2.7.4. X-ray Diffraction (XRD)

The crystalline characteristic of lyophilized nanoparticles and selected 7,8-DHF were recorded on an X-ray diffractometer (Bruker D8, Karlsruhe, Germany). This diffractometer was carried at 40 kV accelerating voltage and 40 mA tube current to produce copper K α radiation. Soller slit and divergence slit were set at 2.5◦ and 0.5◦, respectively, the 2θ angle was ranged from 5◦ to 90◦.

#### 2.7.5. Transmission Electron Microscopy (TEM)

10-fold diluted fresh nanoparticle dispersions were deposited on a copper grid with formvar-carbon coating. Then, the samples were air-dried for 5 min and stained with 2% uranyl acetate. TEM (JEM-1200 EX, Tokyo, Japan) was performed for microscopic observation at 120 kV accelerating voltage.

2.7.6. Field Emission Scanning Electron Microscope (FE-SEM)

The surface morphology of polysaccharide samples and freeze-dried nanoparticles was captured by FE-SEM (GeminiSEM 300, ZEISS, Germany). Before analysis, 3–6 nm of a thick gold layer was covered on the sample surfaces. The electron microscope acceleration voltage was 15.0 kV.

*2.8. Storage Stability of 7,8-DHF*

7,8-DHF, DHF-S/Z, DHF-ALG/S/Z, and DHF-CMC/S/Z were performed at 5 ◦C for 72 h under dark and 25 ◦C for 15 days under light, respectively. At an appropriate point in time, the samples were acquired with 7,8-DHF presence being measured by UPLC. The storage stability was calculated based on the following equation:

Retention rate (%) = retained 7,8-DHF concentration/initial 7,8-DHF concentration × 100 (3)

#### *2.9. In Vitro Simulated Gastrointestinal Digestion*

Based on Yuan et al.'s study via some amendments [28], briefly, 10 mL of simulated gastric fluid (SGF, 3.2 mg/mL pepsin and 2 mg/mL NaCl, pH = 2.5) and 10 mL of 7,8- DHF, DHF-S/Z, DHF-ALG/S/Z, and DHF-CMC/S/Z were mixed for incubating in a 37 ◦C water bath shaker for 60 min at 100 rpm. After SGF digestion, 10 mL of abovementioned simulated gastric digestive fluids were rapidly adjusted to pH 7.4 using 2 M NaOH. Whereafter, 10 mL of simulated intestinal fluid (SIF, 4 mg/mL pancreatin, 5 mg/mL bile salts, 6.8 mg/mL K2HPO4 and 8.8 mg/mL NaCl, pH = 7.4) was added into above-mentioned simulated gastric digestive fluids and incubated for 120 min at same temperature and revolving speed. Finally, the final digestive solution was centrifuged by 20,000× *g* centrifugal force for 1 h, and the supernatant (the mixed micelle phase containing 7,8-DHF) was collected. The bioaccessibility (%) was calculated based on the following equation:

Bioaccessibility (%) = 7,8-DHF concentration in the micelles phases/7,8-DHF concentration in the formulation × 100 (4)
