Diethyl ether

Sodium acetate (molecular
biology grade), acetic acid, diethyl ether (anhydrous, ≥99.7%,
with 1 ppm BHT as inhibitor), hydroquinone (HQ, ≥99%), acetonitrile
(ACN, ≥99.9%, HPLC gradient grade), 2,5-dihydrobenzoic acid
(DHB), N-isopropylacrylamide (NIPAm), deuterated
water (D₂O), 2,2′-azobis(isobutyronitrile) (AIBN),
potassium persulfate (KPS), and N,N,N′,N′-tetramethylethylenediamine (TEMED), HPLC grade
ethanol, 2,2′-azobis(isobutyronitrile) (AIBN), diethyl ether,
2-hydroxyethyl methacrylate (HEMA) 97% containing 200 ppm hydroquinone
(HQ) were all obtained from Sigma-Aldrich (St. Louis, MO, USA). Low-viscosity
locust bean gum (LBG, >94% (dry weight basis)) was supplied by
Megazyme
(Bray, Ireland) (LOT 150901a) (galactose:mannose ratio, 24:76). All
chemicals were used as received except for HEMA, which was passed
through an alumina column prior to polymerization to remove the inhibitor.^{18 (link)}

Total lipids were extracted from adipose tissue as described by Folch et al. [19 (link)]. Certain lipid classes, such as phospholipids (PLs), free fatty acids (FFAs), diacylglycerol (DAG), triacylglycerol (TAG) and cholesterol ester (CE), were separated on a thin-layer chromatography (TLC) plate (Merck KGaA, Darmstadt, Germany) using a mixture of petroleum ether, diethyl ether and acetic acid (113:20:2, v/v/v) as the mobile phase. Lipids were visualized by dipping the plate in a solution of 3% cupric acetate and 8% phosphoric acid and then charring the plate for 5 min at 140°C in an oven. petroleum ether, diethyl ether, acetic acid and cupric acetate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Different spots corresponding to the lipid classes were scraped off into scintillation vials and counted as described above.

Streptomyces Levis ABRIINW111 strain -isolated from soil samples- was extracted in the Department of Microbial Biotechnology, AREEO, Tabriz, Iran and was cultured in Nutrient Agar medium (70148, Sigma, Germany) at 29 °C for 7 days. The loops full of bacteria were inoculated into 25 ml of Mueller Hinton Broth medium (70192, Sigma, Germany) and incubated while agitating on shaker incubator set as 70 rpm at 29 °Cfor 36 h.^{17 (link)} After fermentation time, 1 ml of pre-culture was applied to inoculate 1,000-ml Erlenmeyer flasks, each containing 150 ml of fresh Mueller Hinton Broth medium. The fermentation was done at 29 °C for 7 days on shaker incubator set as 70 rpm, centrifuged at 4000 rpm for 20 min. The Cell free filtrate was mixed with equal volume of Diethyl ether (1:1 V/V) shaken for 10 min at 175 rpm, extracted by Diethyl ether (100921, Merck, USA), by the use of separating funnel. Finally, the obtained organic extract was undertaken to be concentrated at room temperature to achieve 0.01 gr crude extract which was maintained at 4 °Cuntil being utilized.^{17 (link)} As previously described, the turbidity 620 nm, 0.08 O.D. was considered appropriate for inoculation.^{17 (link)}

The rats were put down using a high dose of diethyl ether (MERK, Germany) after completing each experiment. After 72 h, the rats’ brains were placed in 10% formalin. The positions of the guide cannula in the ventricle and the probes of microdialysis in the PAG area of all the brains were verified according to the Paxinos Atlas (18 ).