extraction and hplc of hypericin courtney arnott intermediate lab fall 2001

Extraction and HPLC of Extraction and HPLC of HypericinHypericin

Courtney Arnott

Intermediate Lab

Fall 2001

Hypericin – Background and Hypericin – Background and UsesUses

C30H16O8

Marker for active compounds in St. Johns Wort

Uses: AIDS, cancer, afterbirth pains, cramps, depression, bacterial and viral infections

PurposePurpose

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 HypericinExtraction of Hypericin

Three lot numbers of three different brands of St. Johns Wort

750 mg tablet powder in 40 mL methanolSonicate for 15 minutes, dilute to volume

(50 mL)Remove 10 mL, centrifuge for 15 minutesExpose to fluorescent light for 30 minutes

HPLCHPLC

High Pressure Liquid ChromatographyStationary phase and mobile phaseDiodes measure absorbance across broad

band of spectraCan look at complete UV-vis spectra or

chromatograph at particular wavelength

HPLC TheoryHPLC Theory

Molecules move through column, “stick” to chains of carbon (system is under extreme pressure – 2000+ psi)

Retention time based on size of moleculeColumn separates molecules by size, reach

detector at different times for absorbance readings

HPLC ColumnHPLC Column

C18 column – 18-carbon chains (stationary phase) attached to silica beads on inside of column

Mobile Phase SolutionMobile 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 MethodHPLC Method

Run solvent blank of methanolInject samples10 minute methodObserve chromatography at 590nm (visible

light range)

Problems and SolutionsProblems and Solutions

Pressure problems with Pump C

Small particles in solvents – changed filters

Sonicated frits to remove debris

Changed pre-column

Methanol BlankMethanol Blank

HPLC Data - StandardHPLC Data - Standard

HPLC Data - SampleHPLC Data - Sample

Data AnalysisData 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 OverlayUV-vis Overlay

Further Data AnalysisFurther Data Analysis

Millennium Software integrates peak areaMore absorbance = larger peak area = more

concentrated sampleUnknown area of sample is put into

y=mx+b equation from standard curveSolve equation to find concentration of

sample in ppm

Standard Concentration CurveStandard Concentration Curve

Concentrations of SamplesConcentrations of Samples

Deviation of SamplesDeviation of SamplesCVS Food Lion Natures

Resource

Concentration 31.63 22.97 34.06

(ppm) 25.18 25.38 37.79

24.27 24.46 38.48

Ave ppm 27.03 24.27 36.78

Ave Deviation 3.07 0.87 1.81

Daily Deviation 9.21 2.60 5.43

High Dose 90.29 75.41 115.76

Low Dose 71.87 70.21 104.90

DiscussionDiscussion

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

ConclusionConclusion

Recommended safe daily dosage for humans is 90 ppm – anywhere from 2.34 to 3.92 300 mg tablets

Data on lethal human dosage unavailable

FDA regulations necessary to ensure safe and consistent dosages

Further ResearchFurther Research

Compare results with an FDA regulated medication to further quantify data

Research into properties/uses of hypericin

ReferencesReferences

DialogueWeb – Hypericin www.dialogueweb.com Dictionary of Organic Compounds – Hypericin (p. 3270) Featured Herb Review – St. John’s Wort

www.viable-herbal.com Institute for Nutraceutical Advancement – Methods

“Hypericin and Pseudohypericin by HPLC” www.nutraceuticalinstitute.com

Planta Natural Products – Hypericin (structure and HPLC graph) http://www.planta.at/hyper/hyper.htm