Silent crusher m

Tumor necrotic factor (TNF‐α), prostaglandin-E₂ (PGE₂), interleukin-8 (IL-8), and phosphorylated nuclear factor kappa B (p-NF-κB) detection was conducted according to the manufacturer’s instructions (Elabscience). The stomach tissues (n = 3) were kept in bio freezer (−80°C) for storage, homogenize at 15 rpm × 1,000 using SilentCrusher M (Heidolph) and supernatant was collected after centrifugation (at 1,350 × g for 1 h). Supernatant was then analyzed for TNF‐α (Catalog No: E-EL-R0019), PGE₂ (Catalog No: E-EL-0034), IL-8 (Catalog No: EKF57830), and p-NF-ƙb (Catalog No: E-EL-R0674) quantification through Elabscience Rat ELISA kit.

Kidney was weighed and 10% of tissue homogenate was prepared in phosphate buffer (0.1 m, pH 7.4) using a glass-Teflon homogenizer (Heidolph Silent Crusher M, Germany). Homogenates were then centrifuged for 10 min, 500 g at 4°C. Supernatant was collected and recentrifuged at 2000 g for 10 min. Supernatant was again collected and recentrifuged at 12000 g for 10 min at 4°C and pellet was resuspended in 200 mM mannitol, 50 mM sucrose, and 10 mmol/L Hepes-KOH (pH 7.4). The final supernatant was taken and centrifuged for 1 h at 40000 g [26 ]. The cytosolic fraction was frozen at −80°C until further used.

In the present study, chemicals with analytical purity were used. Organic acid standards (citric acid, tartaric acid, oxalic acid, malic acid, succinic acid and fumaric acid), sugar standards (glucose, fructose, and sucrose), and vitamin C standards (l-ascorbic acid) were obtained from Sigma–Aldrich (St. Louis, MO, USA). The other chemicals were obtained from Merck (Darmstadt, Germany).
For organic acid extraction, the method by Bevilacqua and Califano [18 (link)] was modified. About 200 g of samples was fragmented and 10 g from each sample was delivered to centrifuge tubes. The 10 ml of 0.009 N H₂SO₄ was added to the samples and the samples were homogenized with Heidolph Silent Crusher M, Germany. Then, the samples were mixed for an hour with a shaker (Heidolph Unimax 1010, Germany) and centrifuged at 14.000 × rpm for 15 min. The supernatants were passed through coarse filter paper, then twice through a 0.45 mm membrane filter (Millipore Millex-HV Hydrophilic PVDF, Millipore, USA), and last in a SEP-PAK C18 cartridge. The concentration of organic acids was determined by HPLC using an Aminex column (HPX-87H, 300 mm × 7.8 mm, Bio-Rad) fitted on an Agilent 1100 series HPLC G 1322 A, Germany) [18 (link)]. Organic acids were detected at 214 and 280 nm wavelengths. The mobile phase, 0.009 N H₂SO₄ was passed through a 0.45 μm filter membrane.

First, lecithin (200 mg), buprenorphine hydrochloride (40 mg) and terpene or mixture of terpenes (variable up to 4 mg) were dissolved in 2 mL of absolute ethanol. Next, the resulting solution was transferred to a round-bottom flask and the full solvent evaporation process was carried out. Thin film formation was performed using a rotary evaporator (Teif Azma Teb, Iran) for 30 minutes. Then, the dried thin film was hydrated through stirring and vortex process using an aqueous solution (10 mL) of bupivacaine hydrochloride (40 mg) at 60°C. The resulting solution was homogenized by (SilentCrusher M, Heidolph, Germany) at 20 000 rpm. Finally, the nano-invasome was produced and stored in the refrigerator for the next experiments.

The samples were kept in
a deep freezer (−80 °C) until the analysis. In the study,
citric acid, tartaric acid, malic acid, succinic acid, and fumaric
acid contents of organic acids were determined in a service tree fruit.
The method given by Bevilacqua and Califano^{26 (link)} was modified and used for the extraction of organic acids. 50 g
of the service tree samples were taken and transferred to centrifuge
tubes. 20 mL of 0.009 N H₂SO₄ was added to these
samples and homogenized (Heidolph Silent Crusher M, Germany). Then,
it was mixed for 1 h on a shaker (Heidolph Unimax 1010, Germany) and
centrifuged at 15,000 rpm for 15 min. The aqueous fraction separated
in the centrifuge was first passed through a coarse filter paper,
then twice through a 0.45 μm membrane filter (Millipore Millex-HV
Hydrophilic PVDF, Millipore, USA), and finally through the SEP-PAK
C18 cartridge. Samples were analyzed in an HPLC instrument (Agilent
HPLC 1100 series G 1322 A, Germany). An Aminex HPX-87 H, 300 mm ×
7.8 mm column (Bio-Rad Laboratories, Richmond, CA, USA) was used in
the HPLC system, and the device was controlled by a computer with
an Agilent package program. The DAD detector (Agilent, USA) in the
system is tuned to 214 and 280 nm wavelengths. In the study, 0.009
N H₂SO₄ passed through a 0.45 μm membrane
filter was used as the mobile phase.

Organic acids identified by the technique reported by Bevilacqua and Califano [56 (link)]. Organic acid standards used in research; oxalic acid, tartaric acid, malic acid, citric acid and fumaric acid standards were prepared at the levels of 100, 200, 300, 400, 500, 600, 700 and 800 ppm and formed curves. Each sample (50 g) mixed with 80 mL of 0.009 N H₂SO₄ (Heidolph Silent Crusher M, Berlin, Germany), then homogenized for 1 h with a shaker (Heidolph Unimax 1010, Berlin, Germany). The mixture centrifuged for 15 min at 15,000 rpm, and supernatants filtrated twice with 0.45 µm membrane filter following filtration with coarse filter (Millipore Millex-HV Hydrophilic PVDF, Millipore, Burlington, MA, USA) and run through a SEP-PAK C18 cartridge. Organic acid readings performed with HPLC using Aminex column (HPX—87 H, 300 mm × 7.8 mm, Bio-Rad Laboratories, Richmond, CA, USA) at 214 and 280 nm wavelengths, on Agilent package program (Agilent, Santa Clara, CA, USA).

Rat Nrf2, p-NF-κB, HO-1, and TNF-α ELISA kits were used to quantify the expression of the respective proteins. Expression was measured as per manufacturer’s instructions (Shanghai Yuchun Biotechnology, Shanghai, China). An appropriate amount of tissues (50 mg) were first homogenized while using Silent Crusher M (Heidolph) at 15,000 RPM in PBS (2500 μL) containing PMSF as protease inhibitor and then centrifuged at 4000× g for 10 min and the supernatant was separated. The total protein concentration in each group was determined by the BCA method (Elabscience), and the equivalent quantity of protein was then loaded to determine the protein expression of Nrf2, p-NF-κB, TNF-α, and HO-1 while using ELISA microplate reader (BioTek ELx808) and the concentration (pg/mL) were then normalized to total protein content (pg/mg total protein).