Comparison and Validation of Bioactive flavonoids by Reverse Phase High Performance Liquid Chromatography in Crataegus Species Related to Pharmaceutical Crataegus Product

The use of Crataegus species for the treatment of cardiovascular ailments is widely distributed. Even then only a few species are included in the Pharmacopoeias. In this study bioactive flavonoids of Crataegus azarolus and Crataegus pallasii were identified and quantified in comparison with the well-known pharmaceutical product of Crataegus; Crataegutt (R) Tropfen using reverse phase-high performance liquid chromatography. The method developed is simple, fast, reliable and sensitive. In an attempt to reduce matrix effect prior to high performance liquid chromatography analysis, a novel approach is proposed as an efficient simple clean-up technique termed "indirect-dispersive liquid-liquid microextraction". Validation parameters of the method were calculated as follows: Limit of detection ranged from 0.4 to 3.4 mg/g and limit of quantitation from 1.3 to 11.3 mg/g, intraday and interday precision expressed as percentage relative standard deviation ranged from 1.0 to 2.8 and 1.5 to 4.3, respectively and r(2) values were above 0.9950 for all analytes. The relative recovery of all analytes was more than 98 %. Four predominate peaks were identified using certified standards as Vitexin 2''-O-rhamnoside, rutin, vitexin and hyperoside, with mass concentration (% w/w) in Crataegus azarolus as 4.4 %, 2.9 %, 1.7 % and 4.4 %, in Crataegus pallasii as 4.4 %, 2.6 %, 1.4 % and 4.8 % and in Crataegutt (R) Tropfen as 1.6 %, 1.0 %, 0.6 % and 0.4 % respectively. Thus, the values met the criteria of the United States Pharmacopeia and the European Pharmacopeia monographs. Our investigations postulate that Crataegus azarolus and Crataegus pallasii could be a good source for the production of Crataegus phytomediciness.