Luna c18 2

The analysis was conducted using an LC 1260 Infinity system equipped with a 1260 FC-AS G1364C fraction collector. The system was operated under the control of Open Lab CDS 2.7 software (Agilent, Santa Clara, CA, USA). The separation was optimized on a Luna C18(2) (150 × 4.6 mm ID, 5 µm particle, batch No.: 5291-0217) column (Phenomenex, Torrance, CA, USA) guarded by a pre-column. Isolation was performed using a Luna C18(2) (150 × 10 mm ID, 5 µm particle, batch No.: 5291-0227) column (Phenomenex, Torrance, CA, USA) that was fitted with a pre-column. Parameters were scaled up according to the increase in the column diameter. The collection of fractions was carried out on the basis of fixed time windows considering the latency and threshold recorded at 280 and 340 nm. For the optimized separation of HP5, a two-step gradient was used: 0–5 min at 10% B, followed by 45 min at 20% B extended for equilibration. A flow rate of 0.6 mL/min was used. For the HP8 fraction, an isocratic of 45% B was used. In both cases, the mobile phase was ultra-pure water (A) and acetonitrile (B) with 0.1% formic acid. The thermostat temperature was set at 25 °C. Parameters were scaled up according to the increase in the column diameter.

The method was adapted from Kaltsa et al. [29 (link)]. In brief, a Shimadzu liquid Chromatograph (CBM-20A) and a Shimadzu detector (SPD-M20A) were used. A Phenomenex Luna C18(2) (100 Å, 5 μm, 4.6 × 250 mm) (Phenomenex, Inc., Torrance, CA, USA) retained at 40 °C, a flow rate was 1 mL min⁻¹, and an injection volume 20 μL were used. The mobile phases and the elution program used have been described previously [29 (link)]. Quantification calibration curves were prepared using three points (0, 10, and 50 mg mL⁻¹), for caffeic acid (quantified at 320 nm, y = 0.000009x + 0.8755, R² = 0.9986), rosmarinic acid (at 320 nm, y = 0.00002x + 0.3334, R² = 0.9998), and luteolin-7-O-glucoside (at 345 nm, y = 0.00002x + 1.0794, R² = 0.9980). The estimation of the total area was carried out at 245 nm and 350 nm.

Arbutin, Tetraethylammonium bromide (TBAB), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP), 11-bromoundecanoic acid and cholesterol were purchased from Adamas Reagent (China) Company (Shanghai, China). Anhydrous potassium carbonate, Dichloromethane, Methanol, Petroleum ether, Ethyl acetate and dimethylformamide were bought from Shanghai Titan Chemical Co., Ltd (Shanghai, China). Ligustrazine Hydrochloride (TMP) was purchased from Hubei Xing Yinhe Chemical Co., Ltd. Artesunate (ATS) was purchased from Kunming Pharmaceutical Group Chongqing Wulingshan Pharmaceutical Group (Chongqing, China). Soy lecithin was purchased from Shanghai Taiwei Pharmaceutical Co., Ltd (Shanghai, China). DSPE-mPEG2000 was purchased from A.V.T. (Shanghai) Pharmaceutical Co., Ltd (Shanghai, China). ATS injection was got from Guilin Pharmaceutical Co., Ltd (Guilin, China). TMP Injection was obtained from Beijing Yongkang Pharmaceutical Co., Ltd (Beijing, China). HPLC column (Phenomenex Luna C18(2), 4.6 × 100 mm, 3 μm) was purchased from Phenomenex (California, USA). Acetonitrile HPLC grade was purchased from Tianjin Concord Technology Co., LTD (Tianjin, China). Centrifugal ultrafiltration tubes were purchased from Merk Millipore Ltd (Darmstadt, Germany). LC–MS (Exion LC-20AC, SCIEX Triple Quad 6500 + mass spectrometer, AB SCIEX, USA).