Studies On The Characteristic Components’ Separation And Content Determination Of Lonicera Similes Which Are Lonicera’s Mainstream Varieties In Sichuan

Lonicera similes Hemsl, which was contained in<Sichuan Traditional Chinese medicine standards>(2008), is one of the Lonicera’s basic source plant, and it is also the Lonicera’s mainstream variety in Sichuan. In order to coordinate the key projects "Studies on the characteristic components’separation and Content Determination of Lonicera similes Hemsl which are Lonicera’s mainstream varieties in Sichuan" of the education department in Sichuan Province and on the basis of previous research work, this paper focused on the separation and purification of flavonoids and saponins which are effective part of Lonicera similes Hemsl, and chose the specific index component to establish the qualitative and quantitative analysis method. The research has provided technical support for perfecting and improving the existing quality evaluation criteria of honeysuckle in Sichuan, promoted the standards of herbs in Sichuan from the "Local standards" to the "National standards", expanded the use of superior resources, made the transformation from natural resources advantages into economic advantages in Nanjiang which is the poor area in Sichuan, and promoted the local economic development. The results were as follows:1In order to find the specific ingredients, this paper directly extracted and separated two effective parts--flavonoids and saponins, from the known honeysuckle herbs except for organic acids, rather than used the traditional mode——system solvent extraction separation pharmacodynamic screening-active separation and purification.1.1It was the first time to research on a purification process for the total flavonoids from Lonicera similes Hemsl by macmporous resin.This paper which focused on the purification process of total flavonoids in Lonicera similes Hemsl, used the static adsorption rate and the resolution rate as indicators to inspect the different macroporous adsorption resin in five polar, and selected the suitable type of macroporous resin. It also determined the optimum purification process for the total flavonoids from Lonicera similes Hemsl by examining the related factors which inflenced the dynamic adsorption and desorption. The optimum purification process was as follows:D101macroporous resin was used to purify the extract of Lonicera similes Hemsl, and the sample solution concentration is0.10g crude drug/ml and the rate amount is2.74g crude drug/1g dry resin.1.2Every flow of macroporous resin got from Lonicera similes Hemsl was isolated and purified with macroporous resin, silica gel column chromatography, Gel chromatography, ect. then11compounds were got. Many spectrum techniques such as1H NMR,13C NMR combined with chemical reactions, reference substance and literature contrast were used to confirm structures of9chemical compound, which respectively were5-hydroxyl-7,4’-dimethoxyflavone(Ⅰ), rutin(Ⅲ), quercetin(Ⅳ), amentoflavone(Ⅴ), aesculetin(Ⅶ), β-sitosterol(Ⅷ),5-hydroxyl-7,3’,4’-trimethoxyflavone(Ⅸ), oleanolic acid(Ⅹ), β-Daucosterin(Ⅺ). Among the nine isolated compounds, compounds Ⅰ Ⅲ、Ⅳ、Ⅴ、Ⅸ are flavonoids. Compounds Ⅰ,Ⅴ,Ⅶ,Ⅸ,Ⅹ,Ⅺ were isolated from Lonicera similes Hemsl. for the first time.2TLC identification for the5-hydroxyl-7,3’,4’-trimethoxyflavone in Lonicera similes Hemsl was used for the first time, and its microemulsion TLC identification method was established.Microemulsion TLC identification method of5-hydroxyl-7,3’,4’-trimethoxyflavone in Lonicera similes Hemsl had been established through inspecting the extraction method, the hydrolysis time, the system, the sample volume, the expand distance and temperature, the saturation time of TLC plate, etc. The moisture content75%of microemulsion-formic acid (9:1) for developing solvent was considered having a good separation effect, a moderate Rf value and clear chromatography spots. Lonicera similes Hemsl counterparts medicines and commodities herbs TLC tested on10batches of different origin, indicated that this method had better daptability and reproducibility to dentify5-hydroxyl-7,3’,4’-trimethoxyflavone.At the same time this method was used for contrasting Lonicera similes Hems1with Lonicerae Japonicae Flos(Lonicerae Japonica Thunb)、Lonicerae Flos (Lonicerae macranthoides Hand.-Mazz, Lonicera fulvotomentosa Hsu et S.C.Cheng,Lonicera confusa DC.) which were collected in the China pharmacopoeia(2010). The5hydroxyl-7,3’,4’-trimethoxyflavone was not found in these varieties except in Lonicerae Japonica Thunb. Althought this compound can be detected in Lonicerae Japonica Thunb, the other spots have differences between Lonicerae Japonica Thunb and Lonicera similes Hemsl. The result indicated this compound used for Lonicera similes Hemsl TLC identification had certain specificity.3The HPLC method to determine the5-hydroxyl-7,3’,4’-trimethoxyflavone in flower buds of Lonicera similes Hemsl was established for the first time.In order to establish The HPLC method to determine the5hydroxyl-7,3’,4’-trimethoxyflavone in flower buds of Lonicera similes Hemsl, we optimized the maximum absorption wavelength and chromatographic conditions. Meanwhile, preparation methods for test solutions, the precision, the stability, the reproducibility and the added recovery test were respectively investigated. The method is simple, fast and easy to operate. The contents of5-hydroxyl-7,3’,4’-trimethoxyflavone in flower buds of Lonicera similes Hemsl from ten different habitats were determined by HPLC respectively. The results showed that the average contents of5-hydroxyl-7,3’,4’-trimethoxyflavone in L.similis was0.68%, while the highest and lowest content is1.08%,0.36%respectively.The contents of5-hydroxyl-7,3’,4’-trimethoxyflavone in Lonicera japonica flos (Lonicera japonica Thunb.) and Lonicerae flos (Lonicera macranthoides Hand.Mazz, Lonicera Confusa DC, Lonicera fulvotomentose Hsu et S.C.Cheng) which are collected in the "Chinese Pharmacopoeia"2010edition, were determined by HPLC respectively. The results showed that only Lonicera japonica Thunb. contains5hydroxyl-7,3’,4’-trimethoxyflavone. The contents were0.14%and0.11%, respectively, which were lower than that in Lonicera similes Hemsl obviously. The result indicated the5-hydroxyl-7,3’,4’-trimethoxyflavone used for content determination of Lonicera similes Hemsl was specific to some extent.