identifying active compounds extracted from hypericum perforatum to characterize a traditional...

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: [email protected] 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.

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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

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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)

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NL:1.02E7

Base Peak F: FTMS - p ESI Full ms [150.00-1500.00] MS 32816-1

NL:8.34E6


NL:8.61E6


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


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


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 acid9

*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