Rp 18 column

Azadirachtin (purity, ≥97%) was purchased from Yunnan Zhongke Biological Industry Co., Ltd. (Kunming, China). SF9 cells were purchased from Qingqi Biotechnology Development Co., Ltd. High performance liquid chromatography (HPLC)-grade acetonitrile (J.T. Baker, Phillipsburg, NJ, USA) and ultra-pure water prepared from a Milli-Q purification system (Millipore, Burlington, MA, USA) were used for semipreparative HPLC analysis. Contact Angle and Surface Tension were measured using the JC2000C1 contact angle/surface tension measuring instrument (Shanghai Zhongchen Digital Technology Equipment Co., Ltd., Shanghai, China).
Electrospray ionization (ESI) was recorded on an Agilent 1290 UPLC/6540, and the 1D and 2D NMR spectra were measured on a Bruker 500 MHz spectrometer, with TMS as the internal standard. An RP-18 column (50 μm, YMC Co. Ltd., Kyoto, Japan) gel, MCI gel (75–150 μm, Sci-Bio Chem Co. Ltd., Chengdu, China), and Sephadex LH-20 (40–70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden), were used for column chromatography. Semipreparative HPLC was performed on a YMC Luna C18 (5 μm; 10 × 250 mm) reversed-phase column.

Azadirachtin (purity, ≥81%) was purchased from Yunnan Zhongke Biological Industry Co., Ltd. (Kunming, Yunnan, China). The Insect GABA enzyme-linked immunosorbent assay (ELISA) kit, insect glutamate decarboxylase (GAD) ELISA kit, insect CarE ELISA kit, insect GST-s ELISA kit, and insect cytochrome P450 ELISA kit were purchased from Jianglai Biological Co., Ltd. (Shanghai, China). HPLC-grade acetonitrile (J.T. Baker, Phillipsburg, NJ, USA) and ultra-pure water prepared from a Milli-Q purification system (Millipore, MA, USA) were used for semi-preparative HPLC analysis.
Electrospray ionization (ESI) were recorded on Aglient 1290 UPLC/6540, and the 1D and 2D NMR spectra were measured on the Bruker 500 MHz spectrometer, with TMS as the internal standard. RP-18 column (50 μm, YMC Co., Ltd., Kyoto, Japan) gel, MCI gel (75–150 μm, Sci-Bio Chem Co., Ltd., Chengdu, China), and Sephadex LH-20 (40–70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden), were used for column chromatography. Semi-preparative HPLC was performed on an YMC Luna C18 (5 μm; 10 × 250 mm) reversed-phase column.

Dried V. trifolia L. fruit powder (5 kg) was subjected to methanol-based sonication (three times, each with 15 L MeOH). After solvent evaporation, the resulting methanol extract (150 g) was reconstituted in water and subsequently partitioned with n-hexane (H), CH₂Cl₂ (D), and EtOAc (E) to yield the H (30 g), D (7 g), and E (5 g) fractions, and an aqueous layer.
Fraction D was initially subjected to silica gel column chromatography using a stepwise hexane/acetone gradient, yielding seven sub-fractions (D1–D7). Subsequently, Fraction D2 was chromatographed on a YMC RP-18 column using a 1:1 v/v mixture of MeOH and water, leading to the isolation of D2Ⅰ (0.8 g). D2Ⅰ was further subjected to silica gel column chromatography using a hexane and acetone (10:1 v/v) mixture to yield D2Ⅰ1 (0.1 g). Finally, prep-HPLC employing a J’sphere ODS H-80 column (250 mm × 20 mm) was performed with a mobile phase of 25% aqueous acetonitrile at a flow rate of 3 mL/min to isolate vitekwangin B (15 mg).