Article pubs.acs.org/jnp

Antioxidative Compounds from Garcinia buchananii Stem Bark Timo D. Stark,*,† Mathias Salger,† Oliver Frank,† Onesmo B. Balemba,‡ Junichiro Wakamatsu,† and Thomas Hofmann† †

Lehrstuhl für Lebensmittelchemie und Molekulare Sensorik, Technische Universität München, Lise-Meitner-Straße 34, 85354 Freising, Germany ‡ Department of Biological Sciences, University of Idaho, Moscow, Idaho 83844, United States S Supporting Information *

ABSTRACT: An aqueous ethanolic extract of the stem bark of Garcinia buchananii showed strong antioxidative activity using H2O2 scavenging, oxygen radical absorbance capacity (ORAC), and Trolox equivalent antioxidant capacity (TEAC) assays. Activity-guided fractionation afforded three new compounds, isomanniflavanone (1), an ent-eriodictyol-(3α→6)dihydroquercetin-linked biflavanone, 1,5-dimethoxyajacareubin (2), and the depsidone garcinisidone-G (3), and six known compounds, (2″R,3″R)preussianon, euxanthone, 2-isoprenyl-1,3,5,6-tetrahydroxyxanthone, jacareubin, isogarcinol, and garcinol. All compounds were described for the first time in Garcinia buchananii. The absolute configurations were determined by a combination of NMR, ECD spectroscopy, and polarimetry. These natural products showed high in vitro antioxidative power, especially isomanniflavanone, with an EC50 value of 8.5 μM (H2O2 scavenging), 3.50/4.95 mmol TE/mmol (H/L-TEAC), and 7.54/14.56 mmol TE/mmol (H/L-ORAC).

O

preussianon, the xanthones euxanthone, 2-isoprenyl-1,3,5,6tetrahydroxyxanthone, and jacareubin, and the polyisoprenylated benzophenones isogarcinol and garcinol, were described for the first time in G. buchananii. All compounds were evaluated for their antioxidative activities using H 2 O 2 scavenging, H-/L-TEAC, and the H-/L-ORAC assays. Herein, the isolation, structure elucidation, and antioxidative evaluation of the isolates are described.

verproduction of reactive oxygen species (ROS) in the human body causes undesirable oxidative stress, which is associated with chronic metabolic and degenerative diseases. The destructive power of these free radicals causing oxidative damage is associated with coronary heart disease, atherosclerosis, aging, cancer, and inflammatory conditions.1 Thus, it is imperative to develop new and effective antioxidative drugs to combat animal and human diseases caused by ROS. In recent studies,2,3 an aqueous ethanolic extract of the stem bark of Garcinia buchananii (Clusiaceae) showed strong antioxidative activity, and activity-guided separation yielded 12 compounds with extraordinarily high antioxidative power (Chart 1, Supporting Information). It was assumed that the MPLC fractions M4 and M8 might contain secondary metabolites with high antioxidative activity. G. buchananii, a plant native to eastern, central, and southern Africa is used by the indigenous population to treat dysentery, abdominal pain, and a range of infectious diseases.4,5 Balemba and co-workers6 showed that the aqueous extract from the stem bark of G. buchananii is a nonopiate preparation, which reduces peristalsis by inhibiting neurotransmission and 5-HT3 and 5HT4 receptors.7 Previous chemical investigations on this species resulted in the isolation of compounds including xanthones,8 flavanone-C-glycosides, and (3→8)-linked biflavanones.2,3,9−11 In this study, the chemical constituents of the aqueous ethanolic extract of the stem bark of G. buchananii were investigated to find potential antioxidative lead compounds. As a result, three new compounds, isomanniflavanone (1), an ent-eriodictyol-(3→6)-dihydroquercetin-linked biflavanone, 1,5-dimethoxyajacareubin (2), and the depsidone garcinisidone-G (3), and six known compounds, (2″R,3″R)© XXXX American Chemical Society and American Society of Pharmacognosy



RESULTS AND DISCUSSION The aqueous ethanolic extract of the stem bark of G. buchananii was purified by MPLC and reversed-phase preparative and semipreparative HPLC to afford three new and six known compounds. The structures of the known compounds were identified as (2″R,3″R)-preussianon,12 euxanthone,1,3 2-isoprenyl-1,3,5,6-tetrahydroxyxanthone,14 jacareubin,13,15 isogarcinol, and garcinol16−19 by comparison of their spectroscopic data with published values and were described for the first time in G. buchananii. The absolute configurations of garcinol and isogarcinol were proven for the first time via electronic circular dichroism spectroscopy (ECD) and comparison to published data.20,21 Isomanniflavanone (1) (Figure 1) was isolated as a white, amorphous powder and showed a pseudomolecular ion peak m/z 589.0988 [M − H]− (calcd 589.0982) in the HRESIMS, which in conjunction with 13C NMR data indicated a formula of C30H22O13. Characteristic mass fragments m/z 463, 435, 419, Received: October 9, 2014

A

DOI: 10.1021/np5007873 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Chemical structures of compounds 1−9.

cetin-linked biflavanone. The similarity of the NMR data (Table 1) to manniflavanone isomers2,3 and the fingerprint correlations in the HMBC experiment between H-3 (δH 4.66) and C-5″, C-6″, and C-7″ as well as no correlation to C-8a″ indicated the intramolecular C-3→C-6 linkage of the two flavanone constituent units. Other key HMBC correlations are indicated in Table S3 (Supporting Information). The absolute configuration of 1 was defined by comparison of the experimental and published ECD curves of (2R,3S,2″S,3″S)and (2R,3S,2″R,3″R)-manniflavanone, (2S,3R,2″R,3″R)-GB-1, (2R,3S,2″R,3″R)-GB-4, and (2S,3R,2″R,3″R)-GB-4a.2,3,22 The results showed that the ECD curve of 1 was consistent with the ECD spectrum of (2R,3S,2″R,3″R)-manniflavanone (Figure 3) as well as (2R,3S,2″R,3″R)-GB-4.22 Thus, the structure 1 was assigned as (2R,3S,2″R,3″R)-isomanniflavanone, ent-eriodictyol-(3α→6)-dihydroquercetin. 1,5-Dimethoxyajacareubin (2) (Figure 1) was obtained as a white, amorphous powder. The HRESIMS data of 2 exhibited an ion at m/z 355.1179 [M − H]− (calcd for 355.1182), which in conjunction with 13C NMR data suggested a molecular formula of C20H20O6. The 1H NMR spectrum revealed the presence of a trisubstituted benzene ring [δH 7.39 (H-10a, d, J = 2.0 Hz), 7.11 (H-8, dd, J = 2.0, 8.3 Hz), 6.81 (H-7, d, J = 8.3 Hz)], an aromatic methine proton [δH 6.15 (H-4, s)], two cisolefinic configured protons [δH 6.41 (H-11, d, J = 9.9 Hz) and 5.61 (H-12, d, J = 9.9 Hz)], two methyl groups [δH 1.39 (6H, s)], and two O-methyl resonances [δH 3.80 (3H, s) and 3.53 (3H, s)]. The 13C NMR and phase-sensitive gHSQC spectra showed resonances for one carbonyl carbon (δC 192.4), six methine carbons (δC 127.3, 125.5, 116.3, 114.8, 110.9, 99.2), two O-methyl carbons (δC 62.6, 55.5), two methyl carbons [δC 27.5 (×2)], and eight further carbons (δC 155.9, 154.5, 154.0, 152.3, 147.7, 129.3, 114.7, 105.9, 76.1). These data (Table 1) indicated that compound 2 shared similarities with the

285, 151, and 125 and the UV spectrum were similar to those of (2R,3S,2″R,3″R)- and (2R,3S,2″S,3″S)-manniflavanone and indicated an isomer of manniflavanone.2,3 The 1H NMR spectrum of compound 1 showed rotational isomerism at room temperature (RT). Therefore, in the 1H NMR spectrum a duplicated set of signals with two sharp exchangeable resonances at 12.18 and 12.20 ppm for HO-5 and two broad exchangeable singlets at 12.32 and 12.39 ppm for HO-5″ and at 10.82 and 11.14 ppm for the HO-7 and HO-7″ moieties were observed. Several broad exchangeable resonances between 8.98 and 9.06 ppm for HO-3′, -4′, -3‴, and -4‴ and two exchangeable doublets for HO-3″ at 5.64 and 5.73 ppm were present. Typical A-ring aromatic singlets for H-6, -8, and -8″ from 5.78 to 5.91 ppm as well as characteristic B-ring aromatic protons resonating at 6.61−6.85 ppm were observed. Coupling of H-2 and -3, and H-2″ and -3″, respectively, confirmed a GB-type biflavonoid. In comparison to (2R,3S,2″R,3″R)-manniflavanone (Figure 2A), the 1H NMR spectrum of 1 in DMSO-d6 at 18 °C showed signal duplication only for certain protons, e.g., H-2 (Figure 2B). As these two rotational isomers were similar and therefore resonances partially overlapped, NMR measurements at elevated temperatures were performed. The 1H NMR spectrum recorded at 50 °C (Figure 2C) enabled the separation and proved the existence of two rotational isomers in a ca. 1:1 ratio. The vicinal coupling constants for H-2 and H-3, as well as H-2″ and H-3″ of 11.3−12.6 Hz, indicated their trans-diaxial orientations. The 13C NMR and phase-sensitive gHSQC spectra indicated the presence of two carbonyl carbons (δC 197.0, 198.3), 13 methine carbons [δC 46.5, 71.4, 81.6, 83.1, 94.6, 95.0, 96.0, 115.1 (×2), 115.5, 115.7, 119.0, 119.6], and 15 further carbons [δC 101.3 (×2), 102.0, 128.1, 129.1, 144.7, 144.9, 145.7, 145.8, 161.0, 161.5, 163.0, 163.6, 165.0, 166.5]. On the basis of these data, compound 1 was assigned as an eriodictyol-dihydroquerB

DOI: 10.1021/np5007873 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 2. Excerpt of 1H NMR (DMSO-d6) spectra of (A) (2R,3S,2R,3R)-manniflavanone (18 °C, 500 MHz) and 1 ((B) 18 °C, 500 MHz and (C) 50 °C, 400 MHz).

pyranoxanthone jacareubin (Supporting Information).13,15 Differences comprised the two additional methoxy groups located at C-5 and C-1, assigned by means of their HMBC correlations (Figure 4), as well as the additional aromatic proton [δH 7.39 (H-10a, d, J = 2.0 Hz)]. Other key HMBC correlations are indicated in Figure 4 as well as Table S5 (Supporting Information). Comparing the chemical shifts of the carbons C-4a, C-8a, C-9a, and C-10a in jacareubin13,15 and 2 indicated that C-8a and C-9a were deshielded, whereas C-10a was strongly shielded in 2. The key HRMS fragment assignment of 2 in the positive and negative ionization modes (Figures S13 and S14, Supporting Information) confirmed this seco-xanthone structure, which, in comparison to jacareubin13,15 (Figure S53, Supporting Information), revealed the typical fragments at m/z 123.0447 (C7H7O2) and 106.0212 (C4H4O2). Thus, the structure of 1,5-dimethoxyajacareubin was assigned as shown in 2. Garcinisidone-G (3) (Figure 1) was isolated as a white, amorphous powder and showed a pseudomolecular ion peak at m/z 357.0977 [M − H]− (calcd 357.0974) in the HRESIMS, which in conjunction with 13C NMR data indicated a molecular formula of C19H18O7. The presence of the depsidone (11Hdibenzo[b,e][1,4]dioxepin-11-one) structure, having a hydrogen-bonded 1-hydroxy group, was suggested by the similarity of the NMR data with those of garcinisidones A−F.14,23 The 1H NMR spectrum showed an OCH3 group [δH 3.82 (3H, s)], a

prenyl moiety [δH 1.64 (3H, s), 1.76 (3H, s), 3.52 (2H, d, J = 6.9 Hz), and 5.22 (1H, t, J = 6.9 Hz)], ortho-coupled aromatic doublets [δH 6.71 (1H, d, J = 9.0 Hz) and 6.86 (1H, d, J = 9.0 Hz)], and an aromatic singlet [δH 6.16 (1H, s)]. The 13C NMR and phase-sensitive gHSQC spectra showed resonances for one carbonyl carbon (δC 163.7), one methylene group (δC 21.5), four methine carbons (δC 123.7, 114.8, 112.6, 100.3), one methoxy group (δC 61.0), two methyl (δC 25.5, 17.8), and 10 further carbons (δC 165.0, 161.0, 158.7, 148.8, 144.2, 139.5, 137.7, 130.0, 112.5, 96.3). On the basis of these data (Table 1), compound 3 revealed strong similarity to garcinisidone-F.23 Differences were observed at the linkage of the prenyl moiety in the A ring and subsequently the position of the aromatic proton singlet assigned by means of their HMBC correlations (Figure 5). Other key HMBC correlations are indicated in Figure 5 as well as Table S7 (Supporting Information). The key HRMS fragment assignment of 3 in the positive and negative ionization modes (Figures S22 and S23, Supporting Information) confirmed the structure as garcinisidone-G. All isolates were evaluated for their in vitro antioxidative activities using H2O2 scavenging, H/L-TEAC, and H/L-ORAC assays, respectively. When compared to reference compounds (2R,3S,2″R,3″R)-manniflavanone, (2R,3R)-taxifolin, ascorbic acid, quercetin, epicatechin, BHT, and rac-naringenin, compound 1 showed strong antioxidative capacity in all assays. Values of 8.5 μM (EC50, H2O2 scavenging), 3.50/4.95 mmol C

DOI: 10.1021/np5007873 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H and 13C NMR Data (500 and 125 MHz, DMSO-d6) for Compounds 1−3a 1 2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′, 4′ 5′ 6′ 2″ 3″ 4″ 4a″ 5″ 6″ 7″ 8″ 8a″ 1‴ 2‴ 3‴, 4‴ 5‴ 6‴ 3″-OH 3′, 4′, 3‴, 4‴-OH 7-OH 7″-OH 5-OH 5″-OH a

2

δH (J in Hz)

δC

5.70, d (12.6), 5.72, d (12.6) 4.66, d (12.0)

81.6 46.3, 46.9 197.0 101.3b 163.6 96.0 166.5 95.0 163.0 129.1 115.4,c 115.6,c 115.7c 144.7, 145.0, 145.7, 145.8

position

5.91, s 5.88, s

6.82, m

6.61, 6.61, 4.91, 4.50,

m m d (11.3) m

5.78, brs, 5.82, brs

6.85, m 6.72, m 6.72, m 5.64, d (5.0), 5.73, d (4.8) 8.98−9.03, 3×brs 10.82, brs 11.14, brs 12.18, s, 12.20, s 12.32, brs, 12.39, brs

δH (J in Hz)

115.1d 118.9, 119.1 82.8, 83.4 71.4 198.3 101.3b 161.4, 161.6 102.1 165.0 94.6 160.9 128.1 115.4,c 115.6,c 115.7c 144.7, 145.0, 145.7, 145.8 115.1d 119.6

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 14 15 16 17

Assignments based on HSQC and HMBC experiments. Chemical shifts in ppm.

6.81, d (8.3) 7.11, dd (2.0, 8.3)

7.39, d (2.0) 6.41, d (9.9) 5.61, d (9.9) 1.39, 1.39, 3.53, 3.80,

s s s s

154.1 105.9 154.5 99.2 155.9 147.7 152.3 114.8 125.5 129.3 192.4 114.7 110.9 116.3 127.3 76.1 27.5 27.5 62.6 55.5

δH (J in Hz) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1′ 2′ 3′ 4′ 5′

6.16, s

6.71, d (9.0) 6.86, d (9.0)

3.82, s 3.52,d (6.9) 5.22, t (6.9) 1.64, s 1.64, s

δC 158.7 112.5 165.0 100.3 139.5 148.8 112.6 114.8 161.0 96.3 144.2 137.7 163.7 61.0 21.5 123.7 130.0 25.5d 17.8d

Signals interchangeable in the same column.

Phenomenex), Nucleodur Sphinx (10 × 250 mm, 5 μm; MachereyNagel, Düren, Germany), or ThermoHypersil ODS (10 × 250 mm, 5 μm; Kleinostheim, Germany) as the stationary phase. MPLC separations were performed on a Büchi Sepacore (Flawil, Switzerland) system using a YMC (YMC Europe, Dinslaken, Germany) DispoPackAT ODS-25 flash cartridge (120 g, i.d. 40 mm, l.150 mm). Plant Material. Garcinia buchananii stem bark was collected from trees in their natural habitats in Karagwe, Tanzania, and processed as described previously.2,3,6 A voucher specimen of bark powder was deposited at the University of Idaho Stillinger herbarium (voucher #159918). Extraction and Isolation. Chromatography and extraction of G. buchananii bark powder was done as described earlier.2,3 Eight MPLC fractions were collected, concentrated under reduced pressure, and freeze-dried (M1−M8; 160, 70, 401, 63, 101, 51, 51, and 52 mg). Fractions M4 and M8, which showed higher levels of antioxidant activities, were subjected to purification by HPLC. M4: Preparative HPLC started with a mixture (70/30, v/v) of aqueous HCO2H (0.1% in H2O, pH 2.5) and MeCN, and the MeCN content was increased to 43% within 13 min, followed by column washing and re-equilibration. Fractions were concentrated under reduced pressure and freeze-dried twice, affording five fractions, M4-1 to M4-5. Compound 1 was obtained from M4-5 by semipreparative HPLC using a phenylhexyl column (4.0 mL/min) as the stationary

TE/mmol (H/L-TEAC), and 7.54/14.56 mmol TE/mmol (H/ L-ORAC) are in the range of the biflavanone (2R,3S,2″R,3″R)manniflavanone (Table 2). Elevated activity was also observed for (2″R,3″R)-preussianon (4), while compounds 2, 3, and 5−9 showed moderate in vitro antioxidative activity.



b,c,d

6.15, s

3 δC

EXPERIMENTAL SECTION

General Experimental Procedures. 1H, gCOSY, gHSQC, gHMBC, ROESY, and 13C spectra were recorded on an Avance III 500 MHz spectrometer with a CTCI probe or an Avance III 400 MHz spectrometer with a BBO probe (Bruker, Rheinstetten, Germany). Mass spectra were measured on a Waters Synapt G2-S HDMS mass spectrometer (Waters, Manchester, UK) coupled to an Acquity UPLC core system (Waters, Milford, MA, USA). For ECD spectroscopy, methanolic solutions of the samples were analyzed by means of a Jasco J-810 spectropolarimeter. Polarimetric measurments were performed in ethanolic solutions of the samples by means of a Jasco P-2000 digital polarimeter and 1 mL cell. HPLC separations were performed using a preparative HPLC system (PrepStar, Varian, Darmstadt, Germany). Preparative HPLC (278 nm) was performed using an RP column (21.2 × 250 mm, phenylhexyl, 5 μm; 18 mL/min; Phenomenex, Aschaffenburg, Germany) and semipreparative HPLC (278 nm) using an RP phenylhexyl column (10 × 250 mm, 5 μm, D

DOI: 10.1021/np5007873 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 3. ECD spectra of (2R,3S,2R,3R)-manniflavanone and 1. M8: Preparative HPLC started with a mixture (50/50, v/v) of aqueous HCO2H (0.1% in H2O, pH 2.5) and MeCN, and the MeCN content was increased to 100% within 20 min, followed by column washing for 10 min with 100% MeCN and re-equilibration. Collected fractions were concentrated under reduced pressure and freeze-dried twice, affording 18 fractions, M8-1 to M8-19. Compound 2, euxanthone, and 2-isoprenyl-1,3,5,6-tetrahydroxyxanthone were obtained from M8-5 by semipreparative HPLC using ThermoHypersil (4.0 mL/min) as the stationary phase using gradient elution starting with a mixture (50/50, v/v) of aqueous HCO2H (0.1% in H2O, pH 2.5) and MeOH, and the MeOH content was increased to 90% within 20 min, followed by column washing and reequilibration. Compound 3 and jacareubin were obtained from M8-6 by semipreparative HPLC using ThermoHypersil (4.0 mL/min) as the stationary phase and gradient elution starting with a mixture (30/70, v/v) of aqueous HCO2H (0.1% in H2O, pH 2.5) and MeOH; the MeOH content was increased up to 90% within 20 min, followed by column washing and re-equilibration. Isogarcinol and garcinol were purified from M8-15 and M8-18 via isocratic semipreparative HPLC using ThermoHypersil (4.0 mL/min) and a mixture (10/90, v/v) of aqueous HCO2H (0.1% in H2O, pH 2.5) and MeCN. (2R,3S,2″R,3″R)-Isomanniflavanone (1): white, amorphous powder; [α]25D = −38 (c 0.1, EtOH); UV (MeOH) λmax (log ε) 230 (4.72), 288 (4.52), 340 (3.93) nm; ECD (c 5.1 × 10−4 M, MeOH) λmax nm (Δε) 349 (+0.2), 316 (−1.7), 306 (−2.7), 283 (+6.5), 245 (−1.6), 218 (−14.5), 202 (−1.4); 1H NMR (DMSO-d6, 18 °C, 500 MHz) data, see Table 1; 13C NMR (DMSO-d6, 18 °C, 500 MHz) data, see Table 1; HRESIMS m/z 589.0988 [M − H]− (calcd for C30H21O13, 589.0982). 1,5-Dimethoxyajacareubin (2): white, amorphous powder; 1H NMR (DMSO-d6, 27 °C, 500 MHz) data, see Table 1; 13C NMR (DMSO-d6, 27 °C, 500 MHz) data, see Table 1; HRESIMS m/z 355.1179 [M − H]− (calcd for C20H19O6, 355.1182). Garcinisidone-G (3): white, amorphous powder; 1H NMR (DMSOd6, 27 °C, 500 MHz) data, see Table 1; 13C NMR (DMSO-d6, 27 °C,

Figure 4. Key correlations in the HMBC spectrum of 2.

Figure 5. Key correlations in the HMBC and COSY spectra of 3. phase using the solvents mentioned above and gradient elution starting with a mixture (70/30, v/v) of aqueous HCO2H (0.1% in H2O, pH 2.5) and MeCN; the MeCN content was increased to 50% within 30 min, followed by column washing and re-equilibration. (2″R,3″R)Preussianone was obtained from M4-2 by semipreparative HPLC using Nucleodur Sphinx (4.0 mL/min) as the stationary phase with gradient elution starting with a mixture (60/40, v/v) of aqueous HCO2H (0.1% in H2O, pH 2.5) and MeOH, and the MeOH content was increased to 55% within 20 min, followed by column washing and re-equilibration. E

DOI: 10.1021/np5007873 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 2. Antioxidant Activities of Compounds 1−9 and Reference Compounds compound 1 2 3 4 5 6 7 8 9 ascorbic acid (2R,3S,2R,3R)manniflavanone (2R,3S)-taxifolin epicatechin rac-naringenin quercetin BHT

H-TEACa,b [mmol/ TE/mmol]

L-TEACa,b [mmol/ TE/mmol]

H-ORACa,b [mmol/ TE/mmol]

L-ORACa,b [mmol/ TE/mmol]

3.50 0.40 0.89 1.48 0.40 0.89 0.84 0.48 0.81 1.16 5.58

± ± ± ± ± ± ± ± ± ± ±

0.08 0.03 0.09 0.10 0.04 0.05 0.08 0.09 0.02 0.03 0.31g

4.95 1.04 1.68 2.92 1.33 2.96 2.06 1.67 1.92 1.09 5.29

± ± ± ± ± ± ± ± ± ± ±

0.02 0.09 0.05 0.07 0.01 0.04 0.08 0.24 0.09 0.01 0.20

7.54 2.44 2.50 4.38 2.43 1.06 1.16 0.60 1.28 0.62 13.73

± ± ± ± ± ± ± ± ± ± ±

0.03 0.09 0.04 0.14 0.05 0.03 0.12 0.15 0.08 0.04 0.43g

14.56 4.87 4.34 6.96 2.95 3.81 2.74 3.68 3.44 0.73 15.00

± ± ± ± ± ± ± ± ± ± ±

0.57 0.04 0.05 0.10 0.08 0.04 0.08 0.05 0.05 0.04 0.15

2.48 3.71 1.98 4.85 n.d.l

± ± ± ±

0.16k 0.37k 0.15k 0.30k

2.84 5.01 2.94 4.84 0.76

± ± ± ± ±

0.05 0.25 0.18 0.27 0.03

6.61 9.98 6.20 9.36 n.d.l

± ± ± ±

0.20k 0.33k 0.18k 0.33k

9.30 15.69 7.35 11.94 0.20

± ± ± ± ±

0.11 0.44 0.41 0.86 0.01

H2O2c,d [EC50 μM] 8.5 ≤53.4 ≤45.4 17.5 80.2 19.7 26.3 83.3 15.7 24.2 2.8

(7.7−9.5)

(15.5−19.4) (68.5−93.1) (17.7−21.8) (13.9−60.8) (75.0−93.3) (14.5−17.0) (21.5−27.1) (2.4−3.2)g

11.3 (9.7−13.2)g 4.1 (3.7−4.6)g 8.6 (6.8−11.9)g 6.1 (5.3−7.1)g n.d.l

literature n.r.e n.r. n.r. n.r. n.r. n.r. n.r. n.r. n.r. 1.05f n.r. n.r. n.r. 1.14f 3.10f/10.4−13.5h 0.27i/0.12j

Each sample was analyzed by means of ORAC and ABTS assay by quadruplicate studies. bValues represent the mean ± SD. cEach sample was analyzed by means of H2O2 assay by triplicate studies. dRange in parentheses represents 95% confidence interval. en.r. not reported. fL-TEAC values from Re et al.26 gValues from Stark et al.2,3 hL-ORAC values from Mercader-Ros et al.27 iL-TEAC value from Pereira-Caro et al.28 jL-ORAC value from Madrona et al.24 kValues comparable to Stark et al.2,3 ln.d. not determined. a



500 MHz) data, see Table 1; HRESIMS m/z 357.0977 [M − H]− (calcd for C19H17O7, 357.0974). Antioxidant Assays. H2O2 scavenging, hydrophilic oxygen radical absorbance capacity (H-ORAC), and hydrophilic Trolox equivalent antioxidant capacity (H-TEAC) with ABTS assays were performed as described earlier (Supporting Information).2,3 Lipophilic Oxygen Radical Absorbance Capacity (L-ORAC) Assay. The lipophilic ORAC assay was adapted from the Madrona procedure24 with some modifications. Trolox standards (200, 100, 50, 25, and 12.5 μM) as well as dilutions of each sample solution were prepared using 7% (w/v) randomly methylated methyl-β-cyclodextrin (RMCD) in acetone/H2O (1:1 v/v). Fluorescein sodium salt (FL) and 2,2′-azobis(2-methylpropinamidine) (AAPH) solutions were prepared in the same way as the H-ORAC method (Supporting Information) using phosphate buffer (10 mM, pH 7.4). After FL solution (150 μL) was added in a black 96-well microplate, Trolox standards, the diluted samples, and the 7% RMCD solution as blank, 25 μL each, were applied with mixing. Incubation of the microplate was done as described for the H-ORAC assay, and an AAPH solution (25 μL) was immediately added to all wells. All conditions of the measurement as well as data handling and export were the same as the H-ORAC assay (Supporting Information). Lypophilic Trolox Equivalent Antioxidant Capacity Assay with ABTS (L-TEAC). In order to measure the TEAC of lypophilic compounds, the H-TEAC method25 was modified. In brief, Trolox was dissolved in 50% DMSO (DMSO/H2O, v/v) for standard solutions (1000, 500, 250, 125, and 62.5 μM), and dilution of sample solutions was also performed using 50% DMSO. Pure DMSO (20 μL) was added in a transparent 96-well microplate, and subsequently all the wells (final DMSO concentration 10%) were processed in the same way as described for the H-TEAC method. Finally, all conditions of the measurement as well as data handling and export were the same as the H-TEAC assay (Supporting Information).



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] Fax: +49-8161-712949. Tel: +498161-712911. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We thank S. Lösch for excellent technical assistance. REFERENCES

(1) Finkel, T.; Holbrook, N. J. Nature 2000, 40, 239−247. (2) Stark, T. D.; Matsutomo, T.; Lösch, S.; Boakye, P. A.; Balemba, O. B.; Pasilis, S. P.; Hofmann, T. J. Agric. Food Chem. 2012, 60, 2053− 2062. (3) Stark, T. D.; Germann, D.; Balemba, O. B.; Wakamatsu, J.; Hofmann, T. J. Agric. Food Chem. 2013, 61, 12572−12581. (4) Chinsembu, K. C.; Hedimbi, M. J. Ethnobiol. Ethnomed. 2010, 6, 25. (5) Kisangau, D. P.; Lyaruu, H. V.; Hosea, K. M.; Joseph, C. C. J. Ethnobiol. Ethnomed. 2007, 3, 29−37. (6) Balemba, O. B.; Bhattarai, Y.; Stenkamp-Strahm, C.; Lesakit, M. S. B.; Mawe, G. M. Neurogastroent. Motil. 2010, 22, 1332−1339. (7) Boakye, P. A.; Stenkamp-Strahm, C.; Bhattarai, Y.; Heckman, M. D.; Brierley, S. M.; Pasilis, S. P.; Balemba, O. B. Neurogastroent. Motil. 2012, 24, e27−40. (8) Jackson, B.; Locksley, H. D.; Moore, I.; Scheinmann, F. J. Chem. Soc. 1968, 20, 2579−2583. (9) Jackson, B.; Locksley, H. D.; Scheinmann F. Wolstenholme, W. A. Tetrahedron Lett. 1967, 9, 787−792. (10) Jackson, B.; Locksley, H. D.; Scheinmann, F. Chem. Commun. 1968, 18, 1125−1127. (11) Jackson, B.; Locksley, H. D.; Scheinmann, F.; Wolstenholme, W. A. J. Chem. Soc. 1971, 22, 3791−3804. (12) Messi, B. B.; Ndjoko-Ioset, K.; Hertlein-Amslinger, B.; Lannang, A. M.; Nkengfack, A. E.; Wolfender, J.-L.; Hostettmann, K.; Bringmann, G. Molecules 2012, 17, 6114−6125. (13) Westerman, P. W.; Gunasekara, S. P.; Uvias, M.; Sultanbawa, S.; Kazlauskas, R. Org. Magn. Reson. 1977, 9, 631−636.

ASSOCIATED CONTENT

S Supporting Information *

UPLC-ESI-TOF-MSe and antioxidative analysis. UPLC-HRESIMS, ECD, and NMR of compounds 1−9. These materials are available free of charge via the Internet at http://pubs.acs.org. F

DOI: 10.1021/np5007873 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(14) Ito, C.; Miyamoto, Y.; Takayasu, J.; Nakayama, M.; Kawai, Y.; Rao, K. S.; Furukawa, H. Chem. Pharm. Bull. 1997, 45, 1403−1413. (15) King, F. E.; King, T. J.; Manning, L. C. J. Chem. Soc. 1953, 3932−3937. (16) Gustafson, K. R.; Blunt, J. W.; Munro, M. H. G.; Fuller, R. W.; McKee, T. C.; Cardellina, J. H., II; McMahon, J. B.; Cragg, G. M.; Boyd, M. R. Tetrahedron 1992, 48, 10093−10102. (17) Sahu, A.; Das, B.; Chatterjee, A. Phytochemistry 1989, 28, 1233− 1235. (18) Rama Rao, A. V.; Venkatswamy, G.; Pendse, A. D. Tetrahedron Lett. 1980, 21, 1975−1978. (19) Rama Rao, A. V.; Venkatswamy, G.; Yemul, S. S. Indian J. Chem. 1980, 19B, 627−633. (20) Zhang, H.; Tao, L.; Fu, W. W.; Liang, S.; Yang, Y. F.; Yuan, Q. H.; Yang, D. J.; Lu, A. P.; Xu, H. X. J. Nat. Prod. 2014, 77, 1037−1046. (21) Zhang, H.; Zhang, D. D.; Lao, Y. Z.; Fu, W. W.; Liang, S.; Yuan, Q. H.; Lang, L.; Xu, H. X. J. Nat. Prod. 2014, 77, 1700−1707. (22) Ferrari, J.; Terreaux, C.; Kurtán, T.; Szikszai-Kiss, A.; Antus, S.; Msonthi, J. D.; Hostettmann, H. Helv. Chim. Acta 2003, 86, 2768− 2778. (23) Ito, C.; Itoigawa, M.; Mishina, Y.; Tomiyasu, H.; Litaudon, M.; Cosson, J. P.; Mukainaka, T.; Tokuda, H.; Nishino, H.; Furukawa, H. J. Nat. Prod. 2001, 64, 147−150. (24) Madrona, A.; Pereira-Caro, G.; Bravo, L.; Mateos, R.; Espartero, J. L. Food Chem. 2011, 129, 1169−1178. (25) Van den Berg, R.; Haenen, G. R. M. M.; van den Berg, H.; Bast, A. Food Chem. 1999, 66, 511−517. (26) Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Free Radical Biol. Med. 1999, 26, 1231−1237. (27) Mercader Ros, M. T.; Lucas-Abellán, C.; Fortea, M. I.; Gabaldón, J. A.; Núñez-Delicado, E. Food Chem. 2010, 118, 769−773. (28) Pereira-Caro, G.; Madrona, A.; Bravo, L.; Espartero, J. L.; Alcudia, F.; Cert, A.; Mateos, R. Food Chem. 2009, 115, 86−91.

G

DOI: 10.1021/np5007873 J. Nat. Prod. XXXX, XXX, XXX−XXX

Antioxidative compounds from Garcinia buchananii stem bark.

An aqueous ethanolic extract of the stem bark of Garcinia buchananii showed strong antioxidative activity using H2O2 scavenging, oxygen radical absorb...
1MB Sizes 0 Downloads 16 Views