Determination of Lobetyolin, Total Polysaccharides and Total Flavonoids in Miao Medicine Campanumoea Javanica from Different Harvesting Periods and Determination of the Optimal Harvesting Period of Campanumoea Javanica

Lobetyolin, total polysaccharides and total flavonoids in Campanumoea javanica from different harvesting periods (January to December) were determined by HPLC and UV-Vis, and the optimal harvesting period was obtained by the comprehensive evaluation normalization method. The Campanumoea javanica sample were mixed with methanol for extraction with ultrasound for 40 min. Aftering passing through 0.45 μm filter membrane, lobetyolin in the filtrate was determined by HPLC. The ethanol solution of 80% (volume fraction) was added into the Campanumoea javanica sample, and the mixture was refluxed twice for 1 h each time. After filtering, the filtrate was digested twice with water as media in the microwave oven for 5 min each time, diluted and mixed with 50 g·L^-1 phenol solution and sulfuric acid. The above solution was heated in the water bath at 100 ℃ for 10 min, cooled in an ice water bath for 20 min, and total polysaccharides was determined by UV-Vis at 490 nm, with glucose as reference substance for preparation of standard curve. The Campanumoea javanica sample together with 60% (volume fraction, the same below) ethanol solution was refluxed twice for 1 h each time. After filtration, concentration and dilution, 50 g·L^-1 sodium nitrite solution, 100 g·L^-1 aluminum nitrate solution and 40 g·L^-1 sodium hydroxide solution were added. The solution obtained was diluted to 10 mL with 60% ethanol solution, and total flavonoids was determined by UV-Vis at 509 nm, with rutin as reference substance for preparation of standard curve. The normalized values of lobetyolin, total polysaccharide and total flavonoids were calculated by Hassan method, and the optimal harvesting period was obtained by the total normalized (OD) value. As found by results, linear relationships between mass concentrations of lobetyolin, total polysaccharides and total flavonoids and their corresponding peak areas (for lobetyolin) and absorbances (for total polysaccharides and total flavonoids) were kept in definite ranges, and detection limits of lobetyolin (3S/N), total polysaccharides (3.143s) and total flavonoids (3.143s) were 0.131, 0.046, 0.071 mg·L^-1, respectively. The proposed method was applied to the analysis of actual samples, RSDs (n=6) of the peak areas (for lobetyolin) and absorbances (for total polysaccharides and total flavonoids) were not more than 3.0%, and the average values of test for spiked recovery were 98.8%, 99.1% and 98.5%, respectively. The OD values in October were higher, which could be identified as the optimal harvesting period for Campanumoea javanica, with mass fractions of lobetyolin, total polysaccharides and total flavonoids of 3.08, 185.15, 5.85 mg·g^-1, respectively.