Rotary evaporator

Antimicrobial activities of strain TW-1^T against E. coli KACC 10,185 and P. aeruginosa KEMB 121–234 were examined by disc-diffusion and spotting method, respectively. Screening were done against these Gram-negative pathogens by spotting the colonies of strain TW-1^T on R2A agar plates and incubated at 28 °C for 48 h. Crude product of culture extract was prepared by the culture supernatant of strain TW-1^T to evaluate disc-diffusion test. Strain TW-1^T was cultured in 300 mL of R2A broth at 28 °C (180 rpm for 5 days) into a 500 mL Erlenmeyer flask. Culture supernatant of strain TW-1^T was extracted by equal volume of ethyl acetate (2 ×) with pH 2.0 and 10, respectively⁴⁷ . Organic layer collected from extraction was completely evaporated by Rotary evaporator, (Eyela) and remained residue of crude product was dissolved in 500 µL of methanol. Then, 15 µL of crude product was diffused to a paper-disc (6 mm, Whatman) and antimicrobial activities against P. aeruginosa and E. coli KEMB were checked by measuring the inhibition zones. Trimethoprim/sulfamethaxazole (15 µg) and only methanol (15 µL) were used for positive and negative controls, respectively.

The following three strains (SMS_SU21, SMS_SU13 and SMS_7) with the best inhibitory activity were cultivated in 2.5 liter of modified cross streak media [yeast extract: 3 g; peptone: 3 g; casein: 3 g; starch: 8 g; glycerol: 3 g; CaCO_3: 0.75 g; K₂HPO: 0.5 g; MgSO_4,7H₂O: 0.5 g; NaCl: 12 g, pH 7.4] at 28 °C with a shaking speed of 150 rpm during an incubation period of 8 days for SMS_SU13 and SMS_SU21 whereas 12 days for SMS_7. After incubation all the cultures were centrifuged at 6000 × G (Sorvall Legend ×1R, Thermo Scientific, rotor: F15-6X-100Y) for 15 mins and supernatants were collected, filtered with 0.45 μmWhatman filter paper and subsequently extracted with ethyl acetate (1:1) twice. Residual ethyl acetate was evaporated with rotary evaporator (Eyela, Tokyo, Japan) at 40 °C temperature. Finally the extracts were dissolved in HPLC grade methanol to maintain a concentration of 50 mg ml⁻¹approximately.

Vigna radiata (L.) R. Wilczek (mung bean) was provided by King’s Ground Biotech Co., Ltd. (Pingtung, Taiwan) with a batch number NTU01. Vigna radiata extract (VRE) was prepared from the seed coats of mung bean via physical, chemical and biological processes. After mixing with organic acids under high temperature and pressure, the seed coats of mung beans were dried by hot air and ground to a powder. The VRE product contained 13% crude protein, 70% carbohydrate, and 4% ash. The VRE powder was then extracted with 95% ethanol (1:10 w/v) and sonicated for 1 h. The filtered solvent of the extract was concentrated using a rotary evaporator (Eyela, Japan). The recovery of the dried extract was approximately 15–25% (w/w). The dried extract was stored at −20°C until use.

Samples were prepared in accordance with a previous method [18 (link)], with a slight modification. Schisandra chinensis (Turcz.) Baill (SC) and Lycium chinense Mill. (LC) were obtained from a local herbal market (Seoul, Korea). Botanical identification was performed by Prof. Hong-Jun Kim (College of Oriental Medicine, Woosuk University, Jeonju, Korea). Dried SC and LC were roasted at 220°C for 9 min in a 30% aqueous ethanol using a CBR-101A roaster (Genesis, Korea). Each sample (5 g) was extracted twice in 5 mL of distilled water for 3 h at 95°C. The extracts were filtered and evaporated using a rotary evaporator (EYELA, Japan) at (40–50)°C. Finally, the extracts were lyophilized in a freeze drier (Il Shin, Korea). Samples were prepared at 100 mg/mL concentration in dimethyl sulfoxide for further study.

Whole plants of GT, which were grown and collected in Gyeongsangbuk-do province in Korea in 2011, were purchased from Omniherb Co. (Daegu, Korea) and authenticated by Professor S. Lee at the College of Korean Medicine, Sangji University, Wonju, Korea. A voucher specimen (no. sjomph003) is kept at the College of Korean Medicine, Sangji University. The air-dried whole plant of GT (50 g) was cut and extracted with 50% EtOH (500 ml, twice) at 60°C for 3 h, assisted by ultrasonic waves (40 kHz). The extract was filtered with filter paper (6 μm; Whatman PLC, Kent, UK) and concentrated using a rotary evaporator (Eyela, Tokyo, Japan). Subsequently, the extract was lypophilized using a freeze dryer (Labconco, Kansas City, MO, USA) to yield 14.2 g of powder (28.4%). The powder was dissolved in distilled water for stock solution (1 mg/ml) and diluted with culture medium prior to use in the experiments.

Polysiphonia japonica was collected, rinsed with freshwater to remove the salt, epiphytes, and sand, and stored at −75°C. The frozen samples were lyophilized and finely ground. To prepare the extract, 1 g (dry weight) of the alga was solubilized in 100 mL of 80% methanol for 24 h under continuous shaking at 20°C, and then the extracts were filtered and concentrated under a vacuum in a rotary evaporator (EYELA, Tokyo, Japan) at 40°C.

CS samples were obtained from the field near Hanaro Farm (Haenam County, Jeollanam-do, Republic of Korea) (34°23′00.4″ N 126°33′59.0″ E; 25 April 2019). CS identity was confirmed by Jong-Cheol Yang from the Division of Forest Biodiversity and Herbarium, Korea National Arboretum (Republic of Korea). A voucher specimen (no. CS-0001) was kept at the Herbarium of the College of Life and Health Science, Hoseo University (Republic of Korea). Absolute was extracted from CS flowers by solvent extraction as previously described [27 (link)]. In brief, flowers (5.27 kg) were completely immersed in hexane (Samchun, Pyeongtaek, Republic of Korea) at room temperature (RT) for 1 h. Solvent was removed from extracts using a rotary evaporator (EYELA, Tokyo, Japan) at 25 °C under vacuum to yield a dark yellow waxy residue (concrete), which was mixed with ethanol and left at −20 °C overnight. The mixture was then filtered through a sintered glass funnel, and the ethanol was evaporated at 35 °C. The light yellow wax obtained (absolute) (CSFAb; 1.50 g, yield 0.028%, w/w) was stored at −80 °C until required.