Two Sarcoviolins with Antioxidative and α-Glucosidase Inhibitory

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Two Sarcoviolins with Antioxidative and α‑Glucosidase Inhibitory Activity from the Edible Mushroom Sarcodon leucopus Collected in Tibet Ke Ma,†,‡,⊥ Junjie Han,†,⊥ Li Bao,† Tiezheng Wei,† and Hongwei Liu*,† †

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, People’s Republic of China ‡ University of Chinese Academy of Sciences (UCAS), 19A Yuquan Road, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Edible mushrooms are known as an important source of natural antioxidants. The ethyl acetate extract of the edible mushroom Sarcodon leucopus (Zangzi mushroom) possesses strong antioxidative activity. Bioactivity-guided isolation afforded 10 compounds from its fruiting bodies, including two new sarcoviolins, sarcoviolin β (1) and episarcoviolin β (2), and one new p-terphenyl derivative (3) along with seven known compounds. The structures of the new compounds were elucidated by spectroscopic methods and comparison with the known compounds. Compounds 1−10 were found to have antioxidant effects in the DPPH scavenging assay, the total antioxidant capacity assay, the reducing power assay, and the lipid peroxidation assay. Further study indicated that they could protect DNA strands from free radical-induced cleavage at 200 μM. Compounds 1−10 also presented strong α-glucosidase inhibitory activity. Of all tested compounds, compound 1 exhibited the strongest inhibitory activity, with an IC50 value of 0.58 μM.

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Sarcodon leucopus is known as a valuable edible mushroom in the region of Tibet. In our search for natural antioxidants from mushrooms, the EtOAc extract of S. leucopus was found to have strong DPPH scavenging ability (IC50 = 41 ± 1 μg/mL). Chemical investigation on Sarcodon has led to the identification of a number of bioactive secondary metabolites. Examples include scabronines A−G from S. scabrosus, which are the stimulators of nerve growth factor (NGF) synthesis,11 three diterpenes, glaucopines A−C, from S. glaucopus,12 sarcodonins with cytotoxicity from S. leucopus,13 and p-terphenyl derivatives from S. laevigatum.14 The antioxidative activity guided separation of S. leucopus collected in Tibet, China, resulted in the isolation of two new sarcondonins (1 and 2), one new pterphenyl derivative (3), as well as seven known p-terphenyl products. The antioxidant activities of 1−10 were evaluated using five bioassay methods: DNA protection assay, DPPH scavenging assay, total antioxidant capacity assay, lipid peroxidation assay, and reducing power. Herein, we describe

xidative stress is closely related to pathophysiological conditions such as aging, cancer, and neurodegeneration. Free radicals and reactive oxygen species (ROS) are believed to be a major cause of oxidative stress due to their oxidative damage on biologically important targets. Intake of natural antioxidants especially those derived from edible materials should be beneficial to human health. Antioxidants in food protect organisms from the oxidative damage to lipids, proteins, and nucleic acids. Mushrooms have been used as food as well as being a significant source of bioactive secondary metabolites. Many species of mushrooms including Agaricus bisporus, Boletus badius, Lepista nuda, Pleurotus ostreatus, Polyporus squamosus, Russula delica, and Verpa conica have been reported to have strong antioxidant activity.1−7 Phenolic compounds usually detected in mushrooms are recognized as metabolites responsible for strong antioxidant activity. Flavoglaucins isolated from the fungus Eurotium chevalieri and terphenyl derivatives from the mushroom Thelephora ganbajun showed good antioxidative effects.8−10 The people living in the Tibet Province of China have a long history of consuming wild mushrooms. The mushroom © 2014 American Chemical Society and American Society of Pharmacognosy

Received: December 9, 2013 Published: March 19, 2014 942

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Figure 1. Structures of compounds 1−10.

Figure 2. Key HMBC correlations of 1−3.

HRTOFMS. The IR spectrum of 1 showed absorptions corresponding to the presence of hydroxyl (3311 cm−1), carbonyl (1769 cm−1), and aromatic (1633 and 1505 cm−1) moieties. 1H and 13C spectra of 1 showed marked similarities to those of sarcoviolins and sarcodonins, indicating the characteristic benzodioxazine core structure as well as an N,N-dioxide ring junction.18,19 A para-benzoquinone structural moiety of 1 was confirmed by the signals due to six ortho- and one metacoupling aromatic protons, as well as 18 aromatic carbons, which was further confirmed by HMBC correlations from the aromatic protons as shown in Figure 2 and the NMR data comparison between 1 and 6 that was obtained in this work.16 Two aliphatic side chains were elucidated as a sec-butyl group by 1H−1H COSY correlations of H-4α(β)−H2-5α(β)−H36α(β) and H-4α(β)−H3-7α(β) and HMBC correlations from H3-7α(β) and H3-6α(β) to C-4α(β) and C-5α(β), which were located at C-2α and C-2β by the HMBC correlations between H-4α and C-2α and H-4β and C-2β. Detailed interpretation of the 2D NMR spectrum of 1 determined the planar structure of

the isolation, structural elucidation, and biological activities of 1−10.

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RESULTS AND DISCUSSION The EtOAc crude extract was chromatographed on a D101macroporous resin column to afford four fractions. The fractions with strong antioxidant activity (fraction B, IC50 = 23 ± 1 μg/mL; fraction C, IC50 = 24 ± 1 μg/mL) were further separated by ODS column chromatography (CC), Sephadex LH-20 CC, and RP-HPLC purification to give compounds 1− 10. The structures of 4−10 were established as 2′,3′-diacetoxy4,4″,5′,6′-tetrahydroxy-p-terphenyl (4),15 2′,3′-diacetoxy3,4,4″,5′,6′-pentahydroxy-p-terphenyl (5),13 leucomelone (6),16 Bl-V (7),17 episarcodonin α (8), episarcodonin (9), and sarcodonin α (10)13 by NMR data analysis and comparison with the literature data. Sarcoviolin β (1) was obtained as red powder. It has a molecular formula of C30H30N2O11, as determined by the molecular ion peak for [M + H]+ at 595.1950 in its 943

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138.7, and 157.4 and carbon signals belonging to an acetyl moiety at δC 19.8 and 167.8. These NMR data in combination with its molecular formula suggest a symmetrical structural feature in 3, which was further confirmed by HMBC correlations from aromatic and methyl protons as indicated in Figure 2. Compound 3 is an acetyl product of 4 at C-5′ and C-6′. To verify that the isolated secondary metabolites 3−5 and 7−10 are authentic natural products, a portion of freeze-dried fruiting body of S. leucopus was extracted with EtOAc, and the resulting extract was subjected to reversed-phase HPLC analysis with H2O and MeCN as solvents. Compounds 1−10 were all detected on the HPLC chromatogram of the crude extract (Figure S29, Supporting Information), indicating that these compounds are indeed naturally occurring metabolites. Compounds 1−10 were evaluated for their antioxidant activity using four different bioassays, the DPPH scavenging assay, the total antioxidant capacity assay, reducing power assay, and the lipid peroxidation assay (Table 2). In the DPPH assay,

1 as shown in Figure 2. Since the isolation of sarcodonin in 2000,20 a great deal of effort has been made to conclusively establish the connectivity and relative stereochemistry of the benzodioxazine ring junction in the sarcodonins.13,18,19 This problem was finally resolved by convergent synthesis of phellodonin and sarcodonin ε18 and X-ray analysis of the methylated derivative of sarcodonin ε.19 Sarcodonins and sarcoviolins are biosynthesized by a hetero-Diels−Alder reaction between the 3,4-benzoquinone of terphenyl and the 1β−2β double bond of N-oxopyrazine, which would readily generate epi-isomerization at N-1β. Three pairs of sarcodonins, namely, sarcodonin and episarcodonin, sarcodonin α and episarcodonin α, and sarcodonin β and episarcodonin β, were previously isolated from the fruiting bodies of S. leucopus. The NMR data of sarcodonins and the corresponding episarcodonins closely resemble each other, except for the 1H and 13C signals assigned to C-4α. The marked upfield shift of H-4α (Δδ > 0.5 ppm) and downfield shift of C-4α (Δδ > 1.0 ppm) were observed for episarcodonins in comparison with those of sarcodonins.13 In this report, the 1H and 13C signals assigned to C-4α in 1 (δH/δC 2.98/34.7) are nearly consistent with those of sarcodonins α and β, indicating the same relative configurations for these products. On the basis of the above analysis, the complete structure of 1 was assigned, and this compound was named sarcoviolin β. Episarcoviolin β (2), obtained as a red powder, showed an HRTOFMS peak for [M + H]+ at 595.1953, which together with the NMR data revealed the same molecular formula (C30H30N2O11) as that of 1. Compound 2 showed quite similar UV and IR spectra to those of 1. The NMR data acquired for 2 was almost identical to those of 1, with the main difference at δH 2.19 ppm, which was assigned to H-4α on the basis of 1 H−1H COSY correlations. Similarly, the biggest chemical shift difference was observed for C-4α at δC 38.9. Following these analyses, the same planar structure as that of 1 was assigned for 2 by the detailed interpretation of its HMBC spectrum (Figure 2). The relative configuration in 2 was elucidated by its NMR data comparison with those of 1. An upfield shift of H-4α (Δδ = 0.79 ppm) and a downfield shift of C-4α (Δδ = 4.2 ppm) were shown, which indicated that 2 is an epimeric isomer of 1 at N-1β. The difference in the chemical shift at C-4α between 1 and 2 might arise from their difference in conformation.13 In the preferred conformation of 1 generated by MM2 calculations (Figures S27 and S28 in the Supporting Information), a planar five-membered ring (ON−C-1β−C3α−OH) is present, with the formation of an intramolecular hydrogen bond between the hydroxyl group at C-3α and the oxygen atom of the N-oxide group. Compound 2 lacks the planar five-membered ring in its preferred conformation. The distance between the hydroxyl group at C-3α and the oxygen atom of the N-oxide group in 2 (3.078 Å) is bigger than that in 1 (2.523 Å). Compound 3 was obtained as a green powder. Its molecular formula was determined to be C26H22O10 on the basis of the positive HRTOFMS peak for [M + Na]+ at 517.1107. The IR spectrum of 3 showed absorptions corresponding to the presence of hydroxyl (3404 cm−1), carbonyl (1779 and 1748 cm−1), and aromatic (1613 and 1526 cm−1) moieties. The 1H and 13C NMR spectra of 3 were similar to those of 4,15 showing signals due to ortho-coupling aromatic protons at δH 6.83 (d, J = 8.5 Hz) and 6.99 (d, J = 8.5 Hz), methyl protons at δH 2.03 (s), and an active hydrogen signal at δH 9.69 (br s), as well as six aromatic carbon signals at δC 115.3, 121.0, 129.3, 130.1,

Table 1. 1H and 13C Spectral Data of 1 and 2 in CD3OD 1 position

δC

1 2 3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″, 6″ 3″, 5″ 4″ 2α 3α 4α 5α 6α 7α 2β 3β 4β 5β 6β 7β

129.0 120.5 141.9 141.4 116.8 126.9 115.2 169.7 169.7 117.1 169.7 169.7 122.5 133.0 115.5 158.2 159.1 169.7 34.7 27.6 12.4 16.3 92.9 161.1 43.3 24.5 12.7 14.5

δH (m, J in Hz) 7.24 (s)

7.10 (d, 7.0) 7.23 (d, 7.0)

7.35 (d, 8.4) 6.82 (d, 8.4)

2.98 1.25 0.82 0.92

(m) (m); 1.52 (m) (t, 7.6) (d, 7.0)

2.51 1.34 1.03 1.30

(m) (m); 1.87 (m) (t, 7.4) (d, 6.8)

2 δC 128.9 121.3 141.5 142.1 116.7 127.5 115.2 168.6 168.6 117.0 168.6 168.6 122.5 133.0 115.5 158.2 157.4 168.6 38.9 27.6 11.8 17.2 92.3 161.0 42.8 24.4 12.9 14.4

δH (m, J in Hz) 7.26 (s)

7.08 (d, 8.3) 7.24 (d, 8.3)

7.32 (d, 8.3) 6.80 (d, 8.3)

2.19 1.40 0.79 0.83

(m) (m); 1.17 (m) (t, 7.3) (br)

2.50 1.40 1.02 1.33

(m) (m); 1.85 (m) (t, 7.4) (d, 6.8)

compounds 1, 2, 5, 6, and 8−10 showed strong radical scavenging activity with IC50 values less than that of the positive control ascorbic acid (IC50 = 27 μM). Compound 3 showed weak radical scavenging activity with IC50 values larger than 50 μM. Similar antioxidant results were also observed in the total antioxidant capacity assay (Table 2). Compounds 2, 4, and 5 presented relatively higher antioxidant capacity. In a reducing power assay, compounds 1, 2, and 5−10 exhibited stronger reducing ability than that of ascorbic acid. In the lipid peroxidation assay, the content of malondialdehyde (MDA)944

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Table 2. Antioxidant and α-Glucosidase Inhibition Activities of Compounds 1−10 compound 1 2 3 4 5 6 7 8 9 10 ascorbic acid tocopherol acarbose

DPPH

T-AOC

reducing power

MDA inhibition

α-glucosidase inhibition

IC50 (μM)

(U)

EC50 (μM)

IC50 (μM)

IC50 (μM)

26 12 >50 44 11 9 28 11 13 18 27

± 0.1 ± 0.1 ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.10 0.1 0.1 0.1

180 146 34 225 192 84 129 135 85 167 187 208

± ± ± ± ± ± ± ± ± ± ± ±

4 2 0.2 4 3 2 1 1 2 4 3 1

18 17 >50 >50 31 13 37 22 17 29 48

± 0.1 ± 0.2

± ± ± ± ± ± ±

0.1 0.2 0.2 0.3 0.1 0.3 0.3

27 27 30 >200 29 49 71 31 54 73

± 0.3 ± 0.2 ± 0.4 ± ± ± ± ± ±

0.6 0.2 0.6 0.5 0.4 0.2

0.58 1.07 35 19 3.35 3.53 6.22 3.62 4.20 1.23

± ± ± ± ± ± ± ± ± ±

0.01 0.04 0.1 0.1 0.09 0.03 0.03 0.06 0.02 0.06

16 ± 0.9 752 ± 0.9

generated by lipid peroxidation was significantly decreased by compounds 1−3 and 5−10, with IC50 values between 20 and 100 μM. Compound 4 showed weak inhibitory activity against lipid peroxidation in mice liver homogenate induced by the Fe2+/ascorbate system with IC50 values greater than 200 μM. Hydroxyl radicals generated by the Fenton reaction are known to cause oxidative cleavage of DNA to yield its open circular or relaxed forms. Natural antioxidants have been documented to protect DNA from free radical-induced breaks.21,22 In the DNA damage protection assay, compounds 1−10 showed protection of DNA damage caused by free radicals at 200 μM (Figure 3). However, they did not show a Figure 4. α-Glucosidase inhibitory activity of compounds 1−10 at different concentrations.

configuration at N-1β and C-2β greatly influences the αglucosidase inhibitory activity of sarcodonins and sarcoviolins. Compounds 1 and 10 with cis configuration at N-1β and C-2β showed stronger activity than compounds 2 and 8 with trans configuration. With the p-terphenyl derivatives (3−7), the number of phenolic hydroxyl groups in the structures greatly contributes to their α-glucosidase inhibitory activity. In summary, the antioxidant and α-glucosidase inhibitory activity detected in the extract and the secondary metabolites from the edible mushroom S. leucopus substantially demonstrates its functional property and potential medicinal usages. In vivo biological evaluation and detailed SAR analysis need further study.

Figure 3. Protective effects of compounds 1−10 on DNA damage caused by hydroxy radicals. Lane C: native pBR322 DNA; lane 0: Fenton’s reagent + DNA; lane 1−10: Fenton’s reagent + DNA + compounds 1−10 (200 μM), respectively.

protective effect at 100 μM. Sarcodonins (hydnellin A and sarcodonin δ) from the mushroom Hydnellum geogerirum have been reported with DPPH scavenging activity. 23 The antioxidant activity of sarcodonins and sarcoviolins supports their potential usage as natural antioxidants. The extracts and secondary metabolites from mushrooms such as the culinary mushrooms Grifola f rondosa and Hericium erinaceus and the medicinal mushroom Coriolus versicolor have demonstrated a hypoglycemic effect.24−26 α-Glucosidase inhibitors such as acarbose, miglitol, and voglibose have been successfully applied as oral antidiabetic drugs for the treatment of type 2 diabetes mellitus.27−29 To evaluate the hypoglycemic potential of the edible mushroom S. leucopus, the α-glucosidase inhibitory activity of compounds 1−10 was determined. As seen in Table 2 and Figure 4, all these metabolites showed in vitro inhibitory activity against α-glucosidase. Sarcoviolin β (1) showed the strongest inhibition, with an IC50 value of 0.58 μM. Compounds 2 and 5−10 exhibited moderate inhibitory activity, with IC50 values in the range 1−10 μM. Compounds 3 and 4 showed relatively weak activity with IC50 values at 35 and 19 μM, respectively. A preliminary structure−activity relationship (SAR) was deduced on the basis of above activity. The

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Perkin-Elmer 241 polarimeter. UV spectra were recorded on a Shimadzu Biospec-1601 spectrophotometer. IR data were recorded using a Nicolet Magna-IR 750 spectrophotometer. 1H and13C NMR data were acquired with a Bruker Avance-500 spectrometer. Chemical shifts were expressed in δ using solvent signals (dimethyl sulfoxide-d6: δH 2.49/δC 39.7) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HR-TOF-MS data were obtained using an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument. Sephadex LH-20 (Amersham Biosciences), and ODS (Lobar, 40−63 μm, Merck) were used for column chromatography. HPLC analysis was performed on an Agilent 1200 HPLC system using an ODS column (C18, 250 × 4.6 mm, YMC Pak, 5 μm; detector: UV) with a flow rate of 1.0 mL/min. 945

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Fungal Material. Fruiting bodies of Sarcodon leucopus were collected from Lizhi County (Tibet, China) in August 2012 and identified by one of the coauthors (Prof. Tiezheng Wei). The characteristic features of the pileus, stipe, context, and spores in the specimen is fully consistent with the morphological description for S. leucopus.30 The fungal specimen was deposited in Mycological Herbarium of Institute of Microbiology, Chinese Academy of Sciences (HMAS No. 252750). Extraction and Isolation. 780 g of dry fruiting bodies of S. leucopus were extracted with ethyl acetate (3 × 3 L), and 30g crude extract was obtained through evaporating to dryness under vacuum. The organic extract (20 g) was fractionated on D101 macroporous resin eluting with gradient of ethanol−water (20%, 35%, 50%, 75%, 90%, 100%) to offer four fractions (A-D). Fraction B and C showing strong DPPH scavenging activity were subjected to further separation. Fraction B was separated by ODS column chromatography (CC) using a gradient of increasing methanol in water (30%, 50%, 60%, 80%, 100%) to give five subfractions (B1−B5). Compound 4 (4.0 mg, tR 25.5 min) and 7 (4.0 mg, tR 28 min) was obtained from fraction B2 by RP-HPLC using 29% acetonitrile in water. Fraction B3 was further separated on Sephadex LH-20 CC eluted with methanol to give compound 3 (14.0 mg). Fraction B4 was eluted with a gradient of increasing methanol in water (40%, 60%, 80%, 100%) on ODS CC to afford compound 5 (51.0 mg) and 6 (63.0 mg). Fraction C was separated using an ODS column eluted with a gradient of increasing methanol in water (30%, 50%, 60%, 80%, 100%) to afford five subfractions (C1−C5). Fraction C2 was subjected to Sephadex LH-20 CC eluted with 70% methanol in water to give compounds 1 (1.1 g) and 2 (150.0 mg). Fraction C3 was separated on Sephadex LH-20 CC eluted with 70% methanol in water to afford compound 8 (180.0 mg) and 10 (32.0 mg). Compound 9 (17.6 mg, tR 24 min) was purified by RP-HPLC using 53% acetonitrile in water from fraction C4. Sarcoviolin β (1): red, amorphous powder; [α]25D −14.55 (c 1.1, methanol); UV (methanol) λmax nm (log ε) 198 (4.54), 270 (4.38); IR (neat) νmax 3311, 2971, 2939, 1769, 1633, 1505, 1326, 1274, 1177, 997 cm−1; for 1H NMR and 13C NMR data see in Table 1; positive HRTOFMS m/z [M + H]+ 595.1950 (calcd for C30H31N2O11, 595.1922). Episarcoviolin β (2): red, amorphous powder; [α]25D −32.59 (c 1.4, methanol); UV (methanol) λmax nm (log ε) 198 (4.48), 270 (4.32); IR (neat) νmax 3300, 2973, 2939, 1781, 1627, 1506, 1325, 1254, 1177, 997 cm−1; for 1H NMR and 13C NMR data see in Table 1; positive HRTOFMS m/z [M + H]+ 595.1950 (calcd for C30H31N2O11, 595.1922). 2′,3′,5′,6′-Tetracetoxy-4,4″-dihydroxy-p-terphenyl (3): green powder; UV (methanol) λmax nm (log ε) 200 (4.36), 275 (4.19); IR (neat) νmax 3404, 2937,1779, 1748, 1613, 1595, 1526, 1378, 1216, 1028, 908 cm−1; 1H NMR (500 MHz, in DMSO-d6) 9.69 (s, 2H) for two hydroxyl protons, 6.99 (d, J = 8.5 Hz, 4H) for H-2, H-6, H-2″, and H-6″, 6.83 (d, J = 8.5 Hz, 4H) for H-3, H-5, H-3″, and H-5″, 2.03 (s, 12H) for methyl protons; 13C NMR (125 MHz, in DMSO-d6) 167.8 (4C) for carbonyl carbons, 157.4 (2C) for C-4 and C-4″, 138.7 (4C) for C-2′, C-3′, C-5′, and C-6′, 130.1 (4C) for C-2, C-6, C-2″, and C6″, 129.3 (2C) for C-1′ and C-4′, 121.0 (4C) for C-1 and C-1″, 115.3 (4C) for C-3, C-5, C-3″, and C-5″, and 19.8 (4C) for acetate methyl carbons; positive HRTOFMS m/z [M + Na]+ 517.1107 (calcd for C26H22O10Na 517.1105). HPLC Analysis of the Extract of S. leucopus. The fresh fruiting bodies were extracted with EtOAc. The EtOAc extract was dissolved in EtOAc (10 mg/mL) and subjected to RP-HPLC analysis . The detection wavelength was set to 254 nm. The mobile phase consisted of MeCN (A) and H2O (B) using a gradient elution of 10−40% A at 0−10 min, 40−50% A at 10−18 min, 50−55% A at 18−35 min, 55− 100% A at 35−45 min, and 100% A at 45−55 min. The flow rate was kept at 1 mL/min. Compounds 1−10 were all detected on the HPLC chromatogram of the crude extract. DPPH Scavenging Assay. The mixture containing 100 μL of each compound in DMSO (final concentrations of 6.25, 12.5, 25, 50 μM) and 100 μL of DPPH (sigma) radicals in ethanolic solution (final concentration of 0.2 mM) was shaken vigorously and left to stand for

30 min in the dark, and the absorbance was then measured at 517 nm using a Spectra Max 190 microplate reader (Molecular Devices Inc.).31 The scavenging ability was calculated as follows: scavenging ability (%) = [(A517 of control − A517 of sample)/A517 of control] × 100. Ascorbic acid was used as positive control with an IC50 of 27.36 ± 0.10 μM. Total Antioxidant Capacity (ref 32). The total antioxidant capacity of samples was determined using a commercial kit (Jiancheng Biological Engineering Institute, Nanjing, China), and the result was calculated by the equation below. Total antioxidant capacity (unit) = (ODV − ODC) × N/0.3, where ODV is the absorbance value of the sample, ODC is the absorbance value of the control, and N is the dilution of the reaction system. Ascorbic acid and tocopherol were used as positive controls. Determination of Lipid Peroxidation (ref 32). Male mice weighing 18−20 g were purchased from Vital River Laboratories, Beijing, China (No. SCXK (Beijing) 2006−0009). Procedures involving animals and their care were conducted in compliance with institutional guidelines in accordance with NIH Guide for the Care and Use of Laboratory Animals. The mice were anesthetized using diethyl ether, and their abdomen was opened and their liver quickly removed. The liver was then cut into small pieces and then homogenized in phosphate buffer (50 mM, pH 7.4) to give a 10% (w/v) homogenate. The liver homogenate was further centrifuged at 5000 rpm for 10 min. Supernatant of the liver homogenate was collected, and the amount of protein was determined using the Coomassie brilliant blue method. The extent of lipid peroxidation was evaluated by measuring the product of thiobarbituric acid reactive substances in the homogenate. The reaction mixture was composed of 10% tissue homogenate (1 mL), FeSO4 (1 mM, 50 μL), ascorbic acid (1 mM, 50 μL), and 1 mL of different concentrations of the compounds (final concentrations of 25, 50, 100, 200 μM). The reaction mixture was incubated at 37 °C for 1.5 h and then centrifuged at 3000 rpm for 10 min; the supernatant was used to detect MDA content with commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the manufacturer’s instructions at 532 nm. The inhibiting ability was calculated as follows: inhibiting ability (%) = [(Acontrol − Asample)/ Acontrol] × 100. Tocopherol was used as positive control with an IC50 of 16 μM. Reducing Power. A mixture containing 20 μL of each compound (final concentrations of 6.25, 12.5, 25, 50 μM) in distilled water, 50 μL of phosphate buffer (0.2 M, pH 6.6), and 50 μL of potassium ferricyanide [K3Fe(CN)6] (1%) was incubated at 50 °C for 20 min. A 20 μL portion of trichloroacetic acid (10%) was added to the mixture, and then the mixture was centrifuged at 4000 rpm for 10 min. The upper layer of the solution (100 μL) was mixed with distilled water (100 μL) and FeCl3 (20 μL, 0.1%), and the absorbance was measured at 700 nm. Ascorbic acid was used for comparison. DNA Damage Protection Assay (refs 21 and 22). The DNA damage protection assay was conducted according to the previously reported method with some modification. A mixture of 2 μL of test compound (100 and 200 μM) and 3 μL of supercoiled pBR322 DNA (1.5 μg) was incubated at room temperature for 10 min followed by the addition of 15 μL of Fenton’s reagent (30 mM H2O2, 50 μM ascorbic acid, and 80 μM FeCl3). The mixture was then incubated for 30 min at 37 °C. The DNA was analyzed on 0.8% agarose gel using ethidium bromide staining. α-Glucosidase Inhibitory Assay (ref 33). A mixture containing 25 μL of different compounds (final concentrations of 0.78, 1.56, 3.13, 6.25, 12.50, 25.00 μM), 25 μL of α-glucosidase (0.2 U/mL, from Baker’s yeast, Sigma), and 175 μL of phosphate buffer (50 mM, pH 6.8) was left to stand for 10 min at room temperature. The reaction was started by the addition of 25 μL of 23.2 mM p-nitrophenyl-α-Dglucopyranoside (2.5 mM, Sigma) and incubated for 15 min at 37 °C. The assay was conducted in a 96-well plate, and the absorbance was determined at 405 nm using a Spectra Max 190 microplate reader (Molecular Devices Inc.). The control was prepared by adding phosphate buffer instead of the sample in the same way as the test. The blank was prepared by adding phosphate buffer instead of αglucosidase using the same method. The inhibition rates (%) = 946

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[(ODcontrol − ODcontrol blank) − (ODtest − ODtest blank)]/(ODcontrol blank − ODcontrol blank) × 100%. Acarbose was utilized as the positive control with an IC50 of 793.79 ± 0.41 μM.

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra for compounds 1−3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-10-62566577. Fax: 86-10-64807515. E-mail: liuhw@ im.ac.cn. Author Contributions ⊥

K. Ma and J.-J. Han contributed equally to this article.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support of the Ministry of Science and Technology of China (2014CB138304) and the National Natural Science Foundation (Grant Nos. 31000036 and 21072219) is gratefully acknowledged.

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dx.doi.org/10.1021/np401026b | J. Nat. Prod. 2014, 77, 942−947