancer and revealed the molecular basis of their cytotoxic activity against human lung cancer [6]. Furthermore, Kou Figure 2A). These data show that when anions are the same, the total triterpenoid content first increases and then decreases with increasing carbon chain length regardless of using UAE or HAE. This may be because with increasing carbon chain length, the viscosity of ILs gradually increases, and the effective components cannot be extracted by using the overly viscous ILs. The extraction effect of [BMIM] PF6 was the strongest; hence, by using the same cation ([BMIM] + ), the extraction effect of different anions (TfO − , BF4 − , Br − , PF6 − ) was studied. The results showed that [BMIM] Br exerted the best extraction effect ( Figure 2A).  Under the same extraction conditions, the amounts of total triterpenoids obtained after performing ultrasonic-assisted extraction (UAE) were as follows: 9.45 mg [EMIM]PF 6 , 9. (Figure 2A). These data show that when anions are the same, the total triterpenoid content first increases and then decreases with increasing carbon chain length regardless of using UAE or HAE. This may be because with increasing carbon chain length, the viscosity of ILs gradually increases, and the effective components cannot be extracted by using the overly viscous ILs. The extraction effect of [BMIM] PF 6 was the strongest; hence, by using the same cation ([BMIM] + ), the extraction effect of different anions (TfO − , BF 4 − , Br − , PF 6 − ) was studied. The results showed that [BMIM] Br exerted the best extraction effect ( Figure 2A).  Figure 2A). These data show that when anions are the same, the total triterpenoid content first increases and then decreases with increasing carbon chain length regardless of using UAE or HAE. This may be because with increasing carbon chain length, the viscosity of ILs gradually increases, and the effective components cannot be extracted by using the overly viscous ILs. The extraction effect of [BMIM] PF6 was the strongest; hence, by using the same cation ([BMIM] + ), the extraction effect of different anions (TfO − , BF4 − , Br − , PF6 − ) was studied. The results showed that [BMIM] Br exerted the best extraction effect ( Figure 2A).    Figure 2B). Therefore, this IL/dispersant combination was used for follow-up experiments.

Effects of the Central Composite Design
According to the principles of the "central composite test reagent, the experiments were arranged in the form of codes; the codes were obtained based on the actual operation value, and the difference between any two physical quantities was equal to the difference between the corresponding codes. By establishing relevant model fitting effects and factors, the three-dimensional effect surface could be visualized, and the best extraction process was quickly obtained based on the surface area with the best effect.
Experimental results of the central composite design using two extraction methods are shown in Table 1. The three factors (A, solid-liquid ratio; B, concentration of the extraction solution; and C, extraction time) were fitted using the total triterpenoid content as the evaluation index. The fitting equations of UAE and HAE are shown in Table 2. The fitting degree of each equation was good (high R 2 values), and thus, they were used to analyze and predict the extraction process. The results of the binomial fitting analysis of variance ( Table 2) showed that the total model equation was significant (p < 0.05), indicating that the model had a good fit and a small experimental error, both of which are valuable for accurate predictions. Furthermore, this model was also used to predict and optimize the solid: liquid ratio, concentration of the extraction solution, and extraction time. The p-value showed that the primary term of factors B, AB, and BC in UAE and factor A in HAE had a significant effect on the comprehensive score (p < 0.05), whereas the other elements had no significant effect on the result (p > 0.05). As the extraction effect was better for HAE than for UAE, HAE was used for subsequent analyses. According to the established polynomial model, through Design-Export 10.0 software statistics, factors A, B, and C were drawn to create the corresponding surface map ( Figure 3). The area of each factor has been optimized in the figure, and the following conditions for extracting total triterpenoids from I. obliquus   Figure 2B). Therefore, this IL/dispersant combination was used for follow-up experiments.

Effects of the Central Composite Design
According to the principles of the "central composite test reagent, the experiments were arranged in the form of codes; the codes were obtained based on the actual operation value, and the difference between any two physical quantities was equal to the difference between the corresponding codes. By establishing relevant model fitting effects and factors, the three-dimensional effect surface could be visualized, and the best extraction process was quickly obtained based on the surface area with the best effect.
Experimental results of the central composite design using two extraction methods are shown in Table 1. The three factors (A, solid-liquid ratio; B, concentration of the extraction solution; and C, extraction time) were fitted using the total triterpenoid content as the evaluation index. The fitting equations of UAE and HAE are shown in Table 2. The fitting degree of each equation was good (high R 2 values), and thus, they were used to analyze and predict the extraction process. The results of the binomial fitting analysis of variance (Table 2) showed that the total model equation was significant (p < 0.05), indicating that the model had a good fit and a small experimental error, both of which are valuable for accurate predictions. Furthermore, this model was also used to predict and optimize the solid: liquid ratio, concentration of the extraction solution, and extraction time. The p-value showed that the primary term of factors B, AB, and BC in UAE and factor A in HAE had a significant effect on the comprehensive score (p < 0.05), whereas the other elements had no significant effect on the result (p > 0.05). As the extraction effect was better for HAE than for UAE, HAE was used for subsequent analyses. According to the established polynomial model, through Design-Export 10.0 software statistics, factors A, B, and C were drawn to create the corresponding surface map ( Figure 3). The area of each factor has been optimized in the figure, and the following conditions for extracting total triterpenoids from I. obliquus were determined for HAE: extraction solution, [BMIM]Br with MeOH; concentration of the extraction solution, 1.0 mol/L; solid: liquid ratio, 1:30; and extraction time, 83.64 min.

Process Parameter Optimization and Model Validation
To verify the reliability of the model, the experiment was performed according to the optimized extraction conditions described in Section 3.2. Under the same conditions, the total triterpene amounts extracted from 1 g of I. obliquus were 16.81, 16.79, and 16.75 mg, respectively. Compared with the predicted value of 16.96 mg, the relative deviations were as less as 0.88, 1.00, and 1.24%, respectively, indicating that the mathematical model has good predictability.

Evaluation of the Potential Inhibitory Activity of LDH
The potential LDH inhibitors in I. obliquus were screened using UF-LC. The enzymeligand complexes were separated from the small unconjugated molecules through the UF membrane. The binding of I. obliquus at 50 mg/mL to LDH at different concentrations was evaluated, and two compounds were considered as potential LDH ligand compounds ( Figure 4). The retention time of compound 1 was 17.98 min, and the inhibition rates of 5, 10, and 20 U/mL LDH were 23.38, 39.06, and 54.58%, respectively. For compound 2, the retention time was 26.17 min, and the inhibition rates at 5, 10, and 20 U/mL LDH were 27.72, 48.45, and 69.44%, respectively. Because compounds 1 and 2 had potent inhibitory effects on LDH, HSCCC was used to separate them.
membrane. The binding of I. obliquus at 50 mg/mL to LDH at different concentrations was evaluated, and two compounds were considered as potential LDH ligand compounds ( Figure 4). The retention time of compound 1 was 17.98 min, and the inhibition rates of 5, 10, and 20 U/mL LDH were 23.38, 39.06, and 54.58%, respectively. For compound 2, the retention time was 26.17 min, and the inhibition rates at 5, 10, and 20 U/mL LDH were 27.72, 48.45, and 69.44%, respectively. Because compounds 1 and 2 had potent inhibitory effects on LDH, HSCCC was used to separate them.

HSCCC Separation of Potential LDH Inhibitors from I. Obliquus
The separation effect of HSCCC mainly depends on the selection of the solvent system. In the present study, different proportions of n-hexane-ethyl acetate-MeOH-water were used as the solvent system. The K values for compounds 1 and 2, and the resolutions of both compounds, were calculated as described in Section 3.9. The K values of compounds 1 and 2 were calculated as K = A u /A l , and their resolution was the ratio of K 1 to K 2 .
When the K value is in the ideal range (0.5-2.5), the greater the resolution, the better the separation. In the present study, n-hexane-ethyl acetate-MeOH-water (5:5:3:7) was used as the solvent system for the separation of compounds 1 and 2, with K 1 = 1.29, K 2 = 0.99, and a resolution of 1.30 (Table 3). The mobile phase flow rate determines the retention rate of the stationary phase and the separation of the chromatographic peaks. At a column rotation speed of 850 rpm and a flow rate of 1.0 mL/min, the retention rate was 72%, and the separation time of 180 min allowed compounds 1 and 2 to separate. When the flow rate was increased to 2.5 mL/min, the retention rate was 60%, and the separation time was only 100 min. Although the separation time was shortened, compound 1 could not be separated from other impurity peaks. At a flow rate of 1.5 mL/min, compounds 1 and 2 were distinctly separated within 120 min with a 68% retention rate. Hence, the HSCCC separation conditions used were as follows: column rotation, 850 rpm; solvent system, n-hexane-ethyl acetate-MeOH-water (5:5:3:7); flow rate, 1.5 mL/min; and injection volume, 100 mg ( Figure 5).

HSCCC Separation of Potential LDH Inhibitors from I. Obliquus
The separation effect of HSCCC mainly depends on the selection of the solvent system. In the present study, different proportions of n-hexane-ethyl acetate-MeOH-water were used as the solvent system. The K values for compounds 1 and 2, and the resolutions of both compounds, were calculated as described in Section 3.9. The K values of compounds 1 and 2 were calculated as K = Au/Al, and their resolution was the ratio of K1 to K2.
When the K value is in the ideal range (0.5-2.5), the greater the resolution, the better the separation. In the present study, n-hexane-ethyl acetate-MeOH-water (5:5:3:7) was used as the solvent system for the separation of compounds 1 and 2, with K1 = 1.29, K2 = 0.99, and a resolution of 1.30 (Table 3). The mobile phase flow rate determines the retention rate of the stationary phase and the separation of the chromatographic peaks. At a column rotation speed of 850 rpm and a flow rate of 1.0 mL/min, the retention rate was 72%, and the separation time of 180 min allowed compounds 1 and 2 to separate. When the flow rate was increased to 2.5 mL/min, the retention rate was 60%, and the separation time was only 100 min. Although the separation time was shortened, compound 1 could not be separated from other impurity peaks. At a flow rate of 1.5 mL/min, compounds 1 and 2 were distinctly separated within 120 min with a 68% retention rate. Hence, the HSCCC separation conditions used were as follows: column rotation, 850 rpm; solvent system, n-hexane-ethyl acetate-MeOH-water (5:5:3:7); flow rate, 1.5 mL/min; and injection volume, 100 mg ( Figure 5).

Purity Determination and Identification of Potential LDH Inhibitors Using High-Performance Liquid Chromatography (HPLC), Electrospray Ionization-Mass Spectrometry (ESI-MS), 1 H Nuclear Magnetic Resonance (NMR), and 13 C NMR
Potential LDH inhibitors in I. obliquus extracts were analyzed via HPLC. The purity of compounds 1 and 2 were 95.9% and 97.8%, respectively ( Figure 6). In the positive ion mode, the mass spectra of compounds 1 and 2 were recorded. Compounds 1 and 2 were identified as betulin and lanosterol, respectively, based on their HPLC retention times, MS/MS data, as well as 1 H and 13 C NMR data [22,23]. The chemical structures of betulin and lanosterol are shown in Figure 7.

Purity Determination and Identification of Potential LDH Inhibitors Using High-Performance Liquid Chromatography (HPLC), Electrospray Ionization-Mass Spectrometry (ESI-MS), 1 H Nuclear Magnetic Resonance (NMR), and 13 C NMR
Potential LDH inhibitors in I. obliquus extracts were analyzed via HPLC. The purity of compounds 1 and 2 were 95.9% and 97.8%, respectively ( Figure 6). In the positive ion mode, the mass spectra of compounds 1 and 2 were recorded. Compounds 1 and 2 were identified as betulin and lanosterol, respectively, based on their HPLC retention times, MS/MS data, as well as 1 H and 13 C NMR data [22,23]. The chemical structures of betulin and lanosterol are shown in Figure 7.  The study has certain limitations, which can be overcome in the future. First, since some ionic liquids are expensive, it is crucial to perform the least number of experiments to determine the best scheme of ionic liquid extraction in the extraction process. Additionally, studies have shown that long alkyl chain ILs exert toxicity; therefore, the selection and design of ILs should be carefully considered. Second, ultrafiltration liquid chromatography can only be used to detect the inhibitory enzyme activity of compounds in vitro and cannot be used to represent the medicinal effect of compounds in vivo.

General Experimental Procedures
A BS-124S electronic balance (Sartorius, Gottingen, Germany) was used for weight measurements. An ultraviolet-visible spectrophotometer (Thermo Fisher Scientific, San Jose, CA, USA) was used for absorbance measurements. HPLC was conducted on a

Purity Determination and Identification of Potential LDH Inhibitors Using High-Performance Liquid Chromatography (HPLC), Electrospray Ionization-Mass Spectrometry (ESI-MS), 1 H Nuclear Magnetic Resonance (NMR), and 13 C NMR
Potential LDH inhibitors in I. obliquus extracts were analyzed via HPLC. The purity of compounds 1 and 2 were 95.9% and 97.8%, respectively ( Figure 6). In the positive ion mode, the mass spectra of compounds 1 and 2 were recorded. Compounds 1 and 2 were identified as betulin and lanosterol, respectively, based on their HPLC retention times, MS/MS data, as well as 1 H and 13 C NMR data [22,23]. The chemical structures of betulin and lanosterol are shown in Figure 7.  The study has certain limitations, which can be overcome in the future. First, since some ionic liquids are expensive, it is crucial to perform the least number of experiments to determine the best scheme of ionic liquid extraction in the extraction process. Additionally, studies have shown that long alkyl chain ILs exert toxicity; therefore, the selection and design of ILs should be carefully considered. Second, ultrafiltration liquid chromatography can only be used to detect the inhibitory enzyme activity of compounds in vitro and cannot be used to represent the medicinal effect of compounds in vivo.

General Experimental Procedures
A BS-124S electronic balance (Sartorius, Gottingen, Germany) was used for weight measurements. An ultraviolet-visible spectrophotometer (Thermo Fisher Scientific, San Jose, CA, USA) was used for absorbance measurements. HPLC was conducted on a The study has certain limitations, which can be overcome in the future. First, since some ionic liquids are expensive, it is crucial to perform the least number of experiments to determine the best scheme of ionic liquid extraction in the extraction process. Additionally, studies have shown that long alkyl chain ILs exert toxicity; therefore, the selection and design of ILs should be carefully considered. Second, ultrafiltration liquid chromatography can only be used to detect the inhibitory enzyme activity of compounds in vitro and cannot be used to represent the medicinal effect of compounds in vivo.

General Experimental Procedures
A BS-124S electronic balance (Sartorius, Gottingen, Germany) was used for weight measurements. An ultraviolet-visible spectrophotometer (Thermo Fisher Scientific, San Jose, CA, USA) was used for absorbance measurements. HPLC was conducted on a Waters 2695 instrument (DAD, Milford, CT, USA) coupled to a Waters 2998 diode array detector (DAD). A Sigma 1-14 centrifuge (Sigma Zentrifugen, Osterode am Harz, Germany) equipped with an ultra-membrane filter Microcon YM-100 (Millipore, Bedford, MA, USA) was also used. ESI-MS was performed using an LCQ Fleet ion-trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an ESI source (Thermo Fisher Scientific, San Jose, CA, USA). HSCCC was performed on a TBE-300B Spectrum HSCCC (Shanghai Tauto Biotech Co., Ltd., Shanghai, China). 1 H and 13 C NMR spectra were recorded on a Bruker AV-400 and Avance-600 spectrometer (Bruker BioSpin, Ettlingen, Germany).

Chemicals and Reagents
HPLC-grade MeOH for HPLC and LC-MS was purchased from Fisher Scientific (UK). Ultra-pure water (18.2 MΩ-cm) was produced using a Milli-Q water purification system (Millipore, Burlington, MA, USA). LDH was obtained from Sigma-Aldrich (St. Louis, MO, USA), and phosphate-buffered saline (PBS) from Bueke (Switzerland). All organic solvents used for I. obliquus extraction and HSCCC separation were of analytical grade and purchased from Beijing Chemical Works (Beijing, China). The I. obliquus specimen used for extraction was stored in the Central Laboratory of Changchun Normal University, China (voucher specimen number: CCSFU-W1702). All ILs, vanillin, oleanolic acid, and acid standards were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

Determination of Total Triterpenoids
The total triterpenoid content was determined via vanillin-glacial acetic acid-perchloric acid spectrophotometry using oleanolic acid as the standard. Oleanolic acid (10 mg) was dissolved in MeOH in a 50-mL flask to prepare a 0.2 mg/mL standard solution. Standard solutions (0.0-0.7 mL) in test tubes were evaporated in a 90 • C water bath. Thereafter, 0.3 mL 5% vanillin-glacial acetic acid solution and 1 mL perchloric acid were added to each tube and mixed evenly. After 20 min in a water bath at 60 • C, mixtures were rapidly cooled to room temperature (25 • C) with cold water, diluted with glacial acetic acid in a 10-mL flask, and shaken. The absorbance (Y, ordinate) of each mixture was measure at 550 nm and plotted against the mass (X, abscissa) of oleanolic acid. The total triterpenoid content was obtained in mg oleanolic acid per gram of dried I. obliquus. obliquus. During the preliminary screening, when the solid: liquid ratio increased from 1:30 to 1:40, the total triterpenoid content of I. obliquus did not increase significantly. To optimize solvent usage, 1:30 was selected as the maximum solid: liquid ratio. When the concentration of the extract increased from 1.0 mol/L to 1.2 mol/L, the total triterpenoid content of I. obliquus decreased, and when the concentration of the extraction solution was too low, the ILs could not be fully utilized; hence, a concentration of the extraction solution range from 0.1 mol/L to 1.0 mol/L was selected. Furthermore, the total triterpenoid content of I. obliquus decreased slightly at 110 min and greatly after 120 and 150 min [24]. UAE was performed using a 750 W ultrasonic processor (BS-250; Kunshan Ultrasonic Instrument, Co., Ltd., Kunshan, China) at 80% amplitude [25] and 31 • C. Because ultrasonic waves increase the solvent temperature, ice coating was used to control the temperature during the extraction process. The extracts were centrifuged at 10,000× g for 5 min; then, 0.1 mL of the supernatant was evaporated until dryness in a water bath at 60 • C, and a post-treatment test was conducted as described below. All the samples were prepared and analyzed in triplicate [26].

HAE
Total triterpenoids were also extracted from I. obliquus via HAE, and the factor level was consistent with that of UAE. In our preliminary screening, the total triterpenoid yield gradually increased with increasing temperature; therefore, 70 • C was selected as the HAE temperature [27] and the extract treatment was the same as that used for UAE.  Table 4.
The total triterpenoid content in each experimental group was determined using the central composite design, and the experimental data for each group were fitted and analyzed. The experimental scheme and results are shown in Table 1. The same experimental conditions were repeated six times to verify the central point of the experiment, and the results were used to investigate the error of the model. Using Design-Expert 10.0 software (Stat-Ease, Minneapolis, MN, USA) for UAE and HAE statistical analyses, the effect surface map of the two factors in the model was drawn to each evaluation index. The model often reflected the change in the response value with a smaller error, had a significant impact, and better analyzed and predicted the extraction process, based on which optimal scheme was selected.

Statistical Analysis
Design-Expert 10.0 software was used to analyze the extraction conditions of triterpenoids from I. obliquus.

Determination of the Potential Inhibitory Activity of LDH
The potential LDH-inhibitory activity of I. obliquus was determined by UF-LC. The ultrafiltration process included culture, washing, and separation [28]. I. obliquus extract (50 mg) was dissolved in 50% MeOH (1 mL) and filtered using a 0.45-µm membrane. The filtrate (10 µL; 50 mg/mL), PBS (pH 7.4; 100 µL), and LDH (90 µL; 5, 10, and 20 U/mL) were mixed evenly. LDH was replaced with 90 µL PBS in the blank solution. All the four groups (blank, 5, 10, and 20 U/mL LDH) were incubated at 37 • C for 30 min. The mixture was then centrifuged at 10,000× g and 25 • C for 10 min with an ultrafiltration membrane (molecular weight cut-off, 100 kDa) to remove any unbound compounds. Thereafter, 100 µL MeOH: water mixture (50:50, v/v, pH 3.3) was added to the UF membrane and centrifuged at 10,000× g for 10 min (×3) to release the bound ligand [29]. The LDH inhibition rate was calculated using the equation (Aa−Ab)/Aa × 100%, where Aa and Ab are the absorbances of the blank and the experimental group, respectively [30]. Finally, the solvent was removed under vacuum, and the released ligand was used for further analyses.

High-Performance Liquid Chromatography-Diode Array Detector-Mass Spectrometry (HPLC-DAD-MS) Analysis
The HPLC-DAD-MS technique was used to analyze the I. obliquus extract. A Waters 2695 C 18 column (250 × 4.6 mm, 5 µm) was used with a MeOH (A) and water (B) mobile phase at a 0.5 mL/min flow rate. The elution procedure was as follows: 0-30 min, 24-56% solvent A; 30-35 min, 56-76% solvent A. Online ultraviolet spectra were detected at 300 nm. An LCQ Fleet ion-trap mass spectrometer was connected to the photo-diode array instrument by an ESI interface and used to perform the MS and MS n analyses. The mass spectrometer was operated in the positive ion mode, and the flow rate was 0.15 mL/min. The capillary voltage was set at −20 V in the positive ion mode. A full scan was performed using selected reaction monitoring mode-based identification at 4.5 kV spray voltage, 350 • C capillary temperature, and 10 psi pressure. A 100-1200 m/z scan range at a resolution of 17,500 was employed.

Separation of LDH Inhibitors by HSCCC
According to the partition coefficient (K) and the resolution of target compounds (compounds 1 and 2), the HSCCC two-phase solvent system was selected. The two-phase solvent system was thoroughly mixed at 25 • C; then, 10 mg of I. obliquus extract was added, and 2 mL of each of the upper and lower phases were collected. After the solvent was evaporated, the phases were analyzed using HPLC. Under the same operating conditions, the peak areas of the upper and lower phases were recorded as A u and A l , respectively. The K values of compounds 1 and 2 were calculated as K = A u /A l , and their resolution was the ratio of K 1 to K 2 .
For this study, the stationary phase was the upper phase, and the mobile phase was the lower phase. During each separation process, the coiled column was filled with the stationary phase. Following column rotation at 850 rpm for 30 min, the mobile phase was pumped into the column at a flow rate of 1.5 mL/min. When the hydrodynamic equilibrium was reached, the sample solution (100 mg) was injected. The detection wavelength was set at 300 nm, and the eluted compound was collected in the fraction collector. The solvents were then removed from the collected components using a rotary evaporator and analyzed. Following the elution of the required peaks (compounds 1 and 2), the program was stopped, and the stationary phases remaining on the column were collected. It should be noted that the retention rate of the stationary phase is equal to the volume of the stationary phase divided by the total volume of the chromatographic column.

Analytical Data for Compounds 1 and 2
The main features of compounds 1 and 2 are summarized in Table 5.

Conclusions
The total triterpenoids in I. obliquus were extracted in UAE and HAE modes using ILs, different anions, and dispersants.
[BMIM]Br with MeOH served as the extraction solution for subsequent analyses. The solid:liquid ratio, concentration of the extraction solution, and extraction time were optimized using the central composite design and verified. The concentration of the extraction solution was 1.0 mol/L, the solid:liquid ratio was 1:30, the extraction time was 83.64 min, and the temperature was 70 • C, in which the highest total triterpene content was obtained from I. obliquus using HAE. Potential LDH inhibitors in I. obliquus were screened via UF-LC-MS and identified as betulin and lanosterol, which were then separated via HSCCC within 120 min with purities of 95.9% and 97.8%, respectively. Thus, we demonstrated that IL-HAE could be applied for triterpenoid extraction from I. obliquus, and UF-LC-MS is a fast and feasible method for studying the potential medicinal value of these compounds.
Author Contributions: Conceptualization, data curation, and eriting-original draft preparation, Y.W.; validation and writing-review and editing, L.G.; funding acquisition, project administration, and writing-review and editing, C.L.; formal analysis, investigation, supervision, and visualization, Y.Z.; resources, software, and methodology, S.L. All authors have read and agreed to the published version of the manuscript.