HPLC analysis of chlorogenic acid from Flos Lonicerae
Flos Lonicerae, also called Jinyinhua, is one of the widely used herbs in many Chinese medicine prescription. It has latent-heat-clearing, antipyretic, detoxicant and anti-inflammatory actions. It has been, therefore, traditionally used as a herbal medicine in the treatment of a wide range of ailments, including syphilitic skin diseases, tumors, bacterial dysentery, colds, enteritis, pain, swellings, etc
In many other studies, chlorogenic acid, one of the major components in Flos Lonicerae, has been widely adopted to evaluate the quality of Flos Lonicerae owing to its high content and antibiotic property. Several kinds of the other components, such as iridoid glucosides ,flavonoids, and saponins havealso been discovered by using advanced analytical techniques. Chlorogenic acid is identified and further purified by 70% ethanolic extract with high performance liquid chromatography (HPLC) and its anti-oxidant capacity is identified.
1. Preparation of Flos Lonicerae extracts
Flos Lonicerae sample was dried to 50 °C keep constant weight. About 10 g of pulverized sample was added into a round-bottomed flask containing 250 ml different solvents including distilled water, 70% ethanol and methanol. The mixture was heated under reflux for 4 h. The extracts were filtered and the residue was re-extracted under the same conditions. The extracts were combined, concentrated and evaporated to dried powder in rotary evaporator under reduced pressure. The residue was dissolved with 30 ml of water into a 100 ml flask and the product was stored at 4 °C for 24 h followed by centrifugation at 10 000 r/min for 30 min. The supernatant fraction was Flos Lonicerae crude extract and lyophilized to fine powder for long term storage and dissolved in methanol for identification and quantification analysis with HPLC.
2. HPLC analysis
The dissolved extracts were filtered through 0.22 μm membrane filter before loaded into the HPLC system. Chromatography was carried out on C18 analytical / preparative column at 25 °C. The sample loop of 20 μl or 1 ml was used for sample injection. The mobile phase consisted of two solvent components: water (solvent A) and acetonitrile-citrate acid water (20:40:40, v/v, solvent B). In the gradient elution solvent B was increased from 5%~100% within 30 min. The flow rate was 0.2 ml/min for analysis and 0.8 ml/min for preparation. The wavelength used to detect chlorogenic acid was 324 nm. Twenty microlitres of different concentrations (0.02~0.50 mg/ml) of chlorogenic acid reference was loaded into HPLC system for construction of the calibration curve by plotting the peak areas (Y) versus the concentrations (X). Each extract powder was dissolved with methanol to a concentration of 0.5 mg/ml and 20 μl of the crude extract was loaded into HPLC system for identification and quantification of chlorogenic acid in Flos Lonicerae. The content of chlorogenic acid in Flos Lonicerae was determined by the corresponding calibration curve and was expressed as milligram of chlorogenic acid per gram of Flos Lonicerae. The peak area of chlorogenic acid in each extract was the mean of three parallel measurements in chlorogenic acid quantification. The chromatography peak was identified by comparing the retention time with that of chlorogenic acid reference. The chlorogenic acid used for antioxidant activity assay was produced by preparative column separation and manually collected according to the retention time and chromatography peak of chlorogenic acid under the same chromatography conditions.