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Chemical constituents and antidepressant activity of the new species Hypericum enshiense occurring in China
ARTICLE IN PRESS Phytomedicine 17 (2010) 410–413
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Phytomedicine journal homepage: www.elsevier.de/phymed
Chemical constituents and antidepressant activity of the new species Hypericum enshiense occurring in China Dongmei Wang, Jie Bai, Feng Sun, Depo Yang School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
a r t i c l e in f o
Keywords: Hypericum enshiense Hypericacae HPLC-MS Antidepression Forced swimming test Tail suspension test
a b s t r a c t Hypericum enshiense L. H. Wu et F. S. Wang is a new species of Hypericum occurring in China, which was first identified and denominated by our laboratory. No research has been reported on the antidepressant activity and chemical constituents of this new species. In this study, the qualitative and quantitative analyses of the chemical constituents in the hydroalcoholic extract of this species were performed using HPLC/DAD/ESI-MS online method. Hypericin, pseudohypericin and some flavonoids were identified or tentatively identified. Furthermore, H. enshiense had a high content of hypericins than H. perforatum. In addition, the antidepressant activity of the hydroalcoholic extract of the species was investigated using forced swimming test (FST) and tail suspension test (TST). The extract significantly shortened the immobility time in FST and TST, while did not alter the locomoter activity of mice. These results suggested for the first time that the hydroalcoholic extract of H. enshiense might possess potential antidepressant-like activity in the animal behavioral models, and this species might act as a new potential resource for developing antidepressants to treat depressive disorders. & 2009 Elsevier GmbH. All rights reserved.
Introduction Hypericum L. is a large cosmopolitan genus, chiefly distributed in temperate zone. The genus is rich in plant resources, which contains about 400 species mainly herbs and shrubs, more than 60 of which are distributed in China. Hypericum enshiense L. H. Wu et F. S. Wang is a perennial herb native to Badong (Shennongjia, Hubei), a distribution center of Hypericum in China. The plant was first identified as a novel species of Hypericum genus and denominated by our laboratory (Wu et al. 2002). This new species, often seen in forest margin, roadside, cultivated margin, is abundant in the collected areas. In our previous study of its pharmacognosy, we found the lamina of leaf with loose black glands, the sepals and the petals with linear black glands (Wu et al. 2004). It has been proposed that hypericin and pseudohypericin are primarily localized in black glands on the margins of leaves and flower petals of H. perforatum (St. John’s wort) (Curtis and Lersten 1990; Fornasiero et al. 1998). H. perforatum is widely used in many countries for the treatment of mild to moderate depression (Yager et al. 1999; Schrader 2000), and hypericin and pseudohypericin are thought to function as two of the main components for its antidepressant activity. No research has been reported on the antidepressant activity and chemical constituents of this new species. Therefore, in this study, we aimed to
Corresponding author. Tel./fax: +86 20 39943043.
E-mail address: [email protected] (D. Yang). 0944-7113/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.07.015
investigate the potential antidepressant activity of the new species H. enshiense collected in blossom, as well as to identify the hypericins and other main constituents from this plant.
Materials and methods Chemicals and plant materials Reference substances of quercetin (95%), rutin (95%) and chlorogenic acid (95%) were purchased from Sigma (USA), and hyperoside (98%) was purchased from Merck (Germany). Hypericin was synthesized in our laboratory and the purity was proved to be not lower than 98% by HPLC analysis. Imipramine-HCl was obtained from JianFu (China). Amitriptyline-HCl was obtained from Shizhou (China). The aerial parts of Hypericum enshiense L. H. Wu et F. S. Wang were collected in Badong (Hubei, China) during the flowering stage in August 2001, and dried at ambient temperature avoiding direct sunlight. The voucher specimen (No. 01018) has been deposited in the Herbarium of Sun Yat-sen University, China. Preparation of the extract The dried aerial parts (ca. 10-30 cm in length) were powdered and macerated in the dark for one day at room temperature with 6-fold amount of 90% ethanol aqueous solution (v/v). The
ARTICLE IN PRESS D. Wang et al. / Phytomedicine 17 (2010) 410–413
extraction was repeated three times. After being filtered and evaporated to remove the solvent under reduced pressure below 45 1C, the extract was obtained with a yield of 8.4%. Animals and drug administration Male NIH mice (18-22 g) were obtained from the Medical Animal Center, Guangzhou University of Chinese Medicine, Guangdong Province, China. The animals were kept in a 12 h light/dark cycle (light on at 07:00) at ambient temperature of 2571 1C with free access to standard laboratory food and tap water. The extract and standard drugs were suspended in 0.5% carboxymethyl cellulose aqueous solution (vehicle), and were given orally 1 h before the experiments in 15 ml/kg. Control animals received vehicle under the same conditions. Extracts were given orally at dose of 500 or 250 mg/kg. Imipramine at dose of 20 mg/kg was given i.p. in the forced swimming test (FST). Amitriptyline at dose of 20 mg/kg was given i.p. in the tail suspension test (TST). Experiments were conducted between 8:00-15:00 o’clock. The mice were just used once. Each group in the behavioral experiment was consisted of 16 animals. Experiments were carried out in compliance with the Experimental Animal Management Bill of the November 14th 1988 Decree No. 2 of National Science and Technology Commission, China. Constituent identification by HPLC-DAD-MS analysis HPLC-DAD-MS experiments were performed on a Nucleodur C18 (5 mm, 250 4.6 mm) column (Macherey-Nagel) using a Thermo Separation Product P4000 chromatograph (San Jose, CA, USA) equipped with a Thermo 6000LP diode array detector and coupled with a Finnigan LCQTM DECA XP ion trap mass spectrometer (Thermo Quest Finnigan, USA). The mobile phase consisted of water-methanol-acetic acid (94.5:5:0.5, v/v, pH ¼ 4.50 adjusted with triethylamine) (A), methanol (B) and acetonitrile (C). A gradient elution was performed as follows: 0 min, A/B/C (85:0:15); 15 min, A/B/C (40:40:20); 35 min, A/B/C (10:15:75); 60 min, A/B/C (10:15:75). The flow rate of the mobile phase was 0.8 ml/min and the injection volume was 20 ml. LC-MS analysis was performed with an electrospray interface in the negative ion mode. The ionization conditions were as follows: source voltage 4.5 kV; sheath gas flow rate 60 ml/min, capillary voltage 24 V; capillary temperature 350 1C, scan range 200-700 amu. Ion trap conditions: acquisition in automatic gain control. Constituent quantification by HPLC-UV Flavonoids were detected at 254 nm after separation under the above HPLC conditions. Using hyperoside as an external standard, the respective response factors of the flavonoids in the extract relative to hyperoside were calculated. Analysis of hypericins was carried out at the wavelength of 590 nm after separation on the column described above with isocratic elution using solvent A/B/C (30:25:45), and other conditions were the same as those used in HPLC-MS analysis. Locomotor activity One hour after the extract or vehicle administration, each mouse was individually placed in a black circular chamber (10 25 cm i.d.) fitted with photoelectric cells. Locomotor activity was monitored continuously for 5 min and the number of movement was recorded via a computer connected to the monitor. The observation was conducted between 8 and 9 o’clock a.m.
411
Forced swimming test (FST) The forced swimming test was performed according to the methods described by Porsolt et al. (1978) with slight modifications. One hour after oral administration of the test drugs by gavages, the mice were individually placed in a cylindrical glass swim vessel (25 12 cm i.d.) filled with 12 cm depth of water maintained at 23-25 1C. The total immobility time was observed by unaided eyes of a trained observer, who was blind to the experimental conditions, during the last 4 min of a single 6-min test session. Mice were considered immobile when they did not further attempt to escape except the movements necessary to keep their heads above the water. Tail suspension test (TST) The mice were suspended by their tails, and the cumulative period of immobility during an observation period of 6 min was measured (Steru et al. 1985). One hour after oral administration of the test drugs by gavages, mice were individually taken from the vivarium to an adjoining room and immediately suspended by the tail to a horizontal ring-stand bar (distance from the tabletop 60 cm) using adhesive tape affixed 1 cm from the tip of the tail. The testing room was brightly lit. The mouse was 15 cm away from the nearest object and both acoustically and visually isolated. The cumulative period of immobility during an observation period of 6 min was recorded. Mice were considered immobile only when they hung passively and motionlessly. Statistical analysis Data analysis was performed by one-way analysis of variance (ANOVA) with the Dunnett post-hoc test for multiple comparisons by SPSS 10.0 software. Data were expressed as means7SEM. The level of statistical significance was set at po0.05.
Results and discussion Chemical analysis of the hydroalcoholic extract prepared from H. enshiense Several HPLC methods have been developed for the analysis of H. perforatum (Brolis et al. 1998; Liu et al. 2000). Considering the similarity of chemical constituents in plants from the same genus, these methods were employed for the chemical analysis of H. enshiense. Twelve compounds in the new species H. enshiense were detected by HPLC-DAD/(–)ESI-MS. Identification of the main constituents in the hydroalcoholic extract of H. enshiense was carried out by comparison of the HPLC retention times, UV absorption spectra and m/z values of their quasi-molecular ions with those of the reference standards and the literature data. Typical HPLC chromatograms monitored at 254 and 590 nm are shown in Fig. 1, while the retention times, UV absorptions, and MS spectral data and the identity of the peaks numbered in the chromatograms are listed in Table 1. Compared with the reference standards, Peaks 1, 4, 5, 7 and 12 were undoubtedly identified as chlorogenic acid, rutin, hyperoside, quercetin and hypericin, respectively. The ESI-MS spectrum of peak 2 exhibited a quasimolecular ion [M–H] at m/z 421, and peak 3 displayed an [M–H] ion at m/z 477. Compared with the literature values (Brolis et al. 1998; ¨ Jurgenliemk and Nahrstedt 2002), the results suggested that Peaks 2 and 3 might be magniferin and isoorientin, respectively. Peak 6 exhibited an ESI-MS spectrum with an [M–H] ion at m/z 447, and
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D. Wang et al. / Phytomedicine 17 (2010) 410–413
Fig. 1. RP-18 HPLC chromatograms of the hydroalcoholic extract of H. enshiense detected at 254 and 590 nm. Assignment of peaks is shown in Table 1.
Table 1 MS data obtained from the HPLC/DAD/ESI-MS analysis and contents of the identified compounds in the hydroalcoholic extracts of H. enshiense (A) and those of H. perforatum from literature (B). No.
Compound
1 2 3 4 5 6 7 8 9 10 11 12 13
Chlorogenic acid Magniferina Isoorientina Rutin Hyperoside Quercitrin Quercetin I3, II8 Biapigenina Protoseudohypericina Pseudohypericina Protohypericina Hypericin Hyperforin a b c
RT (min)
3.52 7.74 9.74 11.50 12.20 14.19 18.07 19.83 30.27 32.27 36.60 38.83 –
ESI [M–H]
353 421 447 609 463 447 301 537 521 519 505 503 –
Maxima wavelength (nm)
228, 273 218, 281, 379 218, 278 229, 254, 354 218, 266, 354 255, 351 247, 379 268, 329 256, 298, 362, 378, 563, 588 245, 285, 327, 472, 545, 588 254, 297, 368, 530, 557, 581 241, 285, 330, 472, 546, 589 –
RRFb
Content (mg/g) A
B
10.1 – – 2.4 8.1 – 10.4 – – 4.7 – 3.0 ndc
– – – 16.7 32.2 6.5 3.2 4.6 – 4.0 – 1.5 32.2
0.347 – – 0.632 1.000 – 1.147 – – – – – –
Tentatively identified only by the ESI-DAD-MS spectra. RRF relative response factor to hyperoside detected at 254 nm. nd, not detected.
a fragment m/z 301 due to loss of a methyl-pentoside unit, and displayed UV spectra with maxima at 218, 255 and 351 nm. Peak 8 exhibited UV maxima at 268 and 329 nm together with an [M–H] ion at m/z 537 in the ESI-MS. Peaks 6 and 8 were tentatively identified as quercitrin and I3, II8-biapigenin, respectively, by comparison with literature values (Brolis et al. 1998; ¨ Jurgenliemk and Nahrstedt 2002). Peak 10 exhibited an extremely similar UV-VIS spectrum with Peak 12 (hypericin), especially in the visible region, and the ESIMS spectrum showed an [M–H] ion at m/z 519. These results suggested Peak 10 might be pseudohypericin, which was reported to be present in H. perforatum (Kurth and Spreemann 1998). The ESI-MS spectra of Peaks 9 and 11 exhibited [M–H] ions at m/z 521 and 505, which displayed similar UV spectra, exibiting red shifts compared with those of pseudohypericin and hypericin. Moreover, protopseudohypericin and protohypericin were known constituents in Hypericum plants, and they were easy to lose two
hydrogen atoms under light to transform to pseudohypericin and hypericin, respectively (Kurth and Spreemann 1998). Therefore, peaks 9 and 11 were tentatively identified as protopseudohypericin and protohypericin, respectively. In addition, it is noteworthy that there was no hyperforin analogues detected in the extract of H. enshiense under our experimental conditions, while hyperforin was clearly detected in H. perforatum using the same extraction and analysis conditions (data not shown). The contents of naphthodianthrone constituents hypericin and pseudohypericin in the hydroalcoholic extract of H. enshiense were calculated by HPLC analysis at 590 nm using synthetic hypericin as the external standard compound. The contents of the flavonoids determined were calculated using the relative response factors to hyperoside (RRF). The contents of the analytes in H. enshiense were compared with those of H. perforatum in previous study (Butterweck et al. 2003a), and the results are summarized in
ARTICLE IN PRESS D. Wang et al. / Phytomedicine 17 (2010) 410–413
Table 2 Effect of the hydroalcoholic extract of H. enshiense on the total duration of immobility in the FST in mice (means7SEM, n ¼ 16, *Po0.05, **Po0.01 compared with control). Treatment
Dose (mg/kg)
Immobility time (s)
Control Extract
– 250 500 20
105.979.2 63.7715.9** 56.777.6** 79.479.8*
Imipramine
Table 3 Effect of the hydroalcoholic extract of H. enshiense on the total duration of immobility in the TST in mice (means7SEM, n ¼ 16, *Po0.05, **Po0.01 compared with control). Treatment
Dose (mg/kg)
Immobility time (s)
Control Extract
– 250 500 20
107.9710.4 71.0710.0* 88.8719.1 43.2710.6**
Amitriptyline
413
hyperforin was concerned with the drug interference caused by the extract of H. perforatum (Moore et al. 2000; Mannel 2004). The absence of hyperforin in H. enshiense suggested its merit on avoiding the metabolic interference caused by hyperforin. The results obtained in the present study provided us certain information on the plant to act as a new potential resource for developing antidepressants to treat depressive disorders. The relationship between constituents and antidepressant activity, and the possible antidepressant mechanisms of H. enshiense will be investigated in future study.
Acknowledgements The authors wish to thank Dr. L.H. Wu for his contribution in collecting and identifying the plant. We thank Mr. J.H. Yao (Instruments of Analysis & Measurement Center, Sun Yat-sen University) for his help in the HPLC-MS experiment. The study was supported and funded by National Natural Science Foundation of China (2004, No. 30470188). References
Table 1. As shown in Table 1, it is of interest that the extract of H. enshiense was rich in hypericins with a content of 7.7 mg/g, even higher than that (5.5 mg/g) reported by Butterweck et al. Previous studies have indicated that napthodianthrones like hypericin and pseudohypericin, phloroglucinol derivative hyperforin (Muller et al. 2001), and some flavonoids, such as hyperoside, isoquercitrin and rutin (Butterweck et al. 2000) are the important active components of the antidepressant effects of H. perforatum. Since the hydroalcoholic extract of H. enshiense contained hypericin, pseudohypericin (the amount being 0.30% and 0.47%, respectively) and some similar flavonoid constituents, the antidepressant activities of the hydroalcoholic extract of the species were further investigated in behavioral models in mice. Antidepressant activities of the extract prepared from H. enshiense Using FST and TST models, the antidepressant activities of the extract of H. enshiense were investigated. As shown in Tables 2 and 3, the extract at both doses of 250 and 500 mg/kg significantly shortened the immobility time of mice in FST and TST (Po0.05 or Po0.01 vs. control, Tables 2 and 3). However, the extract produced no significant effects on locomotor activity (500 mg/kg: 125710; 250 mg/kg: 119712) in comparison with control group (12279) (data are not shown). Recently, Butterweck et al. (2003a) systematically screened for the antidepressant activities of St. John’s wort preparations, and it was found that the extracts devoid of hyperforin or devoid of both hyperforin and hypericin but enriched in flavonoids were still active in behavioral models. It was also reported that hyperoside significantly improved the water solubility of hypericin, and led to a better bioavailability of napthodianthrones (Butterweck et al. ¨ ¨ 2003b). Noldner and Schotz (2002) thought that rutin was essential for the antidepressant activity of H. perforatum extract in forced swimming test. Hyperforin was not detected in the hydroalcoholic extract of H. enshiense, so the antidepressant activity of the extract might be mainly concerned with hypericins and some flavonoid constituents, while other unidentified constituents might also be involved in its antidepressant activity. Taking together, HPLC-ESI-MS analysis of the extract indicated that H. enshiense contained flavonoid constituents and were rich in hypericins, but free of hyperforin. Several studies reported that
Brolis, M., Gabetta, B., Fuzzati, N., Pace, R., Panzeri, F., Peterlongo, F., 1998. Identification by high-performance liquid chromatography-diode array detection-mass spectrometry and quantification by high-performance liquid chromatography-UV absorbance detection of active constituents of Hypericum perforatum. J. Chromatogr. A 845, 9–16. Butterweck, V., Christoffel, V., Nahrstedt, A., Petereit, F., Spengler, B., Winterhoff, H., 2003a. Step by step removal of hyperiforin and hypericin: activity profile of different Hypericum preparations in behavioral models. Life Sci. 73, 627–639. ¨ Butterweck, V., Jurgenliemk, G., Nahrstedt, A., Winterhoff, H., 2000. Flavonoids from Hypericum perforatum show antidepressant activity in the forced swimming test. Planta Med. 66, 3–6. ¨ Butterweck, V., Lieflander-Wulf, U., Winterhoff, H., Nahrstedt, A., 2003b. Plasma levels of hypericin in presence of procyanidin B2 and hyperoside: a pharmacokinetic study in rats. Planta Med. 69, 189–192. Curtis, J.D., Lersten, N.R., 1990. Internal secretory structures in Hypericum (Clusiaceae): H. perforatum L. and H. balearicum L. New Phytol. 114, 571–580. Fornasiero, R.B., Bianchi, A., Pinetti, A., 1998. Anatomical and ultrastructural observations in Hypericum perforatum L. leaves. J. Herbs Species Med. Plants 5, 21–33. ¨ Jurgenliemk, G., Nahrstedt, A., 2002. Phenolic compounds from Hypericum perforatum. Planta Med. 66, 88–91. Kurth, H., Spreemann, R., 1998. Phytochemical charaterization of various St. John’s Wort extracts. Adv. Ther. 15, 117–128. Liu, F.F., Ang, C.Y.W., Heinze, T.M., Rankin, J.D., Beger, R.D., Freeman, J.P., Lay Jr., J.O., 2000. Evaluation of major active components in St. John’s wort dietary supplements by high-performance liquid chromatography with photodiode array detection and electrospray mass spectrometric confirmation. J. Chromatogr. A 888, 85–92. Mannel, M., 2004. Drug interactions with St John’s wort: mechanisms and clinical implications. Drug Saf. 27, 773–797. Moore, L.B., Goodwin, B., Jones, S.A., Wisely, G.B., Serabjit-Singh, C.J., Willson, T.M., Collins, J.L., Kliewer, S.A., 2000. St. John’s wort induces hepatic drug metabolism through activation of the pregnane X receptor. Proc. Natl. Acad. Sci. USA 97, 7500–7502. Muller, W.E., Singer, A., Wonnemann, M., 2001. Hyperforin-antidepressant activity by a novel mechanism of action. Pharmacopsychiatry 34, S98–102. ¨ ¨ Noldner, M., Schotz, K., 2002. Rutin is essential for the antidepressant activity of Hypericum perforatum extracts in the forced swimming test. Planta Med. 66, 577–580. Porsolt, R.D., Anton, G., Blavet, N., Jalfre, M., 1978. Behavioral despair in rats: a new model sensitive to antidepressant treatment. Eur. J. Pharmacol. 47, 379–391. Schrader, E., 2000. Equivalence of St. John’s wort extract (Ze117) and fluoxetine: a randomized, controlled study in mild-moderate depression. Int. Clin. Psychopharmacol. 5, 61–68. Steru, L., Chermat, R., Thierry, B., Simon, P., 1985. The tail suspension test: a new method for screening antidepressant activity in mice. Psychopharmacology 85, 367–370. Wu, L.H., Yang, D.P., Wang, F.S., 2002. New species and a new subspecies of Hypericum from Guangdong, China. Acta Bot. Yunnan. 24, 609–610. Wu, L.H., Yang, D.P., Wang, F.S., 2004. New taxa of Hypericum (Hypericaceae) in China. Acta Phytotax. Sin. 24, 73–78. Yager, J., Siegfreid, S.L., Di Matteo, T.L., 1999. Use of alternative remedies by psychiatric patients: illustrative vignettes and a discussion of the issues. Am. J. Psychiatry 156, 1432–1438.
Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Chemical constituents and antidepressant activity of the new species Hypericum enshiense occurring in China Dongmei Wang, Jie Bai, Feng Sun, Depo Yang School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
a r t i c l e in f o
Keywords: Hypericum enshiense Hypericacae HPLC-MS Antidepression Forced swimming test Tail suspension test
a b s t r a c t Hypericum enshiense L. H. Wu et F. S. Wang is a new species of Hypericum occurring in China, which was first identified and denominated by our laboratory. No research has been reported on the antidepressant activity and chemical constituents of this new species. In this study, the qualitative and quantitative analyses of the chemical constituents in the hydroalcoholic extract of this species were performed using HPLC/DAD/ESI-MS online method. Hypericin, pseudohypericin and some flavonoids were identified or tentatively identified. Furthermore, H. enshiense had a high content of hypericins than H. perforatum. In addition, the antidepressant activity of the hydroalcoholic extract of the species was investigated using forced swimming test (FST) and tail suspension test (TST). The extract significantly shortened the immobility time in FST and TST, while did not alter the locomoter activity of mice. These results suggested for the first time that the hydroalcoholic extract of H. enshiense might possess potential antidepressant-like activity in the animal behavioral models, and this species might act as a new potential resource for developing antidepressants to treat depressive disorders. & 2009 Elsevier GmbH. All rights reserved.
Introduction Hypericum L. is a large cosmopolitan genus, chiefly distributed in temperate zone. The genus is rich in plant resources, which contains about 400 species mainly herbs and shrubs, more than 60 of which are distributed in China. Hypericum enshiense L. H. Wu et F. S. Wang is a perennial herb native to Badong (Shennongjia, Hubei), a distribution center of Hypericum in China. The plant was first identified as a novel species of Hypericum genus and denominated by our laboratory (Wu et al. 2002). This new species, often seen in forest margin, roadside, cultivated margin, is abundant in the collected areas. In our previous study of its pharmacognosy, we found the lamina of leaf with loose black glands, the sepals and the petals with linear black glands (Wu et al. 2004). It has been proposed that hypericin and pseudohypericin are primarily localized in black glands on the margins of leaves and flower petals of H. perforatum (St. John’s wort) (Curtis and Lersten 1990; Fornasiero et al. 1998). H. perforatum is widely used in many countries for the treatment of mild to moderate depression (Yager et al. 1999; Schrader 2000), and hypericin and pseudohypericin are thought to function as two of the main components for its antidepressant activity. No research has been reported on the antidepressant activity and chemical constituents of this new species. Therefore, in this study, we aimed to
Corresponding author. Tel./fax: +86 20 39943043.
E-mail address: [email protected] (D. Yang). 0944-7113/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.07.015
investigate the potential antidepressant activity of the new species H. enshiense collected in blossom, as well as to identify the hypericins and other main constituents from this plant.
Materials and methods Chemicals and plant materials Reference substances of quercetin (95%), rutin (95%) and chlorogenic acid (95%) were purchased from Sigma (USA), and hyperoside (98%) was purchased from Merck (Germany). Hypericin was synthesized in our laboratory and the purity was proved to be not lower than 98% by HPLC analysis. Imipramine-HCl was obtained from JianFu (China). Amitriptyline-HCl was obtained from Shizhou (China). The aerial parts of Hypericum enshiense L. H. Wu et F. S. Wang were collected in Badong (Hubei, China) during the flowering stage in August 2001, and dried at ambient temperature avoiding direct sunlight. The voucher specimen (No. 01018) has been deposited in the Herbarium of Sun Yat-sen University, China. Preparation of the extract The dried aerial parts (ca. 10-30 cm in length) were powdered and macerated in the dark for one day at room temperature with 6-fold amount of 90% ethanol aqueous solution (v/v). The
ARTICLE IN PRESS D. Wang et al. / Phytomedicine 17 (2010) 410–413
extraction was repeated three times. After being filtered and evaporated to remove the solvent under reduced pressure below 45 1C, the extract was obtained with a yield of 8.4%. Animals and drug administration Male NIH mice (18-22 g) were obtained from the Medical Animal Center, Guangzhou University of Chinese Medicine, Guangdong Province, China. The animals were kept in a 12 h light/dark cycle (light on at 07:00) at ambient temperature of 2571 1C with free access to standard laboratory food and tap water. The extract and standard drugs were suspended in 0.5% carboxymethyl cellulose aqueous solution (vehicle), and were given orally 1 h before the experiments in 15 ml/kg. Control animals received vehicle under the same conditions. Extracts were given orally at dose of 500 or 250 mg/kg. Imipramine at dose of 20 mg/kg was given i.p. in the forced swimming test (FST). Amitriptyline at dose of 20 mg/kg was given i.p. in the tail suspension test (TST). Experiments were conducted between 8:00-15:00 o’clock. The mice were just used once. Each group in the behavioral experiment was consisted of 16 animals. Experiments were carried out in compliance with the Experimental Animal Management Bill of the November 14th 1988 Decree No. 2 of National Science and Technology Commission, China. Constituent identification by HPLC-DAD-MS analysis HPLC-DAD-MS experiments were performed on a Nucleodur C18 (5 mm, 250 4.6 mm) column (Macherey-Nagel) using a Thermo Separation Product P4000 chromatograph (San Jose, CA, USA) equipped with a Thermo 6000LP diode array detector and coupled with a Finnigan LCQTM DECA XP ion trap mass spectrometer (Thermo Quest Finnigan, USA). The mobile phase consisted of water-methanol-acetic acid (94.5:5:0.5, v/v, pH ¼ 4.50 adjusted with triethylamine) (A), methanol (B) and acetonitrile (C). A gradient elution was performed as follows: 0 min, A/B/C (85:0:15); 15 min, A/B/C (40:40:20); 35 min, A/B/C (10:15:75); 60 min, A/B/C (10:15:75). The flow rate of the mobile phase was 0.8 ml/min and the injection volume was 20 ml. LC-MS analysis was performed with an electrospray interface in the negative ion mode. The ionization conditions were as follows: source voltage 4.5 kV; sheath gas flow rate 60 ml/min, capillary voltage 24 V; capillary temperature 350 1C, scan range 200-700 amu. Ion trap conditions: acquisition in automatic gain control. Constituent quantification by HPLC-UV Flavonoids were detected at 254 nm after separation under the above HPLC conditions. Using hyperoside as an external standard, the respective response factors of the flavonoids in the extract relative to hyperoside were calculated. Analysis of hypericins was carried out at the wavelength of 590 nm after separation on the column described above with isocratic elution using solvent A/B/C (30:25:45), and other conditions were the same as those used in HPLC-MS analysis. Locomotor activity One hour after the extract or vehicle administration, each mouse was individually placed in a black circular chamber (10 25 cm i.d.) fitted with photoelectric cells. Locomotor activity was monitored continuously for 5 min and the number of movement was recorded via a computer connected to the monitor. The observation was conducted between 8 and 9 o’clock a.m.
411
Forced swimming test (FST) The forced swimming test was performed according to the methods described by Porsolt et al. (1978) with slight modifications. One hour after oral administration of the test drugs by gavages, the mice were individually placed in a cylindrical glass swim vessel (25 12 cm i.d.) filled with 12 cm depth of water maintained at 23-25 1C. The total immobility time was observed by unaided eyes of a trained observer, who was blind to the experimental conditions, during the last 4 min of a single 6-min test session. Mice were considered immobile when they did not further attempt to escape except the movements necessary to keep their heads above the water. Tail suspension test (TST) The mice were suspended by their tails, and the cumulative period of immobility during an observation period of 6 min was measured (Steru et al. 1985). One hour after oral administration of the test drugs by gavages, mice were individually taken from the vivarium to an adjoining room and immediately suspended by the tail to a horizontal ring-stand bar (distance from the tabletop 60 cm) using adhesive tape affixed 1 cm from the tip of the tail. The testing room was brightly lit. The mouse was 15 cm away from the nearest object and both acoustically and visually isolated. The cumulative period of immobility during an observation period of 6 min was recorded. Mice were considered immobile only when they hung passively and motionlessly. Statistical analysis Data analysis was performed by one-way analysis of variance (ANOVA) with the Dunnett post-hoc test for multiple comparisons by SPSS 10.0 software. Data were expressed as means7SEM. The level of statistical significance was set at po0.05.
Results and discussion Chemical analysis of the hydroalcoholic extract prepared from H. enshiense Several HPLC methods have been developed for the analysis of H. perforatum (Brolis et al. 1998; Liu et al. 2000). Considering the similarity of chemical constituents in plants from the same genus, these methods were employed for the chemical analysis of H. enshiense. Twelve compounds in the new species H. enshiense were detected by HPLC-DAD/(–)ESI-MS. Identification of the main constituents in the hydroalcoholic extract of H. enshiense was carried out by comparison of the HPLC retention times, UV absorption spectra and m/z values of their quasi-molecular ions with those of the reference standards and the literature data. Typical HPLC chromatograms monitored at 254 and 590 nm are shown in Fig. 1, while the retention times, UV absorptions, and MS spectral data and the identity of the peaks numbered in the chromatograms are listed in Table 1. Compared with the reference standards, Peaks 1, 4, 5, 7 and 12 were undoubtedly identified as chlorogenic acid, rutin, hyperoside, quercetin and hypericin, respectively. The ESI-MS spectrum of peak 2 exhibited a quasimolecular ion [M–H] at m/z 421, and peak 3 displayed an [M–H] ion at m/z 477. Compared with the literature values (Brolis et al. 1998; ¨ Jurgenliemk and Nahrstedt 2002), the results suggested that Peaks 2 and 3 might be magniferin and isoorientin, respectively. Peak 6 exhibited an ESI-MS spectrum with an [M–H] ion at m/z 447, and
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Fig. 1. RP-18 HPLC chromatograms of the hydroalcoholic extract of H. enshiense detected at 254 and 590 nm. Assignment of peaks is shown in Table 1.
Table 1 MS data obtained from the HPLC/DAD/ESI-MS analysis and contents of the identified compounds in the hydroalcoholic extracts of H. enshiense (A) and those of H. perforatum from literature (B). No.
Compound
1 2 3 4 5 6 7 8 9 10 11 12 13
Chlorogenic acid Magniferina Isoorientina Rutin Hyperoside Quercitrin Quercetin I3, II8 Biapigenina Protoseudohypericina Pseudohypericina Protohypericina Hypericin Hyperforin a b c
RT (min)
3.52 7.74 9.74 11.50 12.20 14.19 18.07 19.83 30.27 32.27 36.60 38.83 –
ESI [M–H]
353 421 447 609 463 447 301 537 521 519 505 503 –
Maxima wavelength (nm)
228, 273 218, 281, 379 218, 278 229, 254, 354 218, 266, 354 255, 351 247, 379 268, 329 256, 298, 362, 378, 563, 588 245, 285, 327, 472, 545, 588 254, 297, 368, 530, 557, 581 241, 285, 330, 472, 546, 589 –
RRFb
Content (mg/g) A
B
10.1 – – 2.4 8.1 – 10.4 – – 4.7 – 3.0 ndc
– – – 16.7 32.2 6.5 3.2 4.6 – 4.0 – 1.5 32.2
0.347 – – 0.632 1.000 – 1.147 – – – – – –
Tentatively identified only by the ESI-DAD-MS spectra. RRF relative response factor to hyperoside detected at 254 nm. nd, not detected.
a fragment m/z 301 due to loss of a methyl-pentoside unit, and displayed UV spectra with maxima at 218, 255 and 351 nm. Peak 8 exhibited UV maxima at 268 and 329 nm together with an [M–H] ion at m/z 537 in the ESI-MS. Peaks 6 and 8 were tentatively identified as quercitrin and I3, II8-biapigenin, respectively, by comparison with literature values (Brolis et al. 1998; ¨ Jurgenliemk and Nahrstedt 2002). Peak 10 exhibited an extremely similar UV-VIS spectrum with Peak 12 (hypericin), especially in the visible region, and the ESIMS spectrum showed an [M–H] ion at m/z 519. These results suggested Peak 10 might be pseudohypericin, which was reported to be present in H. perforatum (Kurth and Spreemann 1998). The ESI-MS spectra of Peaks 9 and 11 exhibited [M–H] ions at m/z 521 and 505, which displayed similar UV spectra, exibiting red shifts compared with those of pseudohypericin and hypericin. Moreover, protopseudohypericin and protohypericin were known constituents in Hypericum plants, and they were easy to lose two
hydrogen atoms under light to transform to pseudohypericin and hypericin, respectively (Kurth and Spreemann 1998). Therefore, peaks 9 and 11 were tentatively identified as protopseudohypericin and protohypericin, respectively. In addition, it is noteworthy that there was no hyperforin analogues detected in the extract of H. enshiense under our experimental conditions, while hyperforin was clearly detected in H. perforatum using the same extraction and analysis conditions (data not shown). The contents of naphthodianthrone constituents hypericin and pseudohypericin in the hydroalcoholic extract of H. enshiense were calculated by HPLC analysis at 590 nm using synthetic hypericin as the external standard compound. The contents of the flavonoids determined were calculated using the relative response factors to hyperoside (RRF). The contents of the analytes in H. enshiense were compared with those of H. perforatum in previous study (Butterweck et al. 2003a), and the results are summarized in
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Table 2 Effect of the hydroalcoholic extract of H. enshiense on the total duration of immobility in the FST in mice (means7SEM, n ¼ 16, *Po0.05, **Po0.01 compared with control). Treatment
Dose (mg/kg)
Immobility time (s)
Control Extract
– 250 500 20
105.979.2 63.7715.9** 56.777.6** 79.479.8*
Imipramine
Table 3 Effect of the hydroalcoholic extract of H. enshiense on the total duration of immobility in the TST in mice (means7SEM, n ¼ 16, *Po0.05, **Po0.01 compared with control). Treatment
Dose (mg/kg)
Immobility time (s)
Control Extract
– 250 500 20
107.9710.4 71.0710.0* 88.8719.1 43.2710.6**
Amitriptyline
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hyperforin was concerned with the drug interference caused by the extract of H. perforatum (Moore et al. 2000; Mannel 2004). The absence of hyperforin in H. enshiense suggested its merit on avoiding the metabolic interference caused by hyperforin. The results obtained in the present study provided us certain information on the plant to act as a new potential resource for developing antidepressants to treat depressive disorders. The relationship between constituents and antidepressant activity, and the possible antidepressant mechanisms of H. enshiense will be investigated in future study.
Acknowledgements The authors wish to thank Dr. L.H. Wu for his contribution in collecting and identifying the plant. We thank Mr. J.H. Yao (Instruments of Analysis & Measurement Center, Sun Yat-sen University) for his help in the HPLC-MS experiment. The study was supported and funded by National Natural Science Foundation of China (2004, No. 30470188). References
Table 1. As shown in Table 1, it is of interest that the extract of H. enshiense was rich in hypericins with a content of 7.7 mg/g, even higher than that (5.5 mg/g) reported by Butterweck et al. Previous studies have indicated that napthodianthrones like hypericin and pseudohypericin, phloroglucinol derivative hyperforin (Muller et al. 2001), and some flavonoids, such as hyperoside, isoquercitrin and rutin (Butterweck et al. 2000) are the important active components of the antidepressant effects of H. perforatum. Since the hydroalcoholic extract of H. enshiense contained hypericin, pseudohypericin (the amount being 0.30% and 0.47%, respectively) and some similar flavonoid constituents, the antidepressant activities of the hydroalcoholic extract of the species were further investigated in behavioral models in mice. Antidepressant activities of the extract prepared from H. enshiense Using FST and TST models, the antidepressant activities of the extract of H. enshiense were investigated. As shown in Tables 2 and 3, the extract at both doses of 250 and 500 mg/kg significantly shortened the immobility time of mice in FST and TST (Po0.05 or Po0.01 vs. control, Tables 2 and 3). However, the extract produced no significant effects on locomotor activity (500 mg/kg: 125710; 250 mg/kg: 119712) in comparison with control group (12279) (data are not shown). Recently, Butterweck et al. (2003a) systematically screened for the antidepressant activities of St. John’s wort preparations, and it was found that the extracts devoid of hyperforin or devoid of both hyperforin and hypericin but enriched in flavonoids were still active in behavioral models. It was also reported that hyperoside significantly improved the water solubility of hypericin, and led to a better bioavailability of napthodianthrones (Butterweck et al. ¨ ¨ 2003b). Noldner and Schotz (2002) thought that rutin was essential for the antidepressant activity of H. perforatum extract in forced swimming test. Hyperforin was not detected in the hydroalcoholic extract of H. enshiense, so the antidepressant activity of the extract might be mainly concerned with hypericins and some flavonoid constituents, while other unidentified constituents might also be involved in its antidepressant activity. Taking together, HPLC-ESI-MS analysis of the extract indicated that H. enshiense contained flavonoid constituents and were rich in hypericins, but free of hyperforin. Several studies reported that
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