ABSTRACT
A new labdane‐type diterpenoid, Nudiloid Z (1), together with twelve known compounds (2‐13), were isolated and purified from the stems and leaves of Callicarpa nudiflora. Compound 1 possesses a three‐membered epoxide ring at C‐12 and C‐13. The planar structure and absolute configuration of 1 were elucidated by comprehensive spectroscopic analyses. The inhibitory effects of compounds 1‐3 and 8 on lactate dehydrogenase (LDH) release in J774A.1 cell was evaluated. The results indicated that compound 1 exhibited significant anti‐inflammatory activity, with an inhibitory concentration 50% (IC50) value of 3.30 ± 0.47 µM on LDH release and reduced the Interleukin 1β (IL‐1β) levels in a dose‐dependent manner.
Keywords: Callicarpa nudiflora , labdane diterpenoids, sesquiterpenoids, anti‐inflammatory
Nudiloid Z (1), a 3,4‐seco‐labdane‐type diterpenoid, was isolated from Callicarpa nudiflora, which showed significant anti‐inflammatory activity via inhibiting the release of LDH and IL‐1β in J774A.1 cells, with an IC50 value of 3.30 ± 0.47 µM.
1. Introduction
The global incidence of inflammation‐related diseases continues to rise, driving anti‐inflammatory drug research and development to become a priority in modern medicine. According to World Health Organization (WHO) statistics, chronic inflammation, such as rheumatoid arthritis, affects over 10% of the global population, while long‐term use of traditional nonsteroidal anti‐inflammatory drugs (NSAIDs) is prone to side effects such as gastrointestinal damage and cardiovascular risks [1]. Against this backdrop, natural products with multi‐target regulations and high safety profiles have regained attention. In the past decade, 38% of the new anti‐inflammatory drugs approved by the Food and Drug Administration (FDA) originated from natural products [2], highlighting their research value. Natural products exhibit significant potential in the anti‐inflammatory field due to their structural diversity and unique biological activity. Compounds such as flavonoids, terpenoids, and alkaloids derived from plants and microorganisms have been shown to inhibit the release of inflammatory mediators by targeting key nodes in inflammatory signaling pathways [3, 4]. A growing body of in vitro, in vivo, and clinical studies continues to reveal their anti‐inflammatory effects, providing lead compounds for new drug development. The Lingnan‐specific medicinal plant Callicarpa nudiflora, listed in the Chinese Materia Medica, has the effects of clearing heat, detoxifying, and stopping bleeding. It primarily contains flavonoids [5], phenylpropanoids, and terpenoids [6]. Previous studies have confirmed its hemostatic, antibacterial, and antiviral activities, and it has been developed into various formulations for hemorrhagic and infectious diseases. In recent years, its anti‐inflammatory effects have garnered increasing attention. Previously, research discovered that new diterpenoids such as nudifloids and cardorubellas in Callicarpa plants exhibit anti‐inflammatory and antitumor activities [7, 8]. Through bioactivity‐guided fractionation (using LDH release inhibition rate as an indicator), the ethyl acetate (EtOAc) fraction of the 95% ethanol extract of C. nudiflora was identified as having significant anti‐inflammatory potential [9]. Previously, our research group has reported seco‐labdane diterpenoids from this fraction that exhibited potential anti‐inflammatory activity [25]. Therefore, this study further analyzed the chemical composition of this active fraction to identify more anti‐inflammatory diterpenoid compounds. In this report, we describe the isolation and structural elucidation of one new diterpenoid possessing a 3,4‐seco‐labdane‐type skeleton (1) together with twelve known compounds (2–13) (Figure 1) from the stems and leaves of C. nudiflora. Previous reports have shown that some of the known compounds have been evaluated for their anti‐inflammatory properties. Among the reported compounds in this series, only compound 6 has been documented to possess anti‐inflammatory activity [10, 11, 12, 13, 14]. To further expand the anti‐inflammatory profile of monomeric compounds from C. nudiflora, four compounds (1, 2, 3, and 8) were selected for preliminary screening based on the availability of the samples.
FIGURE 1.
The chemical structures of compounds 1–13.
2. Results and Discussion
2.1. Structure Elucidation
Compound 1 was isolated as a colorless oil, with specific rotation of +8.95 (c 0.15, CH3OH) (Figure S9). Its HR‐ESI‐MS detected an ion peak at m/z 369.2036 [M + Na] + (Figure S10), establishing the molecular formula C21H30O4, corresponding to six unsaturations. The UV spectrum (Figure S7) exhibited absorption maxima at 202 nm, and the IR spectrum showed characteristic absorption bands for methyl and methylene groups at 2872, 2936 cm−1, the ester group at 1175, 1733 cm−1, and the terminal olefin group at 894, 990 cm−1 (Figure S8). The 1H and 13C NMR data (Figures S1–S3 and Table S1) showed the presence of eight methylene groups (δ C 23.5 CH2‐11, 27.7 CH2‐2, 30.0 CH2‐6, 32.6 CH2‐1, 37.5 CH2‐7, 108.1 CH2‐17, 114.1 CH2‐18, 117.5 CH2‐15), five methines (δ C 46.9 CH‐9, 50.6 CH‐5, 68.7 CH‐12, 130.0 CH‐14, 199.0 CH‐16), five quaternary carbons (δ C 41.3 C‐10, 65.7 C‐13, 146.8 C‐4 C‐8, 174.4 C‐3), two methyl groups (δ C 17.6 CH3‐20, 23.6 CH3‐19) and one methoxy group (δ C 51.9 OCH3‐3). The planar structure of 1 was determined using 2D NMR experiments. The 1H–1H COSY spectrum exhibited the spin system of H2‐1/H2‐2, H‐5/H2‐6/H2‐7, H‐9/H2‐11, and H‐14/ H2‐15 (Figures 2 and S5). The HMBCs (Figures 2 and S4) showed that H2‐1 and H2‐2 are linked to C‐3, H3‐19 is linked to C‐4, C‐5, and C‐18, and H3‐20 is linked to C‐1, C‐5, C‐9, and C‐10. Additional HMBC correlations were observed from H‐12 to C‐13, showing that C‐12 and C‐13 are linked by an oxygen bridge, and correlations from H‐15 and H‐16 to C‐13 confirmed that the terminal double bonds at C‐15 and the aldehyde group at C‐16 are linked to C‐13. The 2D data confirmed that compound 1 possesses a 3,4‐seco‐labdane‐type skeleton. Thus, the planar structure of compound 1 was confirmed.
FIGURE 2.
Key 2D correlations of compound 1.
The NOESY NMR experiment (Figures 2 and S6) together with the data of compound 2 [9] showed that the proton spatial arrangement patterns of the chiral centers C‐5 (δ C 50.6), C‐9 (δ C 46.9), and C‐10 (δ C 41.3) are S configurations. The NOESY spectrum observed the cross peak of H‐9/H‐12, revealing the S configuration of C‐12 (δ C 68.7). However, due to the presence of a free rotational energy barrier, the stereochemical assignment of the side chain at C‐13 (δ C 65.7) was difficult to resolve using the NOESY experiment. To address this challenge, two possible relative configurations, 13R and 13S (Figure S11), were proposed. To ascertain the correct one, the candidate isomers (Figure S12) were evaluated through a combination of NMR calculation and DP4+ probability analysis. The relative configuration was unambiguously assigned through linear regression analysis of the experimental and computed shifts, which exhibited an excellent correlation for the 13R isomer (R 2 = 0.9976) (Figure 3). Notably, for oxirane ring systems bearing a quaternary carbon, uncertainty may arise when analyzing carbon data alone (whether scaled or unscaled), which is a well‐documented characteristic of DP4+ calculations for this type of structure. The advantage of the DP4+ method lies in its integration of all available data (1H and 1 3C, scaled and unscaled), enabling a “constructive compensation of errors” that leads to a reliable configurational assignment. [15] Accordingly, DP4+ analysis was performed using the full dataset, which afforded a 100% probability for the 13R isomer (isomer 1), while the alternative 13S isomer received 0% (Figure 4 and Tables S1–S3). This unequivocally confirms the 13R configuration for compound 1. Finally, the absolute configuration 13R was confirmed by the comparison of calculated and experimental ECD data (Figure 5). Thus, the chemical structure of 1 was identified and named Nudiloid Z.
FIGURE 3.
Linear relationship between calculated and experimental values for 13C NMR of compound 1.
FIGURE 4.
DP4+ probability analysis of compound 1.
FIGURE 5.
Experimental and calculated ECD curves of compound 1.
The known compounds were identified as Nudifloid H (2) [9], Nudiflopene E (3) [16], Callnudoid F (4) [10], Callinudin E (5) [17], syn‐3, 4‐seco‐12S‐hydroxy‐15, 16‐epoxy‐4 (18), 8 (17), 13 (16), 14 (15)‐labdatetraen‐3‐oic acid (6) [18], Nudiflorawu B (7) [6], (−)‐clovane‐2,9‐diol (8) [19], Paulownin (9) [20], 2α,3β,19α‐Trihydroxy‐olean‐12‐en‐17‐oic acid (Arjunic acid) (10) [21], Euscaphic acid (11) [22], Tormentic acid (12) [23], Serrulatin B (13) [24] by comparing their spectral data (Figures S13–36) with those reported in the literature.
2.2. Biological Activity
Compounds 1‐ 3, 8 were evaluated for their anti‐inflammatory effects using the LDH release assay. Among these compounds, compound 1 demonstrated significant anti‐inflammatory activity, as evidenced by its capacity to inhibit LDH release by over 50% in LDH release assays (Figure 6A). Quantitative analysis of compound 1 revealed an IC50 value of 3.30 ± 0.47 µM (Figure 6B), indicating its potent bioactivity.
FIGURE 6.
In vitro NLRP3 inflammatory inhibitory effects of compounds 1–3, 8. (A) Initial screening assessed the impact of compounds 1–3, 8 on LDH (lactate dehydrogenase) release (n = 3). Andrographolide (Andro., 15 µM, 12.1 ± 1.5 µg/mL) served as a positive control. Compounds demonstrating reduced LDH release are highlighted with a red arrow. (B) The IC50 values were calculated for compound 1, which displayed significant anti‐inflammatory activity in J774A.1 cells (n = 3). Compared to the DMSO control group, statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001. n for sample size, n = 3 for average of three experiments, p for probability value, LDH stands for lactate dehydrogenase, and LPS for lipopolysaccharide.
Subsequently, J774A.1 cells underwent pretreatment with LPS and Nig., and were treated with compound 1 at concentrations spanning from 1 to 10 µM to explore its effect on cellular pyroptosis. Cell viability was evaluated by measuring PI permeability, with cell death quantified as the proportion of PI‐positive cells (stained red) relative to the entire J774A.1 cell population (stained blue). As illustrated in Figure 7, compound 1 exhibited a dose‐dependent inhibitory effect on pyroptosis triggered by LPS and Nig. To further elucidate the mechanism of action of compound 1, J774A.1 cells were pretreated with LPS and Nig., followed by treatment with various concentrations of compound 1. The IL‐1β released into the supernatant was quantified using an enzyme‐linked immunosorbent assay (ELISA). The results demonstrated that compound 1 concentration‐dependently inhibited IL‐1β release, as illustrated in Figure 8.
FIGURE 7.
Inhibition of J774A.1 macrophage pyroptosis by compound 1. (A) Visualization of dead cells stained with propidium iodide (PI, red) and nuclei counterstained with Hoechst 33342 (blue). The scale bar denotes 50 µm. (B) Quantification of the proportion of PI‐positive cells (red) among the total cell population (blue). The data presented are the mean values obtained from three independent replicates. Compared to the dimethyl sulfoxide (DMSO) control group, statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001. LPS, lipopolysaccharide; Nig., nigericin.
FIGURE 8.
The inhibition of compound 1 on IL‐1β released by J774A.1 cell apoptosis (n = 3).
3. Conclusion
In summary, a new diterpenoid (1) was isolated from C. nudiflora, along with twelve known compounds (2–13). The structures of all the isolated compounds were determined based on NMR data analysis and ECD calculation. The evaluation of inhibitory effects of the isolated compounds (1–3, 8) on NLRP3 inflammasome activation was performed, which showed promising IC50 values for compound 1.
4. Experimental
4.1. General Experimental Procedures
NMR spectra were recorded on the Bruker AV‐III‐600 or Bruker AV‐III‐400 spectrometer in CDCl3 (Bruker, Zurich, Switzerland). Optical rotations (OR) were measured in methanol using a JASCO P‐2000 polarimeter (Horiba, Japan). Circular dichroism (CD) spectra were recorded using an Applied Photophysics V100 Chirascan spectropolarimeter (Applied Photophysics, London, UK). Infrared (IR) spectra were obtained via a Thermo Scientific Nicolet IS10 FT‐IR spectrometer (Thermo Fisher Scientific, Massachusetts, USA) using KBr pellets. High‐resolution electrospray ionization mass spectrometry (HRESIMS) data were collected on an Agilent 1100 HPLC‐TOF mass spectrometer (Agilent Technologies, USA). Column chromatography (CC) was carried out using silica gel (80–100 and 200–300 mesh; Qingdao Marine Chemical Inc., China), Sephadex LH‐20 (GE Healthcare Bio‐Sciences AB, Uppsala, Sweden), Middle chromatogram isolated reversed‐phase gel (MCI gel) (10‐35 µm; Mitsubishi Chemical Group), and D‐101 macroporous resin (Tianjin Haoju Science and Technology Co., Ltd., China). Semi‐preparative HPLC was performed on an Agilent 1260 system equipped with a diode array detector (DAD) and a Zorbax SB‐C18 column (9.4 × 250 mm). Preparative HPLC was conducted using a system from Jiangsu Hanbon Science & Technology Co., Ltd., equipped with a dual‐wavelength UV detector and a Megres C18 column (30 × 250 mm), at a flow rate of 3 mL/min with detection at 210 nm. Thin‐layer chromatography (TLC) was performed using pre‐coated plates (Qingdao Marine Chemical Inc., China). Compounds were visualized under UV light at 254 nm and by spraying with 10% H2SO4 in ethanol, followed by heating.
4.2. Plant Material
Stems and leaves of C. nudiflora were collected in Shangsi County, Fangchenggang City, Guangxi Province, China (August, 2021). Taxonomic authentication was performed by Mr. Zhang Honglin of Kunming GenPHYTech Co., Ltd. A voucher specimen (No. 00967907) is deposited in the herbarium of the Key Laboratory of Medicinal Chemistry for Natural Resources, Ministry of Education, Yunnan University, Kunming, China.
4.3. Extraction and Isolation
The air‐dried stems and leaves of C. nudiflora were extracted with 90% EtOH under reflux. The concentrated extract was partitioned with EtOAc, followed by sequential chromatography on D‐101 macroporous resin (MeOH/H2O gradient) and silica gel column (CH2Cl2/MeOH gradient) to afford several fractions. The process for the crude extraction of C. nudiflora can be found in the relevant literature [25]. This article describes the subsequent extraction and isolation procedures. Fr.B.1 (47 g) was first subjected to preliminary separation using an MCI column with a gradient elution of methanol/water (50:50–100:0, v/v), yielding 12 fractions labeled Fr.B.1.1 to Fr.B.1.12. Among these, Fr.B.1.7 and Fr.B.1.8 were further processed using a Sephadex LH‐20 gel column (MeOH) to enrich terpenoids and flavonoids, resulting in terpenoid fractions Fr.B.1.7.1‐Fr.B.1.7.5 and Fr.B.1.8.1‐Fr.B.1.8.3, as well as flavonoid fractions Fr.B.1.7.6 and Fr.B.1.8.4.
Fraction Fr.B.1.8.1 was separated using a 200–300 mesh silica gel column with a petroleum ether/acetone (50:1–5:1, v/v) elution system. The separation process was monitored in real‐time by thin‐layer chromatography (TLC), ultimately yielding eight characteristic subfractions (Fr.B.1.8.1.1‐8). Among these, subfraction Fr.B.1.8.1.1 showed major staining spots on TLC and was further purified by normal‐phase silica gel column chromatography (elution system: petroleum ether/ethyl acetate = 12:1, v/v), successfully isolating compound 3 (138 mg). Subfraction Fr.B.1.8.1.8 was subjected to normal‐phase silica gel column chromatography eluted with dichloromethane/methanol (200:1, v/v), yielding six secondary subfractions. From Fr.B.1.8.1.8.5, compound 7 (3.5 mg, t R = 15.1 min) was obtained using semi‐preparative HPLC (3.0 mL/min, MeCN/H2O, 75:25, v/v).
Fr.B.1.7.1 (1.56 g) was first separated by normal‐phase silica gel column chromatography using a petroleum ether/acetone (50:1–5:1, v/v) elution system. Based on TLC analysis, similar components were combined, resulting in fractions Fr.B.1.7.1 to Fr.B.1.7.10. Fr.B.1.7.7 was purified by semi‐preparative HPLC with a mobile phase of 72% MeCN, yielding compound 1 (20.0 mg, t R = 17.5 min). Fr.B.1.7.2 was isolated using normal‐phase silica gel column chromatography using a petroleum ether/EtOAc (50:1–5:1, v/v) elution system to obtain compound 8 (56.0 mg), and was further purified by semi‐preparative HPLC using 64% MeCN as the mobile phase, yielding compound 2 (2.6 mg, t R = 13.5 min), compound 5 (3.8 mg, t R = 18.9 min), and compound 6 (2.6 mg, t R = 23.5 min).
Fr.B.1.7.5 (800 mg) showed good spots on TLC, mostly blue, which had not been observed in previous separation processes. Therefore, this fraction was selected for further study. After removal of impurities and segmentation using normal‐phase silica gel column chromatography with a petroleum ether/acetone (50:1–5:1, v/v) elution system, Fr.B.1.7.5 was divided into Fr.B.1.7.5.1 to Fr.B.1.7.5.4. Through repeated normal‐phase silica gel column chromatography, Fr.B.1.7.5.2 and Fr.B.1.7.5.3 yielded three terpenoid compounds: compound 10 (3.8 mg), compound 11 (13.3 mg), and compound 12 (18.6 mg). Fr.B.1.7.5.1 was processed using semi‐preparative HPLC with a mobile phase of 78% MeCN, yielding compound 13 (3.6 mg). Fr.B.1.7.6, the flavonoid fraction, was purified by semi‐preparative HPLC using 43% MeCN as the mobile phase, yielding compound 9 (7.2 mg, t R = 16.2 min). Fraction Fr.B.1.6.2 was first enriched for terpenoids using a Sephadex LH‐20 dextran gel column (methanol system), yielding a target fraction of 970 mg. This fraction was then systematically separated into six subfractions (Fr.B.1.6.2.1‐6) using a preparative HPLC system equipped with a dual‐wavelength UV detector (λ = 210/230 nm) and eluted with 85% acetonitrile in water. Subfraction Fr.B.1.6.2.5 showed a single major spot on TLC staining and was further purified by semi‐preparative HPLC using 60% acetonitrile in water (flow rate: 3.0 mL/min). High‐purity monomeric compound 4 (38.0 mg) was successfully isolated at a retention time of 16.0 min.
4.4. Characterization Data of Compound 1
Nudiloid Z (1): Colorless oil; C21H30O4, +8.95 (c 0.15, CH3OH); UV (CH3OH) λ max (log ε): 202 (0.58) nm; CD (CH3OH) λ max (Δε): 210 (−3.511), 226 (−0.538), 228 (−0.472) nm; IR ν max: 2936, 2872, 1733, 1175, 990, 894 cm−1; 1H NMR (400 MHz, CDCl3), and 13C NMR (100 MHz, CDCl3): see Table 1; HR‐ESI‐MS m/z: 369.2036 [M + Na] + (calcd for C21H30O4Na, 369.2040).
TABLE 1.
1H (400 MHz) and 13C (100 MHz) NMR data of compound 1 in CDCl3.
| NO. | δ H, mult. (J in Hz) | δ C |
|---|---|---|
| 1 | 1.77, m | 32.6, CH2 |
| 1.53, m2 | ||
| 2 | 2.49 a , m | 27.7, CH2 |
| 2.46 a , m | ||
| 3 | — | 174.4, C |
| 4 | — | 146.8, C |
| 5 | 2.28, m | 50.6, CH |
| 6 | 1.71, m | 30.0, CH2 |
| 1.59, m | ||
| 7 | 2.39, m | 37.5, CH2 |
| 2.08, m | ||
| 8 | — | 146.8, C |
| 9 | 2.24, d (11.9) | 46.9, CH |
| 10 | — | 41.3, C |
| 11 | 1.99, m | 23.5, CH2 |
| 1.56, m | ||
| 12 | 3.11, dd (9.1, 2.3) | 68.7, CH |
| 13 | — | 65.7, C |
| 14 | 6.26, dd (17.3, 10.9) | 130.0, CH |
| 15 | 5.36, dd (17.3, 1.1) | 117.5, CH2 |
| 5.25, d (10.9) | ||
| 16 | 9.53, s | 199.0, CH |
| 17 | 4.94, s | 108.1, CH2 |
| 4.53, s | ||
| 18 | 4.88, s | 114.1, CH2 |
| 4.70, s | ||
| 19 | 1.73, s | 23.6, CH3 |
| 20 | 0.68, s | 17.6, CH3 |
| 3‐OCH3 | 3.67, s | 51.9, CH3 |
aoverlap.
4.5. ECD Calculations
Conformational searches were originally performed using the Spartan'14 program. The main conformation was then optimized with density functional theory (DFT) calculations at the B3LYP/6‐31+G (d,p) level by Gaussian 09 [26]. The NMR calculations were performed at the mPW1PW91/6‐31+G (d,p) levels in chloroform by the GIAO method with the PCM model [27]. ECD theoretical calculations of compound 1 were performed using time‐dependent density functional theory (TDDFT) at the B3LYP/6‐31+G (d,p) level in the CH3OH and PCM model [28]. The Spec Dis program was used to compare the calculated curves with the experimental ECD spectra.
4.6. Anti‐Inflammatory Activity
4.6.1. Cell Culture and Stimulation
The J774A.1 cell line (IZSLER Cat# BS TCL 83, RRID:CVCL_0358) was obtained from the Kunming Institute of Zoology. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat‐inactivated fetal bovine serum (FBS). Culture was carried out in T‐75 flasks under a controlled atmosphere (5% CO2, 37°C). Subculturing was performed at 80%–90% confluency. For experimental treatments, cells were seeded in 24‐well plates at a density of 2 × 106 cells per well and incubated overnight. After aspiration of the medium, cells were stimulated with 200 ng/mL LPS for 3 h. The medium was then replaced with Ultra‐Fectin solution containing 1–10 µM of compound 1 for 30 min. To activate the NLRP3 inflammasome, cells were further treated with 10 µM nigericin for 1 h [29].
4.6.2. Lactate Dehydrogenase (LDH) Release Assay
NLRP3 inflammasome activation was quantified by measuring LDH release into the extracellular medium. Supernatants from treated cells were transferred to a microplate, and LDH detection reagent was added according to the manufacturer's protocol. After incubation, absorbance was measured at 492 nm with a reference wavelength of 630 nm.
4.6.3. Cell Membrane Permeability Assay
Following stimulation with LPS and nigericin, cells were stained with propidium iodide (PI; 3 µg/mL) and Hoechst 33,342 (0.5 µg/mL) for 10 min at room temperature. Images were acquired immediately using an inverted fluorescence microscope (Axio Observer 3, Zeiss) [30]. Three random fields were captured per sample, and the ratio of PI‐positive cells (indicating loss of membrane integrity) to total cells (Hoechst‐positive) was calculated.
4.6.4. Enzyme‐Linked Immunosorbent Assay (ELISA)
The target protein was detected using a double‐antibody sandwich ELISA: Capture antibody was diluted in PBS and incubated overnight at room temperature, followed by three washes. Samples and standards were diluted accordingly and incubated for 2 h at 37°C. After washing, detection antibody was added and incubated for 1 h at 37°C. Following another wash, HRP‐Streptavidin was added and incubated for 40 min at 37°C. After washing, TMB substrate solution was added and incubated for 20 min in the dark, followed by the addition of stop solution. Absorbance was measured at 450 nm (with background correction at 630 nm). Sample concentrations were calculated using a standard curve.
4.6.5. Statistical Analysis
All results are presented as mean ± standard deviation (SD) values. Statistical analysis was performed using GraphPad Prism 10.0 (GraphPad software). Significant differences between more than two groups were assessed by one‐way ANOVA followed by Tukey's post hoc test. p < 0.05 was considered statistically significant.
Author Contributions
Qi Li: investigation, methodology, writing – original draft. Bo Li: conceptualization, investigation, methodology, writing – original draft. Yan‐Ling Tang: conceptualization, investigation, methodology, writing – original draft. Xue‐Ting Ji: methodology. Muhammad Aurang Zeb: investigation, methodology. Yan‐Zi Ma: methodology. Yuan‐Lin Kong: methodology. Zhong‐Dan Liang: methodology. Xiao‐Li Li: conceptualization, funding acquisition, project administration, supervision. Xing‐Jie Zhang: conceptualization, project administration, supervision. Wei‐Lie Xiao: conceptualization, funding acquisition, project administration, supervision.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Supporting File 1: cbdv71515‐sup‐0001‐SuppMat.docx
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (22477108 U24A20805, and 22467022), Applied Basic Research Foundation of Yunnan Province (202501BC070005, 202603AD140002 and 202401AT070444), Project of Yunnan Characteristic Plant Screening and R&D Service CXO Platform (2022YKZY001), The Practical Innovation Project of Postgraduate Students in the Professional Degree of Yunnan University (ZC‐252513838). Sharing platform of phytochemistry R&D, KIB. The authors would like to express their appreciation to the Advanced Analysis and Measurement Centre of Yunnan University for their assistance in sample testing.
Contributor Information
Xiao‐Li Li, Email: lixiaoli@ynu.edu.cn.
Xing‐Jie Zhang, Email: zhangxj@ynu.edu.cn.
Wei‐Lie Xiao, Email: xiaoweilie@ynu.edu.cn.
Data Availability Statement
The data that support the findings of this study are available in the Supporting Information section of this article.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supporting File 1: cbdv71515‐sup‐0001‐SuppMat.docx
Data Availability Statement
The data that support the findings of this study are available in the Supporting Information section of this article.