Study On Extraction And Biological Activity Of Total Phenols And Flavonoids From Ribes Odoratum Wendl. Leaves

In the present study, deep eutectic solvent (DESs) and negative pressure cavitation-assisted extraction (NPCE) are proposed for the green arid efficient extraction of total phenols and flavonoids from Ribes odoratum Wendl. leaves in different stage. Their bioactivity in vitro were compared using antioxidant and antitumor. Using HPLC, the qualitative and quantitative analysis was made on gallic acid, chlorogenic acid, rutin, ferulic acid and isoquercitrin in the leaf of Ribes odoratum Wendl.. The results as follows:1. Total phenols and flavonoids in the leaf of Ribes odoratum Wendl. was optimized by a central composite design (BBD) and response surface methodology (RSM). The optimal parameters of negative pressure cavitation assisted deep eutectic solvent extraction process were as follows:The component of DESs:lactic acid/choline chloride (2:1, mol/mol); Water ratio in DESs:20%; Solvent to solid ratio:26:1 mL/g; Extraction pressure:-0.08 MPa; Extraction temperature:50℃; Extraction time:25 min; Extraction times:2. Based on the above optimized conditions, the average extraction yield of total phenols and flavonoids in the leaf of Ribes odoratum Wendl.was 95.78 mg/g and 33.63 mg/g. Deep eutectic solvents-based negative pressure cavitation-assisted extraction process is better than deep eutectic solvents-based ultrasound-assisted extraction and 70% ethanol-based negative pressure cavitation-assisted extraction.2. Total phenolic and total flavonoid content in Ribes odoratum Wendl. leaves from May to November was compared, the results indicate that the total phenolic in Ribes odoratum Wendl. leaves was highest in 16th July, and total flavonoid in Ribes odoratum Wendl. leaves was highest in 14th Aug.. An efficient HPLC method for determination of gallic acid, chlorogenic acid, rutin, ferulic acid and isoquercitrin was developed as follows:The chromatographic column:C18(5μm,250×4.6 mm i.d.); mobile phase:water (0.10% phosphoric acid) (A)-methanol (B); flow rate:1 mL/min; column temperature:35℃; injection volume:10 μL; detection wavelength:273 nm and 320 nm; gradient elution conditions:0-6 min,9-18%(B); 6-13 min,18-25%(B); 13-23 min,25-40%(B); 23-25 min,40-60%(B).It was found that the maximal extraction yield of gallic acid (17.23 mg/mL) and isoquercitrin (1.58 mg/mL) appeared at 16th July; the maximal extraction yield of chlorogenic acid (3.68 mg/mL) and rutin (7.42 mg/mL) were appear at 14th Aug., the maximal extraction yield of ferulic acid (3.35 mg/mL) was appears at 14th June.3. DPPH scavenging activity test, anti hydroxyl radical (·OH) test and anti superoxide anion free radical test were applied to evaluate the antioxidant activity of Ribes odoratum Wendl. leaves in different periods. The results all showed that the strongest antioxidant capacity in Ribes odoratum Wendl. leaves was which collected at 14th Aug.. The anticancer activities of these crude extract in vitro to human breast cancer cells(MCF-7) were performed using MTT assay. The result showed Ribes odoratum Wendl. leaves which collected at 14th Aug. had the strongest anticancer activities, ICdo=0.48 g/mL and there was a time-dose correlation between inhibition the growth of MCF-7 and concentration. Therefore, we can say in a preliminary estimate that the biological activity of the crude extract in the leaf of Ribes odoratum Wendl. had higher correlation with total flavonoids than total phenols.Above all, DES-NPCE was chosen to extract the total phenols and flavonoids in the leaf of Ribes odoratum Wendl.. DESs as a new kind of extraction solvent, possessed sustainability, biodegradability, pharmaceutical acceptable toxicity and high extractability for compounds with diverse polarity, and could be used as green solvents for the extraction of active components from plant materials. Combined with biological experiments and HRLC-UV analysis, we determine August is the optimal use stage of Ribes odoratum Wendl. leaves. This paper would provide a theoretical basis for the development and efficient utilization of Ribes odoratum Wendl. leaf resources.