Uv 1900i series ultraviolet visible spectrophotometer

TFC was determined by the method of Yi et al. (15 (link)) with some modifications. Briefly, the AlCl₃-NaOH-NaNO₂ method was used. 2.0 mL of rutin standard solution with different concentrations (6.25, 12.5, 18.75, 25.0, 31.25, and 37.5 μg/mL) and 0.4 mL of 5% NaNO₂ solution were mixed for 6 min, and then 0.4 mL of 10% AlCl₃ solution was shaken and reacted, and 6 min later, 4 mL of 4% NaOH solution and 3.2 mL of distilled water were added and mixed. Distilled water replaced the standard solution as the blank control. After reaction at room temperature for 25 min, the absorbance value was measured at 510 nm using a UV-1900i series Ultraviolet-Visible Spectrophotometer (Shimadzu Co., Ltd, Japan). The calibration curve (y = 0.0115x – 0.0767, where y is the absorbance value of a sample and x is the sample concentration), ranged from 6.25 to 37.5 μg/mL (R² = 0.9990). 0.2 mL of sample extract was added to solvent to make 2.0 mL, and TFC in the sample extracts was quantified using the standard curve method.

An amount of 25 mg of oleanolic acid was weighed and fully dried at 105 °C. The solution was dissolved and diluted with methanol into a 25 mL volumetric flask, shaken to make it completely dissolve and prepared as a reference solution with a mass concentration of 1.0 mg/mL. Amounts of 0, 0.2, 0.4, 0.6, 0.8 and 1 mL of the oleanolic acid reference solution with a mass concentration of 1.0 mg/mL were placed into different test tubes. After completely volatilizing the solvent at room temperature, 1.6 mL of perchloric acid solution and 0.4 mL of 5% vanillin–acetic acid solution were added to each test tube, heated in a 70 °C water bath for 15 min and cooled naturally, and then 5 mL of glacial acetic acid was added and shaken well. The absorbance of the reference solution of different concentrations at the wavelength of 548 nm was determined with a UV-1900i series ultraviolet–visible spectrophotometer (Shimadzu Co. Ltd., Kyoto, Japan), and a blank test was performed. With different concentrations of oleanolic acid as the abscissa and absorbance A as the ordinate, we drew the standard curve. The standard curve equation was y = 0.0862x + 0.0012, R² = 0.9999.
An amount of 1 mL of total triterpene extracts of LIM was taken, and the concentration of total triterpenes in the LIM sample solution was measured according to the method of the standard curve equation.