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89ISSN 0326-2383
Acta Farm. Bonaerense 24 (1): 89-90 (2005)Recibido el 17 de agosto de 2004Aceptado el 19 de diciembre de 2004
Comunicaciones breves
KEY WORDS: HPLC, Hypericin, H. styphelioides, H. tetrapetalum, Hypericum nitidum, TLC.PALABRAS CLAVE: Hipericina, HPLC, H. styphelioides, H. tetrapetalum, Hypericum nitidum, TLC.
* Autora a quien dirigir la correspondencia. E-mail: [email protected]
Determination of Hypericin in Hypericum Species Grown in Cuba
Lauro NUEVAS-PAZ 1, Jorge MOLINA-TORRES 2 and Sylvia PRIETO-GONZÁLEZ 1*
1 Centro de Química Farmacéutica; Calle 200 y 21, Atabey, Playa; P.O.Box 16042,La Habana, Cuba; C.P. 11600; Tel. (537)2713994, Fax (537) 33746.
2 Departamento de Biotecnología y Bioquímica, CINVESTAV-IPN, Unidad Irapuato, México.
SUMMARY . Three species of Hypericum grown in Cuba were collected in different seasons and years,dried and powered. The plant material was extracted with methanol and analyzed by HPTLC and reversephase HPLC for hypericin content. In the species of Hypericum analyzed hypericin was not found.RESUMEN. “Determinación de Hipericina en Especies de Hypericum crecidas en Cuba”. Tres especies de Hype-ricum crecidas en Cuba se colectaron, secaron y molieron en estaciones y años diferentes. El material vegetal seextrajo con metanol y se realizó el análisis para determinar el contenido de hipericina mediante HPTLC y HPLCde fase reversa. No se encontró hipericina en las especies de Hypericum analizadas.
INTRODUCTIONThe genus Hypericum consists of more than
370 species. This genus is widely used in folkmedicine since antiquity. Several pharmacologi-cal uses of Hypericum are known worldwide.The most popular plant of this genus is the Hy-pericum perfotatum L. (Saint John’s Wort) main-ly due to its use as a healing agent and by itsanti-depressive and anti-viral applications 1. StJohn’s Wort extract contains at least 10 con-stituents or groups of components that couldcontribute to its pharmacological effects. Thesecomponents include naphthodianthrones (hy-pericin and pseudohypericin), flavonoids (rutin,hyperoside, isoquercitrin, quercitrin and quer-cetin), phloroglucinols (hyperforin and adhyper-forin), tannins (cathechin and epicatechin),proanthocyanidins (procyanidin B2) and bi-flavonoids (biapigenin and amentoflavone) 2.Hypericin and pseudohypericin have been con-sidered effective against several viruses such asthe influenza virus and the Herpes simplex virus1. These two compounds also have been foundto inhibit retroviral infections such as the HIV 3.
For the determination and identification ofHypericin several methods by HPTLC 4, HPLC 5
and HPLC-MS 6,7,8 have been reported. Among the Hypericum species that grow in
Cuba are found Hypericum styphelioides Rich,H. nitidum Lam., and H. tetrapetalum Lam. In
the literature consulted there are not reports re-lated to phytochemicals studies on thesespecies.
The purpose of this work was the determina-tion of hypericin content in these Hypericumspecies to promote their use as antiviral agentsin Cuban folk medicine.
MATERIAL AND METHODS Sample Collection
All the Hypericum species were collectedfrom various locations in the region of Pinar delRío, Cuba. Specimen samples were all collectedat blossoming time with most of the flowersopened from April to November in 2000 and2001. The samples were dried at room tempera-ture for seven days and powdered. Voucherspecimens of H. tetrapetalum (HPPR 9192), H.nitidum (HPPR 9212 and 9278) and H. styphe-lioides (HPPR 9190, 9120-2 and 9277) were de-posited at the Herbarium of the Pinar del RíoBotanical Gardens, Cuba.Sample preparation
Powered samples (0.5 g ) were extractedwith 10 ml of methanol using an ultrasonic bathfor 10 min. For TLC and HPLC analysis the ex-tracts were first filtered.Standard preparation
Commercial hypericin formulation tablets(Nutricia Manufacturing, Greenville, USA. Lot
90
NUEVAS-PAZ L., MOLINA-TORRES J. & PRIETO-GONZÁLEZ S.
No. 95290) were used. Three tablets (0.9 g)equivalent to 2.7 mg of hypericine were pow-dered and extracted with 10 ml of methanol us-ing an ultrasonic bath for 10 min.TLC Analysis
Separations were achieved in HPTLC silicagel plates (60F254, 10 x 10 cm, Merck). The mo-bile phases used were ethyl acetate-formic acid-glacial acetic acid-water (100:11:11:27) (A) 9,toluene-ethyl formate-formic acid (50:40:10) (B) 9
and ethyl acetate-dichloromethane-formic acid-acetic acid-water (100:25:10:10:11) (C) 4.
The HPTLC plates were scanned with a TLCScanner II Densitometer (CAMAG, Switzerland)at 590 nm. In each plate 10 µl of the standardand samples solutions were applied with a Lino-mat IV (CAMAG, Switzerland).
HPLC AnalysisHPLC separations were achieved following
the method reported by Piperopoulous et al. 6
with modifications. The HPLC system consistedof a ternary pump L-7100 with a detector L-7400(Merk-Hitachi, Darmstadt, Germany). The detec-tion was in the visible range at 590 nm and theinjection volume 20 µl. The chromatographicdata were recorded and processed by theBiochrom software (CIGB, Cuba). A SuperspherRP 18 column (250x4 mm I.D., 5 µm, Merck),protected with a LiChrospher 100 RP 18 (4X4mm I.D., 5 µm, Merck) precolumn was used forchromatographic separation. The solvent systemconsisted of methanol-acetonitrile (5:4) mixture(solvent A) and 0.1 M aqueous triethylammoni-um acetate (solvent B). The starting gradientwas composed of 70% A and 30% B, then theportion of A linearly increased with a flow-rateof 1 ml/min to 90% in 15 minutes. The columnequilibration was achieved by a linear change tothe starting conditions over 10 min.
RESULTS AND DISCUSSIONThe TLC analysis in solvents system A, B and
C of the Hypericum perforatum extract showbands with a red fluorescence at Rf = 0.50,Rf=0.95 and Rf=0.48, observed at UV 366 nm. At590 nm, only two peaks (hypericin and pseudo-hypericin) were observed for the Hypericumperforatum extract. For the others extracts ofHypericum species no peak were observed inthe chromatogram at this wavelength. To deter-mine the detection limits of this method 10, 5, 2,1 and 0.5 µl of the Hypericum perforatum ex-tract (c = 0.27 mg/mL) were applied in the TLClayers and chromatogram were recorded underthe same conditions above described. The de-
tection limits for the HPTLC analysis was foundin 0.54 µg of hypericin /spot.
In the HPLC analysis of the Hypericum perfo-ratum extract four peaks were observed in thechromatogram at 2.18 min. (protopseudohyper-icin), 3.86 min (pseudohypericin), 5.43 min(protohypericin) and 6.39 min (hypericin), re-spectively. In the samples chromatograms notany of the above mentioned peaks were ob-served. This result are in agreement with thoseobserved by Snook et al. 10 in a similar studymade with seventeen Hypericum species nativesfrom Southern of United States. The detectionlimits for the method of HPLC was determinedby injection of the dilutions hypericin standardextract. The detection limits for hypericin was18 ng (n= 5, RSD = 5.8 %).
CONCLUSIONSHypericin was not found in the three species
collected in Cuba. If this compound is presentin this species their concentration level is underthe detections limits of chromatographic tech-niques used.
Acknowledgements. This work was partially suppor-ted by the Ministry of Public Health, Republic of Cu-ba (Project MINSAP/Cuba 0008001) and CONACYT/Mexico (Integral Project: E120.941/2002 andJ200.265/2003).
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