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Acetonitrile, HPLC grade, was purchased from LiChrosolv, Merck, Darmstadt, Germany; the methanol and formic acid (LiChrosolv, Merck, Darmstadt, Germany) were of analytical reagent grade. 2.2. Extraction Extraction was performed according to the already published procedure in the literature [4]. Extraction efficiency was determined based on the total extractive percent value find more using different compositions of aqueous ethanol as 50%, 70%, 80%, and 95% at room temperature for 48 hours. Afterwards, one hundred grams of above ground part of D. heterophyllum was weighed accurately and extracted with 400?mL of 80% aqueous ethanol for 48 hours at room temperature. The extract was filtered, decolorized, and defatted by petroleum ether for several times. The extract was reduced to dryness through rotary evaporator under reduced pressure yielding 20?mg dried extract. The extract was reconstituted in 1?mL of pure methanol and injected 10?��L into the column. 2.3. Determination of Total Flavonoids Total flavonoids contents were determined using the method of Chang et al. 2002 [16]. One?g of plant material was extracted with 50?mL of methanol under reflux and the extract obtained was reduced to dryness. The residue was reconstituted in 10?mL of methanol and this extract was used for the determination of total flavonoids contents. Briefly, 0.5?mL of plant extract/standard solution was mixed with 1.5?mL of methanol in a test tube. 0.1?mL of 10% aluminum chloride, 0.1?mL of 1?M potassium acetate, and 2.8?mL TRIB1 of distilled water were added to the test tube and mixed thoroughly Angiogenesis inhibitor after each addition. It was allowed to react for flavonoid-aluminum complex formation for 30 minutes at room temperature. Methanol was used as blank and preceded in the same way as above. Afterwards, the absorbances of the reaction mixtures were measured at 415?nm using the UV-visible spectrophotometer. Quercetin was used as standard and six working standard solutions were prepared in the concentration range 0.01?mg/mL to 0.1?mg/mL for constructing the calibration curve. 2.4. Preparation of Standard Solutions The four flavonoid standards, that is, luteolin, kaempferol, diosmetin, and chrysosplenetin, were mixed and stock solution of standard flavonoids mixture was prepared in methanol having 1?mg/mL concentration of each standard. From this stock solution, four working standard solutions with a concentration range 0.0001�C1?mg/mL were prepared. 2.5. Instrumentation HPLC analysis was performed using a DIONEX Ultimate 3000 HPLC system (Thermo-Fisher, USA) equipped with autosampler and coupled to variable UV wavelength detector. The chromatography was performed on Sunfire C18 column from Waters, USA, having the following specifications: internal diameter 4.6?mm, height 250?mm, and particle size 5?��m. The chromatographic column was protected by a Sunfire C18 guard column of the following dimensions: internal diameter 4.