Isolation of Steroidal Glycosides from the Caribbean Sponge

Article pubs.acs.org/jnp

Isolation of Steroidal Glycosides from the Caribbean Sponge Pandaros acanthifolium Fabrice Berrué,† Malcolm W. B. McCulloch,† Patricia Boland,† Saskia Hart,† Mary Kay Harper,‡ James Johnston,§ and Russell Kerr*,† †

Department of Chemistry and Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI C1A 4P3, Canada ‡ Department of Medicinal Chemistry, University of Utah, 30 South 2000 East, Salt Lake City, Utah 84112, United States § National Research Council Canada, 550 University Avenue, Charlottetown, PEI C1A 4P3, Canada S Supporting Information *

ABSTRACT: Four new steroidal glycosides, acanthifoliosides G−J (1−4), were isolated as minor constituents from the Caribbean marine sponge Pandaros acanthifolium. These metabolites are characterized by a highly oxygenated D ring and the presence of a disaccharide rhamnose-glucose residue and a rhamnose at positions C-3 and C-15, respectively. Their structures were established on the basis of extensive interpretation of 1D and 2D NMR data and HRESIMS analyses. The absolute configurations of the glucose and rhamnose sugars were determined by preparing aldose o-tolylthiocarbamate derivatives and comparison to authentic standards by LC/HRESIMS. Acanthifolioside G (1) exhibited antioxidant and cytoprotective activities.

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teroids are a well-known large class of biologically active secondary metabolites isolated from many groups of marine invertebrates including sponges. Among these metabolites, steroidal glycosides show a range of biological activities including cytotoxic, antibacterial, and hypocholesterolemic; they have also been used in the treatment of heart disorders.1−3 In the marine environment, steroidal glycosides have most frequently been isolated from echinoderms,4 sponges, and soft corals.5 As part of ongoing research on secondary metabolites from Caribbean marine sponges, we undertook a chemical investigation of Pandaros acanthifolium based on putative cytoprotective activity observed during the primary screening phase of extracts derived from the marine sponge. The polyether carboxylic acid acanthifolicin6 was isolated from P. acanthifolium more than 30 years ago; however, it was only recently that P. acanthifolium was reported as a source of steroid glycosides, namely, the pandarosides and acanthifoliosides. Both the pandarosides and acanthifoliosides were shown to exhibit antiprotozoal, cytotoxic, and moderate antimicrobial activity.7−10 Pandarosides are steroidal glycosides characterized by an uncommon 2-hydroxycyclopentenone D ring, whereas acanthifoliosides possess a rare C-15 and C-16 oxidized D ring saturated at positions 16 and 17. Herein we report the isolation and structure elucidation of four novel steroid glycosides, acanthifoliosides G−J (1−4), and describe their antioxidant potential. © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION A specimen of P. acanthifolium was collected by hand using scuba off the coast of the Florida Keys. The organic extract was initially fractionated by vacuum liquid chromatography on reversedphase C18 to generate six fractions. The acanthifoliosides G−J (1−4) were localized within the fraction eluted with 100% EtOH (F4), and purification was achieved by flash chromatography on C18, followed by RP-HPLC. Acanthifolioside G (1) was obtained as a colorless oil. HRESIMS supported a molecular formula of C47H80O17, indicating eight degrees of unsaturation. The 1H NMR, 13C NMR, and HSQC spectra clearly indicated that 1 is a steroid with oxygenation at C-3, C-15, and C-16. The steroidal skeleton was evident from distinctive methyl signals at δH 1.03 (s, H3-18), 0.87 (s, H3-19), Received: July 26, 2012

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dx.doi.org/10.1021/np300520w | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data for 1 (CD3OD) δC, type

δH (J in Hz)

1

38.3, CH2

2

30.5, CH2

1b 1a; 2a 1a,b; 2b

3 4

78.7, CH 35.1, CH2

5 6 7

46.4, CH 29.9, CH2 32.3, CH2

8 9 10 11

32.5, 56.1, 37.0, 21.3,

1.74, m 1.01, m 1.88, m 1.54, m 3.75, dddd (11.1, 11.1, 4.8, 4.8) 1.74, m 1.35, m 1.15 m 1.33 m 2.05, m 0.94, m 1.79, m 0.72, ddd (11.3, 11.3, 3.5)

12

42.4, CH2

1.52, 1.32, 1.99, 1.11,

m m m m

8; 11b; 12a 8; 11a; 12a 11a,b; 12b 12a

13 14 15 16 17 18 19 20 21 22

44.1, 59.6, 79.2, 76.2, 61.7, 15.9, 12.8, 31.9, 19.0, 37.7,

25.9, CH2

24 25 26 27 OAc 1⁗ 2⁗ L-rhamnose (C-15) 1′ 2′ 3′ 4′ 5′ 6′ L-glucose (C-3) 1″ 2″ 3″ 4″ 5″ 6″

40.8, 29.1, 23.2, 22.9,

m dd (5.7, 5.7) dd (8.1, 5.9) m s s m d (6.5) m m m m m m m m

15; 14; 15; 16;

23

0.97, 4.19, 5.40, 1.26, 1.03, 0.87, 1.80, 0.98, 1.19, 0.88, 1.42, 1.17, 1.12, 1.51, 0.88, 0.87,

position

L-rhamnose 1‴ 2‴ 3‴ 4‴ 5‴ 6‴

a

CH CH C CH2

C CH CH CH CH CH3 CH2 CH CH3 CH2

CH2 CH CH3 CH3

HMBCa

COSY

2a,b; 4a,b 3; 5 3 4

1″

7b; 8; 6 7a 7a,b; 9, 14 8; 11a,b

8 16 17 20

16; 21; 22b 20

ROESY

1″; 1b; 2a; 4a; 5

6; 8; 15 7a; 18

8; 13; 18 1′; 13; 16; 17 1‴′; 13; 14

1′; 7a; 8; 14; 16 14; 17

12; 13; 14; 17 10

8 8

17; 20; 22

20; 23a,b 22b 22b; 23a; 24 23b; 25 24; 26; 27 25 25

172.6, C 21.3, CH3

2.14, s

102.8, 72.7, 72.6, 73.7, 70.6, 17.9,

CH CH CH CH CH CH3

4.40, 3.82, 3.60, 3.39, 3.62, 1.21,

bs dd (3.0, 1.5) dd (9.4, 3.2) dd (9.4, 9.4) m d (6.0)

2′ 1; 3′ 2; 4′ 3′; 5′ 4′; 6′ 5′

100.2, 79.4, 79.3, 71.9, 77.6, 62.7,

CH CH CH CH CH CH2

4.49, 3.34, 3.46, 3.28, 3.25, 3.88, 3.65,

d (7.5) m dd (9.0, 9.0) dd (9.0, 9.0) m dd (11.9, 2.1) m

102.3, 72.2, 72.4, 73.9, 69.8, 17.9,

CH CH CH CH CH CH2

5.16, 3.92, 3.66, 3.39, 4.11, 1.23,

bd (1.5) dd (3.4, 1.5) dd (9.4, 3.4) dd (9.4, 9.4) dq (9.5, 6.4) d (6.0)

1‴′

2′; 1′

25; 2′ or 3′; 5′

6′

15; 2′; 2‴′; 5′ 1′; 3′ 2′ 6′; 7a

4′; 5′

5′; 7a; 8

2″ 1″; 3″ 2″; 4″ 3″; 5″ 4″, 6″a,b 5″; 6″b 5″; 6″a

3 1″; 1‴; 3 2″; 4″ 5″

3; 3″; 2a; 5″; 1a or 4a 1‴; 6″a 1″; 1‴; 6

2‴ 1‴; 3‴ 2‴; 4‴ 3‴; 5‴ 4‴, 6‴ 5‴

2″ or 3″; 5‴

6a″; 1″ 5″; 6″b 6″a

6‴ 4‴; 5‴

2‴; 3″; 2″; 5″ or 4″ 1‴; 3‴ or 6″b; 4‴ 5‴ 6″ 6‴; 1 or 4; 2b; 4b; 3‴ 4‴, 5‴ (weak)

HMBC correlations, optimized for 8 Hz, are from proton(s) stated to the indicated carbon. B



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0.98 (d, J = 6.5 Hz, H3-21), 0.88 (m, H3-26), and 0.87 (m, H3-27) and the methine signal at δH 3.75 (tt, J = 11.1, 4.8 Hz, H-3). Key COSY and HMBC correlations of H-15 (δH 4.19)/ C-13 (δC 44.1), C-16 (δC 76.2), C-17 (δC 61.7) and H-16 (δH 5.40)/C-13 (δC 44.1), C-14 (δC 59.6) identified the locations of the two oxygenated methines at C-15 and C-16 of the cyclopentane ring (see Table 1 and Figure 1). The relative configuration of the cholestane skeleton was deduced by analysis of coupling constants and ROESY data. Indeed, the large vicinal coupling constant for H-3 (dddd, J = 11.1, 11.1, 4.8, 4.8 Hz) necessitated an axial position, and consequently

ROESY data. The HMBC cross-peak between H-1′ and the carbon resonance at δC 79.2 (C-15) unambiguously located this sugar on the steroid skeleton. Similar reasoning led to the characterization of the second rhamnose moiety, which was linked to the third sugar by a 1,2 α-glycosidic linkage, as supported by a key HMBC correlation between H-2″ and the carbon resonance at δC 102.3 (C-1‴). Finally, the two apparent triplet signals at δH 3.46 (dd, J = 9.0, 9.0 Hz, H-3″) and δH 3.28 (dd, J = 9.0, 9.0 Hz, H-4″) and the large coupling constant between H-2″ and H-1″ (δH 4.49, d, J = 7.5 Hz) confirmed the identity of the third sugar as a β-glucopyranose. Strong HMBC correlations (H-1″/C-3 and H-3/C-1″) located this residue at C-3. To confirm the identity of the glucose and rhamnose sugars, and to determine their absolute configuration, we adapted Tanaka’s derivatization method for the identification of aldose enantiomers.11 This method proceeds by conversion of the aldose enantiomers to chromatographically separable diastereomeric thiocarbamates. Rather than using HPLC with UV detection, we adapted the method to utilize LC/HRESIMS with selected ion monitoring (SIM) to detect the diastereomeric derivatives with greater sensitivity. Acid hydrolysis of 1 followed by treatment with L-cysteine methyl ester and o-tolylisothiocyanate generated the corresponding thiocabarmates. The reaction mixture was analyzed by LC/HRESIMS and compared to aldose thiocarbamate standards derived from authentic sugars, which revealed the presence of a 2:1 ratio of L-rhamnose:L-glucose (see Supporting Information, S11). Consequently, the structure of 1 was deduced to be 3β-O-[α-Lrhamnopyranosyl(1→2)-β- L-glucopyranosyl]-15 β-O-(α- Lrhamnopyranosyl)-5α-cholestan-16β-yl acetate. Acanthifolioside H (2) was isolated as a colorless oil. HRESIMS supported a molecular formula of C47H78O17, indicating one additional degree of unsaturation compared to 1. NMR spectra of 2 closely resembled those of 1. The main differences were observed in the steroid side chain with two deshielded signals at δH 5.37 (ddd, 15.4, 7.8, 6.5 Hz, H-23) and δH 5.24 (m, H-22); the strong coupling constant (15.4 Hz) indicated a trans configuration for the Δ22,23 double bond. The location of this unsaturation was further confirmed by key COSY correlations (H-20 (δH 2.50)/H-22/H-23/H2-24a,b (δH 1.84 and 1.76, respectively)/H-25 (δH 1.54)). All other spectroscopic data for the remainder of the molecule were very similar to 1, and thus the structure of 2 was established as (E)-3β-O-[α-L-rhamnopyranosyl(1→2)-β-L-glucopyranosyl]-15 β-O-(α-L-rhamnopyranosyl)5α-cholest-22-en-16β-yl acetate. Acanthifolioside I (3) was isolated as a colorless oil, and HRESIMS supported a molecular formula of C49H84O18, indicating eight degrees of unsaturation. Comparison of NMR data with those of 1 and 2 indicated that these were closely related structures with the major differences occurring in the steroid side chain. HSQC correlations revealed a hydroxylated methine (H-23) (δH 3.85 and δC 69.4) as well as an additional ethyl substituent. These observations suggested a poriferastane backbone for the steroidal aglycone and was supported by COSY correlations confirming the assemblies H20 (δH 2.16)/H2-22a,b (δH 1.45 and 0.82, respectively)/H-23/ H-24 (δH 0.90)/H-25 (δH 1.80) and H-24/H2-24′/H3-24″. This steroid side chain has been reported only in the ancanthifoliosides D−F isolated from P. ancanthifolium.10 The absence of discernible coupling constants for H-23 and the lack of material for chemical derivatization precluded a direct determination of the relative or absolute configuration of the

Figure 1. Key HMBC (H→C, arrow) and key H−H COSY (bold) correlations for 1.

H-3 was located on the α-face of the steroid. Furthermore, the observed chemical shift of H-3 is identical to that of known acanthifoliosides with the glycosidic linkage in the β-position.10 The NOE correlations H-3/H-5 and H-19/H-8, in addition to the coupling constant value of H-9 (ddd, J = 11.3, 11.3, 3.5 Hz), were in agreement with a trans configuration for both A/B and B/C ring junctions. Moreover, the key NOE correlations H-8/ H3-18 and H-14/H-15/H-16/H-17 and the observed coupling constant values of H-15 (dd, J = 5.7, 5.7 Hz) and H-16 (dd, J = 8.1, 5.9 Hz) led us to conclude that the C/D ring junction is trans and the protons H-14, H-15, H-16, and H-17 are located on the α-face of the cholestane skeleton. This proposed relative configuration is consistent with the previously reported structures of acanthifoliosides B−F.10 An acetate group (δH 2.14; δC 172.6/21.3) and three sugar moieties (anomeric carbon chemical shifts: δC 102.8 (C-1′), δC 100.2 (C-1″), and δC 102.3 (C-1‴)) evident from the NMR data accounted for the remaining four degrees of unsaturation in 1. These observations were corroborated by HRMSn data that indicated two consecutive neutral losses of m/z 146.058 (C6H10O4), supporting the presence of two deoxyhexose sugars (see Supporting Information, S10). The third sugar was identified as a hexose due to the presence of nonequivalent methylene signals at δH 3.88 (dd, J = 11.9, 2.1 Hz H2-6a″) and 3.65 (m, H2-6b″). Analysis of the COSY and TOCSY data allowed for the identification of three glycosidic spin systems: C-1′ to C-6′, C-1″ to C-6″, and C-1‴ to C-6‴. Furthermore, the apparent triplet signal δH 3.39 (dd, J = 9.4, 9.4 Hz, H-4′) with a large coupling constant placed H-3′, H-4′, and H-5′ in axial positions. The small coupling constants between H-3′ and H-2′ (3.2 Hz), and H-2′ and H-1′ (1.5 Hz), suggested that this unit was a rhamnose residue, which was supported by C



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Figure 2. Evaluation of the antioxidant and cytoprotective activities of acanthifoliosides. (A) Human U373 astrocytoma cells expressing ARE (black) or XRE (white) reporter cassettes were treated with the indicated concentration of test acanthifolioside for 24 h prior to evaluation of luciferase activity. Values are expressed as fold changes in mean activity ± error relative to TBHQ standard + DMSO vehicle control (V). Mock-treated cells (M) served as an untreated control, which is TBHQ alone. (B−D) U373 cells were pretreated with the indicated concentration of test acanthifolioside for 24 h prior to exposure to 300 μM H2O2 for an additional 24 h. Following oxidative stress, cell lysates were prepared for evaluation of SOD (B), GPx (C), or TBARS (D) activity. Values are expressed as the mean ± standard deviation from four replicates and represent the percent inhibition of xanthine oxidase activity (B), the rate of GSSH turnover in nmol/min/ml (C), and the production of MDA reactive equivalents (D, nM/ml). *p < 0.05, **p < 0.01, ***p < 0.001 Student’s t-test. In graphs B−D, mock-treated cell (M) and vehicle control (V) are defined by the real experimental control conditions.

As all other spectroscopic data were very similar to those for 3, the structure of 4 was established as 23-hydroxy-3β-O-[α-Lrhamnopyranosyl(1→2)-β- L -glucopyranosyl]-15β-O-(α- L rhamnopyranosyl)poriferast-5-en-16β-yl acetate. The fraction derived from the organic extract of P. acanthifolium by eluting with 100% EtOH (F4) was determined to possess putative phase 2 protein and antioxidant potential during primary screening for bioactivity in a cell-based assay. In an attempt to identify the molecular species responsible for this activity, the two most abundant pure compounds, acanthifoliosides G (1) and J (4), were assessed for antioxidant activity. The antioxidant response element (ARE) is a transcriptional element that regulates the expression of phase 2 proteins involved in the defense against oxidative stressors and toxins.12 Compounds that activate the ARE have strong potential for use as antioxidants by increasing the endogenous level of beneficial proteins within cells and tissues. However, many compounds that activate the ARE also activate the closely related xenobiotic response element (XRE), a detrimental response.12 Thus, it is necessary to separate beneficial monofunctional ARE inducers from less desirable bifunctional ARE−XRE inducers. As shown in Figure 2A, acanthifolioside G (1) increased ARE activity in a

two stereocenters C-23 and C-24. However, comparison of 1 H NMR data and 13C chemicals shifts δC 69.4 (C-23), δC 54.1 (C-24), δC 20.3 (C-24′), and δC 14.9 (C-24″) with those for acanthifoliosides D−F suggested that 3 has the same configuration as these known compounds. Acanthifolioside I (3) was thus established as 23-hydroxy-3β-O-[α-L-rhamnopyranosyl(1→2)-β-L-glucopyranosyl]-15β-O-(α-L-rhamnopyranosyl)-5αporiferastan-16β-yl acetate. Acanthifolioside J (4) was isolated as a colorless oil. HRESIMS supported a molecular formula of C49H82O18 and implied nine degrees of unsaturation. The NMR data were very similar to those for 3, with differences consistent with additional unsaturation on the B ring. Indeed, a Δ5,6 double bond was evidenced by a quaternary carbon at δC 141.8 (C-5) and the vinyl signals at δC 122.2 and δH 5.41 (H-6), the key COSY correlation H-6/H-7(δH 2.37 and 1.60), and the HMBC correlations H-19 (δH 1.07)/C-5. As with 3, the lack of material for chemical derivatization of 4 precluded a direct determination of the relative or absolute configuration of the two stereocenters C-23 and C-24. However, comparison of 1H and 13 C NMR data with those for acanthifoliosides D−F suggested that 4 has the same configuration as these known compounds. D



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dose-dependent manner (2.27 ± 0.19-fold at 25 μg/mL) in the absence of significant concurrent increases in XRE activity. Indeed, XRE activity typically is a counterscreen for adverse activity above a 2-fold baseline threshold. Acanthifolioside J (4) also influenced ARE activity; however this effect was less intense (1.52 ± 0.05-fold at 25 μg/mL). The ARE response can be exhibited by the cytotoxicity caused by treatment with the tested compounds, but it should be noted that acanthifolioside G (1) induced ARE activity in the absence of observable cytotoxicty (see Supporting Information, S19). To evaluate potential cytoprotective effects, an oxidative stress model was employed in which cells that were pretreated with test acanthifoliosides were exposed to H2O2 as a stressor and levels of antioxidant enzyme activity measured. This study focused on two key protective mediators, superoxide dismutase (SOD) and glutathione peroxidase (GPx). SOD is a metalloenzyme that catalyzes the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide.13 As such, SODs are a critical component of cellular antioxidant defenses and the maintenance of homeostasis. Proper levels of SOD are necessary to restrict the production of peroxynitrite, an oxidizing and nitrating agent formed upon the reaction of nitric oxide and the superoxide anion that has been implicated in diverse pathologies.13 GPx, in turn, catalyzes the reduction of H2O2 into various organic peroxides, alcohols, and water in a manner that provides a mechanism for detoxifying peroxides to avoid free radical generation.14 Thus, it is important in protecting cells from free radical damage and limiting lipid peroxidation. Thiobarbituric acid reactive substances (TBARS) is used in this study as a measure of lipid peroxidation, and it is an indicator that cells have been subject to oxidative stressors.15 Consistent with the results of the ARE assay, pretreatment with 1 significantly increased both SOD (Figure 2B) and GPx (Figure 2C) activity in a dose-dependent manner following exposure to H2O2. Moreover, this activity was associated with a concurrent decrease in indicators of lipid peroxidation and cellular oxidative damage as measured by a TBARS assay (Figure 2D).15 These results suggest that the decrease of damage is correlated to an increase of phase II protein expression. A moderately increased SOD and GPx activity was also observed with 4; however, the increased activity was not as obviously correlated with protection from lipid peroxidation damage as that observed with 1. Acanthifolioside G (1) exhibited ARE-inducing activity that was twice as potent as the gold standard pharmacological inducer tert-butylhydroquinone (TBHQ, 25 μM). It did so in the absence of toxicity and without adverse bifunctional induction of XRE activity. This induction was broad spectrum in that it was not specific to a single phase II protein. As evidenced by SOD and GPx expression, more than one protective species of phase II protein was being influenced, which may be indicative of a more global induction of the cellular defense mechanism and imply the ability to respond to a greater array of insulting stimuli. Finally, this inducing activity was found to correlate with in vitro protection, as evidenced by the TBARS results. Compound 1 therefore meets the criteria of functional efficacy, potential safety, and broad applicability. Taken together, these results suggest that acanthifoliosides have antioxidant and cytoprotective potential, and acanthifolioside G (1) is a candidate for further investigation.

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Infrared spectra were recorded using attenuated total reflectance, with samples deposited as a thin film on a Thermo Nicolet 6700 FT-IR spectrometer (Smart iTR). NMR spectra were obtained on a 600 MHz Bruker Avance III NMR spectrometer. Chemical shifts (δ) are reported in ppm and were referenced to the CD3OD residual peaks at δH 3.31 ppm and δC 49.0 ppm. LC/MS spectra using a thermo gold UPLC column were recorded on Accela Thermo equipment with hyphenated MS-ELSD-UV detection: Finnigan LXQ ion trap mass spectrometer fitted with either an APCI or ESI source, PDA, and LT-ELSD Sedex 80. High-resolution mass spectra were measured on a Thermo Orbitrap Velos mass spectrometer. Automated flash chromatography was performed on a Teledyne Combiflash Rf200 using C-18 RediSep columns. HPLC purifications were carried out on a Thermo Surveyor coupled with a Sedex 55 evaporative light-scattering detector. L-Glucose, D-glucose, L-rhamnose, L-cysteine methyl ester, and D-cysteine were purchased from Sigma Aldrich. D-Cysteine methyl ester was prepared by Fisher esterification. Sponge Material. The marine sponge Pandaros acanthifolium Duchassaing & Michelotti, 1864 was collected at 8 m depth by scuba off the coast of Marathon in the Florida Keys in June 2007 (lat. 24.76, long. −81.16). The specimen was frozen after harvesting and kept at −18 °C until being freeze-dried. A voucher sample (KEY252) has been deposited in the Marine Natural Products Lab at UPEI. P. acanthifolium (order Poecilosclerida, family Microcionidae) is an upright bushy purplish-black sponge that gives off a purple exudate when squeezed. The skeleton is composed of spongin fibers cored by tylostyles (some with microspined heads), rare acanthostyles, and long wispy oxea. Extraction and Isolation. The freeze-dried sponge (10.9 g) was extracted with CH2Cl2/MeOH (1:1), and the crude oil (3.32 g) was fractionated by RP-C18 flash chromatography with the following step gradient: H2O/MeOH (9:1), H2O/MeOH (1:1), H2O/MeOH (2:8), EtOH, Me2CO, and CH2Cl2/MeOH (1:1). Fraction F4 (146 mg), eluted with EtOH, was further fractionated on C18 using an automated chromatography system (Combiflash) and a linear gradient from H2O/MeOH (40:60) to 100% MeOH. All the resulting fractions were then recombined in eight subfractions according to their LC/MS profiles. The subfraction F4-5 (29 mg) was purified by semipreparative reversed-phase HPLC (Phenomenex Gemini C18, 250 × 10 mm, 5 μm) with isocratic conditions, H2O/MeOH/formic acid (15:85:0.1), to afford acanthifolioside G (1, 1.8 mg) along with 20 other fractions. Fraction F4-10 (3.6 mg) was subjected to an additional HPLC purification (Phenomenex Gemini C18, 250 × 10 mm, 5 μm) with an isocratic H2O/MeOH/formic acid (21:79:0.1), yielding acanthifolioside I (3, 0.26 mg) and acanthifolioside J (4, 0.58 mg). The purification of fraction F4-13 (2.6 mg) was identical to F4-10 and afforded acanthifolioside H (2, 0.21 mg). Acanthifolioside G (1): colorless oil; [α]25D −29 (c 0.001, MeOH); IR (film) νmax 3600−3000, 2930, 2854, 1735, 1680, 1597, 1448, 1375, 1236, 1206, 1138,1048 cm−1; 1H and 13C NMR see Table 1; (+) HRESIMS m/z 939.52880 [M + Na]+ (calcd for C47H80O17Na, 939.52877). Acanthifolioside H (2): colorless oil; IR (film) νmax 3600−3000, 2926, 2853, 1731, 1596, 1357, 1283, 1131, 1073, 1047 cm−1; 1H NMR see Table 2; (+) HRESIMS m/z 937.51286 [M + Na]+ (calcd for C47H78O17Na, 937.51312). Acanthifolioside I (3): colorless oil; IR (film) νmax 3600−3000, 2929, 2852, 1598, 1356, 1256, 1133, 1072, 1050 cm−1; 1H NMR see Table 2; (+) HRESIMS m/z 983.55488 [M + Na]+ (calcd for C49H84O18Na, 983.55499). Acanthifolioside J (4): colorless oil; [α]25D −26 (c 0.0005, MeOH); IR (film) νmax 3600−3000, 2934, 2873, 2850, 1715, 1597, 1356, 1255, 1129, 1069, 1048 cm−1; 1H NMR see Table 2; 13C NMR (150 MHz, CD3OD) δC 172.9 (C, C-1‴′), 141.8 (C, C-5),122.2 (CH, C-6), 103.1 (CH, C-1′), 101.9 (CH, C-1‴), 100.1 (CH, C-1″), 79.5 (CH, C-15), 79.0 (CH, C-3″), 78.9 (CH, C-2″), 78.8 (CH, C-3), 77.4 (CH, C-5″), 75.4 (CH, C-16), 73.5 (CH, C-4‴), 73.1 (CH, C-4′), 72.5 (CH, C-2′), 72.2 (CH, C-3′), 72.0 (CH, C-3‴), 71.9 (CH, C-2‴), 71.6 (CH, C-4″), 70.4 (CH, C-5′), 69.5 (CH, C-5‴), 69.0 (CH, C-23), 62.3 (CH2, C-6″), 61.9 (CH, C-17), 59.9 (CH, C-14), 53.6 (CH, C-24),

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Autopol III polarimeter using a 5 cm microcell (1 mL). E



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Table 2. 1H NMR Data (500 MHz, CD3OD) for Compounds 2−4 position 1 2 3 4 5 6 7 8 9 11 12 14 15 16 17 18 19 20 21 22 23 24

2 (J in Hz) 1.73, m 1.01, m 1.88, m 1.54, m 3.75, dddd (11.2, 11.2, 4.9, 4.9) 1.73, m 1.36, m 1.15, m 1.33, m 2.05, m 0.95 m 1.78, m 0.73, ddd (11.3, 11.3, 3.5) 1.52, m 1.32, m 1.95, m 1.14, m 0.98, m 4.17, dd (5.8, 5.8) 5.26, m 1.38, m 1.04, s 0.88, s 2.50, m 1.05, d (6.4) 5.24, m 5.37, ddd (15.4, 7.8, 6.5) 1.84, m 1.76, m

3 (J in Hz) 1.74, 1.01, 1.87, 1.54, 3.74,

m m m m m

position

4 (J in Hz) 1.90, 1.11, 1.92, 1.63, 3.64,

24′ 24″ 25 26 27 OAc 2‴′ L-rhamnose (C-15) 1′ 2′ 3′ 4′ 5′ 6′ L-glucose (C-3) 1″ 2″ 3″ 4″ 5″ 6″

m m m m m

1.72, m 1.35, m 1.12, m 1.34, m 2.04, m 0.94, m 1.78, m 0.73, ddd (11.6, 11.6, 3.7) 1.52, m 1.33, m 1.99, m 1.11, m 0.95, m 4.19, dd (5.8, 5.8) 5.45, dd (8.1, 6.4) 1.23, m 1.06, s 0.89, s 2.16, m 0.98, m 1.45, m 0.82, m 3.85, m

2.46, m 2.29, m 5.41, 2.37, 1.60, 1.88, 1.03,

m m m m m

1.53, 1.49, 2.07, 1.18, 1.00, 4.19, 5.42, 1.25, 1.08, 1.07, 2.17, 1.00, 1.45, 0.83, 3.85,

m m m m m dd (5.9, 5.9) m m s s m d (6.7) m m m

0.90, m

0.91, m

L-rhamnose 1‴ 2‴ 3‴ 4‴ 5‴ 6‴

2 (J in Hz)

3 (J in Hz) m m m m m

4 (J in Hz)

1.54, m 0.86, m 0.87, m

1.33, 0.91, 1.80, 0.87, 0.92,

1.35, 0.92, 1.78, 0.88, 0.94,

m t (7.5) m d (6.9) d (6.8)

2.08, s

2.20, s

4.38, 3.80, 3.60, 3.37, 3.62, 1.21,

bd (1.4) dd (3.0, 1.6) dd (9.6, 3.0) dd (9.6, 9.6) m d (6.3)

4.40, 3.84, 3.61, 3.38, 3.63, 1.21,

bs 4.42, bd (1.5) m 3.80, dd (3.1, 1.6) dd (9.6, 3.0) 3.58, dd (9.4, 3.1) dd (9.6, 9.6) 3.37, dd (9.7, 9.7) m 3.60, m d (6.2) 1.20, d (6.2)

4.49, 3.34, 3.46, 3.26, 3.24, 3.85, 3.65,

d (7.8) m dd (8.8, 8.8) dd (9.7, 9.7) m dd (11.8, 1.9) dd (12.0, 5.5)

4.50, 3.34, 3.46, 3.27, 3.24, 3.85, 3.65,

d (7.8) 4.49, d (7.8) m 3.35, m dd (8.8, 8.8) 3.47, dd (8.8, 8.8) dd (9.6, 9.6) 3.27, dd (9.7, 9.7) m 3.25, m m 3.84, dd (12.0, 2.0) m 3.65, m

5.16, 3.91, 3.66, 3.38, 4.12, 1.23,

bd (1.5) dd (3.4, 1.7) dd (9.7, 3.4) dd (9.6, 9.6) dq (9.6, 6.2) (d, 6.3)

5.17, 3.91, 3.66, 3.38, 4.13, 1.23,

bd (1.5) dd (3.2, 1.7) m dd (9.6, 9.6) dq (9.6, 6.3) (d, 6.3)

2.20, s

5.19, 3.92, 3.67, 3.39, 4.13, 1.24,

bd (1.4) dd (3.3, 1.8) dd (9.5, 3.5) dd (9.6, 9.6) dq (9.6, 6.2) d (6.2)

(70:30:0.1), t = 30 min H2O/MeOH/formic acid (70:30:0.1), t = 32 min 100% MeOH with formic acid (0.1%). L-Cysteine-L-glucose and L-cysteine-D-glucose derivatives (m/z 447.12) were observed at tR(GluLL) = 19.03 min and tR(GluLD) = 16.59 min, respectively, whereas L-cysteine-L-rhamnose and D-cysteine-L-rhamnose derivatives (m/z 431.13) were observed at tR(RhaLL) = 25.30 min and tR(RhaDL) = 11.84 min (see Supporting Information, S11). Phase II Protein Induction. Monofunctional phase 2 protein induction as an indicator of antioxidant capacity was evaluated in reporter assays in which monoclonal human U373 astrocytoma cells were constructed to stably express a firefly luciferase cassette under the transcriptional control of either the antioxidant (ARE) or the xenobiotic (XRE) response elements. Reporter U373 cells were cultured in DMEM media completed with 10% fetal calf serum and antibiotics. Cells (5 × 104/well) were seeded in quadruplicate in a 24well plate and incubated for 24 h with 25, 10, 5, or 2.5 μg/mL of the test glycosides. After incubation, cells were harvested and ARE or XRE activity was evaluated by measuring luciferase activity according to the manufacturer’s instructions (Promega). Mock-treated cells or cells incubated with vehicle alone (DMSO, 0.25% v/v) served as controls. Values for ARE induction were compared to those elicited by the reference standard, tert-butylhydroquinone (25 μM), with activity that is 2-fold greater than that obtained with TBHQ considered a hit. Monofunctional inducers were defined as inputs that were concurrently positive in the ARE assay and negative in the XRE counterscreen. The absence of cytotoxicity associated with glycoside treatment of reporter cell lines was evaluated in parallel cultures under the same conditions using a standard tetrazole reduction (MTT) assay according to the manufacturer’s instructions (Promega).16

51.6 (CH, C-9), 43.9 (C, C-13), 42.7 (CH2, C-22), 41.7 (CH2, C-12), 39.1 (CH2, C-4), 38.3 (CH2, C-1), 37.5 (C, C-10), 32.0 (CH2, C-7), 30.4 (CH2, C-2), 29.2 (CH, C-25), 28.6 (CH, C-8), 27.8 (CH, C-20), 21.4 (CH3, C-2⁗), 21.3 (CH2, C-11), 21.3 (CH, C-27), 20.1 (CH2, C-24′), 20.1 (CH3, C-26), 19.5 (CH3, C-19), 18.2 (CH3, C-21), 17.6 (CH3, C-6′), 17.6 (CH3, C-6‴), 15.3 (CH3, C-18), 14.4 (CH3, C-24″); (+) HRESIMS m/z 981.53918 [M + Na]+ (calcd for C49H82O18Na, 981.53934). Synthesis of Aldose Thiocarbamate Standards. L-Glucose, Dglucose, and L-rhamnose were derivatized with L-cysteine methyl ester and o-tolylisothiocyanate according to the published procedure.11 L-Rhamnose was also derivatized with D-cysteine methyl ester and o-tolylisothiocyanate according to the published procedure.11 Derivatization of Compound 1. The glycoside acanthifolioside G (500 μg) was hydrolyzed overnight by gently refluxing in 0.5 N HCl (CH3CN/H2O, 1:1, 8 mL). The product was extracted with CH2Cl2/ H2O. The H2O layer was dried in vacuo and redisolved in pyridine (1 mL) containing 1 mg/mL L-cysteine methyl ester. The stirred reaction was heated at 60 °C for 1 h, and then 2 μL of o-tolylisothiocyanate was added. Heating was continued for an additional hour, and then the product was concentrated in vacuo. The residue was suspended in 5% MeOH in H2O and fractionated over a C18 SPE cartridge (500 mg). Five fractions were generated by successive elution with 5% (F1), 25% (F2), 50% (F3), 75% (F4), and 100% MeOH (F5). LCMS analysis revealed the glycoside derivatives in F3. LC/HRMS Analyses. F3 was spiked with aldose thiocarbamate standards and subjected to LC/HRMS analyses (Thermo Hypersil Gold C18, 50 × 2.1 mm, 1.9 μm) with the following eluent system: t = 0 H2O/MeOH/formic acid (87:13:0.1), t = 20 min H2O/MeOH/ formic acid (87:13:0.1), t = 28 min H2O/MeOH/formic acid F



Article

Oxidative Stress Assays. Human U373 astrocytoma cells (2 × 104/well) were seeded in quadruplicate in a 24-well plate and incubated for 24 h with 25, 10, 5, or 2.5 μg/mL of the test glycosides. Media was removed, and cells were stressed by incubation for an additional 24 h with fresh media containing glycoside alone or glycoside and 300 μM H2O2 as an oxidative stressor. Mock-treated cells or cells incubated with vehicle alone (DMSO, 0.25% v/v) served as controls. After incubation, cells were washed three times with phosphate-buffered saline (PBS) and then lysed by three rapid freeze− thaw cycles in 100 μL of PBS to obtain cell extracts for subsequent analysis. Lipid peroxidation was evaluated in cell lysates that have not been centrifuged by standard TBARS assay, in which malondialdehyde (MDA) and thiobarbituric acid (TBA) adduct formation was elucidated as colorimetric changes in absorbance (λ = 532 nm).15 MDA (0−100 nmol/mL) served as reference for generating calibration curves, and absorbance results were expressed as MDA equivalents (nmol/mL). Antioxidant enzyme activity in centrifuged cell lysates was evaluated for superoxide dismutase and glutathione peroxidase using commercially available colorimetric assay kits according to the manufacturer’s instructions (Sigma). SOD activity was evaluated as a decrease in absorbance (λ = 532 nm) associated with inhibition of the xanthine oxidase reduction of WST-1 formazan by SOD present in test samples.17 SOD activity was determined by comparing samples to a standard curve of known SOD activity. GPx activity was evaluated indirectly as changes in absorbance (λ = 340 nm) over a three-minute time course associated with the reversible oxidation reaction in which glutathione (GSH) is converted to oxidized glutathione (GSSG) by GPx.18

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(8) Regalado, E. L.; Tasdemir, D.; Kaiser, M.; Cachet, N.; Amade, P.; Thomas, O. P. J. Nat. Prod. 2010, 73, 1404−1410. (9) Regalado, E. L.; Turk, T.; Tasdemir, D.; Gorjanc, M.; Kaiser, M.; Thomas, O. P.; Fernandez, R.; Amade, P. Steroids 2011, 76, 1389− 1396. (10) Regalado, E. L.; Jimenez-Romero, C.; Genta-Jouve, G.; Tasdemir, D.; Amade, P.; Nogueiras, C.; Thomas, O. P. Tetrahedron 2011, 67, 1011−1018. (11) Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I. Chem. Pharm. Bull. 2007, 55, 899−901. (12) Hur, W.; Gray, N. S. Curr. Opin. Chem. Biol. 2011, 15, 162−173. (13) Miller, A. FEBS Lett. 2012, 586, 585−595. (14) Brigelius-Flohe, R. Biol. Chem. 2006, 387, 1329−1335. (15) Yagi, K. Methods Mol. Biol. 1998, 108, 107−110. (16) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63. (17) Sun, Y.; Oberley, L. W.; Li, Y. Clin. Chem. 1988, 34, 497−500. (18) Mannervik, B. Methods Enzymol. 1985, 113, 490−495.

ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR spectra, HRMS, and MS/MS data for acanthifolioside G (1) as well as 1H spectra and HRMS analyses for acanthifoliosides H−J (2−4) are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +1 902 566 0565. Fax: +1 902 566 7445. E-mail: rkerr@ upei.ca. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from Natural Sciences and Engineering Council of Canada (NSERC), the Canada Research Chair Program, the University of Prince Edward Island, the Atlantic Innovation Fund, and the Jeanne and Jean-Louis Lévesque Foundation. The authors also acknowledge NMR services provided by Dr. C. Kirby and L. Kerry (AAFC).

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REFERENCES

(1) Negi, J. S.; Singh, P.; Pant, G. J.; Rawat, M. S. M. Pharmacogn. Rev. 2011, 5, 155−158. (2) Prassas, I.; Diamandis, E. P. Nat. Rev. Drug Discovery 2008, 7, 926−935. (3) Francis, G.; Kerem, Z.; Makkar, H. P. S.; Becker, K. Br. J. Nutr. 2002, 88, 587−605. (4) Iorizzi, M.; De Marino, S.; Zollo, F. Curr. Org. Chem. 2001, 5, 951−973. (5) Ivanchina, N. V.; Kicha, A. A.; Stonik, V. A. Steroids 2011, 76, 425−454. (6) Schmitz, F. J.; Prasad, R. S.; Gopichand, Y.; Hossain, M. B.; Van, der Helm., D.; Schmidt, P. J. Am. Chem. Soc. 1981, 103, 2467−2469. (7) Cachet, N.; Regalado, E. L.; Genta-Jouve, G.; Mehiri, M.; Amade, P.; Thomas, O. P. Steroids 2009, 74, 746−750. G


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