Extraction and HPLC of Hypericin Courtney Arnott Intermediate Lab Fall 2001
Hypericin – Background and Uses C 30 H 16 O 8 Marker for active compounds in St. Johns Wort Uses: AIDS, cancer, afterbirth pains, cramps, depression, bacterial and viral infections
Purpose To determine the amount of hypericin in different lot numbers of brands of St. Johns Wort to see if there is a significant variation from dose to dose.
Extraction of Hypericin Three lot numbers of three different brands of St. Johns Wort 750 mg tablet powder in 40 mL methanol Sonicate for 15 minutes, dilute to volume (50 mL) Remove 10 mL, centrifuge for 15 minutes Expose to fluorescent light for 30 minutes
HPLC High Pressure Liquid Chromatography Stationary phase and mobile phase Diodes measure absorbance across broad band of spectra Can look at complete UV-vis spectra or chromatograph at particular wavelength
HPLC Theory Molecules move through column, “stick” to chains of carbon (system is under extreme pressure – psi) Retention time based on size of molecule Column separates molecules by size, reach detector at different times for absorbance readings
HPLC Column C 18 column – 18-carbon chains (stationary phase) attached to silica beads on inside of column
Mobile Phase Solution 66.6% methanol, 17.2% phosphate buffer (pH 2.1), 16.2% ethyl acetate pH of buffer keeps OH groups from protonating Can adjust concentration to change retention times of peaks
HPLC Method Run solvent blank of methanol Inject samples 10 minute method Observe chromatography at 590nm (visible light range)
Problems and Solutions Pressure problems with Pump C Small particles in solvents – changed filters Sonicated frits to remove debris Changed pre-column
Methanol Blank
HPLC Data - Standard
HPLC Data - Sample
Data Analysis All sample graphs showed two peaks – one at 2-3 minutes and one at 6 minutes First peak suspected to be methanol, second peak hypericin (based on standard data) Confirmed second peak as hypericin by extracting UV-vis spectra on standard and sample at 6 minutes – spectra matched
UV-vis Overlay
Further Data Analysis Millennium Software integrates peak area More absorbance = larger peak area = more concentrated sample Unknown area of sample is put into y=mx+b equation from standard curve Solve equation to find concentration of sample in ppm
Standard Concentration Curve
Concentrations of Samples
Deviation of Samples CVSFood LionNatures Resource Concentration (ppm) Ave ppm Ave Deviation Daily Deviation High Dose Low Dose
Discussion Generic brands showed less hypericin than stated, name brand showed more Generic brand (Food Lion) had most consistent dosage All samples showed some significant variation between dosages Most were still within recommended daily dosage amounts
Conclusion Recommended safe daily dosage for humans is 90 ppm – anywhere from 2.34 to mg tablets Data on lethal human dosage unavailable FDA regulations necessary to ensure safe and consistent dosages
Further Research Compare results with an FDA regulated medication to further quantify data Research into properties/uses of hypericin
References DialogueWeb – Hypericin Dictionary of Organic Compounds – Hypericin (p. 3270) Featured Herb Review – St. John’s Wort Institute for Nutraceutical Advancement – Methods “Hypericin and Pseudohypericin by HPLC” Planta Natural Products – Hypericin (structure and HPLC graph)