Constituents of the Edible Leaves of Melicope pteleifolia with Potential

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Constituents of the Edible Leaves of Melicope pteleifolia with Potential Analgesic Activity Ba-Wool Lee,† Jung-Geun Park,† Thi Kim Quy Ha,† Ha Thanh Tung Pham,† Jin-Pyo An,† Jung-Ran Noh,‡ Chul-Ho Lee,‡ and Won-Keun Oh*,† †

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Korea Bioactive Natural Material Bank, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea ‡ Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon 34141, Republic of Korea S Supporting Information *

ABSTRACT: Melicope pteleifolia has long been consumed as a popular vegetable and tea in Southeast Asian countries, including Malaysia and southern mainland China, and is effective in the treatment of colds and inflammation. In the search for active metabolites that can explain its traditional use as an antipyretic, six new phloroacetophenone derivatives (3−8) along with seven known compounds (1, 2, and 9−13) were isolated from the leaves of M. pteleifolia. Their chemical structures were confirmed by extensive spectroscopic analysis including NMR, IR, ECD, and HRMS. All compounds isolated from the leaves of M. pteleifolia (1−13) have a phloroacetophenone skeleton. Notably, the new compound 8 contains an additional cyclobutane moiety in its structure. The bioactivities of the isolated compounds were evaluated, and compounds 1, 6, and 7 inhibited tumor necrosis factor-α-induced prostaglandin E2. Moreover, the major constituent, 3,5-di-C-β-D-glucopyranosyl phloroacetophenone (1), was found to be responsible for the antipyretic activity of M. pteleifolia based on in vivo experiments.

T

the body temperature above the normal range, and it occurs mainly when the concentration of prostaglandin E2 (PGE2) increases.11 PGE2 is a key mediator of the elevation of body temperature as well as inflammation and pain, especially in response to infection,12 and it is synthesized by constitutive cyclooxygenase-1 (COX-1) and inducible cyclooxygenase-2 (COX-2).13 As COX-2 is responsible for the febrile temperature elevation as an immune response, this form has been the target of selective nonsteroidal anti-inflammatory drug (NSAID) development to reduce PGE 2 production.14 Although fever is a natural biological response to maintain homeostasis during nonspecific immune responses, antipyretics are commonly used to control fever due to the discomfort that occurs. As fever is regarded as an important symptom of various abnormal conditions, the need to develop antipyretics with reduced adverse effects is increasing.15 Most antipyretics, such as aspirin and other NSAIDs and paracetamol (acetaminophen), generally inhibit the enzyme cyclooxygenase and lower the concentration of PGE2 to suppress the body temperature as their mechanism of action.16 However, they have many side effects, including hepatotoxicity, gastrointestinal discomfort (e.g., dyspepsia), and complications (e.g., gastrointestinal bleeding). From this point of view, the current antipyretic medications are not suitable for treating

he genus Melicope comprises approximately 233 species distributed throughout Australia, the Pacific islands, and Asia, including Vietnam and mainland China.1 Melicope pteleifolia (Champ. ex Benth.) T.G. Hartley (Rutaceae), known also as Evodia lepta,2 is a popular vegetable and herbal remedy in southern China and Southeast Asia.3,4 M. pteleifolia is a deciduous shrub that grows up to 14 m in height and is commonly known as “tenggek burung”, “uam, sam ngam”, “sampang uam”, and “san-cha-ku” in Malaysia, Thailand, Indonesia, and China, respectively.2,5 As an edible plant resource, the young leaves of M. pteleifolia are consumed as a main ingredient in a traditional salad called “ulam”, which is rich in minerals and vitamins and is a popular appetizer in Malaysia. As a common food, the leaves of M. pteleifolia are readily available in night markets and farmer’s markets and in small restaurants in Malaysia.6,7 Similarly, the leaves of M. pteleifolia are the main raw materials for Guangdong herbal tea, which is well known in China, and the tender stems are also consumed as food.4,8 Interestingly, “999 Ganmaoling”, which comprises the leaves of M. pteleifolia, is currently used in the People’s Republic of China as a formula for treating colds.9 In Vietnam, the same plant is traditionally used as a medicine known as “ba chac” for the treatment of high fever and various inflammatory disorders.10 Fever is a complex but fundamental biological response caused by a variety of stimuli, including bacterial invasion, inflammation, and cancer. Fever is defined as an elevation of © XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 12, 2019

A

DOI: 10.1021/acs.jnatprod.9b00224 J. Nat. Prod. XXXX, XXX, XXX−XXX

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patients with certain diseases or for treating infants and young children. Therefore, screening and developing antipyretics from natural resources that are used as foodstuffs seems to be a useful alternative approach for this purpose. Previous studies on the biological activities of M. pteleifolia have shown that its extracts and isolated compounds exhibit anti-inflammatory effects, protect against high glucose-induced oxidative stress, serve as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activators, and have antiproliferative effects.17−19 In addition, benzopyrans,20,21 glycosidic compounds,8 furoquinoline alkaloids,19 and various acetophenone derivatives10,18 have been reported as chemical constituents. However, although the leaves of M. pteleifolia have been used to prepare various foods with antipyretic properties, there have been no reports of the bioactive compounds responsible for the traditional uses of this plant. The purpose of this study, therefore, was to identify the active components and their effects on the production of PGE2 in HaCaT cells and body temperature in ICR mice and ultimately to explain the antipyretic activity of this plant. Furthermore, to characterize the constituents responsible for the antipyretic activity of M. pteleifolia, isolation techniques involving highperformance liquid chromatography (HPLC) coupled with quadrupole-time-of-flight mass spectrometry (qTOF/MS) and NMR spectroscopy were used.

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RESULTS AND DISCUSSION In this study, relative mass defect (RMD) filtering was implemented to find derivatives of compound 1, which is an active antipyretic component. The term “mass defect” is

Table 1. 1H NMR Data of Compounds 3−8 (δ in ppm, J in Hz) position 8 (8V) 1′ (1VI) 2′ (2VI) 3′ (3VI) 4′ (4VI) 5′ (5VI) 6′ (6VI)

1″ 2″ 3″ 4″ 5″ 6″

(1VII) (2VII) (3VII) (4VII) (5VII) (6VII)

2‴ (2VIII) 3‴ (3VIII) 4‴ (4VIII) 5‴ (5VIII) 6‴ (6VIII) 7‴ (7VIII) 8‴ (8VIII) 2IV 3IV 4IV 5IV 6IV 7IV 8IV OCH3 OCH3′

3a

4a

5b

6b

7b

8a

2.56, s 4.73, d (9.7) 3.46, m 3.26, overlap 3.33, m 3.26, overlap 3.61, overlap

2.54, s 4.70, d (10.0) 3.46, t (8.7) 3.64, m 3.32, overlap 3.25, m 3.62, overlap 3.59, overlap 3.59, overlap 4.76, d (9.9) 3.66, overlap 3.30, overlap 3.42, m 3.24, overlap 4.49, overlap

2.56, s 4.97, d (9.8) 3.63, dd (9.4, 9.4) 3.52, dd (8.8, 8.8) 3.57, dd (8.9, 8.9) 3.67, ddd (9.6, 3.2, 3.2) 4.47, d (3.2)

2.57, s 4.97, d (9.8) 3.64, m 3.52, dd (8.7, 8.5) 3.57, m 3.68, m 4.48, overlap

2.57, s 4.97, d (9.8) 3.63, dd (9.5, 9.2) 3.52, dd (9.3, 9.0) 3.58, dd (9.5, 9.3) 3.68, m 4.51, dd (12.0, 2.4) 4.47, d (12.0, 4.0)

2.49, s 4.73, d (9.8) 3.43, dd (9.1, 9.1) 3.26, overlap 3.35, overlap 3.28, overlap 3.62, m

4.97, d (9.8) 3.63, dd (9.4, 9.4) 3.52, dd (8.8, 8.8) 3.57, dd (8.9, 8.9) 3.67, ddd (9.6, 3.2, 3.2) 4.47, d (3.2)

4.98, d (9.8) 3.64, m 3.53, dd (8.7, 8.5) 3.57, m 3.68, m 4.51, overlap

7.47, d (8.5) 6.80, d (8.5)

7.47, d (8.6) 6.81,d (8.6)

4.97, d (9.8) 3.63, dd (9.5, 9.3) 3.52, dd (9.3, 9.0) 3.58, dd (9.5, 9.3) 3.68, m 4.51, dd (12.0, 2.4) 4.47, d (12.0, 4.0) 7.20, br s

4.71 (9.8) 3.57, dd (9.4, 9.4) 3.26, overlap 3.31, overlap 3.52, m 4.40, d (11.2) 4.23, dd (12.0, 4.8) 7.05, d (8.6) 6.64, d (8.6)

6.80, d (8.5) 7.47,d (8.5) 7.65, d (16.0) 6.36, d (16.0) 7.47,d (8.5) 6.80, d (8.5)

6.81,d (8.6) 7.47,d (8.6) 7.64, d (16.0) 6.36, d (16.0) 7.19, d (2.0)

6.82, d (8.8) 7.08, br d (8.0) 7.65, d (16.0) 6.40, d (15.2) 7.20, br s

6.80, d (8.5) 7.47,d (8.5) 7.65, d (16.0) 6.36, d (16.0)

6.82, d (8.2) 7.08, dd (8.3, 1.9) 7.65, d (15.2) 6.40, d (16.0) 3.88, s

6.82, d (8.8) 7.08, br d (8.0) 7.65, d (16.0) 6.40, d (15.2) 3.88, s 3.88, s

4.70, d (9.7) 3.66, m 3.26, overlap 3.22, overlap 3.53, m 4.47, d (10.4) 4.20, dd (12.0, 6.4) 7.33, d (0.8)

6.78, d (8.5) 7.09, dd (8.0, 1.5) 7.55, d (15.0) 6.51, d (15.0)

7.97, d (8.4) 7.54, t (7.9) 7.67, t (7.3) 7.54, t (7.9) 7.97, d (8.4)

3.79, s

6.64, 7.05, 3.47, 3.23,

d d d d

(8.6) (8.6) (9.1) (9.1)

a

Data were measured in DMSO-d6 at 800 MHz. bData were measured in CD3OD at 800 MHz. B

DOI: 10.1021/acs.jnatprod.9b00224 J. Nat. Prod. XXXX, XXX, XXX−XXX

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13

C NMR Data of Compounds 3−8 (δ in ppm)

position IV

Article

1 (1 ) 2 (2IV) 3 (3IV) 4 (4IV) 5 (5IV) 6 (6IV) 7 (7IV) 8 (8IV) 1′ (1V) 2′ (2V) 3′ (3V) 4′ (4V) 5′ (5V) 6′ (6V) 1″ (1VI) 2″ (2VI) 3″ (3VI) 4″ (4VI) 5″ (5VI) 6″ (6VI) 1‴ (1VII) 2‴ (2VII) 3‴ (3VII) 4‴ (4VII) 5‴ (5VII) 6‴ (6VII) 7‴ (7VII) 8‴ (8VII) 9‴ (9VII) 1IV 2IV 3IV 4IV 5IV 6IV 7IV 8IV 9IV OCH3

3a 105.0, C 162.9, C 103.0, C 162.5, C 103.5, C 162.6, C 202.5, C 32.6, CH3 75.2, CH 72.9, CH 78.2, CH 69.0, CH 81.1, CH 59.9, CH2 74.7, CH 71.6, CH 78.2, CH 70.0, CH 78.4, CH 63.8, CH2 125.5, C 110.9, CH 148.0, C 149.4, C 115.3, CH 123.5, CH 145.3, CH 114.1, CH 166.7, C

4a 104.9, C 162.0, C 103.6, C 162.0, C 103.7, C 162.0, C 203.0, C 32.7, CH3 74.9, CH 72.5, CH 78.0, CH 69.1, CH 81.0, CH 59.9, CH2 74.9, CH 71.8, CH 77.8, CH 69.5, CH 77.9, CH 64.0, CH2 126.6, C 129.2, CH 128.8, CH 133.4, CH 128.8, CH 129.2, CH 165.7, C

5b 106.4, C 163.3, C 104.1, C 162.3, C 104.1, C 163.3, C 205.4, C 33.4, CH3 76.8, CH 74.1, CH 78.8, CH 71.2, CH 79.9, CH 63.9, CH2 76.8, CH 74.1, CH 78.8, CH 71.2, CH 79.9, CH 63.9, CH2 127.1, C 131.3, CH 116.9, CH 161.4, C 116.9, CH 131.3, CH 147.0, CH 114.7, CH 168.9, C 127.1, C 131.3, CH 116.9, CH 161.4, C 116.9, CH 131.3, CH 147.0, CH 114.7, CH 168.9, C

55.6

6b

7b

8a

106.4, C 163.1, C 104.1, C 162.9, C 104.1, C 163.3, C 205.3, C 33.4, CH3 76.8, CH 74.1, CH 78.8, CH 71.2, CH 79.9, CH 63.9, CH2 76.9, CH 74.1, CH 78.8, CH 71.3, CH 80.0, CH 64.0, CH2 127.1, C 131.3, CH 116.9, CH 161.4, C 116.9, CH 131.3, CH 147.0, CH 114.7, CH 168.9, C 127.7, C 111.6, CH 149.4, C 150.8, C 116.5, CH 124.5, CH 147.3, CH 115.0, CH 168.9, C 56.5

c

104.8, C 161.6, C 103.5, C 161.5, C 103.7, C 161.3, C 203.3, C 32.8, CH3 74.9, CH 72.4, CH 77.7, CH 69.0, CH 81.0, CH 59.8, CH2 75.0, CH 71.6, CH 77.7, CH 69.5, CH 78.3, CH 63.8, CH2 131.3, C 127.8, CH 115.3, CH 156.3, C 115.3, CH 127.8, CH 46.4, CH 44.7, CH 171.9, C

163.3, C 104.1, C 162.9, C 104.1, C 163.1, C 206.1, C 33.4, CH3 76.8, CH 74.1, CH 78.8, CH 71.2, CH 80.0, CH 64.3, CH2 76.8, CH 74.1, CH 78.8, CH 71.2, CH 80.0, CH 64.3, CH2 127.6, C 111.6, CH 149.4, C 150.8, C 116.5, CH 124.5, CH 147.3, CH 115.0, CH 168.9, C 127.6, C 111.6, CH 149.4, C 150.8, C 116.5, CH 124.5, CH 147.3, CH 115.0, CH 168.9, C 56.5 56.5

a

Data were measured in DMSO-d6 at 200 MHz. bData were measured in CD3OD at 200 MHz. cPeak too small to be observed.

produce new compounds (3−8), and their RMD values were in the determined range (Table S1 and Scheme S2, Supporting Information). Compound 3 was isolated as a brownish gum and found to have a molecular formula of C30H36O17 based on its highresolution electrospray ionization mass spectrometric (HRESIMS) ion peak at m/z 667.1866 [M − H]− (calcd for C30H35O17, 667.1880), corresponding to 13 degrees of unsaturation. The 1H NMR data (Table 1) showed the presence of one singlet methyl group (δH 2.56, s, H-8), two anomeric protons [δH 4.73 (d, J = 9.7, H-1′) and 4.70 (d, J = 9.7, H-1″)], two olefinic protons [δH 7.55 (d, J = 15.0, H-7‴) and 6.51 (d, J = 15.0, H-8‴)], three aromatic protons at δH 6.78−7.33 (H-2‴, 5‴, and 6‴), and sugar ring protons at δH 3.22−3.66 (m, H-2′−H-5″). The 13C NMR spectrum (Table 2) exhibited 30 resonances, including signals for 15 sp2 carbons in the δC 100−170 ppm range. Among them, nine carbons could be attributed to the presence of a trans-ferulic acid,

defined as the deviation of each atomic mass from the nominal mass, and the mass defects of analogous metabolites fall within a narrow range. Mass defect filtering (MDF) was originally introduced for the identification of drug metabolites from various matrices22 and has been applied to natural product chemistry to identify novel metabolites.23 RMD, which was derived from the concept of MDF, was designed to help mine novel metabolites without a parent structure at the initial stage.24 As the RMD is calculated in ppm as (mass defect/ measured monoisotopic mass) × 106, it reflects the fractional hydrogen content, and values of analogues in the same classes remain constant regardless of their monomer or polymer form. In the early isolation stage, the n-BuOH fraction was subjected to HRMS measurement and RMD analysis. The range of the RMD values of the target compounds was set at 270−330 based on previously reported data24 and the RMD value of the active constituent, compound 1 (RMD: 300.9). As a result, the constituents from n-BuOH were dereplicated effectively to C

DOI: 10.1021/acs.jnatprod.9b00224 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. (A) Key HMBC (arrows) and COSY (bold) correlations of 3 and 8. (B) ROESY correlations (dashed arrows) of compound 8a. (C) Experimental and calculated ECD spectra of 8a (MeOH).

which was supported by the UV absorption maximum at 325 nm and the large coupling constant between H-7‴ and H-8‴ (d, J = 15.0 Hz). This result was confirmed by the HMBC correlations between H-2‴/5‴/6‴ (δH 7.33/6.78/7.09) and

C-3‴/C-4‴ (δC 148.0/149.4). Moreover, the methoxy group at δC 55.6 was determined to be linked to C-3‴ based on their HMBC correlation (Figure 1A). The 13C NMR and HSQC spectra indicated that six carbons out of the 15 downfield D

DOI: 10.1021/acs.jnatprod.9b00224 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Compound 6 was isolated as a brown, amorphous solid, with the molecular formula C39H42O19, established from its HRESIMS, which showed a deprotonated peak at m/z 813.2243 [M − H]− (calcd for C39H41O19, 813.2248). The resonances in the 13C NMR spectrum of 6 were similar to those of 5. However, there were more peaks in similar chemical shift ranges, suggesting that 6 has an asymmetric structure, which can be attributed to the presence of a methoxy group on either side of the p-coumaric acid moieties. This was supported by the 30 amu difference in their molecular weights and the HMBC correlation between the OMe (δH 3.88, s) and the carbon at 149.4 (C-3IV) (Tables 1 and 2). The HMBC correlations were used to construct the fragment from H-6″ (δH 4.51, overlap) to C-9IV (δC 168.9) of this derivative and indicated that a trans-ferulic acid moiety is bound to H-6″, while the other side had a trans-p-coumaric acid substituent. Thus, compound 6 was established as 5-C-(6′-O-trans-pcoumaroyl)-β-D-glucopyranosyl-3-C-(6″-O-trans-feruloyl)-β-Dglucopyranoside phloroacetophenone. Compound 7 was isolated as a dark brown solid with the molecular formula C40H44O20 determined from its HRESIMS, which showed a deprotonated peak at m/z 783.2140 [M − H]− (calcd for C40H43O20, 783.2142). Similar to compound 5, the 24 resonances in its 13C NMR spectrum, the proton signals from the phenolic acid moiety integrating for multiples of two (Figure S25, Supporting Information), and the 30 amu difference in molecular weight compared to that of compound 6 suggested strongly that compound 7 also has a symmetrical structure with trans-ferulic acid moieties on both of the glucose fragments. Accordingly, upon an analysis of all of the data, compound 7 was deduced to be 5-C-(6′-O-trans-feruloyl)-β-Dglucopyranosyl-3-C-(6″-O-trans-feruloyl)-β-D-glucopyranoside phloroacetophenone. Compound 8 was isolated as a brown, amorphous solid, with [α]25 D 73.9 (c 0.1, MeOH). Its HRESIMS data showed a deprotonated peak at m/z 1275.3680 [M − H]− (calcd for C58H67O32, 1275.3621), suggesting a molecular formula of C58H68O32. The UV maximum absorptions at 230, 285, and 333 nm of 8 were similar to those of 1, implying that it is an analogue of 1. Interestingly, only one set of signals (Tables 1 and 2) was observed in the 1H and 13C NMR spectra of 8. Moreover, another deprotonated molecular ion at m/z 637.1753 [M − 2H]−2 was also observed in the HRESIMS, and this peak had the second highest abundance. The 1D NMR signals of compound 8 were very similar to those of 2 except for the linkage between the phloroacetophenone moiety and the p-hydroxyphenyl group.10 All of these data suggested that compound 8 is a dimer of phloroacetophenone Cglycoside with a unique symmetrical structure. The 1H and 13C NMR spectra of compound 8 did not show proton signals with a large coupling constant characteristic of an α,β-unsaturated ketone in the downfield region. Instead, only a pair of methine signals [δH 3.47 (1H, br d, J = 9.1) and 3.23 (1H, br d, J = 9.1)] with corresponding carbon resonances at δC 46.4 and 44.7 were observed. The COSY correlation (Figure 1A) showed that the two methine carbons are connected to each other. The HMBC correlations from the methine proton at δH 3.23 (H-8‴) to the carbon at δC 44.7 (C-8VII) and from the other methine proton at δH 3.47 (H-7‴) to the corresponding carbon at δC 46.4 (C-7VII) suggested the presence of a cyclobutane ring (Figure 1A). Compound 8 was thus considered to be a dimer of 2 produced via a [2 + 2]cycloaddition reaction in the plant.28 The HMBC correlations

carbons were part of a fully substituted benzene ring and that three of them were oxygenated aromatic carbons (δC 162.9, 162.6, 162.5, 105.0, 103.5, and 103.0). These signals and the appearance of a downfield singlet methyl group at δH 2.56 together with a keto carbonyl signal at δC 202.5 suggested the presence of a phloroacetophenone moiety. Twelve carbons in the 59−82 ppm range were attributed to two hexose sugars. These two sugars were determined to be C-glycosides and were found to be linked to C-3 and C-5 based on HMBC correlations from H-1″ (δH 4.70) and H-1′ (δH 4.73) to C-2/ 3/4 (δC 162.9, 103.0, and 162.5) and C-4/5/6 (δC 162.5, 103.5, and 162.6), respectively.25 The presence of a transferulic acid unit connected to C-6″ was determined based on the key HMBC correlations from H-6″ (δH 4.47, d, J = 10.4 Hz and 4.20, dd, J = 12.0, 6.4 Hz) to C-9‴ (δC 166.7) (Figure 1A). Moreover, all these observations were reasonably supported when comparing these spectroscopic data to previously reported data of 5-C-β-D-glucopyranosyl-3-C-(6″O-trans-p-coumaroyl)-β-D-glucopyranoside phloroacetophenone.10 Thus, compound 3 was determined as 5-C-β-Dglucopyranosyl-3-C-(6″-O-trans-feruloyl)-β-D-glucopyranoside phloroacetophenone. Compound 4 was isolated as a brownish gum, with the molecular formula C27H32O15 established from its HRESIMS data, which showed a deprotonated peak at m/z 595.1666 [M − H]− (calcd for C27H31O15, 595.1668). Compound 4 was found to be different from 3 in terms of the derivatized part. The absence of the α,β-unsaturated ketone and the five aromatic protons of the unsubstituted phenyl ring corresponding to H-2‴,6‴ (δH 7.97, d, J = 8.4 Hz), H-3‴,5‴ (δH 7.54, t, J = 7.9 Hz), and H-4‴ (δH 7.67, t, J = 7.3 Hz) suggested that the benzoyl group is bound to a di-C-β-D-glucosidic phloroacetophenone fragment. The HMBC correlation between H-6″ (δH 4.49, overlap) and C-7‴ (δC 165.7) confirmed the connectivity of the benzoyl moiety. Therefore, compound 4 was established as 5-C-β-D-glucopyranosyl-3-C-(6″-O-benzoyl)-β-D-glucopyranoside phloroacetophenone. Compound 5 was isolated as a brown, amorphous solid. The molecular formula, C38H40O18, was determined from its HRESIMS data, which showed a deprotonated molecular ion at m/z 783.2140 [M − H]− (calcd for C38H39O18, 783.2142). The presence of 23 resonances in the 13C NMR spectrum and the 1H NMR peaks integrating for multiples of two (Table 1) suggested that 5 has a highly symmetrical chemical structure. The large coupling constant between H-7‴ and H-8‴ (J = 16.0 Hz), the splitting pattern, and the integration of the aromatic protons as well as the HMBC correlations from H-2‴ (δH 7.47, d, J = 8.5 Hz) to C-4‴ (δC 161.4) and from H-3‴ (δH 6.80, d, J = 8.5 Hz) to C-1‴ (δC 127.1) suggested that two trans-pcoumaric acids are bound to a di-C-β-D-glucosidic phloroacetophenone core. The HMBC correlation from H-6′ (δH 4.47, d, J = 3.2 Hz) to C-9‴ (δC 168.9) proved that this fragment is linked to C-6′ of a glucose unit. Once compound 5 was purified, it was found to be very vulnerable to sunlight due to the photoisomerization of the cinnamic acid moiety.26 As the isolation procedure provided the trans form mixed with a small amount of the cis form, the mixture was treated with I227 to convert the cis form to the trans form (Figure S35, Supporting Information). Accordingly, compound 5 was assigned as 5-C-(6′-O-trans-p-coumaroyl)-β-D-glucopyranosyl3-C-(6″-O-trans-p-coumaroyl)-β-D-glucopyranoside phloroacetophenone. E

DOI: 10.1021/acs.jnatprod.9b00224 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 2. Effects of compounds isolated from M. pteleifolia on PGE2 production in HaCaT cells stimulated with TNF-α. After 2 h of pretreatment with compounds 1−13 (A) and compounds 1, 6, and 7 at various concentrations (B), the cells were then incubated with 20 ng/mL TNF-α for 24 h. PGE2 levels in the supernatants were measured using a PGE2 ELISA kit. The results are presented as the means ± SD (n = 3); *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the group that received only TNF-α treatment.

of H-7‴ with C-6‴/C-9‴/C-1VII and of H-8VII with C-9‴ also supported the above conclusions. Moreover, after the alkaline hydrolysis of 8 using KOH, 4,4′-dihydroxytruxinic acid (or 4,4′-dihydroxytruxillic acid), which has a cyclobutane unit as its core, was obtained, confirming these results (Figure 1B). However, whether the compound is a truxinyl-([7.7′,8.8′]lignan) or a truxillyl-([7.8′,8.7′]-lignan) derivative could not be determined from the 1H−1H COSY and HMBC data due to the symmetry of the cyclobutane derivative. The ESIMS ion at m/z 213.2 [C14H12O2 + H]+ corresponding to the 4,4′-

ethylenediphenol structure suggested that the cyclobutane ring is a truxinyl structure (8a, head to head) rather than a truxillic structure (head to tail).29 For the relative configuration of 8, several reports have suggested that perfect symmetry in the NMR spectra and a distinct splitting pattern of H-7‴ and H-8‴ (broad doublet) along with a typical coupling constant (J = 9.1 Hz) are characteristic of δ-truxinate-type dimers.30,31 The ROESY correlations of H-2 (2′)/H-8 (8′) and H-6 (6′)/H-7′ (7) in 8a also supported a δ-truxinate-type structure in 8 (Figure 1B). The absolute configuration of the cyclobutane F

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Figure 3. Antipyretic activity of 1 from M. pteleifolia on LPS-induced pyrexia in mice. Compound 1, the major constituent of fraction MP-001, was suspended in 0.5% carboxymethyl cellulose and administered orally 30 min before LPS injection. Treatment with acetaminophen (100 mg/kg, APAP) and dexamethasone (5 mg/kg), which are known antipyretic or anti-inflammatory agents, were used as positive controls. The Tb of each test animal was measured for 6 h after the LPS injection. The results were analyzed using Student’s t-test and compared to the LPS group data at each hour, and the threshold of significance was set at p < 0.05.

similar antipyretic effects at a lower concentration, and, notably, compound 1, the major constituent of M. pteleifolia, showed an efficacy similar to that of aspirin at the same concentration. These results also suggest some structure− activity relationship (SAR) information on the compounds isolated from M. pteleifolia. Among the monosubstituted 3,5-diC-β-D-glucopyranosyl phloroacetophenones such as 2−4, ferulic acid derivatization resulted in a slightly increased inhibitory activity compared to that of 1. Interestingly, the inhibitory activities on PGE2 production differed significantly among the disubstituted compounds. The ferulic acid moieties in 6 and 7 may have enhanced their abilities to reduce PGE2 levels, whereas the two p-coumaric acid moieties of compound 5 weakened its activity. Our results and previous studies on the abilities of cinnamic acid derivatives to inhibit COX-2 also implied that cinnamic acid fragments in the isolated compounds can influence their overall activities.32 Before the assessment of an active pure compound in a mouse model, the activity of the fraction in which the active component was predominantly contained was evaluated in ICR mice. As shown in Table S2 (Supporting Information), the injection of lipopolysaccharide (LPS) caused a fever that reached a peak of 0.6 ± 0.05 °C at 6 h. The values of body temperature (Tb) in the LPS group were significantly higher than those in the normal group (phosphate-buffered saline (PBS)-treated) 3 h after treatment. Treatment with acetaminophen (APAP), a well-known antipyretic drug, had a powerful antipyretic effect. Among the samples tested, MP-001 at a dose of 300 mg/kg markedly suppressed the LPS-induced febrile response for up to 6 h after treatment. These findings suggest that the major constituent of MP-001 would also attenuate the LPS-induced febrile response. Although APAP is one of the most commonly used analgesic and antipyretic drugs and represents a multibillion-dollar product including Tylenol, ironically, its safety has been questioned and is highly controversial. It has been reported that 46% of all acute liver failure in the U.S. and 40% to 70% of all cases in the UK and Europe can be attributed to dose-related APAP toxicity.33,34 As the safety window is relatively narrow, it is easy to overdose on APAP, and this leads to hepatocellular necrosis via metabolic pathways in which the drug is metabolized into a highly

ring in 8 was further determined by an ECD analysis of (7S,8R,7′S,8′R)-truxinic acid (δ-truxinic acid) and (7R,8S,7′R,8′S)-truxinic acid ((−)-δ-truxinic acid). After a conformational search using a molecular mechanics force field (MMFF), 10 major conformers of (7S,8R,7′S,8′R)-truxinic acid were selected for density functional theory (DFT) geometry optimizations at the B3LYP/def-SV(P) level (Figure S37, Supporting Information). ECD calculations were conducted for all optimized conformers with time-dependent DFT (TDDFT) at the B3LYP/def-SV(P) level. The Boltzmann-averaged calculated spectrum of 7S,8R,7′S,8′R-8a was generated (Table S3, Supporting Information), and it was in good agreement with the experimental ECD spectrum of 8a (Figure 1C). Therefore, compound 8 was elucidated as a δtruxinate-type dimer of 2 and accordingly named bimelicoside A. The structures of the known compounds 1, 2, and 9−13 were identified by means of spectroscopic analysis (ESIMS, 1D and 2D NMR) and by comparison of their NMR and MS data with previously reported data of the following compounds: 3,5di-C-β-D-glucopyranosyl phloroacetophenone (1), 5-C-β-Dglucopyranosyl-3-C-(6″-O-trans-p-coumaroyl)-β-D-glucopyranoside phloroacetophenone (2), and melicospiroketals A−E (9−13).10 To verify the efficacy of the isolated compounds as antipyretic agents, compounds 1−13 were assayed for their ability to reduce the level of PGE2 production when HaCaT cells were treated with tumor necrosis factor-α (TNF-α), an inflammation mediator. In a cell viability test, the test compounds were not found to be cytotoxic, except for 6 and 7 at a concentration of 100 μM (Figure S38A, Supporting Information). Compounds 6 and 7 were used for this experiment because they did not show any cytotoxicity at 50 μM in an additional cell viability assay (Figure S38B, Supporting Information). As shown in Figure 2A, most of the compounds tested reduced PGE2 production, and compounds 6 and 7 exhibited potent activity at 50 μM. Therefore, 6, 7, and the major constituent, 1, were subjected to a PGE2 assay at various concentrations and showed dosedependent inhibitory activities (Figure 2B). Compared to the positive control (100 μM aspirin), compounds 6 and 7 exerted G

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flow rate of 2 mL/min with UV detection at 205 and 254 nm and an Optima Pak C18 column (10 × 250 mm, 5 μm particle size; RS Tech, Seoul, Korea) or a YMC-Triart phenyl column (10 × 250 mm, 5 μm particle size; YMC Co., Ltd., Kyoto, Japan). Fluorescence measurements were recorded with a fluorescence microplate reader (Spectra Max Gemini XPS, Molecular Devices, San Jose, CA, USA). Biological data were obtained using a microplate reader (VersaMax, Molecular Devices, San Jose, CA, USA). Regular column chromatography (CC) was carried out with silica gel (particle size: 63−200 μm, Zeochem AG, Rüti, Switzerland), RP-C18 (particle size: 75 μm, Nacalai Tesque, Kyoto, Japan), and Sephadex LH-20 (GE Healthcare, Little Chalfont, UK). Silica gel 60 F254 and RP-18 F254S TLC plates were obtained from Merck (Darmstadt, Germany). Industrial-grade ethanol, ethyl acetate, and n-butanol were used for extraction and purification. Analytical-grade acetonitrile and methanol were used for isolation and analysis. All solvents were purchased from Daejung Chemical (Siheung, Korea). Plant Material. The leaves of Melicope pteleifolia were purchased from Ba Vi district, Hanoi, Vietnam, in June 2016. The samples were identified based on morphological characteristics by Ha Thanh Tung Pham of Seoul National University. A voucher specimen (SNU-122016) was deposited in the herbarium of the College of Pharmacy at Seoul National University. Extraction and Isolation. The leaves of M. pteleifolia (2 kg) were extracted with 10% EtOH (3 × 10 L, for 6 h each) at 60 °C. The combined extract was concentrated by an evaporator to yield a dry residue (364.6 g). The crude extract was suspended in H2O and then sequentially partitioned with EtOAc and n-BuOH (3 times each). The n-BuOH fraction (7.0 g) was subjected to Sephadex LH-20 open CC with a gradient solvent system from 30% to 100% MeOH, and 12 subfractions (F.1 − F.12) were obtained. The n-BuOH fraction was further analyzed with HRESIMS for dereplication. F.2 (2.1 g) was subjected to semipreparative HPLC with CH3CN/H2O (v/v, 10:90) containing 0.1% formic acid and afforded compounds 9 (0.5 mg, tR = 25.1 min), 10 (0.4 mg, tR = 17.1 min), 11 (0.2 mg, tR = 21.6 min), 12 (0.3 mg, tR = 25.9 min), and 13 (0.2 mg, tR = 20.5 min). F.3 (1.5 g) was subjected to preparative HPLC (MeOH/H2O gradient 15% → 30%) to afford 1 (135.4 mg, tR = 33.5 min). Compound 8 (8.5 mg, tR = 42.3 min) was obtained from subfraction F.8 (0.6 g) by semipreparative HPLC using a phenyl column with CH3CN/H2O (v/v, 21/79) containing 0.1% formic acid. F.10 (0.8 g) was subjected to semipreparative HPLC using a phenyl column with CH3CN/H2O (v/v, 26/74) containing 0.1% formic acid to obtain compounds 2 (1.5 mg, tR = 19.3 min) and 3 (6.5 mg, tR = 20.4 min). Compound 4 (4.0 mg, tR = 51.5 min) was isolated from subfraction F.11 (0.5 g) by semipreparative HPLC using a phenyl column with CH3CN/H2O containing 0.1% formic acid (v/v, 24/76). F.13 (0.7 g) was subjected to semipreparative HPLC using a phenyl column with CH3CN/H2O containing 0.1% formic acid (v/v, 31:69) and afforded compounds 5 (1.2 mg, tR = 41.4 min), 6 (1.4 mg, tR = 44.2 min), and 7 (0.7 mg, tR = 45.8 min). 5-C-β-D-Glucopyranosyl-3-C-(6″-O-trans-feruloyl)-β-D-glucopyranoside phloroacetophenone (3): brownish gum; [α]25 D 28.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 233 (4.4), 287 (4.3), 327 (4.2) nm; IR νmax 3359, 2893, 1689, 1516, 677 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 667.1866 [M − H]− (calcd for C30H35O17, 667.1880). 5-C-β-D-Glucopyranosyl-3-C-(6″-O-benzoyl)-β-D-glucopyranoside phloroacetophenone (4): brownish gum; [α]25 D 35.4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (4.4), 285 (4.1) nm; IR νmax 3343, 2892, 1720, 1621, 1365, 1276, 1085 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 595.1666 [M − H]− (calcd for C27H31O15, 595.1668). 5-C-(6′-O-trans-p-Coumaroyl)-β-D-glucopyranosyl-3-C-(6″-Otrans-p-coumaroyl)-β-D-glucopyranoside phloroacetophenone (5): brown, amorphous solid; [α]25 D −3.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (4.5), 291 (4.5), 315 (4.4) nm; IR νmax 3567, 2892, 1689, 1501, 677 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 783.2140 [M − H]− (calcd for C38H39O18, 783.2142).

reactive and toxic intermediate, N-acetyl-p-benzoquinone imine (NAPQI). Once glutathione, which can detoxify NAPQI, is depleted, NAPQI can exert its toxicity.35 On the other hand, MP-001 was the fraction of M. pteleifolia that contained compound 1, and this compound accounted for more than 13.3%36 and 25% of the water extract (data not shown) and MP-001, respectively, and is readily obtained via HP-20 column chromatography. As MP-001 can be prepared industrially in a few simple steps and has advantages over APAP because it originates from a natural edible vegetable and thus may be relatively safe, M. pteleifolia may have great potential in the pharmaceutical industry. Therefore, it is likely that compound 1 is responsible for the antipyretic activity of MP-001, and so this compound was subjected to in vivo experiments to investigate this hypothesis. Mice were pretreated with isolated compound 1, which comprised more than 25% of active fraction MP-001, at various doses before LPS injection. As shown in Figure 3, compound 1 at a dose of 10 mg/kg markedly suppressed the LPS-induced febrile response for up to 6 h after treatment. These findings demonstrated that pretreatment with 1 attenuated the increase in Tb during the LPS-induced febrile response and that 1 is the active constituent in M. pteleifolia and plays a key role in reducing Tb. Chemically, 1 is hydrophilic and is easily extracted by even water extraction; thus, the traditional use of M. pteleifolia among the Malay, Chinese, and Vietnamese populations is reasonable and was justified by the above results. This antipyretic activity of 1 seems to be related to its structural similarity to the well-known antipyretic aspirin. Aspirin is derived from salicin, which is the active antiinflammatory constituent of willow (Salix) bark and is commonly used for treating various conditions, including inflammation, fever, heart attack, stroke, and cancer. Both aspirin and compound 1 possess benzene rings substituted with carbonyl groups at C-1 and by hydroxy groups at C-2. However, unlike aspirin, which has a carboxylic acid functionality at C-1 and an acetoxy group at C-2, compound 1 has an acetyl group at C-1 and three hydroxy groups at C-2, C-4, and C-6 and is C-glycosylated at C-3 and C-5. Aspirin acetylates COX enzymes and disables them by forming hydrogen bonds with the hydroxy groups of Ser-530 and Ser-516 of COX-1 and -2, respectively.37 As another NSAID, ibuprofen exerts its activity by forming a salt bridge between the carboxyl group of ibuprofen and the Arg-120 of COX-2, and the carboxylate group of naproxen contributes to its ability to inhibit COX-2 by forming a hydrogen bond. 38,39 Considering its structural similarities with other NSAIDs and their investigated mechanisms, it is likely that the carboxylic acid group at C-1 and the hydroxy groups of compound 1 play important roles in mediating its ability to inhibit COX enzymes and concomitantly reduce PGE2 levels.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a JASCO P-2000 polarimeter (JASCO International Co. Ltd., Tokyo, Japan). Electronic circular dichroism (ECD) spectra were measured using a Chirascan-Plus (Applied Photophysics Ltd., Surrey, UK). IR data were obtained using a Nicolet 6700 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). 1D and 2D NMR spectra were recorded in deuterated solvents using an AVANCE 800 MHz spectrometer (Bruker, Billerica, MA, USA). HRESIMS values were obtained using an Agilent Technologies 6530 qTOF MS spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA). A Gilson HPLC purification system was used for isolation at a H

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5-C-(6′-O-trans-p-Coumaroyl)-β-D-glucopyranosyl-3-C-(6″-Otrans-feruloyl)-β-D-glucopyranoside phloroacetophenone (6): brown, amorphous solid; [α]D25 −23.2 (c 0.35, MeOH); UV (MeOH) λmax (log ε) 231 (4.0), 292 (3.9), 317 (3.9) nm; IR νmax 3338, 2888, 1705, 1623,, 1447, 1271, 1169 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 813.2243 [M − H]− (calcd for C39H41O19, 813.2248). 5-C-(6′-O-trans-Feruloyl)-β-D-glucopyranosyl-3-C-(6″-O-transferuloyl)-β-D-glucopyranoside phloroacetophenone (7): dark brown solid; [α]25 D −27.0 (c 0.27, MeOH); UV (MeOH) λmax (log ε) 234 (3.7), 292 (3.6), 325 (3.7) nm; IR νmax 3364, 2927, 1745, 1602, 1363, 1277, 1175 cm−1 ; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 843.2349 [M − H]− (calcd for C40H43O20, 843.2353). Bimelicoside A (8): brown, amorphous solid; [α]25 D 73.9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 231 (4.7), 286 (4.5) nm; IR νmax 3335, 2888, 1748, 1621, 1517, 677 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 1275.3680 [M − H]− (calcd for C58H67O32, 1275.3621). HPLC-qTOF-MS Conditions for Dereplication Using RMD. The n-BuOH fraction was analyzed using an HPLC system coupled with a qTOF mass spectrometer (Agilent 6530 qTOF LC/MS, Agilent Technologies Co., Ltd., Santa Clara, CA, USA), and the system was equipped with a G1312C binary pump, a G1322A degasser, a G1316 thermostatically controlled column oven, and a G1329B autosampler. Liquid chromatographic analyses were carried out on a C18 reversed-phase LC column (YMC-Triart C18, 5 μm particles, 3.0 mm × 150 mm). The mobile phase consisted of H2O with 0.1% formic acid (A) and CH3CN with 0.1% formic acid (B), and a gradient elution of 0−3 min (10% B), 3−15 min (10−20% B), 15−40 min (20−60% B), 40−41 min (60−100% B), 41−51 min (100% B), and 51−55 min (10% B) at a flow rate of 0.3 mL/min was used. The n-BuOH fraction of the 10% EtOH extract of M. pteleifolia was analyzed in the negative ionization mode with a 6530 qTOF mass spectrometer equipped with an ESI interface. The conditions of the ESI source were a gas temperature of 350 °C, a drying gas flow rate of 10 L/min, a fragmentor voltage of 180 V, a nebulizer pressure of 30 psi, a skimmer of 60 V, a sheath gas temperature of 350 °C, a sheath gas flow rate of 12 L/min, an OCT IRF Vpp voltage of 750 V, and a Vcap voltage of 4000 V. The raw data were processed with MassHunter software (developed by Agilent Technologies). Acid Hydrolysis of 8. Compound 8 (8.0 mg, 0.006 mmol) was dissolved in 0.3 mL of a 1% KOH aqueous solution and stirred at room temperature for 2 h. The reaction mixture was then neutralized with aqueous 2 N HCl and dried under a stream of N2. The dried mixture was extracted with EtOAc (3 times), and the dried EtOAcsoluble fraction was subjected to semipreparative HPLC with CH3CN/H2O containing 0.1% formic acid (v/v, 21/79) to afford 8a (0.3 mg) in high purity. Compound 1 was also detected in the H2O fraction by LC/MS analysis as a product of the alkaline hydrolysis of compound 8. Computational ECD Analysis. Conformational analyses for (7S,8R,7′S,8′R)- and (7R,8S,7′R,8′S)-truxinic acid were initially carried out using CONFLEX software (CONFLEX Corporation, Kyoto, Japan) in MMFF to obtain populated conformers. Geometry optimization and TDDFT calculations were implemented using TURBOMOLE 7.2 (COSMOlogic GmbH & Co. KG, Leverkusen, Germany) with the combination of B3LYP/def-SV(P) (functional/ basis set) for 10 major conformers from each isomer. Then, the Boltzmann-averaged spectra of each isomer were generated using a Gaussian band shape with a 0.10 eV exponential half-width (shift = ±0 nm). Cell Culture and Cytotoxicity Assay. Spontaneously immortalized human keratinocytes (HaCaT cell line) were maintained in Roswell Park Memorial Institute 1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (HyClone), penicillin (100 U/mL), and streptomycin (100 μg/mL) (Gibco, Grand Island, NY, USA) at 37 °C under a 5% CO2 atmosphere. A 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to measure the cell viability. HaCaT cells were seeded into 96-well culture plates at a density of 104 cells/well.

After incubation for 12 h, the cells were exposed to the test compounds dissolved in serum-free medium. The cells were incubated for an additional 24 h, 20 μL of MTT solution (Sigma, St. Louis, MO, USA) was added to each well, and the cells were incubated for a further 4 h. The medium was removed, and the formazan product was thoroughly dissolved in dimethyl sulfoxide (100 μL). The absorbance was measured at 550 nm using a microplate reader (VersaMax). Each sample was evaluated in triplicate, and the experiments were repeated at least three times. TNF-α Stimulation in a Skin Cell Model. HaCaT cells were seeded into 24-well plates at a density of 1.5 × 105 cells/well. After overnight incubation in growth medium, the cells were cultivated in serum-free medium for 12 h. The cells were then pretreated with the test compounds for 2 h, after which they were stimulated with 20 ng/ mL of TNF-α (ProSpec, Rehovot, Israel). Negative control cells were not treated with TNF-α. After 24 h of incubation, the supernatant from each was collected and stored at −80 °C. The level of PGE2 in the cell culture supernatants was measured using an enzyme-linked immunosorbent assay (ELISA) kit (PGE2 ELISA kit, R&D Systems, Minneapolis, MN, USA). The absorbance was recorded at 450 nm using a microplate reader (VersaMax) according to the manufacturer’s protocol. Body Temperature Measurement in an Animal Model. Male 8-week-old ICR mice (Koatech, Pyeongtaek, Korea) were used in this study, and each group included 5 mice. They were housed in plastic cages in a temperature-controlled room (22 ± 1 °C) and maintained in a reverse 12 h light/dark cycle with free access to food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (accepted no. KRIBB-AEC-16099) and were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health. A lipopolysaccharide (Sigma)-induced fever test was performed during the light phase of the circadian cycle.40 LPS (0.5 mg/kg) was dissolved in pyrogen-free PBS and then injected intraperitoneally to induce fever. The body temperature (Tb) was measured by gently inserting a small thermoprobe into the rectum using a TH-5 Thermalert Monitoring thermometer (Physitemp Instruments, Clifton, NJ, USA). On the day of the experiment, the baseline Tb was determined from six measurements taken over 2 h at 30 min intervals. The Tb values of the tested animals ranged from 36.6 to 37.5 °C. To determine the antipyretic effect, the 30% EtOH eluate (named MP-001) from the passage of the water extract through a Diaion HP-20 column or compound 1, which was the main constituent of M. pteleifolia, was suspended in 0.5% carboxymethyl cellulose. Acetaminophen (100 mg/kg, APAP; Sigma) and dexamethasone (5 mg/kg, Sigma), known antipyretics or anti-inflammatory agents, were used as positive controls. All test compounds were orally administered 30 min before the LPS injection, and the Tb of the test animal was measured up to 6 h after the LPS injection. The results were analyzed by the Student t-test and compared to the LPS group data for each hour, and the threshold of significance was set at p < 0.05. Statistical Analysis. Data were calculated as the means ± standard deviations (SD) of three independent experiments. The differences between the means of two groups were determined by the one-way analysis of variance (ANOVA) followed by Tukey’s or Duncan’s post hoc test (SPSS Statistics 23, Chicago, IL, USA). Statistical significance was accepted at *p < 0.05, **p < 0.01, and ***p < 0.001.

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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.jnatprod.9b00224. Additional information (PDF) I

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AUTHOR INFORMATION

Corresponding Author

*Tel and Fax: +82-02-880-7872. E-mail: [email protected] (W.-K. Oh). ORCID

Won-Keun Oh: 0000-0003-0761-3064 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was financially supported, in part, by grants from the KBNMB (NRF-2017M3A9B8069409) and from the Basic Science Research Program (NRF-2017R1E1A1A01074674) through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Planning.

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DOI: 10.1021/acs.jnatprod.9b00224 J. Nat. Prod. XXXX, XXX, XXX−XXX