The stem bark powder was extracted with aqueous methanol (50%) using soxhlet apparatus. The extract was concentrated using rotary vacuum evaporator (Yamato rotary vacuum evaporator, Japan). The extract/test compound (AqMeOH-Ba) was subsequently frozen (Operon, Korea), lyophilized (Virtis, USA) and stored at −20 °C until used for experiments.
Rotary vacuum evaporator
Manufactured by Yamato Scientific
Sourced in Japan
A rotary vacuum evaporator is a laboratory equipment used to gently and efficiently remove solvents from samples under reduced pressure. It functions by rotating a round-bottom flask containing the sample while applying a vacuum, allowing the solvent to evaporate at a lower temperature.
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7 protocols using rotary vacuum evaporator
1
Aqueous Methanol Bark Extract
2
Biosurfactant Extraction from Bacterial Isolate
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The isolate MSA31 was inoculated into 1 L of production media composed of minimal salt media enriched with 10 g olive oil, 10 g ammonium nitrate and 19 g NaCl and incubated at 28°C for 144 h with agitation of 180 rpm. After incubation, the CFS was obtained by centrifugation at 14,000 rpm for 20 min at 4°C (Eppendorf). The supernatant was acidified to pH 2.0 with 0.1N HCl and allowed to form precipitate by incubating overnight at 4°C. The acid precipitate was collected by centrifugation at 12,000 rpm for 30 min, 4°C. The precipitated biosurfactant was washed several times with sterile distilled water and the pH was adjusted to 7.0 using 0.1 N NaOH. The precipitate was resuspended in sterile distilled water and solvent optimization was performed by adding equal volume of extraction solvents such as methanol, ethyl acetate, diethyl ether and dichloromethane (v/v). The resultant aliquot was concentrated to dryness in a rotary vacuum evaporator (Yamato). The solvent extract with high emulsification activity was further purified using column chromatography on silica gel (60–120 mesh) with step wise elution with methanol and water ranging from 65 to 100% (v/v) at a flow rate of 0.5 ml/min at room temperature (27°C). The purified fraction was used for chemical characterization and identification of active molecule.
3
Mangrove Leaf Phytochemical Extraction
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Tender foliage of E. agallocha (voucher specimen AEA-8, 2020) was collected from mature trees dotting the mangrove wetland of Ayiramthengu, Kollam district (09°12ʹ N and 76°47ʹ E), Kerala state, India and identified taxonomically with the aid of an eminent mangrove taxonomist. Prior to extraction, the leaves were washed with water and cleaned to remove salts and other associated debris, chopped into small pieces, and dried under shade (to prevent photolysis and thermal degradation). The completely dried plant material was powdered in a coffee grinder and extracted using organic solvents of increasing polarity separately, such as chloroform (trichloromethane [TCM]), dichloromethane (DCM), ethyl acetate (EtOAc), and methanol (MeOH), as well as water.12 (link) All solvents used were of analytical grade and supplied by Merck India. In a typical batch, 100 g powdered material was extracted with 1 L of solvent in a Soxhlet apparatus for 5 hours, filtered using Whatman number one filter paper, concentrated in a rotary vacuum evaporator at 40°C (Yamato), then stored in a freezer at –20°C for further studies.
4
Phytoconstituent Extraction and Preparation
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The phytoconstituents were extracted using a hydroethanolic solvent in a 1:1 ratio, employing a reflux apparatus. The resulting extract was then concentrated at 45°C by using a rotary vacuum evaporator from Yamato Scientific Co., Japan. In preparation for the in vivo study, the concentrated extract was subsequently stored at −20°C.
5
Extraction and Characterization of Harsingar Leaves
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Fresh harsingar (Nyctanthes arbor-tristis) leaves were collected from the campus of GLA University, Mathura, India, and authenticated by the Agharkaar Research Institute in Pune, India. (AUTH-22). For the extraction, shadow-dried leaves were grinded into powder. The phytoconstituents were extracted with hydroethanolic solvent using a reflux apparatus, and the extract was concentrated at 45 °C using a rotary vacuum evaporator (Yamato Scientific Co., Tokyo, Japan) For in vitro tests, the final concentrated extracts were kept at −20 °C.
6
Extraction and Fractionation of AM
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Dried roots of AM were bought from Nuri Herb Co. Ltd. (YoungCheon, Gyeongsangbuk-do, Republic of Korea). AM (200 g) was chopped and extracted for three times with 1.5 L of 80% ethanol for 24 hours at room temperature with shaking (100 rpm) and occasional sonication. The extract was combined, filtered, and concentrated with a vacuum rotary evaporator (Yamato Scientific Co. Ltd., Tokyo, Japan) under reduced pressure and lyophilized. The powder was suspended in distilled water and further fractionated with three different solvents in a stepwise manner. The fractions were the hexane fraction (hexane fraction of AM, HAM), ethyl acetate fraction (ethyl acetate fraction or AM, EAM), and butanol fraction (butanol fraction of AM, BAM). The ethanol, hexane, ethyl acetate, and butanol were all purchased from SK chemicals (Seoul, Republic of Korea). The extract powder was dissolved in dimethyl sulfoxide (DMSO; Amresco, Solon, OH, USA) as a stock solution at a 200 mg/ml concentration, and diluted with medium to the desired concentration prior to use.
7
Extraction and Quantification of Polygonum viviparum
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Polygonum viviparum L. (PV) was obtained from Tibet. Its authenticity was confirmed by Dr Shin-Ming Ku (Herbarium, Biodiversity Research Center, Academia Sinica, Taipei, Taiwan). The herb (PV 100 g) was extracted with 3 L of 2-propanol for 7 days, then the extract was filtered and centrifuged at 13 000 × g for 10 min. The extract supernatant was passed through a 0.22 μm sterile filter (Millipore, Billerica, MA, USA) and first concentrated using a vacuum rotary evaporator (Yamato, Tokyo, Japan) at 40°C. Normally, 8.76 g of dried powder could be obtained from 100 g of PV. The dried extract yield from the crude material was thus approximately 8.76% [4 ].
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