Extraction and HPLC of Hypericin
Courtney Arnott
Background
St. Johns Wort is an herbal medication that is often used to treat depression. It has many
active ingredients in it, such as anthracene derivatives, flavenoids, and xanthones that act as
a monoamine oxidase inhibitor (MAOI). It is also believed to affect serotonin uptake in
the brain, similar to the effects of many antidepressants1. By increasing the brain’s ability to
uptake and use serotonin, a sense of happiness and well-being is produced. Hypericin
(m.w. 504) is an anthracene derivative found in St. Johns Wort. While not believed to be
the active compound, it is directly related to the levels of active compounds in the drug and
thus is commonly used as a marker for the flavenoids that are believed to be the active
compounds. It is a blue-black crystalline substance that produces a cherry-red solution in
organic bases2.
Hypericin has many uses in and of itself. In addition to being used for depression and
anxiety, it can also be prescribed for afterbirth pains, sleep disorders, cuts, and burns. Most
recently, preliminary research has shown that hypericin shows anti-cancer and antiretroviral properties, thus making it look promising as a new AIDS treatment3.
St. Johns Wort, like most other herbal medications, has not been evaluated or approved by
the FDA; thus there are no regulations or standards for its production or safety. However,
many people continue to use the drug as an over-the-counter remedy. Since hypericin is a
marker that is easily extracted and characterized, this experiment was designed to look at
hypericin levels in different lot numbers of St. Johns Wort. Levels of hypericin were
calculated through the use of High Pressure Liquid Chromatography to compare the
consistency of dosages among lot numbers of three different brands. Two generic brands,
from CVS and Food Lion, and one name brand, Nature’s Resource, were selected for
comparison due to availability and price.
Experimental
Sample Preparation
The experimental method used was adapted from “Hypericin and Pseudohypericin by
HPLC.”4 A mobile phase solution was prepared for use in HPLC analysis. The solution
consisted of 66.6% methanol, 17.2% phosphate buffer at pH 2.1, and 16.2 % ethyl acetate
by volume. Standards of hypericin were prepared in methanol at concentrations of 1:1,
1:10, 1:100, and 1:1000 from a stock solution of 1mg/mL hypericin. The standards were
wrapped in foil to avoid excessive light exposure, since hypericin is a photosensitive
compound and can degrade in the presence of light.
The next step was preparation of St. Johns Wort tablets and the extraction of hypericin
from the tablets. Tablets were crushed with a mortar and pestle; capsules were opened and
the plastic casings discarded. 750 mg St. Johns Wort powder was mixed with 40 mL
methanol. The flasks were sonicated for 15 minutes to completely dissolve the powder.
The solution was then diluted to volume. A 10 mL aliquot was removed and centrifuged at
3400 rpm for 5 minutes to separate the liquid from excess tablet material. The supernatant
was removed to a 50 mL beaker. The procedure was repeated to give a total volume of 10
mL extracted sample.
The beakers were covered with plastic wrap and exposed to fluorescent light for 30
minutes to prepare for analysis. The beakers were then covered with aluminum foil and
stored in a dark place until ready for injection.
HPLC Analysis
Chromatography analysis was run using two Waters 510 HPLC Pumps, a Waters 600s
Controller, and a Waters 996 Photodiode Array Detector. The stationary phase was a
Nova-Pak C18 3.9x150mm column, and the mobile liquid phase was a solution of 66.6%
methanol, 17.2% phosphate buffer (pH 2.1) and 16.2% ethyl acetate.
HPLC pumps were primed with filtered methanol according to the procedure described by
S. Piepho and M. Moshier.5 The pumps were then switched over to filtered mobile phase
solution via an equilibrate process. A 50 uL sample of hypericin extract was injected and
run through the HPLC for 10 minutes. The obtained peak, at approximately 6 minutes,
was integrated and the areas fitted to a standard concentration curve to determine the
concentration in each sample.
Results/Conclusions
All lot numbers of St. Johns Wort showed some variation from the actual amounts of
hypericin said to be contained within the tablet (30 ppm).
Concentration
(ppm)
CVS
Food Lion
31.63
25.18
24.27
22.97
25.38
24.46
Nature's
Resource
34.06
37.79
38.48
Table 1. Concentrations of hypericin in St. Johns Wort samples.
Statistical analysis was conducted using Microsoft Excel to calculate the average ppm for
each brand and the average deviation for the samples from the average concentration. The
average deviation was then tripled to find the daily deviation from the average that can be
expected from each brand, since all brands recommended taking the tablets three times a
day.
Average ppm
AveDeviation
DailyDeviation
CVS
Food Lion
27.03
3.07
9.21
24.03
0.87
2.61
Nature's
Resource
36.78
1.81
5.43
Table 2. Average concentrations and statistical deviations.
The daily deviation was added and subtracted to the average concentration of hypericin to
show the range of concentrations that could be ingested in one day’s worth of
medication.
High Dose
Low Dose
CVS
Food Lion
90.29
71.87
75.41
70.21
Nature's
Resource
115.76
104.90
Table 3. Range of concentrations of hypericin in daily dosage.
The Food Lion brand St. Johns Wort showed the most consistent dose among different lot
numbers, with a range of 5.2 ppm between the high dose and the low dose. The CVS
brand showed the highest variation, with a range of 18.42 ppm. The brand name Nature’s
Resource was in the middle, with a range of 10.86 ppm.
Based on these variations, FDA evaluation would be necessary to assure safe and consistent
dosages for patients taking this medication. Further research in this area is necessary,
including repeating this experiment with trials of a single lot number to determine how
much of the variation can be expected from the experimental method, and comparing
these results to a medication that is regulated by the FDA to determine how much variation
can be expected from the industry standard.
Endnotes
1. “Hypericum Perforatum.” PDR for Herbal Medicines, p. 906.
2. “Hypericin.” DialogueWeb. www.dialogueweb.com (accessed 2-9-00).
3. “St. Johns Wort.” Woodland Publishing, Inc. www.nutraceutical.com/education
(accessed 4-26-00).
4. “Hypericin and Pseudohypericin by HPLC.” Institute for Nutraceutical
Advancement – Methods. www.nutraceuticalinstitute.com/methods/index.html
(accessed 4-4-00).
5. “HPLC Set-up.” S. Piepho and M. Moshier, Sweet Briar College. 3-28-01.
Figures
Hypericin Standard Concentration Curve
1400
y = 5E-05x + 19.737
R2 = 0.9985
1200
Concentration (ppm)
1000
800
600
400
200
0
0
5000000
10000000
15000000
20000000
25000000
30000000
Integral Area
Figure 1. Hypericin Standard Concentration Curve.
Figure 2. Comparison of ppm Between St. Johns Wort Samples.
(Red = Nature’s Resource Samples, Yellow = Food Lion Samples, Blue = CVS Samples)