Determination Of Main Active Ingredients And Fingerprint Of Essential Oil In Flos Chrysanrhemi

The flower of Chrysanthemum is one of the most important global cut flowers and pot plants. To date, about 3000 species of Chrysanthemum have been identified worldwide. Officially, the flower of Chrysanthemum morifolium Ramat.(CMR) was documented in China Pharmacopoeia (2005). These species included Hangbaiju, Gongju, Boju, Chuju. However, only Hangbaiju, a significant species, was cultivated on a large scale in Zhejiang province, China.The flowering head of Chrysanthemum was used as a herbal tea or remedy for centuries.In the past,it was proved to be antipyretic,detoxicate, stanch bleed, reduce the swell ,and nourish hepar and view.By now,it have been found to possess antitumor, anti-inflammatory, antibacterial and antioxidant activities and to increase the coronary flow. In particular, the extraction of Hangbaiju has attracted more and more attentions for its therapeutic effects to cardiovascular diseases with few or no side effect. 1. Determination of luteolin and apigenin in hydrolysed Flos Chrysanthemi[Objective] This chapter reports the determination of mainly active ingredients in Flos Chrysanthemi by HPLC, and the influence of sources, grades, processing,harvest time, growth stages and storage life on flavone in Hangbaiju, which is one of the most significant chrysanthemum.[Methods] A new HC1 hydrolysis/HPLC method has been developed to determine the free and conjugated forms of luteolin and apigenin in CMR. The hydrolytic procedure was optimized by a two indexes orthogonal experimental design in which HC1 concentration, methanol concentration, hydrolysis temperature and hydrolysis time were only considered. Quantification of active ingredients was done by HPLC at 35°C using Agilent Zorbax SB-C18 column (4.6mm x 250 mm, 5 urn ), the methanol: 0.25% phosphoric acid aqueous solution (51:49, v/v) mobile phase and DAD at 344 nm.[Results] The optimum hydrolytic conditions for luteolin and apigenin were obtained: 2.4 mol/L HC1, 80% (v/v) methanol, 80 °C hydrolysis temperature and 2h hydrolysis time.The assay was linear from 2.233~71.456 ugml"1 andl.668~ 53.376 ng-ml"1 for luteolin and apigenin. the RSD for their variation coefficients were 2.4%> 1.6%(intra-day) and 3.6% 3.2%(inter-day). Their average method recoveries were 100.3%(RSD 0.83%) and 103.9%(RSD 0.91%), respectively. In this paper, it was observed that variety ,sample sources, grades, processing and harvest time significantly influenced the mainly bioactive ingredients. The content trend of luteolin and apigenin was similar in Hangbaiju.These samples of early harvest time,good grade and initial growth stage were rich in luteolin and apigenin.However,flavone is not influenced by storage life obviously.[Conclusions] The method developed is accurate, sensitive and simple which can be applied to determination of luteolin and apigenin in Flos Chrysanthemi simultaneously. The contents of luteolin and apigenin were influenced by many factors and these two compounds could be used as index ingredients in Flos Chrysanthemi.2. Determination of three components of essential oil in Flos Chrysanthemi simultaneously[Objective] This chapter reports the determination of mainly active ingredients of essential oil in Chrysanthemum morifolium Ramat.(CMR) by GC and the influenceof sources, grades, processing, harvest time, growth stages and storage life on essential oil in Hangbaiju,which is one of the most significant chrysanthemum. Some useful information was provided for quality control.[Methods] Orthogonal experiment is completedly employed in preliminary test. For example, an optimal extraction of essential oil in CMR was obtained by L9 (34) orthogonal test in which only four main factors, i.e. extraction time, soak time,powder size and volume of water concentration were tested. For each variable, the influence on extraction efficiency was considered from three levels. In this paper, n-tetradecane and n-octanol were used as internal standard substances for P-elemene and camphor, borneol respectively. Essential oil was analysed by a SHIMADZU GC-14B equipped with a flame ionization detection (FID) system and a split capillary inlet port. A Hp-5 (30mx 0.32mm i.d., 0.25um film thickness) capillary column was used. The column temperature was 70°C to 260 °C and rose by the program: 70 °C 2.5 °C-min*' 140 °C 10 "C-min"1 260 °C, and the temperatures of the injector and the detector were 280°C and 300°C, respectively. Split ratio was 20:1. Flow rates were lml/min for the nitrogen carrier gas, 350 ml-min"1 for air, and 70 ml-min"1 for hydrogen.[Results] At last, the hydrodistillation was performed at the following contions: 50g powder whose diameter was about 0.355 mm was heated for 5h in 500ml water. The linear ranges of P-elemene,camphor and borneol were 0.6250~10.00mgml"1, 0.02231~0.3570 mg-ml"1 and 0.01143~0.1828 mg-ml"1 respectively. The RSD for their repeatability were 0.90% , 1.7%, 1.9%. Their average recoveries were 98.2% (RSD 1.7%),97.8(RSD 0.45%) and 100.4(RSD 1.6%). The content of p-elemene did not change significantly during the harvest time, while it changed at some degree in Flos Chrysanthemi from different varieties,sources, grades, processing, growth stages and storage life.As for camphor and borneol, their contents were considerably poor and varied irregularly.The content of P-elemene, was influenced by different varieties , sources, grades, processing, harvest time, growth stages and storage life.[Conclusions] The method developed is accurate, sensitive and simple which can be applied to determination of these three components in Flos Chrysanthemisimultaneously. The contents of above three ingredients of essential oil were influenced by many factors in Flos Chrysanthemi and this information was probably helpful for quality control. 3. GC-MS fingerprinting of volatile oils of Flos Chrysanthemi[Objective] To apply GC-MS fingerprints to identification and quality control of essential oil in Flos Chrysanthemi.[Methods] The capillary column was a DB — 5MS (0.25mm*30m*0.25um). Oven temperature increases by 80°C 2°Cmin'' 100 °C (45min) 0.5'C-min'1 132 "C 5 "Omui"1 250 °C (lOmin) . The 1.0 ml-min"1 Nitrogen was used as carrier gas. The injected volume was 2 u 1 .The Agilent6890N/5973 mass spectrometer was used. It is an ion trap. Its detector was used in 70 eV electronic impact ionisation,scanning between 50 and 500 (m/z). In addition, their chromatograms of Chrysanthemum essential oil were evaluated by similarity and hierarchical clustering analysis.[Results] The approach for fingerprint of essential oil is accurate, sensitive and simple. The similarity of genuine Hangbaiju was over 0.92 and clustering analysis could be appllied to the identification of Flos Chrysanthi from different areas or varieties.[Conclusions] the newly developed Fingerprints of essential oil by GC-MS may represent a contribution to identification and quality control of chrysanthemum.