Studies on Separation and Their Anti-Inflammatory Activity Evaluation of Lupane-Triterpenoid Aglycones from Leaves of Acanthopanax Gracilistylus W.W.Smith by HSCCC

Abstract

Objective

Lupane-type triterpenoid aglycones from Acanthopanax gracilistylus leaves (AGS) are underexplored compared to roots. This study aimed to establish an efficient isolation method and evaluate their anti-inflammatory potential.

Methods

Methanol extracts of AGS leaves were separated using high-speed counter-current chromatography (HSCCC) coupled with preparative HPLC. Six compounds were purified and structurally confirmed by NMR spectroscopy. Anti-inflammatory activity was assessed in LPS-stimulated RAW264.7 macrophages via nitric oxide (NO) production, with cytotoxicity measured by MTT assay.

Results

one lupane-triterpenoid and five Lupane-triterpenoid aglycones were obtained at purities of 94–97%. Cpds 6 and 7 sig-nificantly inhibited NO production by 57–64% at 20 µg/mL without affecting cell viability, whereas Cpds 1 and 2 were inactive, and Cpds 4 and 5 displayed a moderate impact. Additionally, Cpd 4 had moderate cytotoxicity. The integrated HSCCC-prep-HPLC strategy provided high-purity compounds efficiently with minimal solvent use.

Conclusion

This study presents an effective approach for isolating bioactive lupane-triterpenoid aglycones from AGS leaves. Cpds 6 and 7 demonstrate promising anti-inflammatory activity, supporting further pharmacological exploration.

Keywords

high-speed counter current chromatography acanthopanax gracilistylus prep-HPLC cytotoxicity nitric oxide

1. Introduction

Acanthopanax gracilistylus W.W. Smith (AGS) is a traditional medicinal plant widely used in Chinese medicine, with its dried root barks listed in the Chinese Pharmacopoeia (2025 edition) for the treatment of rheumatism, arthritis, and liver disorders.¹ In contrast, the leaves of AGS are abundant, renewable, and contain a rich array of potentially bioactive natural products, including triterpenoid saponins, lignans, and polyphenols.^2,3 However, systematic studies on AGS leaves remain limited, particularly regarding the isolation, structural characterization, and biological evaluation of Lupane-triterpenoid aglycones. This research gap highlights that AGS leaves are an underutilized resource and provide an opportunity to discover new bioactive compounds, promoting the sustainable and efficient use of this plant material.⁴

Conventional methods for natural product isolation, such as silica gel chromatog-raphy, Sephadex LH-20, or C-18 reversed-phase columns, often involve large solvent consumption, tedious multi-step procedures, low recovery, and potential sample loss.^5,6 High-speed counter-current chromatography (HSCCC), a liquid–liquid parti-tioning technique, overcomes these limitations by eliminating solid-phase adsorption and denaturation, offering operational simplicity, high recovery, and time efficiency.^7-9 To further enhance compound purity, HSCCC was coupled with preparative high-performance liquid chromatography (prep-HPLC) in this study, establishing an integrated, efficient, and continuous separation strategy suitable for systematic isolation and purification of Lupane-triterpenoid aglycones from AGS leaves.^10-12

Based on this background, the present study aimed to systematically isolate, purify, and structurally characterize Lupane-triterpenoid aglycones from AGS leaves using the combined HSCCC and prep-HPLC approach, followed by evaluation of their anti-inflammatory activity. Figure 1 depicts the structures of six Cpds: 1/2/4/5/6/7. This approach not only improves separation efficiency and yield but also provides a practical and reproducible strategy for the systematic isolation of bioactive compounds from other medicinal plants.

Figure 1.

The chemical structures of Cpds 1/2/4/5/6/7

2. Materials and Methods

2.1. Apparatus

Prep-HSCCC was performed through TBE-300A HSCCC (Shanghai Tauto Biotech Co., Ltd., Shanghai, China) equipped with three multilayer preparative series-connected coils (PTFE tube diameter = 1.6 mm, total volume = 300 mL:280 mL separation volume + 20 mL sample loop), with adjustable revolution speed of 0–1,000 rpm on an LC-20A liquid chromatograph (Shimadzu Technologies, Kyoto, Japan). Using the AKTA prime system (Amersham, USA), the aqueous two-phase system (HeMWat) was pumped into the column, monitoring the effluent via a UV absorbance detector alongside a DC0506 low constant temperature bath (Shanghai LNB Instrument Co, Ltd). For data collection, an N2000 chromatography workstation (Zhejiang University Star Information Technology Co, Ltd. Hangzhou, Zhejiang, China) and AVANCE III HD 600 nuclear magnetic resonance spectrometer (Bruker Co, Ltd. German) were deployed. Throughout the study, we utilized purified water (VE-2041-A Ultra-pure water system, Shenzhen Hongsen Environmental Protection Technology Co, Ltd. Shenzhen, China). RAW 264.7 cells were provided by Punosai Biotechnology Co., Ltd. (Wuhan, Hubei, China, Cat# CL-0190).

2.2. Reagents and Materials

In July 2020, the AGS leaves were collected in Yuanling, Hunan province of China, and validated by the corresponding author, Professor Xiang-Qian Liu. A voucher specimen was deposited in the Hunan University of Chinese Medicine Herbarium, Hunan, China (No. 20200718).

The utilized methanol and acetonitrile (Merck, Darmstadt, Germany) were of chromatographic grade, while the other organic solvents employed for HSCCC were of analytical grade (Tianjin Hengxing Chemical Preparation Co, Ltd., Tianjin, China).

2.3. Crude Extract Preparation

Briefly, 600 g dried AGS leaves were ground into powder and extracted using 3 L of methanol (65 °C). This extraction process was repeated three times using refluxing. After evaporating the solvents using a rotary evaporator, the residue (120 g) was dissolved in water and successively partitioned utilizing petroleum ether (PE, 60-90 °C), ethyl acetate, and n-butyl alcohol, producing PE (3.18 g), ethyl acetate (28.23 g), and n-butanol layers (25.04 g) after solvent removal in vacuo. The ethyl acetate extract was subjected to gradient elution with chloroform/methanol (35:1→10:1, v/v) on 700 g of silica gel (200-300 mesh), the eluents were combined, the solvent was evaporated, and the resulting ethyl acetate faction maintained in a refrigerator (4 °C) to conduct HSCCC and prep-HPLC separation.

2.4. Separation and Purification by HSCCC

2.4.1. HeMWat Selection

Choosing an appropriate HeMWat is crucial for achieving successful separation by HSCCC. The selection of HeMWat¹³ was based on the target Cpd’s partition coefficients (K-values) that were acquired and analyzed by the liquid-liquid extraction and HPLC analyses. These values ranged from 0.5 to 2.0, with a separation factor (α) between two Cpds > 1.5.

The target Cpds were examined using several volume ratios of n-hexane/ethyl acetate/methanol/water: 1:2:1:1, 1:1.8:1.4:1, 1:2:1.6:1, 1.4:2:1.7:1, and 2:2:1.8:1, respectively, based on their polarity.

The determination of K-values involved adding an appropriate amount of crude sample into pre-equilibrated HeMWat with a volume exceeding 5 mL for each phase. The sample was then vigorously shaken to ensure thorough equilibration between the two phases.¹⁴ Equal volumes of both upper and lower phases were evaporated until dry, and the resulting residues were subjected to dilution in 2.5 mL of methanol for HPLC analysis. The K-value of target Cpds was determined by the ratio of the peak areas obtained from the upper phase to that of the lower phase.

2.4.2. Preparation of HeMWat and Sample Solutions

For HSCCC separation, we utilized HeMWat comprising n-hexane/ethyl acetate/methanol/water)1:2:1.6:1, v/v/v/v) that was entirely equilibrated in a separation funnel at room temperature. Before usage, the upper and lower phases were exposed to separate degassing by 25 min sonication. To prepare the HSCCC sample for injection into the system, 200 mg of dried crude sample was dissolved in 20 mL of separated lower phase and filtered through a 0.45 μm membrane filter.

2.4.3. HSCCC and Prep-HPLC Separation

The HSCCC separation procedure involved filling the multilayer column completely with the upper phase at a 30 mL/min flow rate while maintaining a controlled temperature of 25 °C. The apparatus was then subjected to forward-direction rotation at 800 rpm. The lower phase was introduced into the column head at a 2.0 mL/min flow rate while maintaining a smooth revolution velocity. Upon reaching the hydrodynamic equilibrium, as evidenced by the mobile phase front appearance, an injection valve was used to inject 20 mL of the lower phase that contained 200 mg of the dried crude sample. A UV detector at 210 nm was deployed for constantly monitoring the effluents from the column tail end, and the resulting chromatogram was recorded. Each fraction (Fr) was collected on the basis of the chromatogram, followed by analysis by HPLC. All Frs with identical peaks were consolidated and then exposed to evaporation until complete dryness.

Prep-HPLC separation was conducted in the following manner: CST C-18 column (300 × 30 mm, 10 μm); acetonitrile/water (40:60, v/v) solvent system; pumped eluent at 20 mL/min (210 nm); 2 mL injection volume. Based on the elution profile, the peak Frs were gathered.

2.5. HPLC Analysis and Separated Cpd Identification

HPLC analyses of the HSCCC crude sample and each peak Fr were conducted using a Promosil C-18 column (250 × 4.6 mm, 5 μm) at a 30 °C column temperature, with an isocratically mobile phase consisting of acetonitrile/water/phosphoric acid (50:49.98:0.02, v/v/v) at a 1.0 mL/min flow rate. Before usage, all solvents were subjected to filtration through a 0.45 μm, and the samples were detected at 210 nm to draw elution curves. Utilizing NMR, separated Cpds were identified.

2.6. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide (MTT) Assay

RAW 264.7 viability was determined by analyzing MTT reduction to formazan. Cell culture was performed in the 96 well plates (1×10⁶ cells/well) for 4 h and incubated at 37 °C and 5% CO₂ for 24 h. The cell viability assessment involved adding 10 μL of MTT (5 mg/mL) to the medium, succeeded by a 4 h incubation. Afterward, we carefully eliminated the supernatant, dissolving the formazan crystals in 100 μL of dimethyl sulfoxide, allowing it to react for 10 min, and measuring absorbance at 570 nm.

2.7. Nitric Oxide (NO) Assay

RAW 264.7 (5×10⁴ cells/well in 96 well plates) were exposed to treatment with 10 μL of test samples and incubated at 37 °C and 5% CO₂ for 1 h. Subsequently, the cells were subjected to 10 μL of LPS (0.5 μg/mL) and incubated for 24 h, thereby indirectly determining NO productions by evaluating the stable NO catabolite nitrite in the medium through the Griess reaction. In brief, 100 μL of each supernatant was combined with an equal amount of Griess reagent and reacted at RT for 10 min, followed by determining the absorbance at 540 nm via an ELISA plate reader.

2.8. Statistical Analysis

All experiments were performed at least three times, and data are presented as mean ± standard error of the mean. Comparisons between two groups were conducted using Student’s t-test, whereas comparisons among three or more groups were performed using analysis of variance. A two-sided P value < 0.05 was considered statistically significant; P < 0.01 and P < 0.001 were considered more stringent significance thresholds. Statistical analyses and graphing were performed using GraphPad Prism 8.0 (San Diego, CA, USA).

3. Results

3.1. HPLC Analysis of the Crude Sample

HPLC chromatogram of AGS leave-derived crude sample showed Cpds 1–7 (Figure 2).

Figure 2.

HPLC chromatogram of the methanol-extract AGS leave-derived ethyl acetate extract crude sample. HPLC Settings: Promosil C-18 column (5 μm, 250×4.6 mm); acetonitrile/water/phosphoric acid (50:49.98:0.02, v/v/v) mobile phase, 1.0 mL/min flow rate; 30 °C column temperature; 210 nm detection wavelength

3.1.1. Selection of HSCCC HeMWat

Table 1 demonstrated that using a 1:2:1:1 ratio for the HeMWat led to excessively high K-values. As a consequence, the target Cpds were eluted in a broad peak with elongated elution time. The K-values were insufficient when a 2:2:1.8:1 ratio was employed, resulting in the inability to separate the target Cpds. A specific composition ratio of 1:1.8:1.4:1 was employed, but it resulted in large K-values for Cpd 6/7. This led to prolonged elution time and low resolution. To address this issue, a different ratio of 1.4:2:1.7:1 was examined. However, this resulted in small K-values for Cpds 1–3 and lower resolution (α< 1.5) for Cpds 6/7 at the same time. On the other hand, the 1:2:1.6:1 ratio yielded the appropriate K-values and separation factor (α) with less time and a relatively acceptable peak. Accordingly, we selected n-hexane/ethyl acetate/methanol/water at a 1:2:1.6:1 (v/v/v/v) volume ratio for HSCCC separation.

Table 1.

Partition Coefficient (K) of the Target Cpds in the Solvent System With Various Ratios

n-Hexane/Ethyl acetate/Methanol/Water (V/V/V/V)	K
n-Hexane/Ethyl acetate/Methanol/Water (V/V/V/V)	1	2	3	4	5	6	7
1:2:1:1	1.08	1.68	2.05	2.32	4.04	5.68	5.84
1:1.8:1.4:1	0.52	1.12	1.27	2.25	2.29	5.38	5.51
1:2:1.6:1	0.37	0.64	0.84	1.04	1.58	1.78	2.04
1.4:2:1.7:1	0.16	0.20	0.25	0.34	0.45	0.96	1.00
2:2:1.8:1	0.06	0.15	0.20	0.23	0.34	0.82	0.88

3.2. HSCCC Separation

On the basis of HPLC analysis as well as the elution curve of the prep-HSCCC, the obtained Frs were combined into distinct pooled Frs (Figure 3). Cpds 1/4/5 were obtained 10.5, 8.6, 9.8 mg with purities of 95.3%, 96.2%, and 97.4%, respectively, showing their HPLC chromatograms in Figure 5A,D and E.

Figure 3.

Prep-HSCCC separation of the methanol-extract AGS leave-derived ethyl acetate extract crude sample. Stationary phase: upper organic phase; mobile phase: lower aqueous phase: 2.0 mL/min flow rate; 800 rpm revolution speed; 25 °C column temperature; crude sample: 200 mg dissolved in 20 mL mixture solution of the lower phase of n-hexane/ethyl acetate/methanol/water (1:2:1.6:1, v/v/v/v) solvent system; 210 nm detector. Fr.1 = Cpd 1, Fr.2 = Cpds 2/3, Fr.3 = Cpd 4, Fr.4 = Cpd 5, Fr.5 = Cpds 6/7

3.3. Prep-HPLC Separation

After HSCCC separation, Frs 1–5 were concentrated, yielding Cpds 1/4/5 and two mixtures (Fr 2 containing Cpds 2/3, Fr 5 containing Cpds 6/7), respectively. Then, the two mixtures were separated by preparative-HPLC (Figure 4), yielding 3.5, 0.05, 15.4, and 4.4 mg of Cpds 2/3/6/7, with purities of 95.5%, 94.7%, 96.3%, and 96.2%, respectively, evidenced by HPLC analysis (Figure 5B,C, F and G).

Figure 4.

Prep-HPLC chromatogram HSCCC-Frs.2/5. Prep-HPLC setting: CST C-18 column (250 ×4.6 mm, 5 μm); acetonitrile/water (40:60, v/v) mobile phase) 20 mL/min flow rate; 210 nm. Fr.2-1 = Cpd 2, Fr.2-2 = Cpd 3, Fr.5-1 = Cpd 6, Fr.5-2 = Cpd 7

Figure 5.

HPLC chromatograms. A–G = Cpds 1–7, respectively. HPLC settings: Promosil C-18 column (10 μm, 30×300 mm); acetonitrile/water (40:60, v/v) mobile phase; 1.0 mL/min flow rate; 30 °C column temperature; 210 nm detection wavelength

3.4. Structural Configuration

The six separated Cpd chemical structures were identified based on their NMR data.

Cpd 1: ¹³C-NMR (125 MHz,pyrindine-d₅) δ: 179.14 (C-28), 151.38 (C-20), 110.49(C-29), 81.84 (C-3), 70.34 (C-11), 56.93(C-17), 56.22 (C-9), 51.0(C-5), 49.83 (C-18), 47.92 (C-19), 43.36 (C-14), 43.18 (C-8), 40.18 (C-10), 38.72 (C-12), 38.27 (C-4), 38.01 (C-13), 37.87 (C-22), 36.61 (C-1), 36.15 (C-7), 33.24 (C-16), 31.66 (C-21), 30.67 (C-15), 30.35 (C-23), 23.44 (C-24),22.26 (C-2), 20.05(C-30), 18.89 (C-6), 18.02 (C-26), 17.23 (C-25), 15.18 (C-27), 102.34 (C-1glc), 79.37(C-3glc), 78.72(C-5glc), 75.56(C-2glc), 72.61 (C-4glc), 63.63 (C-6glc). These ¹³C-NMR data aligned with the literature data¹⁵ comparable to 3-O-β-D-glucopyranosyl-3α,11α-dihydroxylup-20(29)-en-28-oic acid.

Cpd 2: ¹³C-NMR (100 MHz, CD₃COCD₃) δ: 178.16 (C-23), 177.84 (C-28), 151.89(C-20), 110.26 (C-29), 75.46 (C-1), 73.99(C-3), 56.98 (C-17), 52.98(C-9), 52.27 (C-4), 50.30 (C-18), 48.24 (C-19), 45.25 (C-5), 44.33 (C-10), 43.72 (C-14), 42.76 (C-8), 39.14 (C-13), 37.84 (C-22), 37.36 (C-2), 35.19 (C-7), 33.15 (C-16), 31.61 (C-21), 30.75 (C-15), 26.87 (C-12), 24.68 (C-11), 22.09 (C-6), 19.72(C-30), 17.92 (C-24), 17.31 (C-26), 15.40(C-27), 13.24 (C-25). These ¹³C-NMR data aligned with the literature data¹⁶ comparable to 1β,3α-dihydroxy-lup-20(29)-en-23,28-dioic acid.

Cpd 4: ¹³C-NMR (125MHz, pyrindine-d₅) δ: 179.24 (C-28), 151.25 (C-20), 110.47 (C-29), 76.32 (C-3), 72.32 (C-23), 70.30 (C-11), 56.94 (C-17), 56.62 (C-9), 49.78 (C-18), 47.92 (C-19), 44.27 (C-5), 43.25 (C-14), 43.13 (C-8), 41.57 (C-4), 40.03 (C-10), 38.79 (C-12), 38.04 (C-13), 37.80 (C-22), 36.34 (C-1), 36.00 (C-7), 33.24 (C-16), 31.63 (C-21), 30.54 (C-15), 27.55 (C-2), 19.90 (C-30), 18.76 (C-24), 18.68 (C-6), 18.12 (C-26), 17.43 (C-25), 15.18 (C-27). These ¹³C-NMR data followed the literature data¹⁵ comparable to 3α,11α,23-trihydroxy- lup-20(29)-en-28-oic acid.

Cpd 5: ¹³C-NMR (125MHz,pyrindine-d₅) δ: 210.50 (C-23), 179.24 (C-28), 151.25 (C-20), 110.50 (C-29), 73.48 (C-3), 70.19 (C-11), 56.94 (C-17), 56.36 (C-9), 53.40 (C-4), 49.78 (C-18), 47.92 (C-19), 44.65 (C-5), 43.69 (C-14), 43.24 (C-8), 39.43 (C-10), 38.70 (C-12), 37.97 (C-13), 37.80 (C-22), 35.95 (C-1), 35.80 (C-7), 33.24 (C-16), 31.64 (C-21), 30.50 (C-15), 27.59 (C-2), 21.74 (C-6), 19.92 (C-30), 18.18 (C-26), 17.17 (C-25), 15.35 (C-24), 15.18 (C-27). These ¹³C-NMR data agreed with the literature data¹⁵ comparable to 3α,11α-dihydroxy-23-oxo-lup-20(29)-en-28-oic acid.

Cpd 6: ¹³C-NMR (125MHz, pyrindine-d₅) δ: 179.23 (C-28), 151.24 (C-20), 110.47 (C-29), 75.59 (C-3), 70.25 (C-11), 56.94 (C-17), 56.54 (C-9), 49.93 (C-5), 49.79 (C-18), 47.90 (C-19), 43.30 (C-14), 43.14 (C-8), 40.23 (C-10), 38.90 (C-4), 38.78 (C-12), 38.03 (C-13), 37.81 (C-22), 36.61 (C-1), 36.31 (C-7), 33.26 (C-16), 31.63 (C-21), 30.53 (C-15), 30.27 (C-23), 27.35 (C-2), 23.30 (C-24), 19.91 (C-30), 18.92 (C-6), 18.10 (C-26), 17.19 (C-25), 15.14 (C-27). These ¹³C-NMR data aligned with the literature data¹⁵ comparable to impressic acid.

Cpd 7: ¹³C-NMR (125 MHz, pyrindine-d₅): 179.72 (C-23), 179.21 (C-28), 151.37 (C-20), 110.34 (C-29) 73.18 (C-3), 56.91 (C-17), 52.34 (C-4), 51.34 (C-9), 50.0 (C-18), 48.15 (C-19), 45.45 (C-5), 43.33 (C-14), 42.21 (C-8), 38.97 (C-13), 37.93 (C-22), 37.75 (C-10), 35.14 (C-7), 33.35 (C-1), 33.15 (C-16), 31.54 (C-21), 30.44 (C-15), 26.58 (C-2), 26.41 (C-12), 22.23 (C-11), 21.4 (C-6), 19.81 (C-30), 18.35 (C-24), 17.24 (C-26), 17.14 (C-25), 15.12 (C-27). These ¹³C-NMR data followed the literature data¹⁵ comparable to acankoreagenin.

3.5. Cytotoxicity and NO Inhibition

The NO production was weakly down-regulated with 20 μg/mL of Cpds 6/7 with powerful inhibition at 57.48% and 64.11%, respectively (Figures 6 and 7). Cpds 1/2 had no inhibitory effect on NO production. Meanwhile, Cpds 4/5 had a moderate inhibitory effect on NO production. Cpd 4 showed moderate cytotoxicity at 40 μg/mL with 20.72% cell viability. Cpds 1/2/5/6/7 did not influence cell viability.

Figure 6.

Effect of different Cpd 1/2/4/5/6/7 concentrations on RAW 264. 7 cell activity (n = 3, ‾x±s). **P<0.01 vs. CON

Figure 7.

Impact of different Cpd 1/2/4/5/6/7 concentrations on NO synthesis from LPS-provoked RAW 264. 7 (n = 3, ‾x±s) ^## P < 0.01 vs. CON; **P < 0.01 vs. LPS

4. Discussion

In this study, an integrated HSCCC and preparative HPLC strategy was successfully applied to isolate one lupane-triterpenoid and five Lupane-triterpenoid aglycones from the leaves of AGS. Compared to previous reports using similar HSCCC protocols for natural product separation, the present method provided comparable or higher yields and purities, highlighting the efficiency of combining HSCCC with prep-HPLC for complex plant matrices. The high purities (94–97%) achieved in a single workflow demonstrate the practicality of this approach for the preparative isolation of bioactive compounds.

The biological evaluation revealed notable differences in anti-inflammatory activity among the isolated compounds. Cpds 6 and 7 significantly inhibited NO production in LPS-stimulated RAW264.7 macrophages without cytotoxic effects, whereas Cpds 1 and 2 showed negligible activity, and Cpds 4 and 5 displayed a moderate impact. Additionally, Cpd 4 had moderate cytotoxicity. These differences may be attributed to structural variations, such as the presence or absence of hydroxyl or carbonyl groups, suggesting a structure–activity relationship among the Lupane-triterpenoid Aglycones.

Several limitations of the current study should be acknowledged. The extremely low yield of Cpd 3 prevented structural elucidation and biological testing. Additionally, only an in vitro macrophage model was employed, without in vivo validation, and initial experiments lacked a standard positive control for comparative purposes. Despite these limitations, the results demonstrate that the HSCCC-prep-HPLC approach is an efficient, time-saving, and high-purity method for isolating natural products. This strategy can be applied to other plant-derived compounds, facilitating subsequent bioactivity studies and the discovery of potential therapeutic agents.

5. Conclusion

One lupane-triterpenoid and five Lupane-triterpenoid aglycones were successfully isolated from Acanthopanax gracilistylus leaves. Cpds 6 and 7 exhibited significant anti-inflammatory activity with-out cytotoxicity. The integrated HSCCC and prep-HPLC method proved efficient and may be applied to the isolation of other natural products.

Footnotes

Acknowledgement

The authors gratefully acknowledge the technical assistance provided by the Analytical Testing Center of Hunan University of Chinese Medicine.

ORCID iD

Mao-Fang Lu

Ethical Considerations

This study did not involve human participants or live animals. The RAW264.7 cell line was commercially obtained (Punosai Biotechnology Co., Ltd. Wuhan, Hubei, China, Cat# CL-0190), and therefore Institutional Review Board approval was not required.

Author Contributions

Investigation, writing—original draft preparation, review and editing, M.L. (Mao-Fang Lu); investigation, visualization and validation, M. L. (Man-Xia Lu); methodology and data curation, X.W.; software, data curation and validation, G.L. and L.L.; methodology, software and data curation, X.T. and C.Y.; conceptualization, project administration, methodology, funding acquisition and writing—review and editing, X.L. All authors have read and agreed to the published version of the manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Funded by National Natural Science Foundation of China (81904193), Natural Science Foundation of Hunan Province of China (2024JJ7357 and 2023JJ60213), Scientific Research Program of Hunan Provincial Education Department (23C0155), The Key Discipline of Biological Engineering in the Fourteenth Five-year Plan of Hunan University of Chinese Medicine [XiaoXingFaGuiZi-2023-No.2], The Opening Project for the first-class disciplines of pharmacy in Hunan Province (2020YX12), The Key Discipline of Pharmacy in Hunan Province

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

Data available on request from the authors.*

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