Acta Farm.

Bonaerense 24 (1): 89-90 (2005)

Recibido el 17 de agosto de 2004 Aceptado el 19 de diciembre de 2004

Comunicaciones breves

Determination of Hypericin in Hypericum Species Grown in Cuba

Lauro NUEVAS-PAZ 1, Jorge MOLINA-TORRES 2 and Sylvia PRIETO-GONZLEZ 1* Centro de Qumica Farmacutica; 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 Biotecnologa y Bioqumica, CINVESTAV-IPN, Unidad Irapuato, Mxico.
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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 reverse phase HPLC for hypericin content. In the species of Hypericum analyzed hypericin was not found. RESUMEN. Determinacin de Hipericina en Especies de Hypericum crecidas en Cuba. Tres especies de Hypericum crecidas en Cuba se colectaron, secaron y molieron en estaciones y aos diferentes. El material vegetal se extrajo con metanol y se realiz el anlisis para determinar el contenido de hipericina mediante HPTLC y HPLC de fase reversa. No se encontr hipericina en las especies de Hypericum analizadas.

INTRODUCTION The genus Hypericum consists of more than 370 species. This genus is widely used in folk medicine since antiquity. Several pharmacological uses of Hypericum are known worldwide. The most popular plant of this genus is the Hypericum perfotatum L. (Saint Johns Wort) mainly due to its use as a healing agent and by its anti-depressive and anti-viral applications 1. St Johns Wort extract contains at least 10 constituents or groups of components that could contribute to its pharmacological effects. These components include naphthodianthrones (hypericin and pseudohypericin), flavonoids (rutin, hyperoside, isoquercitrin, quercitrin and quercetin), phloroglucinols (hyperforin and adhyperforin), tannins (cathechin and epicatechin), proanthocyanidins (procyanidin B2) and biflavonoids (biapigenin and amentoflavone) 2. Hypericin and pseudohypericin have been considered effective against several viruses such as the influenza virus and the Herpes simplex virus 1. These two compounds also have been found to inhibit retroviral infections such as the HIV 3. For the determination and identification of Hypericin 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 related to phytochemicals studies on these species. The purpose of this work was the determination of hypericin content in these Hypericum species to promote their use as antiviral agents in Cuban folk medicine. MATERIAL AND METHODS Sample Collection All the Hypericum species were collected from various locations in the region of Pinar del Ro, Cuba. Specimen samples were all collected at blossoming time with most of the flowers opened from April to November in 2000 and 2001. The samples were dried at room temperature for seven days and powdered. Voucher specimens of H. tetrapetalum (HPPR 9192), H. nitidum (HPPR 9212 and 9278) and H. styphelioides (HPPR 9190, 9120-2 and 9277) were deposited at the Herbarium of the Pinar del Ro Botanical Gardens, Cuba. Sample preparation Powered samples (0.5 g ) were extracted with 10 ml of methanol using an ultrasonic bath for 10 min. For TLC and HPLC analysis the extracts were first filtered. Standard preparation Commercial hypericin formulation tablets (Nutricia Manufacturing, Greenville, USA. Lot

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: sylvia.prieto@infomed.sld.cu

ISSN 0326-2383

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NUEVAS-PAZ L., MOLINA-TORRES J. & PRIETO-GONZLEZ S.

No. 95290) were used. Three tablets (0.9 g) equivalent to 2.7 mg of hypericine were powdered and extracted with 10 ml of methanol using an ultrasonic bath for 10 min. TLC Analysis Separations were achieved in HPTLC silica gel plates (60F254, 10 x 10 cm, Merck). The mobile phases used were ethyl acetate-formic acidglacial acetic acid-water (100:11:11:27) (A) 9, toluene-ethyl formate-formic acid (50:40:10) (B) 9 and ethyl acetate-dichloromethane-formic acidacetic acid-water (100:25:10:10:11) (C) 4. The HPTLC plates were scanned with a TLC Scanner II Densitometer (CAMAG, Switzerland) at 590 nm. In each plate 10 l of the standard and samples solutions were applied with a Linomat IV (CAMAG, Switzerland). HPLC Analysis HPLC separations were achieved following the method reported by Piperopoulous et al. 6 with modifications. The HPLC system consisted of a ternary pump L-7100 with a detector L-7400 (Merk-Hitachi, Darmstadt, Germany). The detection was in the visible range at 590 nm and the injection volume 20 l. The chromatographic data were recorded and processed by the Biochrom software (CIGB, Cuba). A Superspher RP 18 column (250x4 mm I.D., 5 m, Merck), protected with a LiChrospher 100 RP 18 (4X4 mm I.D., 5 m, Merck) precolumn was used for chromatographic separation. The solvent system consisted of methanol-acetonitrile (5:4) mixture (solvent A) and 0.1 M aqueous triethylammonium acetate (solvent B). The starting gradient was composed of 70% A and 30% B, then the portion of A linearly increased with a flow-rate of 1 ml/min to 90% in 15 minutes. The column equilibration was achieved by a linear change to the starting conditions over 10 min. RESULTS AND DISCUSSION The TLC analysis in solvents system A, B and C of the Hypericum perforatum extract show bands with a red fluorescence at Rf = 0.50, Rf=0.95 and Rf=0.48, observed at UV 366 nm. At 590 nm, only two peaks (hypericin and pseudohypericin) were observed for the Hypericum perforatum extract. For the others extracts of Hypericum species no peak were observed in the chromatogram at this wavelength. To determine the detection limits of this method 10, 5, 2, 1 and 0.5 l of the Hypericum perforatum extract (c = 0.27 mg/mL) were applied in the TLC layers and chromatogram were recorded under the same conditions above described. The de90

tection limits for the HPTLC analysis was found in 0.54 g of hypericin /spot. In the HPLC analysis of the Hypericum perforatum extract four peaks were observed in the chromatogram at 2.18 min. (protopseudohypericin), 3.86 min (pseudohypericin), 5.43 min (protohypericin) and 6.39 min (hypericin), respectively. In the samples chromatograms not any of the above mentioned peaks were observed. This result are in agreement with those observed by Snook et al. 10 in a similar study made with seventeen Hypericum species natives from Southern of United States. The detection limits for the method of HPLC was determined by injection of the dilutions hypericin standard extract. The detection limits for hypericin was 18 ng (n= 5, RSD = 5.8 %). CONCLUSIONS Hypericin was not found in the three species collected in Cuba. If this compound is present in this species their concentration level is under the detections limits of chromatographic techniques used.
Acknowledgements. This work was partially supported by the Ministry of Public Health, Republic of Cuba (Project MINSAP/Cuba 0008001) and CONACYT /Mexico (Integral Project: E120.941/2002 and J200.265/2003).

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