HPLC analysis of chlorogenic acid from Flos Lonicerae

HPLC analysis of chlorogenic acid from Flos Lonicerae

Flos Lonicerae, also called Jinyinhua, is one of the widely used herbs prescribed in many Chinese formulas. 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, bacterialdysentery, colds, enteritis, pain, swellings, etc

In many other studies, chlorogenic acid, one of the major components in Flos Lonicerae, has been widely adopted to control 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 have been also discovered and characterized using advanced analytical techniques due to their effective activities. Chlorogenic acid is identified and further purified from 70% ethanolic extract with high performance liquid chromatography (HPLC) and its antioxidant capacity is characterized.

Methods

1. Preparation of Flos Lonicerae extracts

Flos Lonicerae sample was dried at 50 °C to constant weight. Approximately 10 g of pulverized sample was added to a round-bottomed flask containing 250 ml of different solvents including distilled water, 70% ethanol and methanol, respectively. 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 dryness with 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 designated as Flos Lonicerae crude extract and lyophilized to fine powder for long term storage and dissolved with methanol for identification and quantification analysis with HPLC.

2. HPLC analysis

The dissolved extracts were filtered through 0.22 μm membrane filter before loading 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 acidwater (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 analytical and 0.8 ml/min for preparative purpose respectively. 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 respectively. The content of chlorogenic acid in Flos Lonicerae was determined from the corresponding calibration curve and the content was expressed as milligram of chlorogenic acid per gram of Flos Lonicerae. The peak area of chlorogenic acid in each extract was mean of three parallel measurements for 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 through preparative column separation and manually collected according to the retention time and chromatography peak of chlorogenic acid under the same chromatography conditions.

 

Flos Lonicerae, also called Jinyinhua, is one of the widely used herbs prescribed in many Chinese formulas. 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, bacterialdysentery, colds, enteritis, pain, swellings, etc

 

In many other studies, chlorogenic acid, one of the major components in Flos Lonicerae, has been widely adopted to control 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 have been also discovered and characterized using advanced analytical techniques due to their effective activities. Chlorogenic acid is identified and further purified from 70% ethanolic extract with high performance liquid chromatography (HPLC) and its antioxidant capacity is characterized.

Methods

1. Preparation of Flos Lonicerae extracts

Flos Lonicerae sample was dried at 50 °C to constant weight. Approximately 10 g of pulverized sample was added to a round-bottomed flask containing 250 ml of different solvents including distilled water, 70% ethanol and methanol, respectively. 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 dryness with 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 designated as Flos Lonicerae crude extract and lyophilized to fine powder for long term storage and dissolved with methanol for identification and quantification analysis with HPLC.

2. HPLC analysis

The dissolved extracts were filtered through 0.22 μm membrane filter before loading 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 acidwater (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 analytical and 0.8 ml/min for preparative purpose respectively. 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 respectively. The content of chlorogenic acid in Flos Lonicerae was determined from the corresponding calibration curve and the content was expressed as milligram of chlorogenic acid per gram of Flos Lonicerae. The peak area of chlorogenic acid in each extract was mean of three parallel measurements for 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 through preparative column separation and manually collected according to the retention time and chromatography peak of chlorogenic acid under the same chromatography conditions.