extraction and hplc of hypericin courtney arnott intermediate lab fall 2001
TRANSCRIPT
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Extraction and HPLC of Extraction and HPLC of HypericinHypericin
Courtney Arnott
Intermediate Lab
Fall 2001
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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
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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.
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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
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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
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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
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HPLC ColumnHPLC Column
C18 column – 18-carbon chains (stationary phase) attached to silica beads on inside of column
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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
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HPLC MethodHPLC Method
Run solvent blank of methanolInject samples10 minute methodObserve chromatography at 590nm (visible
light range)
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Problems and SolutionsProblems and Solutions
Pressure problems with Pump C
Small particles in solvents – changed filters
Sonicated frits to remove debris
Changed pre-column
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Methanol BlankMethanol Blank
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HPLC Data - StandardHPLC Data - Standard
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HPLC Data - SampleHPLC Data - Sample
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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
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UV-vis OverlayUV-vis Overlay
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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
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Standard Concentration CurveStandard Concentration Curve
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Concentrations of SamplesConcentrations of Samples
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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
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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
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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
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Further ResearchFurther Research
Compare results with an FDA regulated medication to further quantify data
Research into properties/uses of hypericin
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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