This is a poster presentation given by an undergraduate researcher in the Quave Lab in April 2016. Hypericum perforatum (St. John’s wort) is a well known medicinal herb often associated with the treatment of anxiety and depression. However, an oleolite preparation of the flowers is also widely used in traditional medicine across Eastern Europe and the Balkans. Recent research has shown that this oleolite reduces both wound size and healing time. H. perforatum has been well characterized chemically. Many secondary metabolites have been identified including: naphthodianthrones (hypericin), phloroglucinols (hyperforin), flavonoid glycosides (hyperoside), biflavones and anthocyanidins. The phloroglucinol hyperforin and its derivatives have also been reported as being responsible for its antibacterial activity.8 However, phloroglucinols are quite unstable with light and heat, and thus should not be present in the aged oleolite preparation of H. perforatum. Additionally, hypericin can cause phototoxic skin reactions if ingested or absorbed into the skin, as evidenced by livestock that develop extreme photosensitivity after grazing on H. perforatum flowers.4 Therefore, the established chemistry presents an interesting paradox to the traditional preparation of H. perforatum. The hyperforin responsible for the antibacterial bioactivty should degrade in the sunlight as the traditional oleolite is prepared. Alternately, if hypericin is present in established bioactive levels, then the traditionally prepared oleolite should cause photosensitivity, yet none is reported. In this research, an organic and aqueous laboratory extract of H. perforatum were compared to a traditional oleolite to better understand the chemical composition of this remedy.

1. Identifying Active Compounds Extracted from Hypericum
perforatum to Characterize a Traditional Oleolite
Austin Kim1*, James T. Lyles2, Cassandra L. Quave2,3
1Department of Chemistry, Emory University, Atlanta, GA; 2Center for the Study of Human Health, Emory University, Atlanta, GA;
3Department of Dermatology, Emory University School of Medicine, Atlanta, GA
*E-mail: akim96@emory.edu Lab Website: http://etnobotanica.us/
This work was supported by Dr. Cassandra Quave’s research development funds. The authors would also like to thank to Dr. Fred Strobel of the
Department of Chemistry for assistance with the mass spectrometry experiments, software, and data interpretation.
1. Bendini, A., Bonoli, M., Cerretani, L., Biguzzi, B., Lercker, G., & Gallina Toschi, T. (2003). Liquid-liquid and solid-phase extractions of phenols from virgin olive oil and their separation by chromatographic and electrophoretic
methods. Journal of Chromatography A, 985(1-2), 425–433. http://doi.org/10.1016/S0021-9673(02)01460-7
2. Blatter, A. (2001). HPTLC Investigations of St . John ’ s Wort. CAMAG Scientific
3. Hajdari, A., Mustafa, B., Nebija, D., Kashtanjeva, A., Widelski, J., Glowniak, K., & Novak, J. (2014). Essential oil composition and variability of Hypericum perforatum L. from wild population in Kosovo. Current Issues in Pharmacy and
Medical Sciences, 27(1), 51–54. http://doi.org/10.2478/cipms-2014-0013
4. Klemow, K. M., Bartlow, A., Crawford, J., Kocher, N., Shah, J., & Ritsick, M. (2011). Chapter 11: Medical Attributes of St. John’s Wort (Hypericum perforatum). Herbal Medicine: Biomolecular and Clinical Aspects., 1–43. Retrieved from
http://www.ncbi.nlm.nih.gov/books/NBK92750/nhttp://www.ncbi.nlm.nih.gov/pubmed/22593920
5. Maisenbacher, P., & Kovar, K. a. (1992). Analysis and stability of Hyperici oleum. Planta Medica, 58(4), 351–354. http://doi.org/10.1055/s-2006-961483
6. Montedoro, G., & Servili, M. (1992). Simple and hydrolyzable phenolic compounds in virgin olive oil. 1. Their extraction, separation, and quantitative and semiquantitative evaluation by HPLC. Journal of Agricultural and Food Chemistry,
40, 1571–1576. http://doi.org/10.1021/jf00021a019
7. Pirisi, F. M., Cabras, P., Cao, C. F., Migliorini, M., & Muggelli, M. (2000). Phenolic compounds in virgin olive oil. 2. Reappraisal of the extraction, HPLC separation, and quantification procedures. Journal of Agricultural and Food
Chemistry, 48(4), 1191–1196. http://doi.org/10.1021/jf991137f
8. Saddiqe, Z., Naeem, I., & Maimoona, A. (2010). A review of the antibacterial activity of Hypericum perforatum L. Journal of Ethnopharmacology, 131(3), 511–521. http://doi.org/10.1016/j.jep.2010.07.034Servili, M., Baldioli, M.,
Selvaggini, R., Miniati, E., Macchioni, A., & Montedoro, G. (1999). High-performance liquid chromatography evaluation of phenols in olive fruit, virgin olive oil, vegetation waters, and pomace and 1D- and 2D-nuclear magnetic
resonance characterization. Journal of the American Oil Chemists’ Society, 76(7), 873–882. http://doi.org/10.1007/s11746-999-0079-2
9. Tatsis, E. C., Boeren, S., Exarchou, V., Troganis, A. N., Vervoort, J., & Gerothanassis, I. P. (2007). Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR and/or LC/MS. Phytochemistry, 68(3), 383–393.
http://doi.org/10.1016/j.phytochem.2006.11.026
ABSTRACT
Hypericum perforatum (St. John’s wort) is a well known medicinal herb often associated with the treatment of anxiety and depression. However, an
oleolite preparation of the flowers is also widely used in traditional medicine across Eastern Europe and the Balkans. Recent research has shown that
this oleolite reduces both wound size and healing time. H. perforatum has been well characterized chemically. Many secondary metabolites have been
identified including: naphthodianthrones (hypericin), phloroglucinols (hyperforin), flavonoid glycosides (hyperoside), biflavones and anthocyanidins. The
phloroglucinol hyperforin and its derivatives have also been reported as being responsible for its antibacterial activity.8 However, phloroglucinols are
quite unstable with light and heat, and thus should not be present in the aged oleolite preparation of H. perforatum. Additionally, hypericin can cause
phototoxic skin reactions if ingested or absorbed into the skin, as evidenced by livestock that develop extreme photosensitivity after grazing on H.
perforatum flowers.4 Therefore, the established chemistry presents an interesting paradox to the traditional preparation of H. perforatum. The hyperforin
responsible for the antibacterial bioactivty should degrade in the sunlight as the traditional oleolite is prepared. Alternately, if hypericin is present in
established bioactive levels, then the traditionally prepared oleolite should cause photosensitivity, yet none is reported. In this research, an organic and
aqueous laboratory extract of H. perforatum were compared to a traditional oleolite to better understand the chemical composition of this remedy.
RESEARCH AIMS
To compare the composition of a traditionally prepared H. perforatum remedial oleolite to an organic
and aqueous extract by means of chromatography and spectrometry in order to determine which
active compounds are present in each, elucidating the oleolite’s paradoxical composition.
REFERENCES
METHODS
Aerial parts (flowers, stems, leaves) of H. perforatum were ground in a Wiley Cutting Mill
through a 2mm mesh, sonicated 2 x 20 min in methanol or deionized water, vacuum
filtered through a coarse and fine filter, taken off in a rotary evaporator, redissolved in
deionized water, freeze dried in a lyophilizer, and scraped out into a scintillation vial.
The extracts were then dissolved in 2mg/mL solutions of methanol and run in thin-
layer chromatography, high performance liquid chromatography, and Fourier transform
mass spectrometry against a number of standards at similar concentrations.
The H. perforatum olive oil oleolite tested in this work was procured by Dr. Cassandra
Quave in Kosovo and was run through HPLC and MS in a 20% ethyl acetate solution.
CONCLUSIONS
• The traditional Kosovar oleolite does contain hyperforin, yet at levels higher than
published in current literature (<5% vs. nearly 15%), continuing to contradict the
oleolite’s remedial properties and hyperforin’s natural oxidative breakdown in light.
• Hypericin is also absent from all three preparations, but the oleolite and methanol
extracts retain their deep red color derived from hypericin’s chromophore system.
• Results from extracts of CQ-379 cannot be directly transposed to exploring the
chemistry of the traditional oleolite (flowers vs. aerial parts)
• Future studies:
- Work with extracts of H. reductum blossoms gathered from South Florida
- Utilize flash chromatography to simplify various extracts into active fractions
- End goal: test active fractions against antibacterial MIC assays adapted for S.
aureus, aimed at finding the MICs of the pure compounds extracted from the
genus Hypericum
FUNDING&
THANKS
FIG. 1. H. perforatum and its red olive oil oleolite
FIG. 2. Hypericin and hyperforin, two of the
active compounds studied in this work
FIG. 3. From left to right, depicting the extraction
of ground CQ-379 aerial parts through sonication
(above case, in methanol), filtration, rotary
evaporation, and lyophilization
RT: 0.00 - 80.03
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80
Time (min)
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
NL:
1.02E7
Base Peak F:
FTMS - p ESI
Full ms
[150.00-
1500.00] MS
32816-1
NL:
8.34E6
Base Peak F:
FTMS - p ESI
Full ms
[150.00-
1500.00] MS
32816-2
NL:
8.61E6
Base Peak F:
FTMS - p ESI
Full ms
[150.00-
1500.00] MS
32816-3
RESULTS
Thin-Layer Chromatography
Mobile: Ethyl acetate, dichloromethane, formic, acetic acid (100:25:10:10)
Visualization Reagent: 2% vanillin in sulfuric acid spray
Standards and methodology were chosen and modified on a previously
published H. perforatum HPTLC protocol by CAMAG Scientific2
FIG. 4. Plate CQ 012-78-01 shows that the oleolite is not
compatible with this technique, and requires a different
chromatographic approach—while plate CQ 012-93-02
demonstrates that certain standards may not appear in a given
extract, facilitating further analytical processes
RESULTS
High Performance Liquid Chromatography
FIG. 5. Dissolving the oleolite in 20% ethyl acetate was
determined to be the most effective method of observing
peaks and matching them to the respective standards
Time (min) A% B%
0.00 95.0 5.0
2.00 95.0 5.0
10.00 75.0 25.0
20.00 60.0 40.0
30.00 50.0 50.0
40.00 0.0 100.0
70.00 0.0 100.0
70.01 95.0 5.0
80.00 95.0 5.0
Column Agilent XDB-C18 250x4.6, 5 μm @ 25°C
Mobile: (A) 0.1% formic in H2O (B) 0.1% formic in ACN @ 1 mL/min
Sample CQ 012-83-06 detected @ 278 nm
TABLE 2. Gradient
protocol was adapted and
extended from
Montedoro et al. (1992)6
for maximal elution
appropriate for both the
standards and the oleolite
RESULTS
Fourier Transform Mass Spectrometry
FIG. 6. FTMS analysis of the oleolite (black), methanol extract (red), and water
extract (green). Selected peaks labeled and known compounds identified.
FIG. 7. Determining the presence and identity of a compound in a sample
Left: a quercitin standard, Right: quercitin peaks identified in the oleolite
Peak RT (m) % Area M+ Δ
2* 20.02 4.71 C15H9O7 0.05
8 25.52 19.20 C18H17O14N9 -0.58
15 41.56 23.33 C26H59O5N10 -1.91
16 41.83 9.91 C26H59O7N10 -2.91
27* 50.84 14.86 C35H51O4 1.45
TABLE 3. Selected peaks charted in
order of retention time, with percent
area, predicted molecular ion, and degree
of accuracy. Identified compounds are
marked with an asterisk on Figure 6
Peak RT (m) % Area M+ Δ
26* 22.91 1.56 C15H9O7 0.01
27 26.47 19.20 C30H13O7N7 -0.27
52 41.14 2.15 C23H53O11N9 -0.73
59 46.43 2.16 C30H43O4 0.24
60 47.19 3.09 C29H43O3N3 1.07
63 50.09 9.95 C22H49O6N9 -1.10
64 51.34 2.10 C23H51O6N9 -2.19
Peak RT (m) % Area M+ Δ
3 3.46 11.72 C14H23O12 0.33
11* 11.17 5.48 C16H17O9 0.60
15* 12.36 3.44 C16H17O8 0.50
18 13.11 7.58 C21H27O6N4 -0.43
26 15.47 19.85 C22H15O8N4 -0.17
29 16.14 11.93 C22H15O8N4 -0.17
30 16.37 12.67 C22H17O11N4 0.36
*153-p-coumaroylquinic acid
9
*11chlorogenic acid
*26quercitin
*2quercitin
*27hyperforin
Standard Found in CQ-379? Avg. Calc. Rf Published Rf
Rutin
(Hydrate)
No 0.110 0.112
Chlorogenic
Acid
Yes 0.241 0.218
Hyperoside No 0.255 0.273
Quercitin
(Hydrate)
Yes 0.468 0.437
Hypericin Yes n.d. 0.648
TABLE 1. List of standards and their presence in CQ-379
8
15
16
27
52 59 60
63
64
3
18
29
26
30