Study On The Separation And Purification Of Amentoflavone From Selaginella Tamariscina (Beauv.) Spring

Amentoflavone is one of the active components in Selaginella tamariscina (Beauv.) Spring. It is well known for displaying a remarkable wide spectrum of biological activities, such as cytotoxic and antitumor activity, antifungal and anti-inflammatory activity, antivirus activity, antioxidant activity, and vasorelaxation activity. Amentoflavone has a potential use in pharmaceutical, food and cosmetic areas. The method of extraction of amentoflavone was studied. A two-step precipitation procedure was established for preliminary separation of amentoflavone extract. We developed the separation and purification procedure of amentoflavone by reversed-phase preparative HPLC, silical gel column chromatography and low-pressure column chromatography.1. HPLC method was developed for analyzing the contents of amentoflavone in Selaginella tamariscina (Beauv.) Spring. The analysis method had a good separation effect and was simple and timesaving. The analytical column was DiamodsilTM C18 (5μm,4.6 mm×250 mm), and the solvent system of HPLC analysis was consisted of methanl and 0.1%(v/v) TFA in water. The flow rate was 1.0 mL/min. An aliquot of 10μL sample was injected into the column and detected by UV at 337 nm. Results showed that is a prompt and concise method for quantification of amentoflavone concentration.2. The conditions for extraction of amentoflavone from Selaginella tamariscina (Beauv.) Spring were optimized. The whole herbs of Selaginella tamariscina (Beauv.) Spring were exacted by reflux using 80% ethanol at 75℃twice. The extracting time was 0.5 h each time, and the ratio of solid to liquid was 1:12 (g/mL). The content of amentoflavone extract was 5.92% and the yield was 93.24%.3. A two-step precipitation procedure was established to primarily separate amentoflavone from extract of Selaginella tamariscina (Beauv.) Spring. After the two-step precipitation, the purity of product was increased from 5.92% to 58.19% and the recovery was 88.66%. Compared with traditional technology, the method is simple, the cost is low and no other solvents have to be added to the product.4. Reversed-phase preparative HPLC (pre-HPLC) was used to separate amentoflavone from amentoflavone-rich extracts obtained from the two-step precipitation. The mobile phase of pre-HPLC was methanol:0.1%TFA (65: 35, v/v). The flow rate was 10 mL/min with the detection wavelength at 337 nm. The purity and recovery of product after pre-HPLC were 99.2% and 94.7%, respectively.5. The separation of amentoflavone with silical gel column chromatography was studied. The purity and recovery of amentoflavone with CHC13:MeOH (8:1, v/v) as eluent were 97.16% and 50.74%, respectively.6. The separation of amentoflavone with low-pressure column chromatography was established and optimized. Comparison of isocratic elution and gradient elution was made and the purity and recovery of product with gradient elution mode were 98.7% and 87.8% when the loading amount was 100 mg, respectively. The collection time of product was decreased to 11min, and solvent consumption was greatly reduced.7. The amentoflavone product was identified by HPLC, MS and NMR, and the mass spectra and NMR spectra were obtained to prove the results.