Freeze dryer

SC-E3 was formulated from five oriental medicinal herbs, that is, Bupleuri Radix (voucher specimen number: DUMCKM2015-107), Coptidis Rhizoma (voucher specimen number: DUMCKM2015-083), Gardeniae Fructus (voucher specimen number: DUMCKM2015-069), Rhei Rhizoma (voucher specimen number: DUMCKM2015-017), and Puerariae Radix (voucher specimen number: DUMCKM2015-001). All were purchased as dried herbs from Omniherb (Daegu, Korea) in accord with the good manufacturing practices (GMP) procedures certified by the Korea Food and Drug Administration (KFDA) and authenticated by Professor Sun-Dong Park (Department of Prescriptions, College of Korean Medicine, Dongguk University). Voucher specimens were deposited at the College of Korean Medicine, Dongguk University.
Briefly, a mixture of dried Bupleuri Radix, Coptidis Rhizoma, Gardeniae Fructus, Rhei Rhizoma, and Puerariae Radix (100 g; weight ratios 3 : 1 : 3 : 1 : 3) was macerated in 800 mL of 70% ethanol, stirred for 24 h at room temperature (RT), and filtered twice through an 8 μm Whatman filter paper. After rotary evaporation at 40~45°C, the concentrate was lyophilized using a freeze dryer (EYELA, Japan). The yield of the SC-E3 extract (dried powder) was 15.2% by weight with respect to the dried starting materials.

The GST was formulated from twelve medicinal herbs: Panax ginseng C. A. Meyer, Atractylodes macrocephala Koidzumi, Poria cocos Wolf, Glycyrrhiza uralensis Fischer, Rehmannia glutinosa Liboschitz ex Steudel, Angelica gigas Nakai, Cnidium officinale Makino, Paeonia lactiflora Pallas, Astragalus membranaceus Bunge, Bupleurum falcatum Linne, Uncaria rhynchophylla, and Gastrodia elata Blume. All herbs were purchased as dried herbs from Omniherb (Seoul, Republic of Korea). Briefly, a mixture of the twelve dried components (100 g; weight ratios shown in Table 1) was extracted in 800 ml of 30% ethanol by heating at 80°C for 4 h, and extracts were filtered twice through a filter paper (40 μm, Whatman, Buckinghamshire, UK). Filtrates were concentrated under reduced pressure using a rotary vacuum evaporator (EYELA, Tokyo, Japan) at 40~45°C, and the concentrate was lyophilized using a freeze dryer (EYELA, Tokyo, Japan). The yield of the GST extract (dried powder) was 22.7% of the weight of the dried starting materials.

Amount of cellulose produced in the matrix of the AL biofilms of the bacterial strains (Pcc PC1 ΔcytR, ΔfliC, flhD::Tn5, and ΔmotA) were quantified as described in Anriany et al. (2006) (link) with a few modifications. In brief, strains were grown at 27°C in SOBG broth for 3 days in static condition. Then 3 g (wet weight) of AL biofilm masses were gently collected with sterile spatula and were dried by Freeze dryer (Eyela, Freeze dryer, FDU-830, Japan). The dry masses were mixed with 4.5 mL of acetic-nitric reagent (8:2:1 acetic acid: nitric acid: distilled water) and boiled for 20 min. The boiled mixture was then centrifuged and the supernatant was discarded. The pellet was transferred to a Corex centrifuged bottles, washed twice with sterile distilled water and dried in a clean bench. The dried pellet was mixed with 150 μL of concentrated H₂SO₄ with gentle shaking for 1 h at 27°C. The amount of cellulose was determined by adding 750 μL anthrone (Sigma-Aldrich, St. Louis, MO, United States) reagent (0.2 g in 100 mL H₂SO₄). The Avicel cellulose (Sigma-Aldrich, St. Louis, MO, United States) was used as standard this case, and the cellulose were quantified at 620 nm.

Trichosanthes kirilowii (Maximowicz) seeds were purchased as dried herbs from Humanherb (Gyeongsan, Korea), complied with the good manufacturing practices (GMP) guidelines issued by the Korea Food and Drug Administration (KFDA), and were authenticated by Professor Sun-Dong Park (Department of Prescriptions, College of Korean Medicine, Dongguk University). A voucher specimen (No. DUMCKM2015-109) was deposited at the College of Korean Medicine, Dongguk University. Briefly, seeds of T. kirilowii (200 g) were coarsely ground and extracted in 70% ethanol (800 mL) by heating at 80 °C for 4 h. Extracts were filtered and concentrated under reduced pressure using a rotary vacuum evaporator (EYELA, Japan). Condensed extracts were lyophilized using a freeze dryer (EYELA) and stored at 4 °C. The yield of dried extract (TKSE) was 4.5% (w/w) of dried herb weight.

A549 and MLE-12 cells were purchased from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China) and maintained at 37°C under 5% CO2 in DME/F-12 and 1,640 medium supplemented with 10% fetal bovine serum, respectively.
HSBDF condensed extract of 1.23 g/ml was lyophilized using a freeze dryer (EYELA, Japan). Finally, 15.38 g of lyophilized powder was obtained (yield, 11.23%). The dry powder was stored in an 80°C refrigerator until use.