LC-MS and LC-PDA Analysis of empetrifolium and Feras Q. Alalia,*, Khaled Tawahab, and Mohammad Gharaibehc a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al Isra Private University, P. O. Box 22, 33, Amman 11622, Jordan. E-mail: [email protected] b Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Jordan, Amman 11942, Jordan c Department of Natural Resources and Environment, Faculty of Agriculture, Jordan University of Science and Technology, P. O. Box 3030, Irbid 22110, Jordan * Author for correspondence and reprint requests Z. Naturforsch. 64 c, 476 – 482 (2009); received February 25/March 26, 2009 Within the framework of our continuous efforts to explore Hypericum species from Jor- dan, we report the analysis of the major active metabolites, naphthodianthrones and phloro- glucinols, in the methanolic extracts of two under-explored Hypericum species; H. empetri- folium Willd. and H. sinaicum Hochst. & Steud. ex Boiss., using LC-(+,–)-ESI-MS (TIC and SIM) and LC-UV/Vis spectroscopy. Based on their LC-UV/Vis profi les, retention times and (+,–)-ESI-MS (TIC and SIM) spectral data, hypericin, protohypericin and pseudohypericin were identifi ed in both of the investigated species. In addition adhyperfi rin was only de- tected in H. empetrifolium, while and protopseudohypericin were only detected in H. sinaicum. This is the fi rst report documenting the presence of hypericin, protohypericin, pseudohypericin, protopseudohypericin, and hyperforin in H. sinaicum, and adhyperfi rin in H. empetrifolium. Key words: Hypericum, Naphthodianthrones,

Introduction species of Hypericum can be identifi ed by: (i) op- posite simple entire exstipulate leaves contain- Hypericum is a genus of about 450 species ing translucent and often black or red glandular of herbs or shrubs belonging to the family Clu- secretions; (ii) fl owers with a 5-merous perianth siaceae, formerly . The genus grows comprising green (sometimes red-tinged) sepals widely in temperate regions, and is used in tradi- and free yellow (often red-tinged) petals, tional medicinal practices in various parts of the in 3 – 5 bundles or fascicles, and an ovary with 3 – 5 world. All members of the genus may be referred slender styles; and (iii) a capsular fruit containing to as “Saint John’ wort”, though they are also many small cylindrical seeds (Robson, 2003). commonly just called ; other names One of the most important and commercially include “Rose of Sharon” and “Tutsan” (Anony- recognized species of the genus Hypericum is H. mous, 2007; Robson, 1990, 2003; Yazaki and Oka- perforatum, commonly known as St. John’s wort. da, 1994). The specimen records at National The activity of this species is the Center for Agricultural Research and Extension cause for the widespread interest in the study of (NCARE), Ministry of Agriculture, Baq’a, Jor- the Hypericum genus (Hu and Sim, 2000). H. per- dan, showed the presence of seven species of Hy- foratum L. is well known for its profound phar- pericum in Palestine fl ora during 1900 – 1910. In macological activities as mild antidepressant, anx- his list of wild in Jordan, Al-Eisawi (1998) iolytic, antiviral, wound healing and antimicrobial reported the presence of 5 species of Hypericum; agent (Barnes et al., 2001; Butterweck et al., 2002; H. hyssopifolium Chaix, H. languinosum Lam., H. Sakar and Tamer, 1990). Prescribed as mild anti- olivieri (Spach) Boiss. H. serpyllifolium Lam., and depressant therapeutic, commercially available H. triquetrifolium Turra. Danin (1997) discovered products of H. perforatum are among the best a population of H. sinaicum Hochst. & Steud. ex selling, most successful and effective herbs in the Boiss. in Jordan at Dana Nature Reserve. Most world.

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The Hypericum genus is rich in secondary me- Results and Discussion tabolites, many of which are biologically active. Of the main constituents are naphthodianthrones The investigation benefi tted from the available (hypericin, pseudohypericin, protohypericin, and literature on spectral data and chromatographic protopseudohypericin), phloroglucinols (hyper- elution patterns of phloroglucinols and naph- forin, adhyperforin, hyperfi rin, and adhyperfi rin), thodianthrones (Fuzzati et al., 2001; Tolonen et and a broad range of fl avonoids (e.g., hyperoside al., 2002; Wolfender et al., 2003). Thus, LC-(+,–)- and rutin) (Nahrstedt and Butterweck, 1997). The ESI-MS (TIC and SIM) and LC-UV data were antidepressant activity of H. perforatum was fi rst utilized in an integrative manner to confi rm the attributed to the naphthodianthrones. Recent identity of these compounds in the crude meth- studies revealed that the hyper- anolic extracts. forin and its derivative adhyperforin inhibit vari- A hypericin reference standard was used to de- ous receptors (Butterweck et al., velop the optimum chromatographic separation 2001; Chatterjee et al., 1998; Laakmann et al., 1998; conditions. A neutral mobile phase was used to Meruelo et al., 1988; Muller et al., 1998, 2001). able the switch between the positive and nega- The objective of the present study was to in- tive ionization modes simultaneously. The condi- vestigate, at the analytical level, the methanolic tions were optimized for resolution and response extracts of two unexplored Hypericum species of as outlined in the experimental section. The to- Jordanian origin, namely H. empetrifolium Willd. tal run time was 35 min, with a retention time of and H. sinaicum Hochst. & Steud. ex Boiss., for 16.1 min for hypericin. their major secondary metabolites, mainly phlo- The methanolic extract of the total aerial parts roglucinols and naphthodianthrones using liquid of H. empetrifolium and H. sinaicum were ana- chromatography-electrospray mass spectrometry lyzed for the presence of phloroglucinols (hyper- and liquid chromatography-UV spectroscopy forin, adhyperforin, hyperfi rin, and adhyperfi rin) [LC-(+,–)-ESI-MS (total ion chromatograms, and naphthodianthrones (hypericin, pseudohyper- TIC, and selected ion monitoring, SIM) and LC- icin, protohypericin, and protopseudohypericin) UV]. Although there were limited preliminary using LC-ESI-MS and LC-UV. LC-MS full scan and analytical studies, none of the TICs, scanning m/z 50 – 1000, were fi rst acquired abovementioned species has been investigated in both positive and negative ionization modes. previously for bioactive constituents using LC- This was followed by positive and negative SIM ESI-MS and LC-UV/PDA (Crockett et al., 2007; analyses. SIM was utilized in compounds’ detec- Kitanov, 2001; Rezanka and Sigler, 2007; Xeno- tion in order to overcome the low sensitivity of phontos et al., 2007). the TIC observed. The following naphthodian- H. empetrifolium and H. sinaicum grow wild in throne molecular ions, [M-H]–, were monitored the northern part of Jordan at Ramtha, Ajloun and in the negative ionization mode: hypericin, m/z Irbid. H. sinaicum is 10 – 30 cm high, altogether 503; pseudohypericin, m/z 519; protohypericin, slightly pubescent. Flowers are few in a terminal m/z 505; and protopseudohypericin, m/z 521. The corymbose panicle. The plant is toxic to livestock negative ionization mode was found to be highly (Batanouny, 1999). H. empetrifolium, also called sensitive for this class of phenolic compounds. Dwarf Hypericum, has deep yellow fl owers in late On the other hand, the positive ionization SIM spring to early summer (Batanouny, 1999). mode was used to monitor the following phloro- Based on their UV profi les, retention times and glucinol molecular ions, [M+H]+: hyperforin; m/z (+,–)-ESI (TIC and SIM) mass spectral data, hy- 537; adhyperforin, m/z 551; hyperfi rin, m/z 469; pericin, protohypericin and pseudohypericin were and adhyperfi rin, m/z 483. Compared to TIC, the identifi ed in both of the investigated Hypericum SIM sensitivity was signifi cantly higher for both species; while adhyperfi rin was only detected in investigated chemical classes. Fig. 1 shows typical H. empetrifolium, hyperforin and protopseudohy- stacked (+,–)-ESI LC-MS TIC chromatograms of pericin were only detected in H. sinaicum. This the methanolic extracts of the aerial parts of H. is the fi rst report documenting the presence of empetrifolium and H. sinaicum. Table I summa-

hypericin, protohypericin, pseudohypericin, pro- rizes the retention times, UVmax data, and the mo- topseudohypericin, and hyperforin in H. sinaicum, lecular ions of the identifi ed compounds. By ana- and adhyperfi rin in H. empetrifolium. lyzing the LC-UV and mass spectral data and by

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A 5 6

1 2 7 13 3 8 9 10 11 12 14 15 16

5 10 15 20 25 30 Time [min]

B 3

1 2 4 6 8 9 5 7 10 5 10 15 20 25 30 Time [min]

Fig. 1. Stacked (+,–)-ESI TIC chromatograms of (A) H. empetrifolium and (B) H. sinaicum.

Table I. Retention times and mass spectral data of the (+)- and (-)-ESI TIC and (+)- and (-)-SIM chromatographic peaks.

Plant tR [min] UVmax [nm] Molecular ion Compound – H. empetrifolium 16.1 288, 325, 465, 580 503.2 [M-H] OH O OH H. sinaicum

HO HO

OH O OH Hypericin (1) – H. empetrifolium 4.6 288, 325, 465, 580 519.3 [M-H] OH O OH H. sinaicum

OH HO HO

OH O OH Pseudohypericin (2) – H. empetrifolium 14.1 285, 375, 530, 590 505.2 [M-H] OH O OH H. sinaicum

HO HO

OH O OH Protohypericin (3)

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Plant tR [min] UVmax [nm] Molecular ion Compound – H. sinaicum 3.2 285, 375, 550 521.3 [M-H] OH O OH

OH HO HO

OH O OH Protopseudohypericin (4) H. sinaicum 15.3 285 537.6 [M+H]+ 535.6 [M-H]–

HO O

OO

Hyperforin (5) H. empetrifolium 18.7 285 483.3 [M+H]+

O OH

O O

Adhyperfi rin (6)

comparison with those reported in the literature, standard of hypericin, where complete matching hypericin (1), pseudohypericin (2), protohypericin was observed. (3), protopseudohypericin (4), and hyperforin (5) The (-)-ESI mass spectra of H. empetrifolium were tentatively identifi ed in H. sinaicum. Hyper- and H. sinaicum showed also parent molecular icin (1), pseudohypericin (2), protohypericin (3), ions at m/z 519.3 [M-H]– and an identical UV and adhyperfi rin (6) were identifi ed in H. empe- spectrum with that of hypericin, but eluted at an

trifolium. earlier time (tR = 4.6 min), i.e. this compound is The (-)-ESI mass spectra of hypericin (1) (tR = more polar. These spectral data suggest the iden- 16.1 min) from H. empetrifolium and H. sinaicum tity of this compound as pseudohypericin (2) showed a parent molecular ion at m/z 503.2 [M- (Brolis et al., 1998; Liu et al., 2000; Mauri and Pi- H]–. The UV spectrum of hypericin showed four etta, 2000; Tolonen et al., 2003).

absorption maxima at 288, 325, 465 and 580 nm, The (-)-ESI mass spectrum of the peak at tR = which are typical values for hypericin providing 14.1 min, from H. empetrifolium and H. sinaicum, an additional proof for the identity of this com- showed a parent molecular ion at m/z 505.2 [M- pound (Brolis et al., 1998; Liu et al., 2000; Mauri H]–, i.e. 2 Da more than the analogous peak in and Pietta, 2000; Tolonen et al., 2002, 2003). The hypericin; it was also eluted at an earlier reten- identity of this compound was verifi ed by com- tion time than hypericin. The UV spectrum of parison of its ESI mass spectrum, UV spectrum, the compound showed the following absorption and the HPLC retention time with an authentic maxima 285, 375, 530, and 590 nm. These data

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suggested that this compound was protohypericin protohypericin, pseudohypericin, protopseudo- (3) (Tolonen et al., 2003). hypericin, and hyperforin in H. sinaicum. The

The (-)-ESI mass spectrum of the peak at tR = literature only reports the presence of sinaici- 3.2 min of H. sinaicum showed a parent mo- none, a complex adamantanyl derivative, in H. lecular ion at m/z 521.3 for [M-H]–, 2 Da more sinaicum (Rezanka and Sigler, 2007). Regarding than the analogous peak in pseudohypericin; it H. empetrifolium, this is the fi rst report docu- was also eluted at an earlier retention time than menting the presence of adhyperfi rin. The pres- pseudohypericin. The UV spectrum of the com- ence of pseudohypericin in H. empetrifolium was pound showed absorption maxima at 285, 375, found to be in agreement with an earlier study and 550 nm. These data suggested that this com- (Xenophontos et al., 2007). Our study adds to pound was protopseudohypericin (4) (Tolonen et the study of Kitanov (2001) in which H. empe- al., 2003). trifolium was among 36 species of Hypericum In H. sinaicum, the (-)- and (+)-ESI mass spec- evaluated for their hypericin and pseudohyper-

tra of the peaks at tR = 15.3 min showed parent icin content where only hypericin was found in molecular ions of 537.6 and 535.6 for [M+H]+ and H. empetrifolium. [M-H]–, in the positive and negative ionization modes, respectively. The UV spectrum showed Experimental an absorption maximum at 285 nm, characteris- General tic of phloroglucinols. These mass spectral data suggested that this compound was hyperforin (5) LC-MS data were determined using an Agi- (Tolonen et al., 2003). lant® (Palo Alto, CA, USA) ion-trap mass spec- Finally, the (+)-ESI mass spectrum of the peak trometer equipped with an electrospray ioniza- ® at tR = 18.7 min, in H. empetrifolium, showed a tion source and an Agilant 100 series HPLC parent molecular ion at m/z 483.3 for [M+H]+, instrument. The separation was achieved using a while the UV spectrum showed an absorption Hypersil ODS (125 mm × 4 mm; 5 µm) column maximum at 285 nm suggesting that the com- (Thermo Electron, Auchtermuchty, UK). The mo- pound was adhyperfi rin (6). bile phases used were: (A) 20 mM ammonium ac- Importantly, there were several other peaks etate; (B) acetonitrile. The fl ow rate was 1 mL/min in the (-)- and (+)-ESI TIC mass spectra of in the following gradient system: 0 – 10 min, 50% H. empetrifolium and H. sinaicum that based B; 10 – 25 min, 90% B; 30 – 35 min 50% B. The in- on their molecular ions, and UV spectra could jection volume was 20 µL, and the total run time not be identifi ed or dereplicated to any obvi- was 35 min. The mass detector conditions were ous structural class reported in the literature for set as follows: ESI positive and negative ioniza- the genus Hypericum. These could be potentially tion modes; full scan mode from 50 to 1000 m/z; new compounds; hence our future efforts will be capillary voltage, 4000 V; ESI temperature, 325 ºC; focused toward their classifi cation and identifi ca- gas fl ow rate, 5 L/min. tion. LC-UV/Vis spectra were obtained on a In summary, we have developed a new strat- Lachrom® Merck-Hitachi (Tokyo, Japan) HPLC egy identify of the major secondary metabo- instrument, equipped with a quaternary gradient lites (phloroglucinols and naphthodianthrones) L-7150 pump, L-7455 photodiode-array (PDA) in H. empetrifolium and H. sinaicum based on detector, L-7200 auto-sampler, and D-7000 inter- integrating data from LC-UV/PDA (distinctive face in the range 250 – 650 nm. Mobile phase, fl ow UV spectra for different classes), LC-ESI-MS rate, analytical column, injection volume, and run [(+,–)-TIC and (+,–)-SIM)] and chromatographic times were identical to those for LC-MS. elution patterns. We were able to identify fi ve Ammonium acetate (extra pure) was obtained major compounds; hypericin (1), pseudohyper- from Scharlau Chemie S.A. (Barcelona, Spain), icin (2), protohypericin (3), and protopseudohy- methanol (HPLC grade) was obtained from Tedia pericin (4), and hyperforin (5) in H. sinaicum, Company Inc. (Fairfi eld, OH, USA), acetonitrile while in H. empetrifolium four compounds were (HPLC grade) was obtained from LEDA, Schar- identifi ed, hypericin (1), pseudohypericin (2), lau Chemie S.A., and (-)-hypericin (standard) was protohypericin (3), and adhyperfi rin (6). This is purchased from Sigma-Aldrich (Buchs, Switzer- the fi rst report on the presence of hypericin, land).

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Plant material ing a rotary evaporator (RE 200, Bibby Steriline Ltd., Stone, UK). The concentrated solutions H. empetrifolium and H. sinaicum were collect- were transferred to a 25-mL fl ask and diluted to ed from Ajloun Nature Reserve, Northern part of volume using methanol. Aliquots were removed Jordan during their fl owering stage in 2007. The and centrifuged at 4500 rpm for 5 min using an collected materials were identifi ed by M. G. The EBA 20 centrifuge (Hettich-Zentrifugen GmbH raw plant materials were cleaned and air-dried & Co. KG, Tuttlingen, Germany). Supernatants at room temperature, then ground to a fi ne pow- ® were transferred into glass vials and stored in a der using a blender (Moulinex , Caen, France), refrigerator until required for analysis (Anony- passed through a 24-mesh sieve to generate a mous, 2000). homogeneous powder, stored at room tempera- For the LC-MS and LC-UV studies, aliquots ture (22 – 23 °C), and protected from light until of the supernatants of the methanolic extracts of required for analyses. each plant were fi ltered through a 0.45-μm Te- fl on fi lter and then transferred into 2-mL amber Samples preparation and analysis HPLC vials. A 20-μL aliquot was injected. Hyper- From each fi nely ground plant material, icin standard was used for retention time match- (1000 ± 0.1) mg were accurately weighed, placed ing. in a 250-mL round bottom fl ask fi tted with a refl ux condenser, and refl uxed for 20 min using 80 mL Acknowledgements of HPLC methanol. The samples were then fi l- The authors acknowledge the fi nancial sup- tered, saving the fi ltrate. The herb materials were port from the Deanship of Scientifi c Research, re-extracted twice with 60 mL HPLC methanol, JUST, Irbid, Jordan and the technical help of Mr. followed each time by fi ltration. The collected Munther Tahtamoni, Princess Haya Biotechnol- fi ltrates and washes were combined. The volume ogy Center and Ms. Tamam El-Elimat, Pharma- was reduced to a fi nal volume of about 3 mL us- ceutical Research Center, JUST, Jordan.

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