1. Extraction and HPLC of Hypericin Courtney Arnott Intermediate Lab Fall 2001

2. 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

3. 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.

4. 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

5. 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

6. HPLC Theory Molecules move through column, “stick” to chains of carbon (system is under extreme pressure – 2000+ psi) Retention time based on size of molecule Column separates molecules by size, reach detector at different times for absorbance readings

7. HPLC Column C 18 column – 18-carbon chains (stationary phase) attached to silica beads on inside of column

8. 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

9. HPLC Method Run solvent blank of methanol Inject samples 10 minute method Observe chromatography at 590nm (visible light range)

10. Problems and Solutions Pressure problems with Pump C Small particles in solvents – changed filters Sonicated frits to remove debris Changed pre-column

11. Methanol Blank

12. HPLC Data - Standard

13. HPLC Data - Sample

14. 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

15. UV-vis Overlay

16. 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

17. Standard Concentration Curve

18. Concentrations of Samples

19. Deviation of Samples CVSFood LionNatures Resource Concentration31.6322.9734.06 (ppm)25.1825.3837.79 24.2724.4638.48 Ave ppm27.0324.2736.78 Ave Deviation3.070.871.81 Daily Deviation9.212.605.43 High Dose90.2975.41115.76 Low Dose71.8770.21104.90

20. 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

21. Conclusion 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

22. Further Research Compare results with an FDA regulated medication to further quantify data Research into properties/uses of hypericin

23. References DialogueWeb – Hypericin www.dialogueweb.comwww.dialogueweb.com Dictionary of Organic Compounds – Hypericin (p. 3270) Featured Herb Review – St. John’s Wort www.viable-herbal.com www.viable-herbal.com Institute for Nutraceutical Advancement – Methods “Hypericin and Pseudohypericin by HPLC” www.nutraceuticalinstitute.com www.nutraceuticalinstitute.com Planta Natural Products – Hypericin (structure and HPLC graph) http://www.planta.at/hyper/hyper.htm