Targeted Isolation of Neuroprotective Dicoumaroyl Neolignans and

Aug 14, 2018 - The integration of LC–MS/MS molecular networking and in silico MS/MS fragmentation is an emerging method for dereplication of natural...
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Article Cite This: J. Nat. Prod. 2018, 81, 1819−1828

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Targeted Isolation of Neuroprotective Dicoumaroyl Neolignans and Lignans from Sageretia theezans Using in Silico Molecular Network Annotation Propagation-Based Dereplication Kyo Bin Kang,*,†,‡,§,¶ Eun Jin Park,†,¶ Ricardo R. da Silva,‡ Hyun Woo Kim,† Pieter C. Dorrestein,*,‡ and Sang Hyun Sung†,# †

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826, Republic of Korea Collaborative Mass Spectrometry Innovation Center, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093, United States

J. Nat. Prod. 2018.81:1819-1828. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 08/24/18. For personal use only.

‡

S Supporting Information *

ABSTRACT: The integration of LC−MS/MS molecular networking and in silico MS/MS fragmentation is an emerging method for dereplication of natural products. In the present study, a targeted isolation of natural products using a new in silicobased annotation tool named Network Annotation Propagation (NAP) is described. NAP improves accuracy of in silico fragmentation analyses by reranking candidate structures based on the network topology from MS/MS-based molecular networking. Annotation for the MS/MS spectral network of the Sageratia theezans twig extract was performed using NAP, and most molecular families within the network, including the known triterpenoids 1−7, could be putatively annotated, without relying on any previous reports of molecules from this species. Based on the in silico dereplication results, molecules were prioritized for isolation. In total, six dicoumaroyl 8-O-4′ neolignans (8−13) and three dicoumaroyl lignans (14−16) were isolated from the twigs of S. theezans and structurally characterized by spectroscopic analyses. Isolates were evaluated for their neuroprotective activity, and compounds 14−16 showed potent protective effects against glutamate-induced oxidative stress in mouse HT22 cells at a concentration of 12.5 μM. Web-based platform.10 The fundamental concept of molecular networking is through establishing a chemical similarity network between metabolites based on their MS/MS spectral similarity.11,12 Molecular networking categorizes secondary metabolites into clusters of similar scaffolds. This technique has been used to prioritize the isolation targets in natural product research.13−20 Molecular networking on GNPS also enables dereplication by matching MS/MS data to spectral libraries; however, dereplication annotates only a limited number of nodes, due to limited coverage of known chemical space by available MS/MS fragmentation libraries, even though the GNPS community has already contributed more than 70 000 annotated MS/MS spectra, a number that is growing rapidly.21 There are some alternative solutions. Some researchers co-injected standards of analogues22 and then used the molecular network to propagate the annotation manually, or they used in-house LC−MS/MS libraries17 to secure “seed nodes” for network annotation. These approaches can significantly improve the dereplication efficiency of the

Sageretia theezans, commonly known as a Chinese sweet plum, is an evergreen shrub of the family Rhamnaceae, which is native to the southern seashores of China. S. theezans is extensively grown in China and Japan as bonsai, an ornamental dwarf tree in a pot as a form of art. Although bonsai is currently the major commercial value of S. theezans, historically, this species was used for treating colds, fevers, and hepatitis in Korean and Chinese folk medicines.1,2 There are some previous reports on medicinal activities of S. theezans extracts, such as reactive oxygen species (ROS) scavenging, increase of low-density lipoprotein (LDL) resistance to oxidation,2 HIV type 1 protease inhibition,3 and α-glucosidase inhibition.4 However, little is known about the chemical constituents of S. theezans; only a few flavonoids, phenolic acids, and phytosterols were reported.5,6 Dereplication, the identification of known compounds, is now an essential process for accelerating natural products drug discovery.7,8 In the past few years, MS/MS molecular networking has emerged as a promising technique for analyzing LC-based mass spectrometry and metabolomics data sets,9 in part, because of its accessibility through the Global Natural Product Social (GNPS; https://gnps.ucsd.edu) © 2018 American Chemical Society and American Society of Pharmacognosy

Received: April 10, 2018 Published: August 14, 2018 1819

DOI: 10.1021/acs.jnatprod.8b00292 J. Nat. Prod. 2018, 81, 1819−1828

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molecular network; however, they required previous knowledge or the reference molecules to be available or have data available from related taxon or chemical classes and therefore cannot be used for dereplication of mixtures of uncharacterized chemicals. Recently, in silico MS/MS fragmentation approaches have been suggested as a possible solution for overcoming such limits of an experimental MS/MS data database.23 Wolfender and co-workers suggested an integrated approach of in silico database and MS/MS molecular networking24 and demonstrated its efficiency for dereplication of natural products.22,25 The GNPS Web platform recently introduced an online molecular network analysis tool named Network Annotation Propagation (NAP), in which MetFrag in silico fragmentation prediction of MS/MS spectra26 is combined with a network topological consensus and reranking of in silico annotations.27 NAP uses the in silico annotation tool MetFrag to search spectra that comprised the MS/MS molecular networks against structural libraries of natural product structure collections such as SUPER NATURAL II28 and DNP (Dictionary of Natural Products) and ranks n best candidates for each node (i.e., within acceptable scores from matching, it is possible to have more than one candidate match). Then, NAP reranks these candidates based on the propagation of the structural similarity of neighboring nodes. When a reference MS/MS spectrum is available within the molecular family (subcluster of nodes), the structure can be propagated directly; however, NAP can work even when there are no experimental library matches. In such cases, a network consensus scoring algorithm can be used to account for structural similarity between in silico candidates. NAP often discovers the molecular family and occasionally differentiates among possible isomers or related structural classes. NAP is therefore used to prioritize molecular families of interest (Figure 1).27 In this study, as a part of a continuing phytochemical investigation on Rhamnaceae plants,29−34 bioactive constituents in the twigs of S. theezans were investigated. For efficient dereplication of known compounds, an LC−MS/MS molecular networking approach was applied and the molecular network was further annotated using NAP. This enabled rapid identification of known compounds/molecular families that have reference MS/MS spectra. It subsequently enabled the prioritization of molecules that do not have MS/MS reference spectra in the public domain but their structures or related structures are present in structural databases.

■

Figure 1. Dereplication and chemical prioritization workflow using MS/MS molecular networking and network annotation propagation (NAP).

RESULTS AND DISCUSSION The MeOH extract of the twigs of S. theezans was analyzed by UHPLC−Q/TOF−MS/MS, and the data were processed into a molecular network using MZmine and the GNPS platform (http://gnps.ucsd.edu).10,35 After removal of nonclustered nodes, 56 spectral nodes were interconnected with 129 edges, forming eight molecular families36 (Figure 2). Among these nodes, only one node in the cluster C was annotated by GNPS MS/MS spectral library matching. The node annotated by library searching was annotated as ceanothic acid (1). The precursor ion for this node had an m/z value of 531.3325 (calcd for C31H47O7, 531.3327), while compound 1 had a molecular formula of C30H46O5; so this node was suggested to be a formate adduct ([M + HCOOH − H]−) of 1, and it was confirmed by comparison of retention time and MS/MS spectrum with the known compound 1.30 From this, another node in D having an identical retention time but a different

precursor ion m/z value of 485.3276 (calcd for C30H45O5, 485.3272) could be also identified to represent a deprotonated ion of 1. Inspection of the neighboring nodes in C and D enabled additional annotations consistent with epiceanothic acid (2) and betulinic acid (3) (Figure 2). For annotating the other clusters without any spectral library match, such as A and B, NAP was applied to this MS/MS molecular network. An additional 49 nodes were annotated by this in silico method and indicated that several members or analogues that are represented by the MS/MS spectra in the network are found in natural product structural databases. The NAP consensus assigned nodes in cluster A as p-coumaroyl and caffeoyl ester derivatives of lupane-, oleanane-, or ursanetype triterpenoids. Inspection of MS/MS spectra in A revealed 1820

DOI: 10.1021/acs.jnatprod.8b00292 J. Nat. Prod. 2018, 81, 1819−1828

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Figure 2. (A) Molecules previously isolated from Ziziphus jujuba and Hovenia dulcis (1−7) and isolated from S. theezans in the present study (8− 16). (B) Dereplication of the S. theezans twig extract using LC−MS/MS molecular networking and network annotation propagation (NAP). The top ranked NAP consensus candidates for some nodes are shown. Spectral nodes corresponding to identified compounds 1−16 are indicated with the numbers.

that fragment ions at m/z 145.029 ([(coumaroyl) − H2O − H]−, calcd for C9H5O2, 145.0295) or 161.023 ([(caffeoyl) − H2O − H]−, calcd for C9H5O3, 161.0244) were found in all the spectra, which supported in silico annotations of NAP. By comparing retention time and MS/MS spectra with known pcoumaroyl lupane- and ceanothane-type triterpenoids, nodes representing compounds 4−7 could be identified in cluster A;30,32 hence, it could be confirmed that the molecular family forming A is triterpenoid esters. Although the detailed triterpenoidal backbone types (lupane, oleanane, or ursane) could not be characterized, probably due to their structural rigidity hindering further fragmentation, this result demon-

strated NAP is an efficient dereplication tool for molecular families without any spectral library matching. The NAP annotation, however, exhibited six different candidate structures for spectral nodes of the molecular families shown as cluster B, and the predictions were not reasonable in structural consistency. Nodes originated from precursor ions with the same m/z values were annotated as identical structures; we interpreted that this was a result of a molecular family that formed cluster B to consist of a group of stereoisomers. Such stereoisomers oftentimes exhibit very similar MS/MS spectra and is the reason that NAP annotates at the level of molecular families without considering retention time or other information that would differentiate among 1821

DOI: 10.1021/acs.jnatprod.8b00292 J. Nat. Prod. 2018, 81, 1819−1828

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Table 1. 1H NMR Spectroscopic Data (δH in ppm) of Compounds 8−13 (methanol-d4) 8a

9a

10a

11b

12b

13a

position

δH mult. (J in Hz)

δH mult. (J in Hz)

δH mult. (J in Hz)

δH mult. (J in Hz)

δH mult. (J in Hz)

δH mult. (J in Hz)

2 5 6 7 8 9

7.09, d (1.8) 6.79, m 6.90, m 4.90, d (5.8) 4.69, m 4.49, dd (11.8, 3.4) 4.43, dd (11.8, 6.9) 7.03, d (1.4) 6.79, m 6.92, m 6.62, d (15.8) 6.26, dt (15.8, 6.4) 4.80, d (6.4) 7.34, d (8.6) 6.79, m 6.79, m 7.34, d (8.6) 7.27, d (15.9) 6.15, d (15.9) 7.48, d (8.6) 6.79, d (8.6) 6.79, d (8.6) 7.48, d (8.6) 7.66, d (15.9) 6.38, d (15.9) 3.80, s 3.84, s

7.07, dd (1.8) 6.79, d (8.1) 6.91, dd (8.1, 1.8) 4.91, d (5.9) 4.67, m 4.28, dd (11.8, 3.4) 4.16, dd (11.8, 6.7) 7.07, d (1.9) 7.02, d (8.3) 6.96, dd (8.3, 1.9) 6.64, d (15.8) 6.27, dt (15.8, 6.4) 4.81, d (6.4) 7.35, d (8.6) 6.80, d (8.6) 6.80, d (8.6) 7.35, d (8.6) 7.28, d (15.9) 6.15, d (15.9) 7.48, d (8.6) 6.80, d (8.6) 6.80, d (8.6) 7.48, d (8.6) 7.66, d (15.9) 6.39, d (15.9) 3.83, s 3.83, s

7.02, d (1.8) 6.74, d (8.1) 6.85, dd (8.1, 1.8) 4.82, d (5.8) 4.59, m 4.43, dd (11.2, 3.1) 4.16, dd (11.2, 5.3) 7.01, d (1.9) 6.77, d (8.3) 6.85, dd (8.3, 1.9) 6.62, d (15.8) 6.26, dt (15.8, 6.4) 4.80, dd (6.4, 1.1) 7.57, d (8.6) 6.74, d (8.6) 6.74, d (8.1) 7.57, d (8.1) 6.83, d (12.7) 5.68, d (12.7) 7.47, d (8.6) 6.81, d (8.6) 6.81, d (8.6) 7.47, d (8.6) 7.65, d (15.9) 6.37, d (15.9) 3.81, s 3.77, s

7.00, d (1.8) 6.76, d (8.1) 6.84, dd (8.1, 1.8) 4.84, d (5.9) 4.56, m 4.31, dd (12.0, 3.6) 4.02, dd (12.0, 5.9) 7.07, br s 6.92, br s 6.92, br s 6.65, d (15.8) 6.29, dt (15.8, 6.4) 4.82, d (6.4) 7.58, d (8.6) 6.73, d (8.6) 6.73, d (8.7) 7.58, d (8.7) 6.85, d (12.7) 5.68, d (12.7) 7.48, d (8.6) 6.81, d (8.6) 6.81, d (8.6) 7.48, d (8.6) 7.66, d (15.9) 6.38, d (15.9) 3.81, s 3.84, s

7.09, d (1.8) 6.77, d (8.1) 6.90, m 4.90, d (5.6) 4.69, m 4.48, dd (11.8, 3.2) 4.43, dd (11.8, 6.9) 7.09, d (1.8) 6.99, br s 6.90, br s 6.55, d (15.8) 6.20, dt (15.8, 6.4) 4.74, dd (6.4, 1.0) 7.34, d (7.8) 6.78, d (7.8) 6.78, d (8.6) 7.34, d (7.8) 7.30, d (15.9) 6.16, d (15.9) 7.61, d (8.6) 6.73, d (8.6) 6.73, d (8.6) 7.61, d (8.6) 6.89, d (12.7) 5.82, d (12.7) 3.83, s 3.80, s

7.07, d (1.9) 6.78, d (8.1) 6.91, dd (8.1, 1.9) 4.91, d (6.0) 4.67, m 4.28, dd (11.8, 3.5) 4.15, dd (11.8, 6.6) 7.03, d (1.9) 7.02, d (8.4) 6.94, dd (8.4, 1.9) 6.57, d (15.8) 6.22, dt (15.8, 6.4) 4.75, dd (6.4, 1.1) 7.36, d (8.6) 6.78, d (8.6) 6.78, d (8.4) 7.36, d (8.4) 7.31, d (15.9) 6.16, d (15.9) 7.62, d (8.6) 6.74, d (8.6) 6.74, d (8.6) 7.62, d (8.6) 6.89, d (12.7) 5.83, d (12.7) 3.83, s 3.84, s

2′ 5′ 6′ 7′ 8′ 9′ 2″ 3″ 5″ 6″ 7″ 8″ 2‴ 3‴ 5‴ 6‴ 7‴ 8‴ 3′-OCH3 3″-OCH3 a

Measured at 800 MHz. bMeasured at 600 MHz.

Compounds 8−13 exhibited their deprotonated molecular ions at m/z 667.218 ([M − H]− calcd for C38H35O11, 667.2184), indicating their identical molecular formula of C38H36O11. As mentioned above, spectral nodes from m/z 667.218 were tentatively identified as dadahol B, a dicoumaroylneolignan that was reported only from Artocarpus dadah (Moraceae).38 Among compounds 8−13, 8 and 9 showed identical 1H and 13C NMR spectra to those of dadahol B in the reference (Tables 1 and 2). This demonstrated that NAP can be a powerful tool for dereplication, even in a case where the bibliographical background is lacking. However, dadahol B was isolated only as a diastereomic mixture previously, so it was decided to further identify configurations of 8 and 9 based on spectroscopic data. The relative configurations at C-7 and C-8 were identified by analyzing the 1H NMR spectrum acquired in CDCl3; it was reported that conformations of 8-O-4′ neolignans are fixed in CDCl3 by intramolecular hydrogen bonding, and this produces a J7,8 of 8 and 3.2 Hz, indicating threo- and erythro-configurations, respectively.42 The 1H NMR spectrum of 8 in CDCl3 exhibited a broad singlet resonance of H-7, which indicates a small coupling constant with H-8 (Table 3). Thus, it was suggested that compound 8 is an erythro-derivative of dadahol B. The absolute configuration of 8 was characterized by electronic circular dichroism (ECD) analysis. It is known that 8S-O-4′-neolignans induce positive Cotton effects at 220−250 nm.43−45 Thus, the positive Cotton effect at 220 nm in the ECD spectrum of 8 suggested the (8S)configuration of 8; thus, compound 8 was identified as erythro(7R,8S)-dadahol B. On the other hand, for compound 9, H-7 and H-8 showed a coupling constant of 7.8 Hz in CDCl3,

isomers. Candidate structures annotated by NAP were as follows: mezerein37 for m/z 653.257, dadahol B38 for m/z 667.218, threo-carolignan K39 for m/z 697.229, a pyranocoumarin dimer40 for m/z 669.234, and syringaresinol 4-Oapiofuranosyl-(1→2)-glucopyranoside41 for m/z 727.244. All of these metabolites have not been commonly observed from plants, and none of these have been previously isolated from Sageretia species or other Rhamnaceae plants. A common MS/ MS fragment ion at m/z 163.039 ([coumaroyl − H]−, calcd for C9H7O3, 163.0400) could be found in manual inspection of the MS/MS spectra in B, consistent with the observation that compounds in B were dicoumaroylneolignans such as dadahol B and threo-carolignan K. Suggested molecular formula (C38H38O10 for 653.238, C38H36O11 for 667.218, C38H38O11 for 669.234, C39H38O12 for 697.229, and C40H40O13 for 727.244) for molecules in B also supported that the major molecular family of B is dicoumaroyl neolignans. NAP is based on in silico fragmentation of structure libraries; thus, we hypothesized that several of the annotations in B (e.g., mezerein or pyranocoumarin dimer) were assigned to incorrect structures due to the absence of correct structures in the libraries but that the correct molecules might share some substructural component to the predicted NAP matches. In other words, as they appeared to be unknown molecules, compounds in B were targeted for large-scale isolation. The water-suspended S. theezans extract was sequentially fractionated with n-hexane, CH2Cl2, and EtOAc. The EtOAc fraction was further separated by MPLC, Sephadex LH-20 column chromatography, and preparative HPLC to yield compounds 8−16. 1822

DOI: 10.1021/acs.jnatprod.8b00292 J. Nat. Prod. 2018, 81, 1819−1828

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Table 2. 13C NMR Spectroscopic Data (δC in ppm) of Compounds 8−13 (methanol-d4) position

8a

9a

10a

11b

12b

13a

1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴ 3′-OCH3 3″-OCH3

134.0 111.9 152.3 147.3 117.0 121.1 74.3 83.7 65.4 132.8 111.7 149.0 149.6 117.0 121.0 135.0 123.4 66.3 127.2 131.4 117.0 161.4 117.0 131.4 146.9 114.9 169.1 127.3 131.4 117.0 161.5 117.0 131.4 146.9 115.3 169.2 56.6 56.5

133.5 111.9 149.1 147.6 116.1 120.9 74.8 84.2 65.4 132.8 111.6 152.2 149.9 119.2 121.2 135.0 123.5 66.3 127.1 131.4 117.0 161.5 117.0 131.4 147.0 114.8 168.9 127.3 131.4 117.0 161.5 117.0 131.4 146.9 115.3 169.2 56.5 56.6

133.9 111.9 148.9 147.3 115.9 121.1 74.1 83.4 64.6 132.8 111.6 152.2 149.3 119.2 121.0 135.1 123.3 66.3 128.8 133.8 116.0 160.2 116.0 133.8 145.3 116.6 168.3 127.3 131.4 117.0 161.5 117.0 131.4 146.9 115.2 169.2 56.5 56.6

133.3 111.8 149.0 147.5 116.1 120.9 74.6 83.9 64.4 132.8 111.6 152.1 149.5 118.8 121.2 135.1 123.4 66.3 127.7 133.9 116.0 160.3 116.0 133.9 145.5 116.4 168.1 127.3 131.4 117.0 161.5 117.0 131.4 146.9 115.3 169.2 56.5 56.6

134.0 111.9 149.0 147.3 115.9 121.0 74.3 83.7 65.3 132.9 111.7 152.3 149.5 111.9 120.0 135.0 123.2 66.0 127.2 131.4 117.0 161.4 117.0 131.4 146.9 115.0 169.1 127.9 133.7 116.0 160.2 116.0 133.7 145.2 116.9 168.3 56.5 56.6

133.5 111.8 149.1 147.6 116.8 120.9 74.8 84.1 65.3 132.8 111.6 152.2 149.8 119.1 121.1 135.0 123.2 66.0 127.1 131.4 117.0 161.5 117.0 131.4 147.0 114.8 168.9 127.8 133.8 116.0 160.2 116.0 133.8 145.2 116.8 168.4 56.6 56.5

observed in a relatively upfield position with a J value of 12.7 Hz, which suggests the presence of a Z-p-coumarate moiety in 10. The HMBC spectra of 10 exhibited correlations from H-9 (δH 4.42) and H-7″ to C-9″ (δC 168.3), indicating the Z-pcoumarate moiety is connected to C-9, not to C-9′. Compound 10 showed a broad singlet resonance of H-7 (δH 4.82) in CDCl3 and the positive ECD Cotton effect at 239 nm, suggesting an erythro-(7R,8S)-configuration of 10. Thus, the structure of compound 10 was defined as 9″-O-(Z)-pcoumaroyl-9‴-O-(E)-p-coumaroyl-(7R,8S)-guaiacylglycerol-8O-4′-coniferyl ether. Compound 11 exhibited quite similar 1H and 13C NMR spectra to those of 10. However, the 1H NMR coupling constant between H-7 and H-8 was observed as 8.2 Hz, which indicated a threo-configuration of 11. The chemical shifts of C7 and C-8 in the 13C NMR spectra of 10 and 11 supported this identification. Resonances of C-7 (δC 74.6) and C-8 (δC 83.9) of 11 were shifted slightly more downfield than those of 10 (δC 74.1, C-7; δC 83.4, C-8), of which similar trends were observed between 8 and 9 as reported.42,46 The ECD spectrum of 11 exhibited a positive Cotton effect at 224 nm; thus, the structure of compound 11 was assigned as 9″-O-(Z)-pcoumaroyl-9‴-O-(E)-p-coumaroyl-(7S,8S)-guaiacylglycerol-8O-4′-coniferyl ether. The 1H and 13C NMR spectra of compounds 12 and 13 were similar to those of 10 and 11, indicating the presence of a Z-p-coumaroyl moiety for each compound. The HMBC spectra revealed that the Z-p-coumaroyl moiety in 12 and 13 is connected to C-9′, instead of C-9 in 10 and 11. The absolute configurations at C-7 and C-8 were identified by coupling constants and ECD spectra as the same as compounds 8−11; compounds 12 and 13 were defined as 9″-O-(E)-p-coumaroyl9‴-O-(Z)-p-coumaroyl-(7R,8S)-guaiacylglycerol-8-O-4′-coniferyl ether (12) and 9″-O-(E)-p-coumaroyl-9‴-O-(Z)-pcoumaroyl-(7S,8S)-guaiacylglycerol-8-O-4′-coniferyl ether (13), respectively. Compound 14 exhibited a deprotonated molecular ion at m/z 653.2388 ([M − H]− calcd for C38H37O10, 653.2392) in the HRESIMS, indicating a molecular formula of C38H38O10. Only 19 resonances were observed in the 13C NMR spectrum of 14, which suggested its symmetrical nature. An AA′XX′ aromatic spin system [δH 7.44 (2H, d, J = 8.6 Hz, H-2″, H-6″), 6.79 (2H, d, J = 8.6 Hz, H-3″, H-5″)] and two conjugated methine signals at δH 7.59 (1H, d, J = 15.9 Hz, H-7″) and 6.34 (1H, d, J = 15.9 Hz, H-8″) in the 1H NMR spectrum revealed the symmetrical presence of two E-p-coumaroyl moieties (Table 4). The 1H NMR spectrum also exhibited resonances of a 1,2,4-trisubstituted aromatic ring [δH 6.61(1H, s, H-2), 6.69 (1H, d, J = 8.0 Hz, H-5), 6.57 (1H, d, J = 8.0 Hz, H-6)], a methylene [δH 2.76 (1H, dd, J = 13.8, 7.4 Hz, H-7a), 2.68 (1H, dd, J = 13.8, 7.4 Hz, H-7b)], a tertiary methine [δH 2.24 (1H, m, H-8)], an oxygenated methylene [δH 4.34 (1H, dd, J = 11.3,

a

Measured at 200 MHz. bMeasured at 150 MHz.

suggesting the threo-configuration (Table 3). The ECD spectrum of 9 exhibited a positive Cotton effect at 230 nm. Hence, the structure of compound 9 was defined as threo(7S,8S)-dadahol B. The 1H and 13C NMR spectra of 10 were also similar to those of 8 and 9; thus, compound 10 was suggested to be another stereoisomer of 8 and 9. In the 1H NMR spectrum of 10, resonances of H-7″ (δH 6.83) and H-8″ (δH 5.68) were

Table 3. 1H NMR Spectroscopic Data (δH in ppm) of H-7, -8, and -9 of Compounds 8−13 in CDCl3 8a

9a

10b

11b

12a

13a

position

δH mult. (J in Hz)

δH mult. (J in Hz)

δH mult. (J in Hz)

δH mult. (J in Hz)

δH mult. (J in Hz)

δH mult. (J in Hz)

7 8 9

4.91, s 4.52, m 4.46, dd, (11.8, 7.2) 4.29, dd (11.8, 3.4)

4.89, dd (7.8, 2.2) 4.30, m 4.17, dd (12.9, 6.4) 4.30, m

4.85, s 4.41, m 4.17, d (8.9) 4.41, m

4.81, dd (8.2, 1.8) 4.20, ddd (8.0, 5.2, 3.2) 4.29, dd (12.3, 3.2) 4.01, dd (12.3, 5.2)

4.94, s 4.52, m 4.46, dd (11.8, 6.9) 4.30, dd (11.8, 3.7)

4.90, d (7.5) 4.33, m 4.33, m 4.15, dd (11.3, 6.4)

a

Measured at 400 MHz. bMeasured at 800 MHz. 1823

DOI: 10.1021/acs.jnatprod.8b00292 J. Nat. Prod. 2018, 81, 1819−1828

Journal of Natural Products Table 4. 1H and

13

Article

C NMR Spectroscopic Data (δH in ppm) of Compounds 14−16 (methanol-d4) 14a

position 1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴ 3-OCH3 3′-OCH3

δH mult. (J in Hz) 6.61, s

6.69, d (8.0) 6.57, d (8.0) 2.76, dd (13.8, 7.4) 2.68, dd (13.8, 7.4) 2.24, m 4.34, dd (11.3, 6.0) 4.20, dd (11.3, 5.4) 6.61, s

6.69, d (8.0) 6.57, d (8.0) 2.76, dd (13.8, 7.3) 2.68, dd (13.8, 7.3) 2.24, m 4.34, dd (11.3, 6.0) 4.20, dd (11.3, 5.4) 7.44, d (8.6) 6.79, d (8.6) 6.79, d (8.6) 7.44, d (8.6) 7.59, d (15.9) 6.34, d (15.9)

7.44, d (8.6) 6.79, d (8.6) 6.79, d (8.6) 7.44, d (8.6) 7.59, d (15.9) 6.34, d (15.9) 3.73, s 3.73, s

15b δC 133.0 113.4 149.1 145.9 116.1 122.7 36.2 41.7 65.8 133.0 113.4 149.1 145.9 116.1 122.7 36.2 41.7 65.8 127.2 131.4 117.0 161.4 117.0 131.4 146.9 115.2 169.5 127.2 131.4 117.0 161.4 117.0 131.4 146.9 115.2 169.5 56.3 56.3

δH mult. (J in Hz) 6.56, d (1.8)

6.66, d (8.0) 6.51, dd (8.0, 1.8) 2.65, m 2.15, m 4.32, dd (11.3, 6.0) 4.12, dd (11.3, 5.4) 6.55, d (1.8)

6.65, d (7.9) 6.51, dd (7.9, 1.8) 2.65, m 2.15, m 4.32, dd (11.3, 6.1) 4.12, dd (11.3, 5.7) 7.45, d (8.6) 6.79, d (8.6) 6.79, d (8.6) 7.45, d (8.6) 7.61, d (15.9) 6.33, d (15.9)

7.62, d (8.7) 6.74, d (8.7) 6.74, d (8.7) 7.62, d (8.7) 6.90, d (12.7) 5.82, d (12.7) 3.69, s 3.71, s

16b δC 133.0 113.4 149.0 145.9 116.1 122.8 36.3 41.5 65.6 133.0 113.4 149.1 145.9 116.1 122.8 36.2 41.5 65.6 127.2 131.4 117.1 161.7 117.1 131.4 146.9 115.2 169.4 127.9 133.8 116.1 160.4 116.1 133.8 145.4 116.9 168.6 56.3 56.4

δH mult. (J in Hz) 6.50, d (1.8)

6.64, d (8.0) 6.45, dd (8.0, 1.8) 2.59, dd (13.8, 7.9) 2.54, dd (13.8, 7.1) 2.06, m 4.26, dd (11.3, 6.0) 4.04, dd (11.3, 5.6) 6.49, d (1.8)

6.64, d (8.0) 6.45, dd (8.0, 1.8) 2.59, dd (13.9, 7.9) 2.54, dd (13.9, 7.1) 2.06, m 4.26, dd (11.3, 6.0) 4.04, dd (11.3, 5.6) 7.61, d (8.7) 6.75, d (8.7) 6.75, d (8.7) 7.61, d (8.7) 6.88, d (12.7) 5.80, d (12.7)

7.61, d (8.7) 6.75, d (8.7) 6.75, d (8.7) 7.61, d (8.7) 6.88, d (12.7) 5.80, d (12.7) 3.66, s 3.66, s

δC 133.0 113.4 149.0 145.8 116.1 122.8 36.2 41.2 65.5 133.0 113.4 149.0 145.8 116.1 122.8 36.2 41.2 65.5 127.9 133.7 116.1 160.3 116.1 133.7 145.3 117.0 168.5 127.9 133.7 116.1 160.3 116.1 133.7 145.3 117.0 168.5 56.3 56.3

a

Measured at 400/100 MHz. bMeasured at 800/200 MHz.

6.0 Hz, H-9a), 4.27 (1H, dd, J = 11.3, 5.4 Hz, H-9b)], and a methoxy group [δH 3.73 (3H, s)]; these signals indicated that compound 14 is a derivative of secoisolariciresinol,47 one of the major lignans commonly observed in plants. The HMBC correlation between H-9 and C-9″ (δC 169.5) revealed the E-pcoumaroyl ester substitutions at C-9 and C-9′. To assign the absolute configuration of 14, alkaline hydrolysis was performed to remove the (E)-p-coumaroyl moieties. The resulting secoisolariciresinol exhibited a negative specific rotation ([α]D20 −16.9), indicating the (8R,8′R)-configuration of (−)-secoisolariciresinol.48 Hence, compound 14 was identified as 9,9′-di-O-(E)-p-coumaroyl-(8R,8′R)-secoisolariciresinol. During the isolation process, it was found that compounds 14−16 were interconverting via photoisomerization; thus, compounds 15 and 16 were assumed to be (E,Z)-isomers of

14. These compounds were successfully purified under darkness and analyzed by spectroscopic methods. The 1H NMR spectra of 15 and 16 confirmed the presence of one (15) or two (16) Z-p-coumaroyl moieties, which exhibited coupling constants of 12.7 Hz (Table 4). Thus, these compounds were assigned as 9-O-(E)-p-coumaroyl-9′-O-Z-p-coumaroyl(8R,8′R)-secoisolariciresinol (15) and 9,9′-di-O-(Z)-p-coumaroyl-(8R,8′R)-secoisolariciresinol (16), respectively. To test the feedback effect of expanding the structural library to the NAP annotation, the structures of isolated compounds 8−16 were added to the library and the NAP analysis was performed using identical parameters. As a result, NAP consensus predicted spectral nodes with m/z 653.237 as 9,9′-di-O-p-coumaroylsecoisolariciresinols, not mezerein, even though only the structural library, not the spectral library, was 1824

DOI: 10.1021/acs.jnatprod.8b00292 J. Nat. Prod. 2018, 81, 1819−1828

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Figure 3. Effect of compounds 8−16 on glutamate-induced death of HT22 cells. Data are means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to 5 mM glutamate-treated group.

experimental conditions of interest, permitting the detection of specialized metabolites.

updated (Figure S1, Supporting Information). This highlights the importance of community contributions of structures to structural databases in addition to providing reference spectra. This also suggests that further discovery of unknown metabolites will accelerate the reliability of NAP-based dereplication, which will reaccelerate the natural product discovery. Many lignans and neolignans were reported to have neuroprotective activity.49,50 Thus, the isolated compounds were evaluated for their neuroprotective effect against glutamate-induced oxidative stress on mouse hippocampal HT22 cells. Although compounds 8−16 showed cytotoxicity over 25 μM, they exhibited protective activity in a concentration range of 1.6 to 12.5 μM. Compounds 14−16 showed significant neuroprotective activity, presenting cell viability of 63 ± 3.67%, 73 ± 0.78%, and 74 ± 2.37%, respectively, at 12.5 μM, while cells treated with 5 mM glutamate showed 23 ± 3.57% and 12.5 μM of Trolox, the positive control, protected cells, showing a cell viability of 52 ± 2.36% (Figure 3). Herein the results show that NAP can accelerate bioactive natural products discovery by providing putative annotations on MS/MS molecular networks, even for MS/MS spectra without any spectral library match. NAP improvement of in silico fragmentation annotation was previously validated;27 however, any new putative annotation should be examined and further confirmed by experts. We anticipate that NAP will significantly decrease time and costs for the dereplication process, by providing insights into the interpretation of fragmentation spectra, especially for metabolites without any spectral references or previous history of isolation from related taxa. Combined with other methods such as bioactivity-based molecular networking,20 NAP can enable the association of structural annotations with quantitative changes under

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-2000 polarimeter (Jasco, Easten, MD, USA) using a 1 cm cell. UV and ECD spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics, Surrey, UK), and IR spectra were acquired using a JASCO FT/IR-4200 spectrometer. 1D and 2D NMR spectra were obtained with Bruker AVANCE III 800, 600, or 400 spectrometers (Bruker, Billerica, MA, USA). HRESIMS was performed using a Waters Xevo G2 QTOF mass spectrometer (Waters MS Technologies, Manchester, UK) equipped with an electrospray interface (ESI). Column chromatography (CC) was performed with Kieselgel 60 silica gel (40−60 μm, 230−400 mesh, Merck, Darmstadt, Germany) and Sephadex LH-20 (25−100 μm, Pharmacia, Piscataway, NJ, USA). Preparative and semipreparative HPLC was performed with a system consisting of a Gilson 321 pump and a UV/vis-151 detector (Gilson Inc., Middleton, WI, USA), equipped with a YMC Hydrosphere C18 column (10 × 250 mm, 5 μm, YMC Co. Ltd., Kyoto, Japan). Extra-pure grade solvents for extraction, fractionation, and isolation were purchased from Dae Jung Pure Chemical Engineering Co. Ltd., Siheung, Korea. Deuterated solvents for NMR analyses were purchased from Merck. Plant Material. S. theezans was cultivated at the Medicinal Plant Garden, College of Pharmacy, Seoul National University, Koyang, Korea (GPS N 37°42′42.9″, E 126°49′10.6″), and its twigs were collected in September 2015. The sample was authenticated by Mr. S. I. Han (The Medicinal Plant Garden, College of Pharmacy, Seoul National University), and a voucher specimen (SUPH-1509-06) was deposited in the Herbarium of the Medicinal Plant Garden. LC−MS/MS Molecular Networking. Chromatographic separation was performed on a Waters Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) column. The flow rate of the mobile phase was 0.3 mL/min, and the column temperature was maintained at 30 °C. The mobile phase consisted of H2O (A) and MeCN (B) with a linear gradient of 10−90% B (0−20 min). The MeOH extract of S. theezans twigs (1.0 μL injected into the partial loop with needle overfill mode) was analyzed using an optimized data-dependent acquisition mode 1825

DOI: 10.1021/acs.jnatprod.8b00292 J. Nat. Prod. 2018, 81, 1819−1828

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288 (−2.82), 320 (2.63); 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 667.2194 [M − H]− (calcd for C38H35O11, 667.2184); MS/MS spectrum is deposited in the GNPS spectral library, https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum. jsp?SpectrumID=CCMSLIB00004679187#%7B%7D. threo-(7S,8S)-Dadahol B (9): colorless gum; [α]20 D −291.2 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 200 (3.89), 220 (3.66), 313 (3.71); ECD (MeOH) λmax (Δε) 202 (−2.35), 209 (−3.30), 230 (3.38), 271 (−3.38), 288 (−3.55), 316 (3.29); 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 667.2179 [M − H]− (calcd for C38H35O11, 667.2184); MS/MS spectrum is deposited in the GNPS spectral library, https://gnps.ucsd.edu/ProteoSAFe/ gnpslibraryspectrum.jsp?SpectrumID= CCMSLIB00004679209#%7B%7D. 9″-O-(Z)-p-Coumaroyl-9‴-O-(E)-p-coumaroyl-(7R,8S)-guaiacylglycerol-8-O-4′-coniferyl ether (10): colorless gum; [α]20 D +59.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 200 (3.87), 220 (3.59), 312 (3.57); ECD (MeOH) λmax (Δε) 206 (1.57), 225 (−1.97), 239 (1.48), 291 (−2.48), 345 (−0.74); 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 667.2184 [M − H]− (calcd for C38H35O11, 667.2184); MS/MS spectrum is deposited in the GNPS spectral library, https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum. jsp?SpectrumID=CCMSLIB00004679211#%7B%7D. 9″-O-(Z)-p-Coumaroyl-9‴-O-(E)-p-coumaroyl-(7S,8S)-guaiacylglycerol-8-O-4′-coniferyl ether (11): colorless gum; [α]20 D −0.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 200 (3.77), 224 (3.42), 311 (3.41); ECD (MeOH) λmax (Δε) 206 (3.76), 224 (3.69), 246 (3.45), 282 (2.57), 312 (3.17); 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 667.2188 [M − H]− (calcd for C38H35O11, 667.2184); MS/MS spectrum is deposited in the GNPS spectral library, https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum. jsp?SpectrumID=CCMSLIB00004679212#%7B%7D. 9″-O-(E)-p-Coumaroyl-9‴-O-(Z)-p-coumaroyl-(7R,8S)-guaiacylglycerol-8-O-4′-coniferyl ether (12): colorless gum; [α]20 D −2.5 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 200 (3.85), 215 (3.58), 310 (3.48); ECD (MeOH) λmax (Δε) 219 (3.14), 314 (2.80), 374 (−2.69); 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 667.2182 [M − H]− (calcd for C38H35O11, 667.2184); MS/MS spectrum is deposited in the GNPS spectral library, https://gnps.ucsd. e d u / P r o t e o S A F e / g n p s l i b r a r y s p e ct r u m . j s p ? S p e c t r u m I D = CCMSLIB00004679213#%7B%7D. 9″-O-(E)-p-Coumaroyl-9‴-O-(Z)-p-coumaroyl-(7S,8S)-guaiacylglycerol-8-O-4′-coniferyl ether (13): colorless gum; [α]20 D −0.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 200 (3.77), 224 (3.42), 311 (3.41); ECD (MeOH) λmax (Δε) 206 (3.76), 224 (3.69), 246 (3.45), 282 (2.57), 312 (3.17); 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 667.2191 [M − H]− (calcd for C38H35O11, 667.2184); MS/MS spectrum is deposited in the GNPS spectal library, https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum. jsp?SpectrumID=CCMSLIB00004679214#%7B%7D. 9,9′-Di-O-(E)-p-Coumaroyl-(8R,8R′)-secoisolariciresinol (14): colorless gum; [α]20 D −11.9 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (2.77), 228 (2.49), 291 (2.49) 313 (2.56); ECD (c 0.01, MeOH) λmax (Δε) 211 (3.79), 232 (−3.53), 249 (−0.29), 293 (3.55), 327 (−3.87); IR νmax 3381, 2943, 1694, 1603, 1513, 1448, 1269, 1168, 1032, 832 cm−1; 1H and 13C NMR, see Table 4; HRESIMS m/z 653.2388 [M − H]− (calcd for C38H37O10, 653.2392); MS/MS spectrum is deposited in the GNPS spectral library, https://gnps.ucsd. e d u / P r o t e o S A F e / g n p s l i b r a r y s p e ct r u m . j s p ? S p e c t r u m I D = CCMSLIB00004679188#%7B%7D. 9-O-(E)-p-Coumaroyl-9′-O-(Z)-p-coumaroyl-(8R,8R′)-secoisolariciresinol (15): colorless gum; [α]20 D −44.9 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 223 (2.39), 291 (2.34) 312 (2.40); ECD (MeOH) λmax (Δε) 211 (3.67), 229 (−3.37), 252 (−2.69), 293 (2.84), 326 (−3.57); IR νmax 2348, 2309, 1680, 1603, 1513, 1455, 1268, 1167, 1032, 832, 673 cm−1; 1H and 13C NMR, see Table 4; HRESIMS m/z 653.2382 [M − H]− (calcd for C38H37O10, 653.2392); MS/MS spectrum is deposited in the GNPS spectral library, https://gnps.ucsd.edu/ProteoSAFe/gnpslibraryspectrum. jsp?SpectrumID=CCMSLIB00004679215#%7B%7D.

consisting of a full MS survey scan in the 100−1500 Da range (scan time: 100 ms), followed by an MS/MS scan for the three most intense ions. The collision energy was applied at a gradient from 20 to 80 V. The molecular network was created using a GNPS platform (https:// gnps.ucsd.edu),10 of which the reliability is enhanced by data preprocessing using MZmine 2 software.35 Raw LC−MS files were converted into mzXML using ProteoWizard 3.0.9935,51 then imported into MZmine 2.28. The mass detection was performed with the noise level at 1000 (for MS) and 40 (for MS/MS). The chromatogram was built with ions showing a minimum time span of 0.01 min, minimum height of 2500, and m/z tolerance of 0.001 (or 5.0 ppm). The chromatographic deconvolution was achieved by the baseline cutoff algorithm, with the following parameters: minimum peak height of 1500, peak duration range of 0.02−0.15 min, and baseline level of 500. Chromatograms were deisotoped using the isotopic peaks grouper algorithm with an m/z tolerance of 0.002 (or 5.0 ppm) and a tR tolerance of 0.1 min. The preprocessed chromatograms were exported to GNPS for molecular networking. MS/MS spectra were window filtered by choosing only the top six peaks in the ±50 Da window throughout the spectrum. A network was then created where edges were filtered to have a cosine score above 0.60 and more than three matched peaks. Further edges between two nodes were kept in the network if and only if each of the nodes appeared in each other’s respective top 10 most similar nodes. The spectra in the network were then searched against the spectral library of GNPS. The library spectra were filtered in the same manner as the input data. The molecular network was visualized using Cytoscape 3.5.1.52 The molecular network was used to compute the propagation with NAP (https://proteomics2.ucsd.edu/ProteoSAFe/ ?params={%22workflow%22:%22NAP_CCMS2%22}) using parameters as follows: 10 first candidates, exact mass searches within 5 ppm, and databases of DNP (laboratory internal use with institutional subscription) and SuperNatural (open to the community). After the propagation computation the ‘structure_graph_alt.xgmml’ containing the layout for structure annotation (green border for GNPS spectral matching and blue for in silico annotation) file was directly imported into Cytoscape, and the structures were visualized with the ChemViz2 plug-in. MS/MS raw data and preprocessed peak list file were deposited in the MassIVE Public GNPS data sets (https://massive. ucsd.edu, MSV000081774). The MS/MS molecular network and NAP annotated networks can be browsed and downloaded on the GNPS Web site with the following links: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task= 3b215c4b25594b9c85d92de547815c0a https://proteomics2.ucsd.edu/ProteoSAFe/status.jsp?task= 405c26a262004299843d73278fb3d24c NAP annotated networks produced by rerunning with augmented structural library with the isolated compounds can be found at the following link: https://proteomics2.ucsd.edu/ProteoSAFe/status.jsp?task= 2d7b00ff710c42438146f2bc311f1785 Extraction and Isolation. Dried twigs of S. theezans (6.1 kg) were extracted with MeOH (2 × 30 L, for 3 h each) with ultrasonication at room temperature, and the extract was concentrated in vacuo. The crude extract (56.6 g) was suspended in H2O and extracted successively with n-hexane (12.1 g), CH2Cl2 (19.7 g), and EtOAc (6.7 g). The CH2Cl2 fraction was further divided into five subfractions by silica gel CC, eluted with mixtures of CH2Cl2−MeOH (50:1, 30:1, 20:1, 15:1, 10:1, and 5:1). The lignan-rich subfraction C3 was further separated by MPLC and Sephadex LH-20 to yield two neolignan- and lignan-rich subfractions C3a and C3b. Compounds 8 (1.3 mg), 9 (1.1 mg), 10 (4.0 mg) 11 (3.5 mg), 12 (1.0 mg), and 13 (0.7 mg) were purified from subfraction C3a using reversed-phase semipreparative HPLC eluted with 67% aqueous MeOH. Compounds 14 (6.1 mg), 15 (2.2 mg), and 16 (0.9 mg) were isolated from subfraction C3b using reversed-phase semipreparative HPLC eluted with 45% aqueous acetonitrile. erythro-(7R,8S)-Dadahol B (8): colorless gum; [α]20 D −2.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 200 (3.92), 220 (3.72), 312 (3.78); ECD (MeOH) λmax (Δε) 204 (2.88), 217 (2.70), 222 (2.58), 1826

DOI: 10.1021/acs.jnatprod.8b00292 J. Nat. Prod. 2018, 81, 1819−1828

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9,9′-Di-O-(Z)-p-Coumaroyl-(8R,8R′)-secoisolariciresinol (16): colorless gum; [α]20 D +7.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (2.42), 290 (2.31), 312 (2.35); ECD (MeOH) λmax (Δε) 226 (−3.55), 248 (−2.96), 327 (−3.48); IR νmax 2348, 2309, 1748, 1508, 1032, 675 cm−1; 1H and 13C NMR, see Table 4; HRESIMS m/z 653.2387 [M − H]− (calcd for C38H37O10, 653.2392); MS/MS spectrum is deposited in the GNPS spectral library, https://gnps.ucsd. edu/ProteoSAFe/gnpslibraryspectrum.jsp?SpectrumID= CCMSLIB00004679217#%7B%7D. Hydrolysis of 14. About 1.6 mg of 14 was hydrolyzed with 0.5 M NaOH (0.1 mL) at room temperature for 6 h. The hydrolysate was extracted with EtOAc (3 × 0.2 mL). The EtOAc fraction was collected and dried over Na2SO4. The EtOAc layer was purified by semipreparative column chromatography on a YMC hydrosphere C18 column (10 × 250 mm, 5 μm, YMC Co. Ltd., Kyoto, Japan) with 0.1% aqueous formic acid and MeCN gradient condition. Specific rotations of product were compared with known secoisolariciresinols.48 Bioactivity Evaluation. The mouse hippocampal HT22 cells were obtained from Dr. Ki-Sun Kwon (Korea Research Institute of Bioscience & Biotechnology, Daejeon, Korea). Cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Hyclone), and 100 μg/mL streptomycin (Hyclone) at 37 °C with 5% CO2 conditions. After 24 h of incubation, cells were seeded onto 96-well plates (1 × 104 cells/well in 100 μL of medium) and incubated 24 h with 5% CO2 conditions at 37 °C. HT22 cells were pretreated with or without different concentrations of compounds (1.6125−50.0 μg/mL) for 2 h and exposed to 5 mM Lglutamate. After 20 h of incubation, the cell viability was determined using the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] (Sigma-Aldrich) assay. Cells were treated with 0.5 mg/mL of MTT solution for 2 h at 37 °C. After that, MTT solution was discarded and 100 μL of DMSO was added to each well to solubilize the formazan crystals. The absorbance was measured at 570 nm via a SpectraMAX M5 multiplate reader (Molecular Devices, Sunnyvale, CA, USA). Trolox (Sigma, >97% purity) was used as a positive control.

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Notes

The authors declare the following competing financial interest(s): P. C. Dorrestein is an advisor for Sirenas, a company that employs molecular networking for the discovery of bioactive natural products from marine resources. The work in that company does not overlap with the work presented in this paper. # Deceased July 24, 2018.

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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), which was funded by the Ministry of Science, ICT and Future Planning (NRF-2017R1A2B2011807). We thank the NIH for supporting this work under NIH-UCSD Center for Computational Mass Spectrometry P41 GM103484 and the NIH grants GMS10RR029121 and R03 CA211211, and tools for rapid and accurate structure elucidation of natural products R01 GM107550. We would like to thank S. I. Han (The Medicinal Plant Garden, College of Pharmacy, Seoul National University) for kindly providing the plant material and also thank M.-Y. Oh, Y.-M. Lee (College of Pharmacy, Seoul National University), and Y.-J. Ko (National Centre for InterUniversity Research Facilities) for the NMR experiments.

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DEDICATION This article is dedicated to the memory of our esteemed colleague Dr. Sang Hyun Sung, a good friend, inspiring mentor, and talented scientist who prematurely passed away on July 24, 2018.

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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.8b00292.

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REFERENCES

NAP result obtained from the reanalysis with the isolates; LC−MS base peak ion chromatogram of the S. theezans twigs extract; raw copies of the MS1, NMR, UV, and ECD data of compounds 8−16 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (K. B. Kang). *E-mail: [email protected]. Tel: (+1)-858-534-6607 (P. C. Dorrestein). ORCID

Kyo Bin Kang: 0000-0003-3290-1017 Pieter C. Dorrestein: 0000-0002-3003-1030 Sang Hyun Sung: 0000-0002-0527-4815 Present Address §

College of Pharmacy, Sookmyung Women’s University, Seoul 04310, Republic of Korea.

Author Contributions ¶

K. B. Kang and E. J. Park contributed equally to this article. 1827

DOI: 10.1021/acs.jnatprod.8b00292 J. Nat. Prod. 2018, 81, 1819−1828

Journal of Natural Products

Article

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DOI: 10.1021/acs.jnatprod.8b00292 J. Nat. Prod. 2018, 81, 1819−1828