Article Cite This: J. Agric. Food Chem. 2019, 67, 7706−7715
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Discovery of New Triterpenoid Saponins Isolated from Maesa japonica with Antifungal Activity against Rice Blast Fungus Magnaporthe oryzae Men Thi Ngo,†,‡ Jae Woo Han,† Sunggeon Yoon,† Sohyun Bae,†,‡ Soo-Young Kim,§ Hun Kim,*,†,‡ and Gyung Ja Choi*,†,‡ †
Center for Eco-friendly New Materials, Korea Research Institute of Chemical Technology, Daejeon 34114, Korea Department of Medicinal Chemistry and Pharmacology, University Science and Technology, Daejeon 34113, Korea § Biological and Genetic Resources Utilization Division, National Institute of Biological Resources, Incheon 22869, Korea
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‡
S Supporting Information *
ABSTRACT: While searching for new antifungal compounds, we revealed that a methanol extract of plant species Maesa japonica has a potent antifungal activity in vivo against rice blast fungus Magnaporthe oryzae. To identify the antifungal substances, the methanol extract of M. japonica was extracted by organic solvents, and consequently, six active compounds were isolated from the n-butanol layer. The isolated compounds were five new acylated triterpenoid saponins including maejaposide I (1), maejaposides C-1, C-2, and C-3 (2−4), and maejaposide A-1 (5), along with a known one, maejaposide A (6). These chemical structures were determined by NMR and a comparison of their NMR and MS data with those reported in the literature. Based on the in vitro antifungal bioassay, the five compounds (2−6) exhibited strong antifungal activity against M. oryzae with MIC values ranging from 4 to 32 μg/mL, except for maejaposide I (1) (MIC > 250 μg/mL). When the compounds were evaluated at concentrations of 125, 250, and 500 μg/mL for an in vivo antifungal activity against rice blast, compounds 2− 6 strongly reduced the development of blast by at least 85% to 98% compared to the untreated control. However, compound 1 did not show any in vivo antifungal activity up to a concentration of 500 μg/mL. Taken together, our results suggest that the methanol extract of M. japonica and the new acylated triterpenoid saponins can be used as a source for the development of natural fungicides. KEYWORDS: Maesa japonica, saponins, rice blast, antifungal, plant disease control
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INTRODUCTION Fungal diseases cause serious damage to plants, resulting in the reduction of the yield and quality of crops and vegetable plants. Chemical control using synthetic fungicides is one of the most effective methods for the management of plant diseases.1 However, during the past few decades, increasing concerns about the hazards of synthetic chemicals to humans, animals, and the environment and the rapid development of fungicide resistance toward the major class of fungicides have led to the requirement of developing safe and eco-friendly fungicides.2,3 Therefore, natural resources containing bioactive compounds have been considered for the development of biofungicides that can replace synthetic fungicides.4 Naturally occurring plant products are important sources of antifungal compounds with low toxicity to mammals and the environment as well as a low bioaccumulation because they rapidly degrade in the soil. Many studies have shown the effects of plant extracts on the control of various plant diseases. For example, the organic solvent extracts of Curcuma longa L. and Cassia tora exhibited fungicidal activities against plant pathogenic fungi, such as Phytophthora infestans, Puccinia recondita, and Erysiphe graminis.5,6 Additionally, the pure compounds isolated from Annona squamosa seeds efficiently suppressed the development of plant disease caused by P. infestans and P. recondita.7 Currently, some plant extracts such © 2019 American Chemical Society
as giant knotweed extract from Reynoutria sachalinensis and thyme oil from Thymus vulgaris have been registered as a natural fungicide agent in the US and EU market.8 Saponin is well-known as an importance class of secondary metabolites in plants with a wide range of bioactivities including antifungal properties.9,10 Recently, oleanane and ursane-type triterpenoid saponins from Trevesia palmata were shown to have a strong antifungal activity in vitro and in vivo against rice blast fungus Magnaporthe oryzae.11 The major saponin α-tomatine in tomatoes also induced the programmed cell death of Fusarium oxysporum by activating phosphotyrosine kinase and monomeric G-protein signaling pathways which resulted in Ca2+ elevation and ROS burst.12 Related with the fungicidal mode of action of saponins, it has been reported that avenacin A-1, a triterpenoid saponin from oat roots, has potent antifungal activity by which it makes an interaction with sterols in fungal membranes which results in the deficiency of the membrane integrity.10 In addition, the saponins of Medicago sativa are known to affect the cell membrane integrity of M. oryzae.13 Thus, the mechanism of the antifungal Received: Revised: Accepted: Published: 7706
April 10, 2019 June 17, 2019 June 17, 2019 June 17, 2019 DOI: 10.1021/acs.jafc.9b02236 J. Agric. Food Chem. 2019, 67, 7706−7715
Article
Journal of Agricultural and Food Chemistry
Table 1. 1H and 13C NMR Spectroscopic Data (500 and 125 MHz) of the Aglycon Parts of Compounds 1−6 in Pyridine-d5a 1
2
3
4
5
6 δH (J in Hz)
δH (J in Hz)
Position
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
1
39.7
1.54 m; 0.82*
39.7
39.8
1.43 m
39.7
1.49 m
27.2
27.1
1.79 m
27.1
1.79 m
3
90.4
2.18 m; 1.78 m 3.21 dd (11.7, 5.1)
1.56 m; 0.85 m 2.19 m; 1.82 m 3.26 dd (11.7, 4.3)
39.7
27.2
1.55 m; 0.84 m 2.20 m; 1.82 m 3.27 dd (11.6, 4.6)
39.8
2
1.54 m; 0.82 m 2.16 m; 1.75 m 3.24 dd (11.7, 4.8)
90.5
3.24 d (11.1)
90.4
3.23 d (10.8)
4 5
40.4 56.2
6
19.7
7
34.7
8 9 10 11 12
43.1 50.8 37.3 18.4 33.7
13 14 15
87.4 44.0 33.6
16 17 18
72.0 54.7 46.0
19
39.0
20 21
37.7 82.4
6.14 d (10.0)
38.0 79.7
6.82 d (10.1)
38.0 79.8
6.81 d (10.1)
38.0 79.8
6.81 d (10.1)
33.9 42.2
22 23 24 25 26 27 28 29 30 R1 1′ 2′ 3′
69.8 28.5 17.1 16.9 19.0 20.3 97.6 30.9 21.0
5.03 1.31 1.19 0.82 1.27 1.37 5.47 1.23 1.31
73.8 28.4 17.1 16.9 19.1 20.2 97.6 30.3 21.3
6.39 d (10.1) 1.22 s 1.10 s 0.84 s 1.37 s 1.64 s 5.24 s 1.16 s 1.27 s
73.9 28.6 17.2 16.9 19.2 20.2 97.6 30.3 21.3
6.39 1.31 1.21 0.84 1.36 1.63 5.23 1.16 1.27
73.9 28.4 17.1 16.9 19.2 20.2 97.6 30.3 21.3
6.39 1.27 1.10 0.83 1.36 1.63 5.24 1.16 1.27
73.1 28.5 17.1 16.9 19.1 20.2 98.3 33.9 26.1
169.7 144.7 120.4
7.90 d (16.7) 6.75 d (16.7)
4′
135.6
16.4
5′ 6′ 7′ 8′ 9′ R2 1″ 2″
129.0 129.8 131.0 129.8 129.0
21.5
0.68 m 1.78 m; 1.44 m 1.37*; 1.08 m
1.23* 1.36* 2.12 m; 1.63 m
2.23 m; 1.63 m 6.16* 2.42 m 2.75 t (13.8); 1.63 m
d (10.0) s s s s s s s s
90.1 40.3 56.1 19.7 34.8 43.2 50.7 37.3 18.4 33.8 87.9 44.3 37.1 69.3 55.0 46.8 38.4
168.2 129.4 138.0
0.71 d (11.3) 1.81 m; 1.46 m 1.60*; 1.28*
1.27* 1.36* 2.14 m; 1.65*
2.21 m; 1.65 m 4.75 m 2.42 dd (14.7, 3.6) 3.14 m; 1.54 m
6.00 dd (7.2, 1.7) 2.11 dd (7.2, 1.7) 2.06 t (1.7)
167.8 129.6
27.2 90.3 40.3 56.2 19.8 34.9 43.2 50.7 37.3 18.5 33.8 87.9 44.3 37.1 69.4 55.0 46.8 38.6
168.2 129.4 138.0 16.4 21.5
0.72 d (11.4) 1.83 m; 1.48 m 1.58*; 1.25*
1.28* 1.36* 2.15 m; 1.65*
2.19 m; 1.64 m 4.74 m 2.41 dd (14.6, 3.6) 3.15 t (13.5); 1.55 m
d (10.1) s s s s s d (4.7) s s
6.00 dd (7.2, 1.6) 2.11 dd (7.2, 1.6) 2.05 t (1.6)
167.8 129.7
3″
136.9
5.86 m
136.9
4″
16.3
2.00 m
5″
21.2
1.86 t (1.7)
27.2 90.4 40.4 56.1 19.8 34.9 43.2 50.8 37.3 18.5 33.8 87.9 44.3 37.1 69.4 55.0 46.8 38.7
168.3 129.4 138.1 16.4 21.6
0.70 d (11.1) 1.82 m; 1.48 m 1.61*; 1.29*
1.28* 1.36* 2.17 m, 1.67*
2.21 m; 1.67 m 4.75 m 2.42 dd (14.2, 3.7) 3.14 t (13.4), 1.55 m
d (10.1) s s s s s d (4.2) s s
16.3
16.3
21.3
1.86 t (1.7)
21.3
7707
137.0
19.7 34.8 43.2 50.8 37.3 18.5 33.8 88.1 44.4 37.3 70.3 52.0 47.9 38.8
0.71 m 1.40 m; 1.81 m 1.26 m; 1.59 m 1.30 m 1.51 m 1.61 m
2.22 m; 1.61 m 4.88 m 2.22 m 2.92 m; 1.38 m 2.87*; 2.10 m 6.15* 1.27 s 1.19 s 0.82 s 1.36 s 1.65 s 5.29 s 1.11 s 1.19 s
40.4 56.2 19.8 34.9 43.2 50.8 37.3 18.5 33.8 88.2 44.5 37.3 70.4 52.1 47.9 38.8 33.9 42.2 73.2 28.6 17.2 16.9 19.2 20.3 98.3 33.9 26.2
0.70 d (11.3) 1.42 m; 1.79 m 1.24 m; 1.57 m 1.30 m 1.50 m 1.60 m
2.24 m; 1.60 m 4.86 m 2.24 m 2.92 m; 1.38 m 2.87 m; 2.10 m 6.14* 1.25 s 1.18 s 0.82 s 1.35 s 1.65 s 5.29 s 1.11 s 1.19 s
6.01 dd (7.2, 1.6) 2.11 dd (7.2, 1.6) 2.06 t (1.6)
167.9 129.7 5.86 dd (7.2, 1.6) 2.00 m
40.4 56.2
δC
166.6 121.7 5.87 dd (7.2, 1.6) 2.01 dd (7.0, 1.6) 1.87 t (1.7)
166.7 121.7 149.8
5.96 d (11.4) 6.14*
31.6
5.95 d (11.4) 6.13 d (11.4) 2.75 m
31.7
2.75 m
23.0
1.36 m
23.0
1.36 m
149.8
DOI: 10.1021/acs.jafc.9b02236 J. Agric. Food Chem. 2019, 67, 7706−7715
Article
Journal of Agricultural and Food Chemistry Table 1. continued 1 Position
δC
δH (J in Hz)
2 δC
δH (J in Hz)
3 δC
4
δH (J in Hz)
6″ R3 1‴ 2‴
δC
δH (J in Hz)
5 δC 14.4
170.5 22.7
6 δH (J in Hz)
0.84 t (7.3)
δC 14.4
δH (J in Hz) 0.84 t (7.4)
2.59 s
Assignment aided by 1H−1H COSY (500 MHz), HMQC, and HMBC (500/125 MHz) experiments. *, overlapped.
a
methanol, and the filtrated extract was concentrated by a rotary evaporator, resulting in 702.9 g of a methanol extract. For the in vitro antifungal activity assay, we used five phytopathogenic fungi: Botrytis cinerea, Colletotrichum coccodes, F. oxysporum, M. oryzae, and P. infestans. Fungal sporulation and maintenance were performed as previously described.17 All phytopathogenic fungi were provided by the Korean Agricultural Culture Collection (Jeonju, Korea). Isolation of the Antifungal Compounds from M. japonica. An isolation scheme for the saponins from M. japonica is shown in the supplemental Figure S1. The methanol extract (702.9 g) of M. japonica was suspended in 700 mL of water and then partitioned with n-butanol (BuOH). The resulting n-BuOH fraction and aqueous fraction were concentrated to dryness to give the n-BuOH (500 g) and water-soluble (202.9 g) layers. For the isolation of the active compounds, the n-BuOH fraction was applied to a silica gel column (40 cm × 30 cm i.d.) with gradient elution of CH2Cl2/MeOH/H2O (90:10:0, 85:15:0, 80:20:2, 60:40:5, 40:60:8, and 0:100:10, v/v/v), yielding six fractions B1−B6. Based on an antifungal activity-guided bioassay against M. oryzae, fraction B3 (153.3 g) showing in vitro antifungal activity was applied to a silica gel 60 column (30 cm × 20 cm i.d.) with a gradient elution of CH2Cl2/MeOH/H2O (1:0:0− 0:1:0.1, v/v/v), yielding seven fractions B31−B37. An antifungal fraction B34 (12.0 g) was applied to an MCI gel CHP20/P120 column (25 cm × 8 cm i.d.) with gradient elution of MeOH/H2O (70:30−100:0, v/v) to yield 13 fractions B341−B3413, and fraction B349 (1.1 g) was further subjected to a silica gel column (20 cm × 3 cm i.d.) with isocratic elution of CHCl3/MeOH/H2O (3:1:0.1, v/v/ v), yielding nine fractions B3491−B3499. An active fraction B3495 (205.5 mg) was purified by prep HPLC eluting with 68% aqueous MeOH to yield pure compounds 1 (59.5 mg, tR = 55.8 min) and 2 (30.0 mg, tR = 96.0 min). Another active fraction B35 (30.0 g) was fractionated on an MCI gel column (25 cm × 8 cm i.d.) using a gradient elution of MeOH/H2O (70:30−100:0, v/v) to yield nine fractions B351−B359. The fraction B357 (4.0 g) was applied to an MCI gel column (80 cm × 3 cm i.d.) with a gradient elution of MeOH/H2O (70:30−100:0, v/v) to yield nine fractions B3571− B3579. Fraction B3573 (536.3 mg) was separated by prep HPLC with an isocratic elution of 68% aqueous MeOH to yield pure compounds 3 (34.6 mg, tR = 86.3 min) and 4 (129.9 mg, tR = 97.1 min). The active fraction B5 (50.7 g) was chromatographed on an MCI gel column (35 cm × 8 cm i.d.) eluting with a stepwise gradient mixture of MeOH/H2O (50:50−100:0, v/v) to obtain ten fractions B51− B510. Fraction B59 (3.93 g) was applied to a Cosmosil 75C18-OPN column (30 cm × 5 cm i.d.) eluting with a mixture of C2H3N/H2O (45:55−100:0, v/v) yielding 11 fractions B591−B5911. The active fraction B596 (316.5 mg) was finally purified by prep HPLC with an isocratic elution of 68% aqueous MeOH containing 0.1% formic acid to yield pure compounds 5 (25.6 mg, tR = 68.1 min) and 6 (35.2 mg, tR = 62.3 min). Maejaposide I (Compound 1). Amorphous powder; for 1H and 13 C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 1333.6191 [M + Na]+ (calculated for C65H98O27Na, 1333.6193); UV (λmax, nm, log ε): 214 (2.89), 275 (3.02); IR (νmax): 3375, 2930, 1700, 1635, 1364, 1244, 1069. Maejaposide C-1 (Compound 2). Amorphous powder; for 1H and 13 C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 1325.6503 [M + Na]+ (calculated for C64H102O27Na, 1325.6506); UV
activity of saponins has been proposed as follows: saponins interact with sterols in fungal membranes which results in pore formation and the disruption of the membrane followed by leakage of the cell components.10,14 While searching for environmentally friendly antifungal compounds from Korean herbs, we found that a methanol extract of Maesa japonica (Thunb.) Morizi & Zoll. has potential for controlling plant diseases. The plant M. japonica is a shrub or small tree distributed throughout China, Vietnam, Korea, and the southern parts of Japan. In China, this plant species has been used as a traditional medicine for curing symptoms associated with the common cold, and investigations of the chemical constituents of the genus Maesa have led to the identification of quinones and triterpenoidal saponin components.15,16 From the plant M. japonica, five triterpenoid saponins, maejaposides A−D, have been identified and characterized; however, there has been no investigation on the antifungal properties against phytopathogenic fungi.15 In the present study, we isolated six compounds (1−6) from M. japonica based on the in vitro antifungal activity, and among the six compounds, five compounds (1−5) were newly identified as acylated triterpenoid saponins. Beyond the identification of the new triterpenoid saponins, our results that compounds (2−6) have an in vitro and in vivo antifungal activity against phytopathogenic fungi can be useful for the development of crop-protecting agents.
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MATERIALS AND METHODS
General Experimental Procedures. Analytical thin-layer chromatography was performed on a silica gel 60 F245 and RP−18 F245S plates (Kiesel gel 60, 20 cm × 20 cm, 0.25 mm thickness; Merck & Co., Inc., Kenilworth, NJ, USA), and the resulting spots were detected under UV light and by spraying with p-anisaldehyde reagent. Column chromatography was performed using silica gel 60 (230−400 mesh; Merck & Co.), MCI gel CHP20/P120 (75−150 μm; Mitsubishi Chemical Co., Tokyo, Japan), and Cosmosil 75C18OPN (75 μm; Nacalai Tesque Inc., Kyoto, Japan) columns. The 1D and 2D nuclear magnetic resonance (NMR) spectra were recorded by a Bruker Advance 500 MHz spectrometer (Bruker Co., Billerica, MA, USA). Chemical shifts were referenced to the solvent peaks (δC 150.35 and δH 8.74 for pyridine-d5). High-resolution electrospray ionization mass spectrometry (HRESIMS) was recorded on an ACQUITY Arc system (Waters Co., Milford, MA, USA). Analysis of high-performance liquid chromatography (HPLC) was performed on a Waters 2690 system (Waters Co.) equipped with a reversed-phase C18 Varian Pursuit XRs column (4.6 mm × 250 mm, 5 μm; Agilent Technologies, Inc., Santa Clara, CA). All solvents were purchased from Samchun Pure Chemical Co., Ltd. (Seoul, Korea) and used without further purification. Plant Material and Phytopathogenic Fungi. The M. japonica (14.1 kg) were kindly provided by Dr. Soo-Young Kim at the National Institute of Biological Resources (NIBR; Incheon, Korea), and voucher specimens were deposited in the laboratory of Dr. Soo-Young Kim at the NIBR. The collected plant materials were extracted with 7708
DOI: 10.1021/acs.jafc.9b02236 J. Agric. Food Chem. 2019, 67, 7706−7715
Article
Journal of Agricultural and Food Chemistry Table 2. 1H and
13
C NMR Data (500 and 125 MHz) of the Sugar Moiety of Compounds 1−6 in Pyridine-d5a 1
Position
δC
1
105.7
2 3 4 5 6
80.1 84.2 70.4 78.4 63.4
δH (J in Hz) glucose 4.78*
2 δC 105.6 79.9 84.0 70.4 78.5 63.5
1
4.64* 4.64 m 4.12 m 3.92 m 4.54 d (11.6); 4.26 dd (11.5, 5.2) galactose (terminal) 103.9 5.79 d (7.4)
2 3 4 5 6
74.1 75.8 70.7 77.3 63.6
4.52 m 4.36 m 4.49* 4.48* 4.54 m; 4.26 m
77.0 78.9 73.1 78.5 64.2
1
101.8
galactose 6.18 d (7.8)
101.8
2 3 4 5 6
77.2 76.4 71.6 77.5 62.4
1 2 3 4 5 6
103.1 73.0 73.2 74.4 70.4 18.8
1
4.75 m 4.53 m 4.49* 4.28 m 4.36 m rhamnose 6.28 s 4.81 m 4.70 m 4.24 m 4.87 dd (9.4, 6.2) 1.47 d (6.2)
δH (J in Hz) glucose 4.78*
4.65 m 4.65 m 4.12 m 3.90 m 4.54 d (11.6); 4.26 dd (11.5, 5.2) glucose (terminal) 103.0 5.93 d (7.6)
76.6 76.5 71.6 77.5 62.4 102.8 73.1 73.1 74.4 70.3 18.8
4.16 m 4.44 m 4.10 m 4.44 m 4.69 d (11.3); 4.33 m galactose 6.19 d (7.8) 4.76* 4.51 m 4.51 m 4.28 m 4.38 m rhamnose 6.28 s 4.81 brs 4.76* 4.26 m 4.91 dd (9.5, 6.1) 1.46 d (6.2)
3
4 δH (J in Hz)
δC
glucose 4.81 d (6.8) 80.3 4.63 m 84.1 4.63 m 70.4 4.12 m 78.4 3.91 m 63.5 4.54 m; 4.28 m galactose (terminal) 103.8 5.74 d (7.5) 73.2 4.52 m 75.7 4.26 m 70.7 4.38 m 74.0 4.54 m 63.5 4.54 m; 4.29 m galactose 101.6 6.16 d (7.8) 77.2 4.69 m 76.3 4.54 m 71.7 4.54 m 77.5 4.28 m 62.5 4.38 m rhamnose 101.9 6.21 s 78.6 4.87 m 73.9 4.54 m 74.6 4.15 m 70.2 4.85 m 18.7 1.45 d (6.3) rhamnose(terminal) 104.1 6.01* 105.6
δC 105.6
5 δH (J in Hz)
glucose 4.78*
80.1 83.8 70.5 78.5 63.5
4.66 m 4.69 m 4.13 m 3.92 m 4.53 m; 4.30 m glucose (terminal) 103 5.87*
77 78.9 73 78.7 64.2
4.14 m 4.39 m 4.05 m 4.39 m 4.66 m; 4.28 m galactose 101.6 6.19 d (8.0) 76.8 4.72 m 76.3 4.53 m 71.8 4.52 m 77.6 4.31 m 62.6 4.38 m rhamnose 101.5 6.21 s 79.2 4.82 m 73.2 4.53 m 74.7 4.14 m 70.2 4.90 m 18.5 1.43 d (6.3) rhamnose(terminal) 104.2 5.91 s
2 3 4 5
73.2 72.9 74.7 70.7
4.78 4.54 4.28 4.54
m m m m
72.7 72.7 74.7 70.7
4.80 4.54 4.24 4.52
m m m m
6
19.2
1.69 d (6.3)
19.2
1.64*
δC
6 δH (J in Hz)
δC
δH (J in Hz)
glucuronic acid 105.8 4.87*
glucuronic acid 105.7 4.86*
80.2 83.3 70.4 77.4 ndb
80.3 83.6 71.8 77.9 nd
4.71 4.73 4.24 4.51
m m m m
galactose (terminal) 104.0 5.79*
4.70 4.72 4.46 4.52
m m m m
galactose (terminal) 104.2 5.70*
73.1 75.7 70.7 74.0 63.4
4.52 m 4.30 m 4.51 m 4.51 m 4.58 m; 4.37 m galactose 101.8 6.18*
4.52 m 4.33 m 4.52 m 4.46 m 4.55 m; 4.36 m galactose 101.9 6.12*
77.6 76.5 71.8 77.4 62.5
77.9 76.2 71.9 77.4 62.6
4.74 m 4.52 m 4.51 m 4.26 m 4.35 m rhamnose 103.1 6.26 s 73.2 4.81 m 73.2 4.66 m 74.4 4.26 m 70.3 4.88 m 18.8 1.47 brs
74.0 75.8 70.7 77.3 63.3
4.72 m 4.52 m 4.52 m 4.27 m 4.36 m rhamnose 101.7 6.29 s 82.7 4.78 m 73.2 4.72 m 74.9 4.19 m 70.0 4.82 m 18.6 1.40*
108.0 76.2 78.9 71.4 67.8
xylose 5.08 d (7.0) 4.00 m 4.03 m 4.14 m 3.52 t (10.6)
Assignment aided by 1H−1H COSY (500 MHz), HMQC, and HMBC (500/125 MHz) experiments. bNot detected. *, overlapped.
a
(λmax, nm, log ε): 212 (2.99); IR (νmax): 3362, 2923, 1700, 1640, 1386, 1238, 1071. Maejaposide C-2 (Compound 3). Amorphous powder; for 1H and 13 C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 1471.7017 [M + Na]+ (calculated for C70H112O31Na, 1471.7085); UV (λmax, nm, log ε): 214 (2.92); IR (νmax): 3375, 2931, 1700, 1382, 1231, 1069. Maejaposide C-3 (Compound 4). Amorphous powder; for 1H and 13 C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 1471.7042 [M + Na]+ (calculated for C70H112O31Na, 1471.7085); UV (λmax, nm, log ε): 214 (2.92); IR (νmax): 3366, 2926, 1701, 1653, 1386, 1238, 1069. Maejaposide A-1 (Compound 5). Amorphous solid; for 1H and 13 C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 1255.6090 [M + Na]+ (calculated for C60H96O26Na, 1255.6088); UV
(λmax, nm, log ε): 207 (2.81); IR (νmax): 3370, 2925, 1685, 1636, 1363, 1069. Maejaposide A (Compound 6). Amorphous powder; for 1H and 13 C spectroscopic data, see Tables 1 and 2; ESIMS m/z 1363 [M − H]−. Spectroscopic data were comparable to those previously reported for maejaposide A.15 Monosaccharide Analysis. The compositional analysis of sugar moieties in the compounds was performed as previously described.18 Briefly, each compound hydrolyzed by 1 M hydrochloric acid was washed by ethyl acetate three times and dried in vacuo. The sugars of each sample were dissolved in 0.5 mL of anhydrous pyridine containing 3.0 mg of L-cysteine methyl ester hydrochloride and heated at 60 °C for 1 h. The resulting mixture was added to 50 μL of phenyl isothiocyanate and kept in a water bath at 60 °C for 1 h. After reaction, the final product was applied to a reversed-phase HPLC system, and the peaks were detected by a UV detector at 250 nm. The 7709
DOI: 10.1021/acs.jafc.9b02236 J. Agric. Food Chem. 2019, 67, 7706−7715
Article
Journal of Agricultural and Food Chemistry
Figure 1. Chemical structures of compounds 1−6 isolated from Maesa japonica.
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analytic condition of the HPLC was isocratic elution of 25% aqueous acetonitrile containing 0.1% formic acid for 40 min and subsequent washing of the column with 90% aqueous acetonitrile at a flow rate of 0.8 mL/min. The samples were compared to the standards monosaccharides D-galactose (tR 15.1 min), D-glucose (tR 16.5 min), D-glucuronic acid (tR 16.7 min), D-xylose (tR 18.7 min), and Lrhamnose (tR 27.2 min). Microdilution Broth Assay for In Vitro Antifungal Activity. To determine minimum inhibitory concentration (MIC) values, we used the broth microdilution assay using 2-fold serial dilutions.19 Briefly, each purified compound was serially diluted by starting with 250 μg/mL into the wells of 96-well microtiter plates that contain a spore suspension (1 × 105 spores/mL) of fungal pathogens. As a positive and negative control, we used the chemical fungicide flusilazole and PDB medium containing 5% methanol. The microtiter plates were incubated for 1−3 days, and the MIC values were determined by visual inspection of complete growth inhibition. The assay was performed two times with three replicates for each compound at all concentrations investigated. In Vivo Antifungal Assay against Rice Blast Fungus M. oryzae. To explore the effects of extracts and compounds on the control of plant disease, the solvent extracts (1000 and 3000 μg/mL) and pure compounds 1−6 (125, 250, and 500 μg/mL) were prepared by dissolving in a 5% aqueous methanol solution.20 As positive and negative controls, we used chemical fungicide blasticidin-S (1 and 50 μg/mL; Daeil Bio Co., Seoul, Korea) and 5% aqueous methanol. Tween 20 (0.025%) was used as a wetting agent. For the host plant, rice (Oryza sativa L, cv. Chucheong) was grown in a greenhouse at 25 ± 5 °C for 4 weeks. After treatment with the solvent extracts or pure compounds, the plants were inoculated with a spore suspension (5 × 106 spores/mL) of M. oryzae as previously described.20 The disease severity based on the lesion area on the leaves was evaluated on the fourth day after the inoculation, and the efficacy of disease control was calculated as described previously.20 All experiments were conducted twice with three replicates for each treatment.
RESULTS AND DISCUSSION In the search for new antifungal compounds from plant materials, we discovered that M. japonica methanol extract suppresses the development of rice blast disease caused by the fungus M. oryzae, suggesting that the extract contains antifungal compounds (Table S1). When the MeOH extracts were partitioned by n-BuOH, the resulting n-BuOH layer showed a strong in vivo antifungal activity against M. oryzae among the tested phytopathogenic fungi, but the aqueous layer had no antifungal activity (Table S1). Structural Elucidation of the Active Compounds Isolated from M. japonica. Considering the potent antifungal activity described above, the active compounds were isolated from the n-BuOH fraction by a series of chromatographic procedures to yield five new compounds (1− 5) and one known compound (6) (Figure 1). The known compound (6) was identified as a triterpenoid saponin maejaposide A for which the spectroscopic data of 6 were compared to those reported in the literature (Tables 1 and 2).15 The other five new compounds were identified as triterpenoid saponins with the same connectivity of a sugar chain in the C-3 position of the aglycons (Figure 1). All the compounds (1−6) commonly contained a 13,28-epoxy oleanane-type triterpenoid skeleton (C30) with a hydroxyl substitution at positions C-3, C-16, and C-22. The spectroscopic data of compounds 1−6 are presented in Tables 1 and 2 as well as in the Supporting Information. The HRESIMS spectrum of compound 1 showed a molecular ion at m/z 1333.6191 [M + Na]+, which is consistent with the molecular formula C65H98O27 (calculated m/z 1333.6193 for C65H98O27Na) (Figure S2). The 1H and 13 C NMR spectra showed that compound 1 is a triterpenoid glycoside with four sugar moieties (Tables 1 and 2; Figures S3 and S4). The 13C NMR and DEPT spectra of 1 showed 41 7710
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Figure 2. Key HMBC (blue arrow) and COSY (red bold line) correlations of compounds 1−5. 1
H−1H COSY, HMQC, and HMBC spectra of 1 revealed the presence of an acetyl and (E)-cinnamoyl moieties esterified to C-21 and C-16 hydroxyls of the oleanane skeleton, respectively (Figures 2, S5, S6, and S7). Accordingly, we concluded that the aglycon part of 1 is a 13β,28-epoxyolean-3β,16α,21β,22α,28αpentaol esterified by acetyl and (E)-cinnamoyl functional groups at C-21 and C-16 hydroxyls. For the sugar moieties of 1, the four anomeric proton resonances at δH 4.78, 5.79, 6.18, and 6.28 that appeared in the 1 H NMR spectrum with the corresponding anomeric carbon signals at δC 105.7, 103.9, 101.8, and 103.1 were assigned from the DEPT and HMQC, respectively (Table 2; Figures S7 and S8). With the results from the acid hydrolysates of 1, our
carbon signals, of which 30 were assigned for the aglycon moiety including seven quaternary sp3 (87.4, 54.7, 44.0, 43.1, 40.4, 37.7, and 37.3), eight methine sp3 (δC 97.6, 90.4, 82.4, 72.0, 69.8, 56.2, 50.8, and 46.0), eight methylene sp3 (δC 39.7, 39.0, 34.7, 33.7, 33.6, 27.2, 19.7, and 18.4), and seven methyls (δC 30.9, 28.5, 21.0, 20.3, 19.0, 17.1, and 16.9), and the remaining 11 signals were assigned to two acyl and four sugar moieties (Table 1). The HMBC correlations of the hemiacetal proton H-28 (δH 5.47) to C-13, C-16, C-17, and C-18 indicated the oxygen bridge between C-13 (δC 87.4) and C-28 (δC 97.6) (Figures 2 and S5). The large 3J coupling constant for H-21 and H-22 (10 Hz) represented the trans-axial arrangement for the two aliphatic protons.15 In addition, the 7711
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Journal of Agricultural and Food Chemistry results revealed that the sugar moieties consist of one β-Dglucose, two β-D-galactose, and one α-L-rhamnose (Figure S9). The intersugar linkages were established from the following HMBC correlations: H-1 (δH 6.28) of rhamnose with C-2 (δC 77.2) of galactose; H-1 (δH 6.18) of the galactose with C-3 (δC 84.2) of the glucose; and H-1 (δH 5.79) of the remaining galactose (terminal) with C-2 of glucose (δC 80.1) (Figures 2 and S5). With the cross peak in the HMBC spectrum between H-1 (δH 4.78) of glucose and C-3 (δC 90.4) of aglycon (Figures 2 and S5), we concluded that an [α-L-rhamnopyranosyl(1→2)-β-D-galactopyranosyl(1→3)]-[β-D-galactopyranosyl(1→2)]-β-D-glucopyranoside sugar chain is attached to the C-3 position of the aglycon part. Considering the identified structure of the sugar and aglycon moieties in this study, the structure of 1 was proposed as a new triterpenoid saponin, designated as maejaposide I. The HRESIMS spectrum of compound 2 showed a quasimolecular ion at m/z 1325.6503 [M + Na]+, which is consistent with the molecular formula C64H102O27 (calculated m/z 1325.6506 for C64H102O27Na) (Figure S10). The 1H and 13 C NMR spectra indicated that the structure of 2 has the same triterpenoid skeleton compared with 1, except for the acyl groups. Instead of the E-cinnamoyl and acetyl groups of 1, two angeloyl groups were observed in the 1H and 13C NMR and 1 H−1H COSY data of 2 (Tables 1 and 2; Figures S11−S13). In the HMBC spectrum, the correlations from H-21 (δH 6.82) and H-22 (δH 6.39) to C-1′ (δC 168.2) and C-1″ (δC 167.8) revealed the site of esterification with two angeloyl groups (Figures 2 and S14). The presence/absence of angeloyl and acetyl functional groups led to the downfield shifts of C-16 (from δC 69.8 to δC 73.8, compared to 1) and C-22 (from δC 72.0 to δC 69.3), respectively. Therefore, we concluded that the aglycon part of 2 is a 13β,28-epoxyolean3β,16α,21β,22α,28α-pentaol esterified by two angeloyl functional groups at C-16 and C-22 hydroxyls. The NMR spectroscopic data of 2 showed four anomeric proton signals at δH 4.78, 5.93, 6.19, and 6.28 with the corresponding anomeric carbon signals at δC 105.6, 103.0, 101.8, and 102.8, respectively (Table 2). The proton and carbon resonances for the sugar moiety of 2 were similar to those of 1, except for the anomeric proton and carbon signals at δH 5.93 and δC 103.0 for a glucopyranose (terminal) unit (Table 2). Based on the analysis of acid hydrolysis, the results showed that the sugar moiety of 2 consists of two β-D-glucose, one β-D-galactose, and one α-L-rhamnose unit (Figure S9). The intersugar linkages were established from the following HMBC correlations: H-1 (δH 6.28) of rhamnose with C-2 (δC 76.6) of galactose; H-1 (δH 6.19) of the galactose with C-3 (δC 84.0) of the glucose; and H-1 (δH 5.93) of the remaining glucose (terminal) with C-2 of glucose (δC 79.9) (Figures 2 and S14). Considering the cross peak in the HMBC spectrum between H-1 (δH 4.78) of the glucose and C-3 (δC 90.1) of the aglycon (Figures 2 and S14), our results suggest that an [α-Lrhamnopyranosyl(1→2)-β-D-galactopyranosyl(1→3)]-[β-Dglucopyranosyl(1→2)]-β-D-glucopyranoside sugar chain is attached to the C-3 position of the aglycon part (Figure S14). Based on these findings, compound 2 was identified as a new derivative of maejaposide C, designated as maejaposide C1. Compound 3 was obtained as an amorphous solid and has a molecular formula of C70H112O31, which was established by HRESIMS from a quasi-molecular ion at m/z 1471.7017 [M + Na]+ (calculated m/z 1471.7085 for C70H112O31Na) (Figure
S15). The 1H and 13C NMR spectra revealed that compound 3 has a 13,28-epoxy oleanane-type triterpenoid structure with two angeloyl groups, which was identical to the aglycon part of compound 2 (Table 1; Figures S16 and S17). Beyond the aglycon moiety, the NMR spectroscopic data for 3 showed five anomeric proton signals at δH 4.81, 5.74, 6.16, 6.21, and 6.01 with the corresponding anomeric carbon signals at δC 105.6, 103.8, 101.6, 101.9, and 104.1, respectively (Table 2). Furthermore, HPLC analysis of the acid hydrolysates of 3 supported that the sugar moiety of 3 has one more sugar unit (an α-L-rhamnose) compared to that of compound 2 in which the sugar moiety of 3 contains one β-D-glucose, two β-Dgalactose, and two α-L-rhamnose units (Figure S9). The intersugar linkages were established from the following HMBC correlations: H-1 (δH 6.01) of rhamnose (terminal) with C-2 (δC 78.6) of rhamnose; H-1 (δH 6.21) of rhamnose with C-2 (δC 77.2) of galactose; H-1 (δH 6.16) of galactose with C-3 (δC 84.1) of glucose; and H-1 (δH 5.74) of the remaining galactose (terminal) with C-2 of glucose (δC 80.3) (Figures 2 and S18). With the cross peak in the HMBC spectrum between H-1 (δH 4.81) of glucose and C-3 (δC 90.3) of aglycon (Figures 2 and S18), we concluded that a [α-L-rhamnopyranosyl(1→2)-α-Lrhamnopyranosyl(1→2)-β-D-galactopyranosyl(1→3)]-[β-Dgalactopyranosyl(1→2)]-β-D-glucopyranoside sugar chain is attached to the aglycon at C-3. Thus, our results suggest that compound 3 is a new derivative of maejaposide C, designated as maejaposide C-2. Compound 4 was obtained as an amorphous solid. The molecular formula C70H112O31 of 4 was determined by HRESIMS analysis from a quasi-molecular ion at m/z 1471.7042 [M + Na]+ (calculated m/z 1471.7085 for C70H112O31Na), which was identical to that of compound 3 (Figure S19). The 1D NMR spectroscopic data for 4 were highly similar to those for 3, except for the significant changes in anomeric proton and carbon signals at δH 5.87 and δC 103.0 for a glucopyranosyl unit (terminal) (Tables 1 and 2; Figures S20 and S21). Based on the HPLC analysis for the acid hydrolysates of 4, we found that compound 4 has a sugar chain consisting of two β-D-glucose, one β-D-galactose, and two α-Lrhamnose units (Figure S9). The intersugar linkages were established from the following HMBC correlations: H-1 (δH 5.91) of rhamnose (terminal) with C-2 (δC 79.2) of rhamnose; H-1 (δH 6.21) of rhamnose with C-2 (δC 76.8) of galactose; H1 (δH 6.19) of the galactose with C-3 (δC 83.8) of the glucose; and H-1 (δH 5.87) of the remaining glucose (terminal) with C2 of glucose (δC 80.1) (Figures 2 and S22). The correlation of H-1 (δH 4.78) of glucose to C-3 (δC 90.4) of aglycon indicated that an [α-L-rhamnopyranosyl(1→2)- α-L-rhamnopyranosyl(1→2)-β-D-galactopyranosyl(1→3)]-[β-D-galactopyranosyl(1→2)]-β-D-glucopyranoside sugar chain is attached to the C3 position of a 13,28-epoxy oleanane-type triterpenoid skeleton (Figures 2 and S22). Thus, we concluded that compound 4 is a new derivative of maejaposide C, named as maejaposide C-3. The HRESIMS spectrum of compound 5 showed a molecular ion at m/z 1255.6090 [M + Na]+, which is consistent with the molecular formula C60H96O26 (calculated m/z 1255.6088 for C60H96O26Na) (Figure S23). The 1H and 13 C NMR spectroscopic data indicated that compound 5 has a structure comprising 13β,28-epoxyolean-3β,16α,21β,22α,28αtetraol coupled with an (Z)-2-hexenoyl group and a four sugar chain at the C-22 and C-3 hydroxyls (Tables 1 and 2; Figures S24 and S25). The entire NMR spectra of 5 had a high similarity with those of maejaposide A (6), except for the 7712
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Journal of Agricultural and Food Chemistry Table 3. In vitro Antifungal Activity of Compounds 1−6 against Phytopathogenic Fungi MIC (μg/mL) Phytopathogenic fungi
1
2
3
4
5
6
Flua
Botrytis cinerea Colletotrichum coccodes Fusarium oxysporum Magnaporthe oryzae Phytophthora infestans
>250 >250 >250 >250 >250
>250 >250 >250 4 >250
>250 >250 >250 4 >250
>250 >250 >250 4 >250
>250 >250 >250 16 >250
>250 >250 >250 32 >250
1 16 16 1 125
Synthetic fungicide flusilazole was used as a positive control.
a
Figure 3. Effects of compounds 1−6 on the development of rice blast disease. (A) Control efficacy of compounds 1−6 isolated from Maesa japonica against rice blast disease. The bars represent the mean ± standard deviation of two runs with three replicates. PC = positive control; blasticidin-S (1 μg/mL, dotted bar; 50 μg/mL, lined bar) was used as a positive control. (B) Representatives of the plants treated with compounds 1−6. Plants were inoculated with spores of Magnaporthe oryzae 1 day after treatment with compounds 1−6. (a) Treatment with a chemical fungicide (blasticidin-S, 50 μg/mL) as a positive control. (b, c, and d) Treatment with 500, 250, 125 μg/mL of compounds 1−6, respectively. (e) Treatment with the 0.025% Tween 20 solution containing 5% methanol as a negative control. Right panel shows a magnified picture through a representative leaf.
absence of proton and carbon resonances (δH 5.08 and δC 108.0) for one xylopyranosyl unit (Table 2). The 1H and 13C NMR spectra showed four anomeric proton signals at δH 4.87, 5.79, 6.18, and 6.26 corresponding to carbon signals at δC 105.8, 104.0, 101.8, and 103.1, respectively. Although a signal for the −COOH group of the β-D-glucuronic acid moiety was missing in the 13C NMR spectrum of 5 (Table 2), its presence was confirmed by HRESIMS as well as by the results of the HPLC analysis for the sugar moiety of 5; thus, a polysaccharide chain of 5 has one β-D-glucuronic acid, two β-D-galactose, and one α-L-rhamnose (Figure S9). The linkage of a sugar chain was confirmed by the cross peaks between the anomeric proton and carbon signals in the HMBC spectrum of 5. H-1 (δH 6.26) of rhamnose was correlated with C-2 (δC 77.6) of galactose; H-1 (δH 6.18) of the galactose was correlated with C-3 (δC 83.3) of the glucuronic acid; and H-1 (δH 5.79) of the remaining galactose (terminal) was correlated with C-2 of glucuronic acid (δC 80.2) (Figures 2 and S26). The cross peak in the HMBC spectrum between H-1 (δH 4.87) of glucuronic acid and C-3 (δC 90.5) of aglycon was observed (Figures 2 and S26). Thus, we concluded that an [α-Lrhamnopyranosyl(1→2)-β-D-galactopyranosyl(1→3)]-[β-Dgalactopyranosyl(1→2)]-β-D-glucuropyranoside is attached to the aglycon of 5 at C-3. Compound 5 was identified as a new derivative of maejaposide A, which was designated as maejaposide A-1. In Vitro and In Vivo Antifungal Activity of the Identified Compounds against Phytopathogenic Fungi. Saponins are known to be involved in the permeability of the fungal cell membrane in which saponins form a complex with sterols in the fungal cell membrane, and thus, saponins exhibit antifungal activity.10,14 To investigate the antifungal activity of the compounds identified in this study, a spore
suspension of phytopathogenic fungi was treated with the pure compounds. Of the five fungi tested in this study, the rice blast fungus M. oryzae was the most sensitive to compounds 2−5, but compound 1 did not show any antifungal activity against M oryzae. The minimum inhibitory concentration (MIC) values for compounds 2, 3, 4, 5, and 6 were 4, 4, 4, 16, and 32 μg/mL against M. oryzae, respectively, which were comparable to that of positive control flusilazole (MIC = 1 μg/mL). However, the MIC of compound 1 was over 250 μg/mL. In contrast to M. oryzae, the other pathogens such as B. cinerea and P. infestans were not sensitive at all to the compounds at a concentration over 250 μg/mL (Table 3). The results that the identified compounds 2−6 inhibit the fungal growth of M. oryzae led us to investigate the disease control efficacy of the compounds against rice blast disease. To this end, rice were treated with each compound and then inoculated with the spore suspension of M. oryzae. At all concentrations, compounds 2−6 exhibited high disease control values ranging from 85% to 99%, which were comparable to that of positive control blasticidin-S; at a concentration level of 1 and 50 μg/mL, blasticidin-S exhibited disease control values of 80% and 100%, respectively (Figure 3). However, compound 1 did not suppress the development of rice blast, which was consistent with the in vitro antifungal assay results (Figure 3). Furthermore, beyond the disease control effects, we did not observe phytotoxic symptoms derived from the compounds (data not shown). Antifungal Activity According to the Structural Features. In terms of the antifungal activity according to the compound structure, it has been reported that the sugar moiety of triterpenoid saponins has an important role in the antifungal activity, and the absence of these saccharide moieties often leads to the impaired activities.10,13 Considering 7713
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Journal of Agricultural and Food Chemistry ORCID
the importance of the sugar chains for the biological activity of saponins, our observation that compounds 1−6 commonly had similar sugar chains at the C-3 position gave rise to the following question: why does compound 1 not exhibit antifungal activity? We cannot exclude the possibility that the esterification of the hydroxyl groups at the C-22 position of 13,28-epoxy oleanane saponins with acyl groups is necessary for the antifungal activity against M. oryzae because the C-22 position of compound 1 did not contain acyl groups (e.g., angeloyl or (Z)-2-hexenoyl) compared to that of compounds 2−6 (Figure 1). Similarly, Saha et al. showed that removal of the acyl groups at the C-28 position of a triterpene backbone, which was isolated from Diploknema butyracea, led to a reduction in the antifungal activity.21 In addition to the C-22 position, the presence of the cinnamoyl group at C-21 and the acetyl group at C-16 of compound 1 was different from the same position on the other compounds 2−6. These differences could lead to different interactions with membranes contributing to the various biological effects, supported by previous reports that reduction of antimicrobial activities by the acetyl group was shown in saponins identified from M. balansae and D. butyracea.21,22 Furthermore, we observed that compounds 2−4 had lower MIC values than those of compounds 5−6, suggesting the possibility that the presence of the angeloyl ester group at the C-21 position of compounds 2−4 may lead to increased antifungal activity (Figure 1). Taken together, our findings suggest that not only the complexity of the sugar moieties but also the functional groups at different positions in the aglycon part are important structural features affecting the antifungal activity. To clarify the structure−activity relationship, further investigation on a wider range of derivatives of compounds 1− 6 would be needed. In conclusion, we isolated and identified five new antifungal saponins, designated as maejaposides I, C-1, C-2, C-3, and A-1, along with a known analogue maejaposide A from the methanol extract of M. japonica. Of the six identified compounds, except for maejaposide I (1), the other maejaposides exhibited promising antifungal activity in vitro and in vivo against the rice blast fungus M. oryzae. Furthermore, maejaposides C-1, C-2, and C-3 (2−4) showed strong antifungal activity with a MIC of 4 μg/mL. Our results suggest that the extract of M. japonica as well as its purified saponin molecules could be used to control rice blast disease caused by M. oryzae.
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Hun Kim: 0000-0001-6727-4469 Author Contributions ∥
Funding
This study was financially supported by Golden Seed Project Vegetable Seed Center (213006-05-3-SB910) funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA) and Korea Forest Services (KFS), and also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2019R1A2C1008103). Notes
The authors declare no competing financial interest.
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b02236. Disease control efficacy of organic solvent extracts from M. japonica, isolation scheme of compounds 1−6, and NMR spectral data and MS spectrum of the compounds 1−6 (PDF)
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M.T.N. and J.W.H. contributed equally to this work.
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (H.K.). Phone: +82-42-860-7436. Fax: +82-42-860-4913. *E-mail:
[email protected] (G.J.C.). Phone: +82-42-860-7434. Fax: +82-42-860-4913. 7714
DOI: 10.1021/acs.jafc.9b02236 J. Agric. Food Chem. 2019, 67, 7706−7715
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DOI: 10.1021/acs.jafc.9b02236 J. Agric. Food Chem. 2019, 67, 7706−7715
