Acylphloroglucinol Derivatives from Hypericum andinum

Sep 29, 2014 - A new dimeric acylphloroglucinol derivative, andinin A (1), was isolated from the underground plant parts of Hypericum andinum, along w...
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Acylphloroglucinol Derivatives from Hypericum andinum: Antidepressant-like Activity of Andinin A Gari V. Ccana-Ccapatinta, Eveline D. Stolz, Paola F. da Costa, Stela M. K. Rates, and Gilsane L. von Poser* Programa de Pós-Graduaçaõ em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Sul, Avenida Ipiranga 2752, Porto Alegre/RS 90610-000, Brazil S Supporting Information *

ABSTRACT: A new dimeric acylphloroglucinol derivative, andinin A (1), was isolated from the underground plant parts of Hypericum andinum, along with three known dimeric acylphloroglucinols, uliginosin A (2), uliginosin B (3), and isouliginosin B (4). The structure of 1 was elucidated using 1D and 2D NMR and MS experiments and by comparison with previously reported data for Hypericum dimeric acylphloroglucinols. Andinin A (1) displayed antidepressant-like activity in a mouse forced-swimming test when administered orally at doses of 3, 10, and 30 mg/kg.

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Previous studies have demonstrated that the dimeric acylphloroglucinol molecular scaffold represents a promising tool for the development of new antidepressants.11−13 Uliginosin B is the most well-known dimeric acylphloroglucinol and is reported to occur in 19 Hypericum species from the sections Brathys and Trigynobrathys.9 Uliginosin B exhibits an antidepressant-like effect in rodents by increasing the availability of monoamines in the synaptic cleft, without binding to the monoaminergic sites on the neuronal carriers, indicating that it acts through a different mechanism from most antidepressants.12 Considering this background information, and as part of a continuing search for novel dimeric acylphloroglucinol compounds with potential antidepressantlike activity, an effort was made to isolate and identify additional constituents present in the n-hexane extract of Hypericum andinum Gleason (Hypericaceae). The air-dried underground plant material of H. andinum (roots and stems) was ground and extracted by maceration at room temperature with n-hexane. The extract was fractionated, and fractions were further processed as described in the Experimental Section to yield a new acylphloroglucinol derivative (1) along with the previously known uliginosin A (2), uliginosin B (3), and isouliginosin B (4). The HRESIMS of 1 showed a pseudomolecular ion peak [M + H]+ at m/z 501.2491, consistent with a molecular formula of C28H37O8. The 1H NMR spectrum of 1 (Table 1) exhibited a characteristic signal at very low field (δH 18.74), assigned to an enolizable β-triketonic system, as previously reported for

ental depressive disorders are major public health issues that encourage the search for new therapeutic agents.1 The herbaceous plant Hypericum perforatum L. (Hypericaceae), commonly known as St. John’s wort, constitutes one of the most renowned phytotherapeutical preparations for the treatment of mild to moderate depression.2 Flavonoids, naphthodianthrones, and acylphloroglucinol derivatives are recognized as major contributors to the overall effect observed.3 The polycyclic polyisoprenylated acylphloroglucinols hyperforin, adhyperforin, and hyperfoliatin are lipophilic constituents of H. perforatum and H. perfoliatum L. that display similar pharmacological profiles in animal and biochemical models predictive of antidepressant activity. They are able to inhibit the synaptosomal uptake of dopamine, serotonin, and norepinephrine. However, they do not bind to any of these monoamine sites on neuronal transporters, thus offering a potentially new mechanism of action, different from the current pharmacological treatments for depressive disorders.4−6 In contrast to Hypericum species from the sections Androsaemum, Drosocarpium, and Hypericum distributed mainly in Africa and Eurasia, which produce polyisoprenylated acylphloroglucinols (hyperforin-like), Hypericum species from the sections Brathys and Trigynobrathys, as found in Central and South America, are sources of dimeric acylphloroglucinol structures, consisting of an acylfilicinic acid and an acylphloroglucinol moiety linked by a methylene bridge.7−9 The occurrence of over 30 dimeric acylphloroglucinols reported for Hypericum species from the sections Brathys and Trigynobrathys was reviewed recently, and the presence of five known dimeric acylphloroglucinols, uliginosin A, uliginosin B, isouliginosin B, hyperbrasilol B, and isohyperbrasilol B, in four Peruvian Hypericum species was shown.9,10 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 25, 2014

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enol tautomerization of the acylfilicinic acid moiety in organic solvent solutions.9 This phenomenon complicates spectra interpretation, but in no case prohibited complete assignments. In order to confirm assignments made in CDCl3, further NMR experiments of 1 were recorded in acetone-d6 (Table 1). A characteristic 1H NMR signal for a methylene bridge at δH 3.53, in addition to two acyl carbon signals clearly detected at δC 208.5 and 207.6 in the 13C NMR spectrum, provided further evidence of the presence of two rings in 1. These observations were also supported by the mass spectrum of 1 (Figure S2, Supporting Information), in which characteristic fragments derived from the breakdown of the methylene bridge were observed.9 The acylphloroglucinol moiety was inferred as the second ring by the observation of two signals for chelated hydroxy groups (OH-8′, δH 11.55; OH-10′, δH 13.88) and three signals for aromatic oxygen-bearing carbons (C-6′, δC 160.3; C-8′, δC 159.7; C-10′, δC 165.6) in the 1H and 13C NMR spectra, thus confirming that 1 is a dimeric acylphloroglucinol, consisting of an acylfilicinic acid and an acylphloroglucinol moiety linked by a methylene bridge. This kind of compound is isolated frequently from Hypericum species occurring in the sections Brathys and Trigynobrathys.9 Furthermore, signals of a prenyl side chain were observed (Table 1). The 1D NMR

acylphloroglucinol derivatives carrying an acylfilicinic acid moiety isolated from Hypericum species and ferns of the genera Elaphoglossum and Dryopteris.9,11,14 This evidence, in addition to the presence of carbon signals of a carbonyl unit (C-1, δC 187.3), four enolic carbons (C-2, δC 111.3; C-3, δC 171.1; C-5, δC 199.0; and C-6, δC 107.0), and a dimethyl-substituted carbon (C-4, δC 44.1) in the 13C NMR spectrum of 1, confirmed the presence of a dimethyl-substituted acylfilicinic acid moiety. The 1H and 13C NMR spectrum of 1 showed certain anomalies that are best explained by the expected keto− Table 1. NMR Spectroscopic Data for Compounds 1 and 2 andinin A (1)a position 1 2 3 4 5 6 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ OH-3 OH-5 OH-8′ OH-10′

δC, type 187.3, 107.0, 171.1, 44.1, 199.0, 111.3, 17.1, 24.8, 24.5, 210.9, 36.7, 18.9, 18.9, 18.2, 25.9, 142.4, 116.8, 66.1, 160.3, 105.2, 159.7, 106.1, 165.6, 92.8, 211.7, 39.4, 18.9, 18.9,

C Cd C C C C CH2 CH3 unres. CH3 unres. Ce CH CH3 CH3 CH3 CH3 C CH CH2 Cf Cd Cf Cd C CH Ce CH CH3 CH3

δH, (mult, J in Hz)

3.53 s br 1.49 s br 1.37 s br 4.22 1.18 1.18 1.79 1.86

m, J = 6.8 d, J = 6.3 d, J = 6.3 s s

andinin A (1)b HMBC

1, 2, 3, 6′, 7′, 8′

10, 12, 13 10′, 11′, 13′ 10′, 11′, 12′ 2′, 3′, 4′ 1′, 2′, 3′

5.54 pseudo t, J = 7.0 4.66 s brg

1′, 2′ 3′, 4′

6.10 s

6′, 7′, 9′, 10′

4.13 m, J = 6.8 1.18 d, J = 6.3 1.18 d, J = 6.3 8.99 s 18.74 s 11.55 s 13.88 s

12′, 14′, 15′ 12′, 13′, 15′ 12′, 13′, 14′ 2, 3, 4 4, 5, 6, 10 7′, 8′, 9′ 9′, 10′, 11′

δC, type 187.8, 107.6, 171.6, 44.5, 199.7, 111.8, 17.3, 24.7, 24.7, 211.6, 37.1, 19.2, 18.2, 17.9, 25.7, 142.2, 117.9, 66.7, 160.2, 106.1, 159.9, 106.0, 166.2, 93.6, 211.7, 39.7, 19.2, 18.2,

C Cd C C C C CH2 CH3 CH3 Ce CH CH3 CH3 CH3 CH3 C CH CH2 Cf Cd Cf Cd C CH Ce CH CH3 CH3

δH, (mult, J in Hz)

3.54 s 1.50 s 1.31 s 4.21 1.16 1.16 1.85 1.86

sept, J = 6.8 d, J = 6.8 d, J = 6.8 s s

5.61 m 4.79 d, J = 7.2

6.18 s 4.10 sept, J = 6.8 1.16 d, J = 6.8 1.16 d, J = 6.8 9.14 s 18.81 s br 11.72 s 13.65 s

uliginosin A (2)c δC, type 187.5, 107.3, 171.8, 44.5, 199.6, 111.6, 17.3, 24.9, 24.8, 210.9, 36.9, 19.2, 19.5, 18.1, 26.0, 136.5, 121.9, 22.3. 160.9, 106.5, 158.1, 106.4, 161.3, 105.8, 211.4, 39.2, 19.2, 19.5,

C Cd C C C C CH2 CH3 CH3 Ce C CH3 CH3 CH3 CH3 C C CH2 Cf Cd Cf Cd Cf Cd Ce CH CH3 CH3

a

Acquired in CDCl3 (400 MHz). bAcquired in acetone-d6 (400 MHz). cAcquired in CDCl3 (60 MHz). d−fValues with the same superscript in each column are interchangeable. g4.65 d, J = 7.2, in the 1H NMR spectrum acquired in CDCl3 (60 MHz). B

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spectroscopic data obtained for 1 were found to resemble those of uliginosin A, a dimeric acylphloroglucinol first isolated from H. uliginosum.15 In contrast to uliginosin A, compound 1 bears an O-prenyl side chain since its methylene signals (δH 4.66; δC 66.1) displayed more deshielded values compared to the Cprenyl side chain of uliginosin A (δH 3.45; δC 22.3). This side chain could be linked to C-6′ of the acylphloroglucinol moiety of 1, as confirmed by HMBC correlations (Figure 1) and by

Figure 3. Effect of different doses of andinin A (1), imipramine (20 mg/kg, po), or vehicle (1 mL/100 g, po) in the mouse forced swimming test. The results are presented as means ± SEM (n = 8−10 mice/group). Significantly different values were detected by one-way ANOVA followed by a Student−Newman−Keuls test. **p < 0.01, compared to the vehicle group. ***p < 0.001, compared to the vehicle group. ###p < 0.001, compared to the andinin A (1) 1 mg/kg group.

Figure 1. Key HMBC (→) and COSY correlations of andinin A (1).

direct comparison of hydroxy group chemical shifts with analogous signals observed in uliginosin B and isouliginosin B (Figure 2).9,16,17 Hence, compound 1 was identified by

stimulation (Figure S1, Supporting Information). These results are in line with other studies on the dimeric acylphloroglucinols crassipin A and uliginosin B (3), which demonstrated that these compounds reduce the immobility time in the mouse FST at doses of 15 mg/kg, po and 10 mg/kg, po, respectively.11,12 These new observations, when added to previous results, suggest that Hypericum species from the sections Brathys and Trigynobrathys are sources of dimeric acylphloroglucinol derivatives with potential antidepressant-like activity and may represent new chemical scaffolds for the design of antidepressants.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded using a PerkinElmer model 341 polarimeter. UV spectra were recorded using a Hewlett-Packard model 8452A diode array spectrophotometer. IR spectra were obtained on a PerkinElmer Spectrum BX FT-IR spectrometer. 1D and 2D NMR spectra were measured at 25 °C on a Varian MR400 (operating at 399.736 MHz for 1 H and 100.523 MHz for 13C) or an Anasazi Eft-60 MHz (operating at 60 MHz for 1H and 15 MHz for 13C) NMR spectrometer. Spectra were recorded in CDCl3 (99.8%, Acros Organics, Fair Lawn, NJ, USA), with tetramethylsilane (TMS) as internal standard, and acetone-d6 (99.9%, Sigma-Aldrich, St. Louis, MO, USA) referenced against residual nondeuterated solvent (acetone-d6: δH 2.05/δC 29.8). 1D (1H, 13C) and 2D NMR (COSY, HMBC, and HSQC) spectra were obtained by using the standard pulse sequences from the Varian and Anasazi user libraries. Spectra of isolated compounds are provided as Supporting Information. HRESIMS were acquired in positive-ion mode on a Q-TOF Premier spectrometer equipped with a nanospray ion source (Waters, Milford, MA, USA). Reagent grade acetone, dichloromethane, ethyl acetate, ethyl ether, and n-hexane (F. Maia, São Paulo, Brazil), formic acid (Vetec, Rio de Janeiro, Brazil), and HPLC grade acetonitrile and methanol (Merck, Darmstadt, Germany) were regularly used in the extraction and isolation procedures. Dry column vacuum chromatography (DVCC) was carried out over silica gel H (10−40 μm, Merck, Darmstadt, Germany). Column chromatography (CC) was carried out over silica gel 60 (70−230 mesh, Merck, Darmstadt, Germany). The extracts and fractions were monitored by TLC on precoated silica gel 60 F254 plates (Merck, Darmstadt, Germany) as a stationary phase using several different mobile phases composed of hexane−CH2Cl2 (50:50), hexane−EtOAc (90:10), hexane−EtOEt (95:5), hexane−acetone (95:5), hexane−EtOAc (95:5), hexane−EtOEt (97.5:2.5), and hexane−acetone (97.5:2.5).

Figure 2. Overlaid (20−8 ppm) 1H NMR spectra of andinin A (1), uliginosin B (3), and isouliginosin B (4) in CDCl3 (400 MHz).

spectroscopic analysis as 3,5-dihydroxy-2-isobutyryl-4,4-dimethyl-6(2,4-dihydroxy-3-isobutyryl-6-((3-methylbut-2-enyl)oxy)benzyl)cyclohexa-2,5-dienone and named trivially andinin A. The 1H NMR (400 MHz, CDCl3) and MS data of 2 were in agreement with those of uliginosin A.15,17 Previously published 13 C NMR data of uliginosin A in DMSO-d6 showed many unresolved signals.17 Likewise, the 13C NMR spectra acquired in CDCl3 and acetone-d6 at 400 MHz were greatly unresolved (Figures S17 and S19, Supporting Information). Nevertheless, when acquired in CDCl3 at 60 MHz (Table 1), the signals were clearly observed and the chemical shift values were in general agreement with those published for uliginosin B (3) and isouliginosin B (4).9,17 The administration of andinin A (1) produced a dosedependent reduction in the immobility time of mice submitted to the forced-swimming test (FST) (Figure 3) (ANOVA: F(5,60) = 16.032, p < 0.001). In the FST, the immobility behavior is assumed as an expression of “behavioral despair”, which is considered a depressive-like symptom. However, test compounds that alter motor behavior can induce false results. Andinin A (1) did not alter the locomotor activity at the highest dose (30 mg/kg, po) in the open field test (Student’s t test: p = 0.249), revealing that the anti-immobility effect observed in the FST was not related to a nonspecific motor C

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The three latter solvent pairs were acidified with 0.25% formic acid. After elution, acylphloroglucinols were detected by fluorescence quenching at 254 nm, as dark-blue-colored spots at 356 nm, and by orange (1), reddish-orange (2), and brown-yellow (3 and 4) spot colors after spraying with anisaldehyde/sulfuric acid reagent and heating. Centrifugal planar chromatography (CPC) was carried out on silica gel 60 G F254 (1 mm plates) using a Chromatotron instrument (model 7924 T, Harrison Research, San Bruno, CA, USA). Plant Material. The underground plant parts of Hypericum andinum were collected in Amparaes, Calca Province, Cusco (May 2008), under consent of the Dirección General Forestal y de Fauna Silvestre of the Republic of Peru (0147-2010-AG-DGFFS-DGEFFS). The plants were identified by Prof. Washington H. Galiano Sánchez, Academic Department of Biology, University of San Antonio Abad del Cusco, Peru. Voucher specimens were deposited in the Herbarium of the Federal University of Rio Grande do Sul (ICN, UFRGS), Brazil (ICN 190419), and in the Herbarium Vargas of the National University of San Antonio Abad del Cusco (CUZ, UNSAAC), Peru (Ccana-Ccapatinta 06). Extraction and Isolation. The air-dried and powdered vegetal material (3.50 kg) was successively extracted by maceration with nhexane over 72 h (five times, plant solvent ratio 1:5). The extracts were pooled and evaporated to dryness under reduced pressure and then treated with cold acetone to obtain an insoluble lipid residue and an acetone-soluble fraction that yielded a dark greenish, viscous oil residue after solvent evaporation (62.5 g). The whole acetone-soluble fraction (62.5 g) was subjected to DVCC on silica gel H using a hexane−EtOAc gradient as mobile phase. An initial bright yellow, broad band was eluted with a hexane− EtOAc mixture (100:0−90:10), while a second, greenish band remained at the application point, eluting only after increasing the mobile phase polarity (90:10−80:20), and was not further worked up since no acylphloroglucinol profile was seen by TLC. The initial fraction (40.2 g) was resubmitted to DVCC using a hexane−EtOAc gradient as mobile phase (100:0−90:10) to obtain an initial bright yellow fraction (100:0−98:2), from which pure uliginosin B (3) was obtained and two further reddish fractions (A, 97:3−95:5, and B, 95:5−90:10). Fraction A (4.2 g) was submitted to CC using a hexane−EtOAc gradient as mobile phase (100:0−90:10) to afford eight subfractions (SbFr. 1−8). SbFr. 3 (980 mg) was subjected to repeated CPC on 1 mm silica gel plates using hexane−EtOAc (100:0− 90:10), hexane−EtOEt (100:0−95:5), and hexane−acetone (100:0− 95:5), with or without 0.25% formic acid as mobile phases, to afford 23 mg of pure 1, together with uliginosin A (2) and isouliginosin B (4). Andinin A (1): yellow oil; [α]20D +3.0 (c 0.5, CHCl3); UV (MeOH) λmax (log ε) 226 (4.16) 282 (4.09) 352 (3.91) nm; IR (neat) νmax 3242, 2927, 2854, 1731, 1619, 1597, 1426, 1359 cm−1; 1H NMR and 13 C NMR see Table 1; ESIMS m/z 501 [M + H]+ (6), 445 (17), 433 (63) 375 (27), 277 (13), 265 (24), 237 (70), 225 (25), 209 (51), 197 (100), 69 (8); HRESIMS m/z 501.2491 [M + H]+ (calcd for C28H37O8, 501.2488). Test Animals. Adult male CF1 mice (25−35 g) were purchased from the Fundaçaõ Estadual de Produçaõ e Pesquisa em Saúde (Porto Alegre, Brazil). The animals were housed as six mice per plastic cage (L: 28 cm, W: 17 cm, H: 13 cm) and were held under a 12 h light/ dark cycle (lights on at 7 A.M.) at constant temperature (23 ± 1 °C) with free access to standard certified rodent diet (Nuvilab CR-1, São Paulo, Brazil) and tap water. All experiments were approved by a local Ethics Committee of Animal Use (UFRGS, number 23825/2012). Experiments were conducted between 10:00 and 16:00 h. The animals were subjected to fasting for 3 h before treatment, with free access to water, and were allowed to acclimate to the experimental room for at least 2 h before experiments. Treatments. Imipramine (Henrifarma, São Paulo, Brazil) and andinin A (1) were suspended in saline (0.9% NaCl) with 2% polysorbate 80 (Merck, Darmstadt, Germany). All solutions were freshly prepared on test day and administered po at 1 mL/100 g body weight. The doses of andinin A (1) used in the forced swimming test were 1, 3, 10, or 30 mg/kg, and only the highest dose (30 mg/kg) was evaluated in the open field test. The vehicle (saline plus 2%

polysorbate 80) and imipramine 20 mg/kg were used as negative and positive controls, respectively. The concentration of imipramine in the vehicle was 2 mg/mL, while andinin A (1) was suspended at four different concentrations: 0.1, 0.3, 1, or 3 mg/mL. Forced-Swimming Test. The FST was conducted using the method described by Porsolt and associates18 with minor modifications previously standardized and validated.12 Briefly, mice were treated with andinin A (1), imipramine, or vehicle. Sixty minutes later the animals were forced individually to swim in a cylinder pool (10 cm diameter, 13 cm hight, 22 ± 1 °C) for 6 min; the total time of immobility during the 6 min test was scored and determined by a blinded observer. Each mouse was considered immobile when it ceased struggling and remained floating motionless in the water, making only those movements necessary to keep its head above water. Open-Field Test. In order to rule out any unspecific locomotor effect, mice were treated with andinin A (1), 30 mg/kg, po, or vehicle and after 60 min were placed individually in an acrylic box (40 × 30 × 30 cm), with the floor divided into 24 equal squares. The number of crossings in the squares with the four paws was measured during a period of 6 min by a blinded observer. Statistical Analysis. The data were evaluated using Student’s t test or one-way analysis of variance (ANOVA) followed by the Student− Newman−Keuls posthoc test. All results were expressed as means ± SEM. The analyses were performed using Sigma Stat 3.2 software (Jandel Scientific, San Rafael, CA, USA). Differences were considered statistically significant at p < 0.05.

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ASSOCIATED CONTENT

S Supporting Information *

NMR spectra of compounds 1−4 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (+55) 51 33085529. Fax: (+55) 51 3308 5437. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the Brazilian agencies CAPES, CNPq (472219-2012-0), and FAPERGS (1016474) for financial support. G.V.Cc. is a Peruvian student who acknowledges a fellowship from the PEC-PG program of the CAPES/ CNPq−Brazil (PEC-PG 2011, 190470/2011-9). The LRNANO/CNANO laboratory of the UFRGS is specially acknowledged for NMR facilities. Special thanks are given to Miss V. Herzfeldt for technical support in bioassays, Mrs. G. N. Wentz (LRNANO/CNANO laboratory, Chemistry Institute, UFRGS) for assistance with recording 2D NMR spectra, Dr. M. H. Holzschuh (Analytic Central, Pharmacy Faculty, UFRGS) for recording mass spectra, and Prof. Mrs. J. F. Gonzales Bellido (Organic Chemistry Laboratory, Chemistry Department, UNSAAC) for assistance with vegetal material preparation.

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REFERENCES

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