Study On Reverse Continuous Cycle Extraction And Quality Control For Trollii Flos Oral Liquid

Trollii Flos Oral Liquid(TFOL) is contained in the "Chinese Pharmacopoeia" 2010 version of the second supplement now,Which is consists only of flos trollii.TFOL is widely used for the treatment of upper respiratory tract infection, tonsillitis, and pharyngitis that is caused by toxic heat.In the field of medicine, flos trollii has a long history of medicinal use, "Supplement to compendium of Materia Medica" refers to the "bitter, cold, non-toxic" treat "aphtha, sore throat, otalgia and ophthalmalgia. Modern with advanced technology apply flos trollii to medicine and tea, in the aspect of anti-inflammatory and cleaning away heat and toxic material played a superb effect.In today’s abuse of antibiotics, drug resistance increased, TFOL may be used as the main drug treatment of upper respiratory infection, pharyngitis, and tonsillitis.TFOL with vertical static extraction tank to decocting flos trollii in the process of production.The traditional method has the following disadvantages: 1. Impurity( such as protein and tannin) increase by several lengthydecocting flos trollii,TFOL is more difficult to purify. 2. Mushy flos trollii make filter clogging,and causes the liquid and dregsdifficult to separate.Frequent replacement of the filter so that thepossibility of contamination of liquid to increase,contaminated liquid isimportant factors affecting the quality of the oral solution. 3. Large amount of solvent leads to long concentrated time and high energyconsumption.The current quality standards of TFOL is orientin content determination,determination of the content of a single component can not indicate the intrinsic quality of the product,TFOL quality standard needs to be improved.This research was carried out in order to solve practical problems in industrial production,improve the level of TFOL extraction process and quality control.Objective:1. Use reverse continuous cycle extraction(RCCE) to extract flos trollii, compared with the current standard of TFOL extraction process, determine the optimum extraction.2. HPLC was used to establish method for determination of glycosides and vitexin of liquid extract of flos trollii; And UV-VIS was used to establish method for determination of total flavonoids of liquid extract of flos trollii.3. HPLC was used to establish characteristic fingerprint of TFOL and simultaneously determine orientin and vitexin, and for commercial and RCCE enlarge production TFOL similarity evaluation and quality analysis.Methods:1.Mass concentration of the total flavonoids, orientin and vitexin as index,with extraction temperature, extraction time, powder type and added water as the experimental factors, each factor design 3 level, The orthogonal design L18(37) and RCCE were used to extract flos trollii, the optimum extraction was determined. Solvent consumption, extraction time, extraction rate and concentration of the total flavonoids, orientin and vitexin as index, compare RCCE(referred to as new technology) and the current standard of TFOL extraction process.2.The determination of Orientin and Vitexin in liquid extract of flos trollii, HPLC conditions: Agilent HC-C18(4.6mm × 250 mm, 5μm) column; methanol(A)- acetonitrile(B)-0.1% phosphoric acid aqueous solution(C) as the mobile phase, gradient elution as follows: 0 ~ 45min(4% A-12% B-84% C), 45 ~ 60min(10% A-20% B-70% C); 349 nm as the detecting wavelength; flow rate 1.0m L ? min-1; column temperature 25 ℃; injection volume 5μL. The determination of total flavonoids in liquid extract of flos trollii, UV-VIS conditions: rutin as reference substance, sodium nitrite- aluminum nitrate-sodium hydroxide complex color reflection, detection wavelength 510 nm.3.Establish characteristic fingerprint of TFOL and determine orientin and vitexin, HPLC conditions: Phenomenex C18(4.6 × 250 mm, 5μm) column, A(acetonitrile)-B(containing 0.1% phosphoric acid and 1% tetrahydrofuran solution) as the mobile phase, gradient elution as follows: 0min ~ 20 min ~ 38 min ~ 55 min ~ 70 min, A 3%~16% ~ 16% ~ 30% ~ 50%; flow rate 1.0μg·m L-1; column temperature 25 ℃; 230 nm as the detecting wavelength; injection volume 10μL. The characteristic fingerprint of commercially and RCCE enlarge production TFOL were evaluated using the “Similarity evaluation system for chromatographic fingerprint of TCM”published by GPC(Version 2004A).Results:1.The optimum extraction conditions of TFOL were as follows: RCCE,extraction temperature 90 ℃, extraction time 2 h, powder type 10 mesh, added water 20 times.2.HPLC was used to determine orientin and vitexin in liquid extract of flos trollii: The linear ranges of orientin and vitexin were 18.46 ~ 295.37(r = 0.999 9) and 5.84 ~ 93.44 μg·m L-1(r = 0.999 9), the average recoveries of orientin and vitexin were 96.07%, RSD = 0.74%(n = 6) and 97.73%, RSD = 1.42%(n = 6), respectively. UV-VIS was used to determine total flavonoids in liquid extract of flos trollii: rutin linear range was 10.56 ~ 63.36 μg·m L-1(r = 0.999 9), total flavonoids average recovery was 99.39%, RSD = 1.52%(n = 6).3.Fingerprint of TFOL determined 20 common peaks, and the similarity fingerprint peaks of commercially available TFOL were all greater than 0.948, the similarity fingerprint peaks of RCCE enlarge production TFOL were all greater than 0.958. The linear ranges of orientin and vitexin were 10.00 ~ 392.80μg·m L-1(r = 0.999 9) and 2.40 ~ 94.90μg·m L-1(r = 0.999 9), the average recoveries of orientin and vitexin were 101.85%, RSD = 1.61%(n = 6) and 102.53%, RSD = 1.45%(n = 6), respectively.Commercially available TFOL orientin and vitexin contents were between 1.99~4.63 and 0.33~1.56mg·m L-1,RCCE enlarge production TFOL orientin and vitexin contents were between 2.39~2.46 and 0.57~0.58mg·m L-1.Conclusion:1.The technology of RCCE is stable and feasible, and less solvent, less time, high extraction rate; Consistent quality of RCCE enlarge products and original technology products, The RCCE methods could be used as production process reference of TFOL.2.The methods that determination orientin and vitexin by HPLC and determination total flavonoids by UV-VIS in liquid extract of flos trollii are accurate, sensitive, simple, stability, and has good reproducibility.The determination methods could be used as quality control reference of TFOL and related products.3.The method that establishing TFOL fingerprint and determination of orientin and vitexin by HPLC is simple, accurate, high sensitive, and also with better stability and reproducibility, so this method can provide basis for quality control of TFOL.The fingerprint of commercially TFOL is similar, but the content of orientin and vitexin has the difference.