Vacuum freeze dryer

Subsequent to obtaining approval from the Ethics Committee of Zhejiang Chinese Medical University and methods were carried out in accordance with the approved guidelines. All the herbal medicines of GFW, including Cinnamomum cassia (GUI ZHI) 15 g, Poria cocos (FU LING) 15 g, Semen persicae (TAO REN) 15 g, Paeonia albiflora (CHI SHAO) 15 g, Cortex moutan (MU DAN PI) 15 g, were purchased from Chinese Herbal Medicine of Zhejiang Chinese Medical University (Hangzhou, China), which were boiled by water. The filtrates of GFW were combined and concentrated to the crude drug concentration of 1.0 g/ml. Then the filtrates were freeze-dried into power by vacuum freeze dryer (Christ, Germany).
20 male 8-week-old SD rats (300–350 g) were kept in Experimental Animal Center of Zhejiang Chinese Medical University (Hangzhou, China).The rats were randomly divided into two groups, the drug serum group was administered by 0.1 ml/100 g water decoction at crude drug concentration of 2.16 g/ml daily and the blank serum group was given the same volume of physiologic saline for 3 days. Blood was acquired from the rats 1 h after the last time of administration. The serum was collected by centrifugation (3000 rpm for 15 min) and then filtered through a 0.22 μm cellulose acetate membrane.

For biomasses assay, conidial suspension of the strains was inoculated into PDB medium. The final concentration of conidia in the PDB medium was 1 × 10⁴ conidia/ml, and then the conidia suspension was inoculated at 26°C on a rotary shaker (200 rpm). The biomasses were measured at the 4 dpi. Mycelia were harvested and dried by Vacuum freeze dryer (Martin Christ, Osterode am Harz, Germany) and weighted by Mettler Toledo XS105 (Switzerland). Biomass relative to Vd991 = Biomass (Vd991, ΔVdDrs2, or Com)/Biomass of Vd991. For conidia yield assay, fungal spores were added into PDA (temperature bellow 60°C) to the final concentration of 5 × 10⁴ conidia/ml. Plates were placed at 26^°C for 10 days. Conidia were collected with a puncher and suspended in water with 0.05% Tween 80. Conidial concentration was determined by hemocytometer. All experiments were performed in triplicates with three independent repeats.

Liver tissue samples of different treatment groups were prepared by freeze-drying at −40 °C for 24 h using a vacuum freeze dryer (Christ, Germany). Two-hundred milligrams of potassium bromide and 2 mg of liver tissue samples were ground into tablets in an agate bowl to prepare infrared detection samples. A Fourier transform infrared spectrometer (FTIR) (PE-Spectrum 65, UK) was used to scan with 4000–400 cm⁻¹ wavenumber range, 4 cm⁻¹ resolution and accumulating 64 scans [35 (link)].