Monitoring the Chemical Profile in Agarwood Formation Within One

molecules

Communication Monitoring the Chemical Proﬁle in Agarwood Formation within One Year and Speculating on the Biosynthesis of 2-(2-Phenylethyl)Chromones

Ge Liao 1,2,†, Wen-Hua Dong 1,2,†, Jin-Ling Yang 1,3 ID , Wei Li 1,3, Jun Wang 1,3, Wen-Li Mei 1,3,* and Hao-Fu Dai 1,3,* 1 Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China; [email protected] (G.L.); [email protected] (W.-H.D.); [email protected] (J.-L.Y.); [email protected] (W.L.); [email protected] (J.W.) 2 College of Horticulture and Landscape Architecture, Hainan University, Haikou 570228, China 3 Hainan Engineering Research Center of Agarwood, Haikou 571101, China * Correspondence: [email protected] (W.-L.M.); [email protected] (H.-F.D.); Tel.: +86-898-6698-7529 (W.-L.M.); +86-898-6696-1869 (H.-F.D.) † These authors contributed equally to this work.

Received: 8 April 2018; Accepted: 20 May 2018; Published: 25 May 2018

Abstract: Agarwood is highly valued for its uses as incense, perfume, and medicine. However, systematic analyses of dynamic changes of secondary metabolites during the process of agarwood formation have not yet been reported. In this study, agarwood was produced by transfusing the agarwood inducer into the trunk of Aquilaria sinensis, and changing patterns of chemical constituents, especially 2-(2-phenylethyl)chromones (PECs), in wood samples collected from the 1st to 12th month, were analyzed by GC-EI-MS and UPLC-ESI-MS/MS methods. Aromatic compounds, steroids, fatty acids/esters, sesquiterpenoids, and PECs were detected by GC-MS, in which PECs were the major constituents. Following this, UPLC-MS was used for further comprehensive analysis of PECs, from which we found that 2-(2-phenylethyl)chromones of ﬂindersia type (FTPECs) were the most abundant, while PECs with epoxidated chromone moiety were detected with limited numbers and relatively low content. Speculation on the formation of major FTPECs was fully elucidated in our context. The key step of FTPECs biosynthesis is possibly catalyzed by type III polyketide synthases (PKSs) which condensate dihydro-cinnamoyl-CoA analogues and malonyl-CoA with 2-hydroxy-benzoyl-CoA to produce 2-(2-phenyethyl)chromone scaffold, or with 2,5-dihydroxybenzoyl-CoA to form FTPECS with 6-hydroxy group, which may serve as precursors for further reactions catalyzed by hydroxylase or O-methyltransferase (OMT) to produce FTPECs with diverse substitution patterns. It is the ﬁrst report that systematically analyzed dynamic changes of secondary metabolites during the process of agarwood formation and fully discussed the biosynthetic pathway of PECs.

Keywords: Agarwood; Aquilaria sinensis; 2-(2-Phenylethyl)chromones; biosynthesis

1. Introduction Agarwood is the fragrant resinous heartwood from trees of the genus Aquilaria. It is widely accepted that the precious and high-priced agarwood has been of great signiﬁcance in Asian cultures for centuries. For example, it has been used as incense in Buddhist, Hindu, and Islamic ceremonies, and also as traditional medicine in Ayurveda and Chinese therapies [1]. Healthy Aquilaria trees, however, hardly contain resin unless they were stimulated by various forms of injury or microbial attack [2–5]. Agarwood-producing plants are timber species which take quite a long time to grow and

Molecules 2018, 23, 1261; doi:10.3390/molecules23061261 www.mdpi.com/journal/molecules Molecules 2018, 23, 1261 2 of 29 the agarwood-formation process is also time consuming. This results in the fact that wild agarwood is extremely rare and cannot satisfy the sustainable market need for wild agarwood. Under certain circumstances, great efforts are under way to produce agarwood by intentional injuries. The first trial initiated by Tunstall was to inoculate trees with fungi isolated from agarwood in 1929 [1]. After that, a series of progresses have been achieved in developing artificial methods of agarwood production such as fungi inoculation, physical wounding, and chemical treatment (e.g., solutions of FeCl3 or acetic acid) [6–8]. Nowadays, experienced farmers in Southeast Asia obtain agarwood by using traditional methods, such as axe chopping, nailing, holing, burning, and the partial trunk pruning method, among others. Although a series of methods aimed at generating agarwood artificially have been developed rapidly and promoted in many plantations of Aquilaria species, the mechanism of agarwood formation has not been fully understood, and no convincing standard method has been established for quality control of the artificial agarwood. As far as we know, only a few studies concerned about the changes happening to Aquilaria trees after stimulation, e.g., investigation on post-infection changes in sugars, ascorbic acid, phenols, and protein during pathogenesis in A. malaccensis, showed that those primary metabolites decreased whereas secondary metabolites increased [9]. Observation of a sequential change in the wood coloration around injury sites revealed that a pale discoloration occurred after 1 month, followed by a darker yellow-brown discoloration after 3 months, which then became dark brown within 8–10 months and changed to black within 20 months and was accompanied by a burning scent [5]. Analyzing agarwood samples after induction of 15th, 30th, and 60th day, respectively, by using GC-MS showed that sesquiterpenes were detected after 6 days and the relative content of sesquiterpenes and aromatic compounds were increased with prolonged time [10]. Unlike previous studies, agarwood samples used in this study were obtained from a relatively new and efficient transfusion method with the agarwood inducer invented by our group [11]. The chemical constituents were monitored for a fixed period by using GC-EI-MS and UPLC-ESI-MS/MS methods for the purpose of revealing the dynamic changes of the chemical profile in the process of agarwood formation. The detected compounds were identified by searching established database like NIST14 and WILEY275, or matching with reference compounds obtained by our previous study to enable us to conduct qualitative analysis of 2-(2-phenylethyl)chromones (PECs) in agarwood samples by using UPLC-MS. Based on the comprehensive comparison of PECs of different origin, as well as by detailed analyses of the obtained results in this study, we firstly proposed the hypothetical biosynthesis of PECs, which provides the basis for further studies on the mechanism of agarwood formation.

2. Results and Discussion

2.1. The Results of GC-MS Analysis The ether extracts of injured A. sinensis, which did not cover all the components contained in the samples, were analyzed by GC-MS leading to the characterization of 52 compounds in eight samples collected monthly from the 1st to 6th, then the 9th and 12th months, respectively (Table1). The identiﬁcation of 2-(2-phenylethyl)chromone derivatives were mainly based on their MS characterization and fragmentation patterns proposed by our group [12]. Other compounds were characterized by matching with the NIST14 and WILEY275 database and by comparing their MS spectra with those of literature data [1]. The relative contents of the compounds were determined by normalization. Molecules 2018, 23, 1261 3 of 29

Table 1. Volatile constituents of agarwood samples produced from the 1st to 12th months. Molecules 2018, 23, x FOR PEER REVIEW 3 of 28

Molecules 2018, 23, x FOR PEER REVIEW Molecular Relative Content (%) 3 of 28 No. Compounds MW Table 1. Volatile constituents of agarwood samples producedFormula from the 1st to 121th months 2. 3 4 5 6 9 12

1 NonanalMolecular C9H18O 142Relative 0.85 Content 0.89 (%) 1.16 0.22 No. TableCompounds 1. Volatile constituents of agarwood samples produced fromMW the 1st to 12th months. 4 2 4-Phenylbutan-2-one (Benzylacetone) FormulaC10H12O 1481 2 1.023 4 0.46 5 0.176 0.099 0.2112 0.14 0.11 Molecular Relative Content (%) 1 Nonanal 4 C9H18O 142 0.85 0.89 1.16 0.22 3 No. PhenylpropanoicCompounds acid methyl ester C10H12O2 MW164 0.11 2 4-Phenylbutan-2-one (Benzylacetone) FormulaC10H12O 148 1 1.022 0.463 0.174 0.095 0.216 0.149 0.1112 4 4 C9 H18 O 150 1.80 0.19 0.09 0.03 0.12 31 PhenylpropanoicPhenylpropionicNonanal acid methyl acid ester △ CC109H1210O2 2 164142 0.85 0.89 1.16 0.22 0.11 2 4-Phenylbutan-2-one (Benzylacetone)△ C10H12O 148 1.02 0.46 0.17 0.09 0.21 0.14 0.11 5 4 2,6-Di-tert-butyl-2,5-cyclohexadiene-1,4-dionePhenylpropionic acid C C914HH10O202O 2 150220 1.80 0.19 0.09 0.040.03 0.12 0.12 △ 10 12 2 53 2,6-DiPhenylpropanoic-tert-butyl-2,5-cyclohexadiene acid methyl△ ester-1,4- dione 4 C14H20O2 220164 0.04 0.12 0.11 6 4-(4-Methoxyphenyl)butan-2-one (Anisylacetone) C11H14O2 178 0.09 0.11 0.09 0.17 0.44 0.69 0.46 4 Phenylpropionic acid △ C9H10O2 150 1.80 0.19 0.09 0.03 0.12 6 4-(4-Methoxyphenyl)butan-2-one (Anisylacetone)4 C11H14O2 178 0.09 0.11 0.09 0.17 0.44 0.69 0.46 7 2,4- Di-tert—butylphenol△ C1414H20 222O 206 0.67 0.17 0.21 0.39 0.66 0.26 0.35 0.08 75 2,6-Di-tert2,4-butyl- Di-2,5tert-—cyclohexadienebutylphenol - 1,4-dione △ CC14HH22OO 206220 0.67 0.17 0.21 0.390.04 0.660.12 0.26 0.35 0.08 4 11 14 2 8 86 4-4-Methoxy-phenylpropanoic4(4-Methoxy-Methoxyphenyl)butan-phenylpropanoic-2-one acid (Anisylacetone) methyl△ ester CC1111HH1414OO3 3 194178194 0.09 0.11 0.09 0.17 0.44 0.69 0.070.46 0.07 7 2,4- Di-tert—butylphenol △ C14H22O 206 0.67 0.17 0.21 0.39 0.66 0.26 0.35 0.08 (1(1SS,3a,3aRR,4,4SS,8a,8aSS))-4,8,8-trimethyl-9-methylenedecahydro--4,8,8-trimethyl-9-methylenedecahydro△ - 9 9 △ CC15HH24 204204 0.10 0.10 8 4-Methoxy1,41,4-methanoazulene--methanoazulenephenylpropanoic (Junipene) acid methyl ※ ester C11H1514O243 194 0.07 (1S,3aR,4S,8aS)-4,8,8-trimethyl-9-methylenedecahydro△ - 2-[(3R,5R,6R)-6,10-dimethylspiro[4.5]dec-9-en-3-yl] 15 24 109 2-[(3R,5R,6R)-6,10-dimethylspiro[4.5]dec-9-en-3-yl]※ CC15HH26O 222204 0.10 0.68 0.09 0.88 0.02 10 1,4-methanoazulene (Junipene)※ C H O 222 0.68 0.09 0.88 0.02 propanpropan-2-ol-2-ol (Agarospirol) 15 26 2-[(3R,5R,6R)-6,10-dimethylspiro[4.5]dec-9-en-3-yl] 2-[(1R,3S,4S)-4-ethenyl-4-methyl-3-prop-1-en-2- 15 26 1110 2-[(1R,3S,4S)-4-ethenyl-4-methyl-3-prop-1-en-※ C15H26O 222 0.36 0.68 0.09 0.88 0.02 11 yl cyclohexyl]propanpropan-2-ol (Agarospirol)-2-ol (Elemol) ※ C H O 222 0.36 2-yl cyclohexyl]propan-2-ol (Elemol) 15 26 2-[(1R,3S,4S)-4-ethenyl-4-methyl-3-prop-1-en-2- 2-[(2R,8S,8aR)-8,8a-dimethyl-2,3,4,4a,7,8-hexahydro- 15 26 1211 ※ C15H26O 222 0.140.36 0.51 0.08 0.94 0.06 2-[(2R,8S,8aR)-8,8a-dimethyl-2,3,4,4a,7,8-hexahydro-1yl cyclohexyl]propan-2-ol (Elemol) H -naphthalen-2-yl]※ 12 1H-naphthalen-2-yl]propan-2-ol [(-)-Jinkoh-eremol] C15H26O 222 0.14 0.51 0.08 0.94 0.06 2-[(2R,8S,8apropan-2-olR)-8,8a-dimethyl [(-)-Jinkoh-eremol]-2,3,4,4a,7,8-hexahydro- 2-[(3S,5R,8S)-3,8-dimethyl-1,2,3,4,5,6,7,8- 15 26 1312 ※ C15H26O 222 0.14 0.51 0.240.08 1.510.94 0.06 1H-naphthalenoctahydroazulen2-[(3S,5R-2,8-Syl]propan)-3,8-dimethyl-1,2,3,4,5,6,7,8-5-yl] propan-2-ol [(--2)--Jinkohol (Guaiol)-eremol] ※ 13 C15H26O 222 0.24 1.51 0.06 -octahydroazulen-5-yl]2-[(3S,5R,8S)-3,8-dimethyl propan-2-ol-1,2,3,4,5,6,7,8 (Guaiol)- (1aR,4S,4aS,7R,7aS,7bS)-1,1,4,7-tetramethyl-octahydro- 15 26 1413 ※ C15H26O 222 0.16 0.81 0.500.24 0.451.51 0.050.06 (1aR,4Soctahydroazulen1a,4aHS-,7cyclopropa[e]azulenR,7aS,7bS)-1,1,4,7-tetramethyl-octahydro-1a-5-yl] propan-4-ol -(Viridiflorol)2-ol (Guaiol) ※ H- 14 C H O 222 0.16 0.81 0.50 0.45 0.05 (1aR,4S,4acyclopropa[e]azulen-4-olS,7R,7aS,7bS)-1,1,4,7-tetramethyl (Viridiﬂorol)-octahydro- 15 26 2-Hydroxy-1,2,3-propanetricarboxylic acid 15 26 1514 ※ CC12HH20OO7 276222 1.59 0.69 1.01 0.16 0.81 0.50 0.45 0.05 1aH2-Hydroxy-1,2,3-propanetricarboxylic-cyclopropa[e]azulentriethyl ester (Triethyl-4-ol citrate)(Viridiflorol) 15 C H O 276 1.59 0.69 1.01 2-4Hydroxy-(4acid-Hydroxy triethyl-1,2,3-3-propanetricarboxylic ester-methoxyphenyl)butan (Triethyl citrate) acid- 12 20 7 15 C12H20O7 276 1.59 0.69 1.01 16 triethyl ester (Triethyl citrate) C11H14O3 194 1.71 0.11 4-(4-Hydroxy-3-methoxyphenyl)butan-2-one (Zingerone) 16 C H O 194 1.71 0.11 (1S,4R,5S4,6-(4R-)Hydroxy-4,7,72-one-trimethylspiro[bicyclo[-3- (Zingerone)methoxyphenyl)butan4△ 4.1.0]heptane- - 11 14 3 16 C1115H1422O3 194 1.71 0.11 17 ※ C H O 218 0.39 0.39 5,3′-cyclopentene]2-one- (Zingerone)1′-carbaldehyde (Vitrenal) 0 (1S,4R,5S,6R)-4,7,7-trimethylspiro[bicyclo[4.1.0]heptane-5,3△ - 17 (1S,4R,5S,6R)-4,7,7-trimethylspiro[bicyclo[0 4.1.0]heptane- C15H22O 218 0.39 0.39 3-(2-Hydroxypropan-2-yl)-6-methylspiro[4.5]dec-9- 15 22 1817 cyclopentene]-1 -carbaldehyde (Vitrenal) ※ C15H22O 218 0.22 0.340.39 0.360.39 0.58 0.26 5,3′-enecyclopentene]-10-carbaldehyde-1′-carbaldehyde (Neopetasane) (Vitrenal) ※ 3-(23-(2-Hydroxypropan-2-yl)-6-methylspiro[4.5]-Hydroxypropan-2-yl)-6-methylspiro[4.5]dec-9- 18 3-(2-Hydroxypropan-2-yl)-6-methylspiro[4.5]dec-9- C1515H2222O 218 0.22 0.34 0.36 0.58 0.26 1918 dec-9-ene-10-carbaldehyde (Neopetasane)※ CC15HH24OO2 236218 2.450.22 1.410.34 1.910.36 7.460.58 1.630.26 eneene--1010--carbaldehydecarbaldehyde (Neopetasane) (Baimuxinal) ※ 3-(2-Hydroxypropan-2-yl)-6-methylspiro[4.5] 20 3-(2-Hydroxypropan-2-yl)-6-methylspiro[4.5]dec☆ -9- C17H34O2 270 0.58 0.47 19 19 Methyl hexadecanoate (Methyl palmitate) CC1515HH2424OO2 2 236236 2.45 1.41 2.45 1.91 1.417.46 1.63 1.91 7.46 1.63 dec-9-ene-10-carbaldehydeene-10-carbaldehyde (Baimuxinal) (Baimuxinal) ※ 21 1-O-butyl 2-O-(2-methylpropyl)benzene-1,2- C16H23O4 278 0.47 0.08 0.47 0.38 0.51 20 20 Methyl hexadecanoate (Methyl pa palmitate)lmitate) ☆ CC1717HH3434OO2 2 270270 0.58 0.58 0.47 0.47 Molecules21 2018, 23, x; doi:1-O FOR-butyl1 -PEERO- 2-butylO REVIEW-(2-methylpropyl)benzene-1,2-dicarboxylate 2-O-(2 -methylpropyl)benzene-1,2- www.mdpi.com/journal/C16H23O4 molecules278 0.47 0.08 0.47 0.38 0.51 21 C H O 278 0.47 0.08 0.47 0.38 0.51 (Isobutyl phthalate) 4 16 23 4 Molecules 2018, 23, x; doi: FOR PEER REVIEW www.mdpi.com/journal/molecules Molecules 2018, 23, x FOR PEER REVIEW 3 of 28

Table 1. Volatile constituents of agarwood samples produced from the 1st to 12th months.

2022 Methyl hexadecanoateHexadecanoic acid(Methyl (Palmitic palmitate) acid) ☆ C17HC3416O2H 32O2270 256 0.58 0.27 0.27 1.85 0.47 3.27 2.59 21 1-O-butyl 2-O-(2-methylpropyl)benzene-1,2- C16H23O4 278 0.47 0.08 0.47 0.38 0.51 23 Propan-2-yl hexadecanoate (isopropyl palmitate) C19H38O2 298 1.94

Molecules24 2018, 23, x; doi: FOR PEERMethyl REVIEW octadecanoate (Methyl stearate) www.mdpi.com/journal/C19Hmolecules38O2 298 0.33 0.06 25 (Z)-octadec-9-enoic acid (Oleic acid) C18H34O2 282 0.62 6.50 0.65 4 26 1,5-Diphenyl-1-penten-3-one C17H16O 236 0.17 0.51

27 Methyl cis-9,10-epoxystearate C19H36O3 312 0.66 0.65

28 2-(2-Phenylethyl)chromone * C17H14O2 250 8.71 20.34 6.31 8.91 6.49 1.18 0.48 2.36 Bis(6-methylheptyl) benzene-1,2- 29 C H O 390 0.85 0.16 0.46 0.22 0.50 1.40 0.45 0.92 dicarboxylate (Diisooctyl phthalate) 4 24 38 4

30 5 or 6 or 7 or 8-Hydroxy-2-(2-phenylethyl)chromone * C17H14O3 266 1.02 0.94 0.59 0.39 0.40 0.22

31 5 or 6 or 7 or 8-Methoxy-2-(2-phenylethyl)chromone * C18H16O3 280 12.63 14.76 8.13 14.40 8.69 4.55 5.72 12.69

32 2-[2-(4-Methoxyphenyl)ethyl]chromone * C18H16O3 280 Overlapped with No. 31 5 or 6 or 7 or 8-Methoxy-2- 33 C H O 280 0.95 0.45 0.47 0.56 0.43 0.16 0.35 (2-phenylethyl)chromone * 18 16 3

34 2-(2-Phenylethyl)chromone: A ring: 1OH, 1OCH3 *C18H16O4 296 0.95 1.27 1.26 1.73 0.80 0.64 1.01 2.40

35 6,8-Dihydroxy-2-(2-phenylethyl)chromone * C17H14O4 282 1.22 4.48 2.55 1.83 1.13 1.04 1.44 1.73

36 5 or 6 or 7 or 8-Hydroxy-2-(2-phenylethyl)chromone * C17H14O3 266 9.28 21.60 12.10 8.09 4.25 5.20 3.03 6.13

37 2-(2-Phenylethyl)chromone: A ring: 1OH, 1OCH3 *C18H16O4 296 0.81 1.01 0.88 0.92 0.36 1.56

38 6-Methoxy-2-[2-(3-methoxyphenyl)ethyl]chromone * C19H18O4 310 4.15 3.35 1.84 3.29 3.34 2.80 1.85 2.25

39 6,7-Dimethoxy-2-(2-phenylethyl)chromone * C19H18O4 310 44.25 18.15 22.12 24.12 17.99 13.99 18.64 24.87

40 5,8-Dihydroxy-2-[2-(4-methoxyphenyl)ethyl]chromone * C18H16O5 312 0.43 0.60 0.57 7.37 0.82 2.73

41 2-(2-Phenylethyl)chromone: A ring: 1OH, 1OCH3 *C18H16O4 296 1.41 1.46 1.36 1.06 2.03 0.96 1.59

42 6-Hydroxy-2-[2-(4-methoxyphenyl)ethyl]chromone * C18H16O4 296 0.95 2.18 1.59 0.45 4.08 1.90 2.10

43 6,7-Dimethoxy-2-[2-(4-methoxyphenyl)ethyl]chromone * C20H20O5 340 1.74 1.26 2.97 2.15 2.24 3.96 5.63 3.96

44 Ergost-5-enol C28H48O 400 0.05 0.36 0.12 0.30 # 45 Stigmasterol C29H48O 412 2.62 0.92 3.48 1.66 1.96 1.27 3.52 5.03 # 46 Clionasterol C29H50O 414 1.76 0.30 2.80 1.23 1.50 1.75 2.42 2.60 # 47 5α-Stigmast-3-one C29H50O 414 0.05 0.28 0.11 # 48 Ergosta-4,6,8(14),22-tetraen-3-one C28H40O 392 0.80 0.25 0.81 # 49 (22E,24R)-Stigmasta-4,22-dien-3-one C29H46O 410 0.06 0.58 0.20 2.26 # Molecules 2018, 23, x FOR PEER REVIEW 3 of 28

Table 1. Volatile constituents of agarwood samples produced from the 1st to 12th months.

Molecular Relative Content (%) No. Compounds MW Formula 1 2 3 4 5 6 9 12 1 Nonanal C9H18O 142 0.85 0.89 1.16 0.22 2 4-Phenylbutan-2-one (Benzylacetone) C10H12O 148 1.02 0.46 0.17 0.09 0.21 0.14 0.11 3 Phenylpropanoic acid methyl ester △ C10H12O2 164 0.11 4 Phenylpropionic acid △ C9H10O2 150 1.80 0.19 0.09 0.03 0.12 5 2,6-Di-tert-butyl-2,5-cyclohexadiene△ -1,4-dione C14H20O2 220 0.04 0.12 6 4-(4-Methoxyphenyl)butan-2-one (Anisylacetone) C11H14O2 178 0.09 0.11 0.09 0.17 0.44 0.69 0.46 7 2,4- Di-tert—butylphenol △ C14H22O 206 0.67 0.17 0.21 0.39 0.66 0.26 0.35 0.08 8 4-Methoxy-phenylpropanoic acid methyl△ ester C11H14O3 194 0.07 (1S,3aR,4S,8aS)-4,8,8-trimethyl-9-methylenedecahydro△ - 9 C15H24 204 0.10 1,4-methanoazulene (Junipene) ※ 2-[(3R,5R,6R)-6,10-dimethylspiro[4.5]dec-9-en-3-yl] 10 C15H26O 222 0.68 0.09 0.88 0.02 propan-2-ol (Agarospirol) ※ 2-[(1R,3S,4S)-4-ethenyl-4-methyl-3-prop-1-en-2- 15 26 11 ※ C H O 222 0.36 Molecules 2018, 23, 1261 yl cyclohexyl]propan-2-ol (Elemol) 5 of 29 2-[(2R,8S,8aR)-8,8a-dimethyl-2,3,4,4a,7,8-hexahydro- 12 Molecules 2018, 23, x FOR PEER REVIEW C15H26O 222 0.14 0.51 0.08 0.94 0.06 3 of 28 1H-naphthalen-2-yl]propan-2-ol [(-)-Jinkoh-eremol] ※ 2-[(3S,5R,8S)-3,8-dimethylTable 1.-1,2,3,4,5,6,7,8Cont. - 15 26 13 ※ C H O 222 0.24 1.51 0.06 octahydroazulen-5-yl] propan-2-ol (Guaiol)Table 1. Volatile constituents of agarwood samples produced from the 1st to 12th months. Molecular Relative Content (%) No. Compounds(1aR,4S,4aS,7R,7aS,7bS)-1,1,4,7-tetramethyl-octahydroMW - 14 C15H26O 222 0.16 0.81 0.50 0.45 0.05 1aH-cyclopropa[e]azulen-4-olFormula (Viridiflorol) ※ 1 2 3 4 5 6Molecular 9 12 Relative Content (%) No. Compounds MW 50 Stigmast-4-en-3-one C29H48O 412 2.99 0.53 1.89 1.41 8.27 1.93Formula 2.21 0.96 1 2 3 4 5 6 9 12 # 2-Hydroxy-1,2,3-propanetricarboxylic acid 15 C12H20O7 276 1.59 0.69 1.01 51 Stigmasta-3,5-dien-7-one 1 C29H46O 410Nonanal 0.31C9H 0.9018O 142 0.85 0.89 1.16 0.22 # triethyl ester (Triethyl citrate) 52 (5α)-Stigmastane-3,6-dione2 4C-Phenylbutan29H48O2 428-2-one 0.81(Benzylacetone) 0.32 1.55 0.79 4.41C10H 0.9812O 0.45148 1.02 0.46 0.17 0.09 0.21 0.14 0.11 #4-(4-Hydroxy-3-methoxyphenyl)butan- 16 C11H14O3 194 1.71 0.11 13 22-one (Zingerone) 3Phenylpropanoic 4 acid methyl 5 ester △ 6 9C10H12O2 12 164 0.11 Aromatic compounds (No./RC, %)(1 3/S,4 1.99R,54S ,6R)- 7/4,7,7 3.50-trimethylspiro[bicyclo[ 7/2.74△ 6/1.86Phenylpropionic4.1.0]heptane- 7/3.47 acid △ 6/2.55 7/3.98C9H10O 8/1.922 150 1.80 0.19 0.09 0.03 0.12 17 C15H22O 218 0.39 0.39 Fatty acids/esters (No./RC, % ) 2/2.245,3′5 -cyclopentene] 2/0.97-1′ 4/2.20-carbaldehyde2,6-Di-tert 2/2.47-butyl (Vitrenal)-2,5-cyclohexadiene ※ 1/1.94△ -1,4-dione 2/9.76 \ C14H20O4/3.772 220 0.04 0.12 Steroids (No./RC, %) 4/8.183-(2-6Hydroxypropan 7/2.23-2 8/11.74-yl)4-(4-6--Methoxyphenyl)butanmethylspiro[4.5]dec 8/5.78-9-- 2-one 7/19.51 (Anisylacetone) 3/4.95 6/10.93C11H14O 5/9.342 178 0.09 0.11 0.09 0.17 0.44 0.69 0.46 15 22 18 ※ C H O 218 0.22 0.34 0.36 0.58 0.26 Sesquiterpenoids (No./RC, %) \\\7 ene-10-carbaldehyde (Neopetasane)2,47/3.74- Di- tert —butylphenol 6/4.14 7/3.57△ 6/11.38C14H22O 6/2.14 206 0.67 0.17 0.21 0.39 0.66 0.26 0.35 0.08 2-(2-phenylethyl)chromones (No./RC, %) 10/83.863-(2-8Hydroxypropan 13/89.46 -15/64.322-yl)4--6Methoxy-methylspiro[4.5]dec15/70.78-phenylpropanoic-9- 14/48.14 acid methyl△ ester 15/49.74 14/42.19C11H14O 13/64.593 194 0.07 19 C15H24O2 236 2.45 1.41 1.91 7.46 1.63 Identiﬁed compounds (No./RC, %) 19/96.28ene 30/96.16-10-carbaldehyde35/81.00(1S,3aR (Baimuxinal),4S,8aS39/84.62)-4,8,8 -※trimethyl -9 35/77.21-methylenedecahydro 33/70.58△ - 33/68.48 36/81.76 15 24 9 4 ※ C H 204 0.10 Note: MW = Molecular Weight,20 * 2-(2-phenylethyl)chromones,Methyl hexadecanoateSteroids, Aromatic (Methyl1,4 compounds,pa-methanoazulenelmitate) ☆ Fatty acids/esters,(Junipene) Sesquiterpenoids.C17H34O2 RC270 is the relative content. 0.58 0.47 # 21 1-O-butyl 2-O-(2-methylpropyl)benzene2-[(3R,5R,6R)-6,10-dimethylspiro[4.5]dec-1,2- -9-en-C316-yl]H23 O 4 278 0.47 0.08 0.47 0.38 0.51 10 C15H26O 222 0.68 0.09 0.88 0.02 propan-2-ol (Agarospirol) ※ Molecules 2018, 23, x; doi: FOR PEER REVIEW 2-[(1R,3S,4S)-4-ethenyl-4-methylwww.mdpi.com/journal/-3-prop-1-en-2- molecules 11 C15H26O 222 0.36 yl cyclohexyl]propan-2-ol (Elemol) ※ 2-[(2R,8S,8aR)-8,8a-dimethyl-2,3,4,4a,7,8-hexahydro- 12 C15H26O 222 0.14 0.51 0.08 0.94 0.06 1H-naphthalen-2-yl]propan-2-ol [(-)-Jinkoh-eremol] ※ 2-[(3S,5R,8S)-3,8-dimethyl-1,2,3,4,5,6,7,8- 13 C15H26O 222 0.24 1.51 0.06 octahydroazulen-5-yl] propan-2-ol (Guaiol) ※ (1aR,4S,4aS,7R,7aS,7bS)-1,1,4,7-tetramethyl-octahydro- 14 C15H26O 222 0.16 0.81 0.50 0.45 0.05 1aH-cyclopropa[e]azulen-4-ol (Viridiflorol) ※ 2-Hydroxy-1,2,3-propanetricarboxylic acid 15 C12H20O7 276 1.59 0.69 1.01 triethyl ester (Triethyl citrate) 4-(4-Hydroxy-3-methoxyphenyl)butan- 16 C11H14O3 194 1.71 0.11 2-one (Zingerone) (1S,4R,5S,6R)-4,7,7-trimethylspiro[bicyclo[△ 4.1.0]heptane- 17 C15H22O 218 0.39 0.39 5,3′-cyclopentene]-1′-carbaldehyde (Vitrenal) ※ 3-(2-Hydroxypropan-2-yl)-6-methylspiro[4.5]dec-9- 18 C15H22O 218 0.22 0.34 0.36 0.58 0.26 ene-10-carbaldehyde (Neopetasane) ※ 3-(2-Hydroxypropan-2-yl)-6-methylspiro[4.5]dec-9- 19 C15H24O2 236 2.45 1.41 1.91 7.46 1.63 ene-10-carbaldehyde (Baimuxinal) ※

20 Methyl hexadecanoate (Methyl palmitate) ☆ C17H34O2 270 0.58 0.47 21 1-O-butyl 2-O-(2-methylpropyl)benzene-1,2- C16H23O4 278 0.47 0.08 0.47 0.38 0.51

Molecules 2018, 23, x; doi: FOR PEER REVIEW www.mdpi.com/journal/molecules Molecules 2018, 23, 1261 6 of 29

These 52 compounds were classified into five types according to their structures, and the number and relative content of each type were counted and listed in Table1. Healthy A. sinensis mainly contained fatty acids, alkanes, and aromatic compounds, while GC-MS analysis of ether extracts of injured A. sinensis revealed the appearance of 2-(2-phenylethyl)chromones, sesquiterpenoids, steroids, fatty acids/esters, and aromatic compounds in the volatile constituents, in which 2-(2-phenylethyl)chromones were the major components, existing in large amounts in the sample of the 1st month after the injury, and their relative content still accounted for more than half of the total relative content in the sample of the 12th month. As one of the characteristic constituents in agarwood, sesquiterpenoids emerged relatively late, accompanied by much lower relative content. The reason why the relative content of sesquiterpenoid was not so high was probably because the wide variety of sesquiterpenoid skeletons made them difficult to be identified, or probably because the formation time for the samples obtained in this study was not long enough for the accumulation of sesquiterpenoids. Besides, unlike the results of previous GC-MS analysis of agarwood [13], there were some steroids detected in this study. This was probably because the column we used has the capability of high-temperature resistance, which enabled the volatilization and identification of steroids under the oven temperature of 310 ◦C for 10 min. Among those known compounds, clionasterol and stigmasterol as the major components of steroids in agarwood and also commonly distributed in other plants. Oleic acid and palmitic acid were the main constituents of the fatty acids, which could be the remaining compounds produced before agarwood formation [14,15]. It is noteworthy that some aromatic compounds were also detected, such as phenylpropionic acid, phenylpropionic acid methyl ester, and 4-methoxy-phenylpropionic acid methyl ester, which possibly have some relationship with the aromatic substrates of PECs biosynthesis, as well as 4-phenylbutan-2-one (benzylacetone), 4-(4-methoxyphenyl)butan-2-one (anisylacetone), and 4-(4-hydroxy-3-methoxy phenyl)butan-2-one (zingerone), which were frequently found in volatile oil of agarwood [1,13]. In conclusion, it was revealed by GC-MS analysis that sesquiterpenoids appeared relatively late and presented quite low relative content in the early stage of agarwood formation after transfusing the agarwood inducer to A. sinensis. On the contrary, the woods around the hole have produced plenty of PECs in the first month (total relative content was 83.86%), and among them, 6,7-dimethoxy-2-(2-phenylethyl)chromone was the dominant one with a relative content of 43.43%, which almost took up half of the total relative content of the sample in the first month. However, GC-MS is only suitable for volatile constituents and the identification via GC-MS can only be achieved within limited library searching. Therefore, given the fact that PECs compromised a high percentage of the identified compounds, while some nonvolatile PECs perhaps failed to be detected by GC-MS, UPLC-MS analysis was carried out to study the dynamic changes of PECs during the process of agarwood formation.

2.2. The Results of UPLC-MS Analysis Sixty-four 2-(2-phenylethyl)chromones (PECs) in total were detected by UPLC-MS from the obtained samples (Figure1, Table2). Among them, 30 compounds were identified by comparing their MS spectra with those of reference compounds (Table3) and the structures of others were deduced on the basis of their MS characterization, fragmentations patterns, and characteristic fragment peaks. According to the characteristic structure of chromone skeleton, 2-(2-phenylethyl)chromones (PECs) can be subdivided into four groups, i.e., 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones with two epoxys linked at the cyclohexane ring shortened as DEPECs, 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromones with single epoxy at the cyclohexane ring named as EPECs, 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromones which possess a highly oxygenated chromone skeleton are called THPECs, and 2-(2-phenylethyl)chromones of ﬂindersia type were abbreviated to FTPECs (Figure2). All of the 64 compounds detected in the collected samples were classiﬁed into the above four types, and statistical analysis of the number and total relative content of each group led us to know that FTPECs were the primary group with numerous diversities and highest relative content and THPECs as the next group, while EPECs and Molecules 2018, 23, 1261 7 of 29 Molecules 2018, 23, x FOR PEER REVIEW 7 of 28 Molecules 2018, 23, x FOR PEER REVIEW 7 of 28

DEPECs had limited numbers and comparatively low content (Table2). To the best of our knowledge, numbersnumbers andand comparativelycomparatively lowlow contentcontent (Table(Table 22)).. TToo thethe bestbest ofof ourour knowledge,knowledge, DEPECsDEPECs andand DEPECs and EPECs were quite rare in nature, and such nonaromatic chromone skeletons have only EPECsEPECs werewere quitequite rarerare inin nature,nature, andand suchsuch nonaromaticnonaromatic chromonechromone skeletonsskeletons havehave onlyonly beenbeen been identiﬁed in agarwood [16–20], and no other known chromones were found to have such an identifiedidentified inin agarwoodagarwood [16[16––2020],], andand nono otherother knownknown chromoneschromones werewere foundfound toto havehave suchsuch anan epoxyepoxy epoxy group in the chromone moiety except PECs. Also, no other plants except Aquilaria species has groupgroup inin thethe chromonechromone moietymoiety exceptexcept PECsPECs.. AAlsolso,, nono otherother plantsplants exceptexcept AquilariaAquilaria speciesspecies hashas beenbeen been reported to contain DEPECs and EPECs. reportedreported toto ccontainontain DEPECsDEPECs andand EPECs.EPECs.

th 1212th

th 99th

th 66th

th 55th

th 44th

rd 33rd

nd 22nd

st 11st

FigureFigureFigure 1.1. 1. TheTheThe UPLCUPLC UPLC spectrumspectrum spectrum ofof of agarwoodagarwood agarwood samplessamples samples producedproduced fromfrom thethe 11 1ststst toto 1212 12ththth months.months.

C O C O A A Phenylethyl moiety Phenylethyl moiety O O Chromone moiety Chromone moiety

FTPECs DEPECsDEPECs FTPECs O O O O O O

O O O O O O

THPECsTHPECs EPECsEPECs OH OH OH OH O HO O HO O O O HO O HO O O

HO HO HO O HO OH O O O or OH O O or OH O OH O Figure 2. The characteristic structures of four types of 2-(2-phenylethyl)chromones. FigFigureure 2 2.. TheThe characteristic characteristic structures structures of four types of 2 2-(2-phenylethyl)chromones.-(2-phenylethyl)chromones. Molecules 2018, 23, 1261 8 of 29

Table 2. The UPLC-MS analysis results of agarwood samples produced from the 1st to 12th months.

Molecular Relative Content (%) No Compounds MW Formula 1 2 3 4 5 6 9 12

1 THPECs: A ring: 4 OH groups, C ring: 1 OH, 1 OCH3 C18H20O8 364 2.27 7.89 5.78 3.11 S1: 5α,6α,7α,8β-Tetrahydroxy-2-[2-(3-hydroxy-4-methoxyphenyl) 2 C H O 364 1.43 ethyl]-5,6,7,8-tetarahydrochromone 18 20 8 S2: 5α,6β,7β,8α-Tetrahydroxy-2-[2-(3-hydroxy-4-methoxyphenyl) 3 C H O 364 0.78 1.57 2.48 0.22 ethyl]-5,6,7,8-tetarahydrochromone 18 20 8 S3: 5α,6β,7β,8α-Tetrahydroxy-2-[2-(4-methoxyphenyl)ethyl]-5,6,7,8- 4 C H O 348 8.27 9.13 7.47 10.56 8.26 14.73 6.05 5.11 tetrahydrochromone 18 20 7 S4: 5α,6β,7β,8α-Tetrahydroxy-2-[2-(4-methoxyphenyl)ethyl)]-5,6,7,8- 5 C H O 318 10.00 0.99 5.08 0.88 0.46 1.35 0.38 0.43 tetrahydrochromone (Agaroretrol) 17 18 6

6 THPECs: A ring: 4 OH groups, C ring: 1 OCH3 C18H20O7 348 0.10

7 THPECs: A ring: 4 OH groups C17H18O6 318 0.41 0.24 0.82 0.85 0.99 1.61 1.50 0.22

8 THPECs: A ring: 4 OH groups C17H18O6 318 1.79 4.65 2.60 4.65 4.63 6.90 2.70 3.71 S5: 5:6-Epoxy-7β,8α-dihydroxy-2-[2-(3-hydroxy-4-methoxyphenyl) 9 C H O 346 0.13 0.08 1.21 0.80 ethyl]-5,6,7,8-tetarahydrochromone 18 18 7

10 THPECs: A ring: 3 OCH3, 1 OH C20H24O6 360 0.07 0.28 0.20

11 THPECs: A ring: 3 OCH3, 1 OH, C ring: 1 OCH3 C18H20O8 390 0.26

12 THPECs: A ring: 3 OH, 1 OCH3 C18H20O6 332 0.11 S6: (5R,6R,7R,8R)-5,6 :7,8-Diepoxy-2-[2-(3-hydroxy-4-methoxyphenyl) 13 C H O 328 0.07 0.87 1.25 0.02 ethyl]-5,6,7,8-tetrahydrochromone (Oxidoagarochromone C) 18 16 6

14 DEPECs: C ring: 1 OCH3 C18H16O5 312 0.05 0.45 1.17 0 15 EPECs: 7 -OH C17H14O4 282 1.15

16 DEPECs: C ring: 1 OCH3, 1 OH C18H16O6 328 0.35 1.21 1.30 0.22

17 THPECs: A ring: 3 OH groups, 1 Cl, C ring: 1 OCH3 C18H19O6Cl 366 0.30

18 THPECs: A ring: 3 OH groups, 1 Cl C17H17O5Cl 336 1.28 0.16 1.08 0.12 0.51 0.19

19 S7: 6-Hydroxy-7-methoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone C18H16O5 312 0.46

20 FTPECs: A ring: 1 OH, 1 OCH3, C ring: 1 OH C18H16O5 312 0.38 0.04

21 FTPECs: A ring: 1 OH, 1 OCH3, C ring: 1 OH, 1 OCH3 C19H18O6 342 0.09

22 S8: 5:6-Epoxy-7β,8α-dihydroxy-2-[2-(4-methoxyphenyl)ethyl]chromone C18H18O6 330 0.05 0.12 0.20 2.80

23 EPECs: A ring: 2 OH groups, C ring: 1 OCH3 C18H18O6 330 0.18 0.16

24 FTPECs: A ring 1 OH, 1 OCH3, C ring: 1 OH, 1 OCH3 C19H18O6 342 0.85 0.95 2.24 1.60 Molecules 2018, 23, 1261 9 of 29

Table 2. Cont.

Molecular Relative Content (%) No Compounds MW Formula 1 2 3 4 5 6 9 12 S9: 7-hydroxy-6-methoxy2-[2-(3-hydroxy-4-methoxyphenyl)ethyl] 25 C H O 342 0.42 0.38 0.93 0.67 chromone 19 18 6

26 S10: 5:6-Epoxy-7β,8α-dihydroxy-2-(2-phenylethyl)chromone C17H16O5 300 1.57 2.12 1.52 0.65 1.12

27 THPECs: A ring: 3 OH groups, 1 Cl C17H17O5Cl 336 0.14 0.15 0.37 0.34

28 FTPECs: A ring: 1 OH, C ring: 1 OH, 1 OCH3 C18H16O5 312 1.35 1.57 2.55 1.67

29 DEPECs: C ring: 1 OCH3 C18H16O5 312 0.07

30 S11: 6-Hydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone C18H16O5 312 0.50 0.48 0.51 0.40

31 S12: 6,8-Dihydroxy-2-[2-(4-methoxyphenyl)ethyl]chromone C18H16O5 312 0.60 0.20 1.13 0.56 1.00

32 S13: 6,8-Dihydroxy-2-(phenylethyl)chromone C17H14O4 282 1.23 2.78 1.50 1.28 1.03 0.54 0.86

33 S14: 6,7-Dimethoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone C19H18O5 326 0.37 0.51

34 S15: 6-Hydroxy-2-[2-(2-hydroxyphenyl)ethyl]chromone C17H14O4 282 0.24 0.27 0.02 S16: (5R,6R,7R,8R)-5,6 :7,8-Diepoxy-2-[2-(4-methoxyphenyl)ethyl]- 35 C H O 312 0.16 0.14 0.76 1.06 1.87 1.81 0.39 1.01 5,6,7,8-tetrahydrochromone (Oxidoagarochromone B) 18 16 5 S17: (5R,6R,7R,8R)-5,6 :7,8-Diepoxy-2-(2-phenylethyl)- 36 C H O 282 5.08 5,6,7,8-tetrahydrochromone (Oxidoagarochromone A) 17 14 4

37 S18: 6,7-Dimethoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone C20H20O6 356 0.47 1.33

38 FTPECs: A ring: 2 OH groups C17H14O4 282 0.20 3.45 1.74 2.29 1.08 1.76 0.98 0.67

39 FTPECs: C ring: 1 OH C17H14O3 266 0.04 0.33 0.13 0.15 0.28 0.07

40 S19: 2-[2-(4-Hydroxy-3-methoxyphenyl)ethyl]chromone C18H16O4 296 0.43 0.32

41 FTPECs: A ring: 2 OCH3 groups, C ring: 1 OH C19H18O5 326 0.96 0.25 0.39

42 S20: 8-Hydroxy-2-(2-phenylethyl)chromone C17H14O3 266 1.02 0.88 1.07 1.06 0.59 0.61 0.80 0.65

43 FTPECs: A ring: 1 OH, 1 OCH3, C ring: 1 OCH3 C19H18O5 326 0.15 0.07 0.75 0.38 0.86 0.92 1.31 2.02

44 S21: 7-Hydroxy-6-methoxy2-[2-(4-methoxyphenyl)ethyl]chromone C19H18O5 326 0.32 0.73 0.64 0.73 0.20

45 FTPECs: A ring: 1 OH, 1 OCH3 C18H16O4 296 1.97 1.41 2.33 1.17 1.75 1.57 1.88 2.46

46 7-hydroxy-6-methoxy2-(2-phenylethyl)chromone C18H16O4 296 0.86 0.50 1.09 1.24 1.38 1.12 0.79 0.42

47 S22: 2-[2-(2-Hydroxyphenyl)ethyl]chromone C17H14O3 266 0.74 0.35 0.47 0.17

48 S23: 6-Methoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone C19H18O5 326 0.06 0.77 0.11

49 S24: 6-Hydroxy-2-[2-(4-methoxyphenyl)ethyl]chromone C18H16O4 296 0.52 1.06 3.58 2.57 7.90 4.46 2.56 5.81 Molecules 2018, 23, 1261 10 of 29

Table 2. Cont.

Molecular Relative Content (%) No Compounds MW Formula 1 2 3 4 5 6 9 12

50 S25: 6-Hydroxy-2-(2-phenylethyl)chromone C17H14O3 266 14.07 19.15 15.60 12.12 14.96 6.86 2.96 10.55 S26: 5-Hydroxy-6-methoxy-2-[2-(3-hydroxy-4- 51 C H O 342 0.25 0.45 0.14 methoxyphenyl)ethyl]chromone 19 18 6

52 FTPECs: A ring: 1 OH, C ring: 1OH C17H14O4 282 0.67 0.10 0.27 0.09

53 6,7-Dimethoxy-2-[2-(4-methoxyphenyl)ethyl]chromone C20H20O5 340 2.19 1.10 3.32 2.67 4.65 6.00 5.58 4.30

54 S27: 6,7-Dimethoxy-2-(2-phenylethyl)chromone C19H18O4 310 23.59 12.74 18.94 17.75 15.06 14.25 17.54 18.56

55 FTPECs: A ring: 2 OCH3 groups C19H18O4 310 0.20 0.25 0.43 3.29

56 FTPECs: A ring: 2 OH, C ring: 1 OCH3 C18H16O5 312 1.31 0.44

57 FTPECs: A ring: 2 OH groups C17H14O4 282 1.21 0.80 1.17 1.36

58 S28: 2-[2-(4-Methoxyphenyl)ethyl]chromone C18H16O3 280 0.27 0.39 0.53 0.38 1.60 0.51 0.46 0.62

59 S29: 2-(2-Phenylethyl)chromone C17H14O2 250 5.83 13.55 4.74 5.18 5.53 1.90 0.81 2.33

60 6-Methoxy-2-[2-(4-methoxyphenyl)ethyl]chromone C19H18O4 310 2.96 2.90 2.21 2.92 3.62 2.56 1.74 3.02

61 6-Methoxy-2-(2-phenylethyl)chromone C18H14O3 280 6.91 10.60 6.54 19.12 8.75 4.87 5.00 9.89

62 FTPECs: A ring: 2 OCH3 groups, 1 OH, C ring: 1 OCH3 C20H20O6 356 0.24 0.59 0.28

63 S30: 5-Hydroxy-6-methoxy-2-[2-(4-methoxyphenyl)ethyl]chromone C19H18O5 326 1.13 0.90 0.70 0.73 0.87 1.06 1.06 0.86

64 5-Hydroxy-6-methoxy-2-(2-phenylethyl)chromone C18H16O4 296 2.86 2.50 2.96 3.38 2.34 2.44 3.68 3.13 1 2 3 4 5 6 9 12 FTPECs 18/67.46 20/76.46 23/69.84 24/77.62 24/76.49 26/58.14 32/62.70 35/75.59 (n/RC, %) THPECs 7/21.96 7/15.60 6/17.42 7/17.60 6/17.39 7/34.56 7/20.32 11/13.76 (n/RC, %) EPECs 1/1.57 2/2.17 4/3.34 4/1.66 1/0.13 1/0.08 1/1.21 4/5.87 (n/RC, %) DEPECs 2/5.24 1/0.14 1/0.76 1/1.06 4/2.34 3/3.89 4/4.21 5/2.49 (n/RC, %) Total 28/96.23 30/94.37 34/91.36 36/97.94 35/96.35 37/96.67 44/88.44 55/97.71 (n/RC, %) Molecules 2018, 23, 1261 11 of 29 Molecules 2018,, 23,, xx FORFOR PEERPEER REVIEWREVIEW 11 of 28 Molecules 2018, 23, x FOR PEER REVIEW 11 of 28 Molecules 2018, 23, x FOR PEER REVIEW 11 of 28 Molecules 20182018, 23, x FOR PEER REVIEW 11 of 28 TableTable 3. Characterization 3. Characterization of 30 reference of 30 reference compounds compounds by using U byPLC/ESI using- UPLC/ESI-MS/MS.MS/MS. Table 3. Characterization of 30 reference compounds by using UPLC/ESI-MS/MS. Table 3.. Characterization of 30 reference compounds byby usingusing UPLC/ESIPLC/ESI--MS/MS.. Table 3. Characterization+ of 30 reference compounds by using UPLC/ESI-MS/MS. Table 3. Characterization[M + H]+ Fragment of 30 reference Ions compounds by using UPLC/ESI-MS/MS. No. Structure tR (min) [M + H]+ Fragment+ Ions Name No.No. Structure tR (min) tR (min) [M + H] [M++ + Fragment H] (m/z) Ions Fragment Ions (m/z)Name Name No. Structure tR (min) [M[M(m ++/ zH]H]) + FragmentFragment(m/z) IonsIons Name No. Structure ttRR (min)(min) [M(m +/ zH]) + Fragment(m/z) Ions Name No. Structure tR (min) [M(m +/ zH]) Fragment(m/z) Ions Name No. Structure OCH3 tR (min) (m/z) (m/z) Name OH OCH3 (m/z) (m/z) OH OCH3 (m/z) (m/z) HO OH O OCHOCH3 HO OHOH O OHOCH3 5α,6α,7α,8β-Tetrahydroxy5α,6-2α-[2,7-α(3,8-βhydroxy-Tetrahydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]--4-methoxyphenyl)ethyl]- HO OH 3 5α,6α,7α,8β-Tetrahydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]- S1 S1 OH O OCH3 9.6 9.6365 365347, 329 5α,6α,7α,8β 347, 329 -Tetrahydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]- S1 HOHO OH OO OH 9.6 365 347, 329 5α,6α,7α,8β-Tetrahydroxy-2-[2-(3-hydroxy-5,6,7,8-tetarahydrochromone4-methoxyphenyl)ethyl]- S1 HO O OHOH 9.6 365 347, 329 5α,6α,7α,8β-Tetrahydroxy5,6,7,8--tetarahydrochromone2-[2-(3-hydroxy-4-methoxyphenyl)ethyl] - S1S1 HO O OH 9.69.6 365365 347347,, 329329 5α,6α,7α,8β-Tetrahydroxy5,6,7,8--tetarahydrochromone2-[2-(3-hydroxy-4-methoxyphenyl)ethyl] - S1 HO OH O OH 9.6 365 347, 329 5α,6α,7α,8β-Tetrahydroxy5,6,7,8--tetarahydrochromone2-[2-(3-hydroxy-4-methoxyphenyl)ethyl] - S1 HOHO OH O 9.6 365 347, 329 5,6,7,8-tetarahydrochromone HO OH O 5,6,7,8-tetarahydrochromone HO OHOH OO 5,6,7,8-tetarahydrochromone OH O OH O OCH3 OH OCH3 OH OCH3 HO OH O OCHOCH3 HO OH O 3 5α,6β,7β,8α-Tetrahydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]- OH OHOCH3 5α,6β,7β,8α-Tetrahydroxy5α,6-2β-[2,7-β(3,8-hydroxyα-Tetrahydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]--4-methoxyphenyl)ethyl]- S2 HO OH O OHOCH3 9.9 365 347, 329, 301 S2 S2 HOHO OH OO OH 9.9 9.9365 365347, 329, 301 347,5α,6β,7β,8α 329, 301 -Tetrahydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]- S2 HO O OHOH 9.9 365 347, 329, 301 5α,6β,7β,8α-Tetrahydroxy5,6,7,8--tetarahydrochromone2-[2-(3-hydroxy-5,6,7,8-tetarahydrochromone4-methoxyphenyl)ethyl] - S2S2 HO O OH 9.99.9 365365 347347,, 329,329, 301301 5α,6β,7β,8α-Tetrahydroxy5,6,7,8--tetarahydrochromone2-[2-(3-hydroxy-4-methoxyphenyl)ethyl] - S2 HO OH O OH 9.9 365 347, 329, 301 5α,6β,7β,8α-Tetrahydroxy5,6,7,8--tetarahydrochromone2-[2-(3-hydroxy-4-methoxyphenyl)ethyl] - S2 HOHO OH O 9.9 365 347, 329, 301 5,6,7,8-tetarahydrochromone HO OH O 5,6,7,8-tetarahydrochromone HO OHOH OO 5,6,7,8-tetarahydrochromone OH O OH O OCH3 OH OCH3 OH OCH3 HO OH O OCH3 HO OH OCH3 5α,6β,7β,8α-Tetrahydroxy-2-[2-(4-methoxyphenyl)ethyl]- OH O OCH3 5α,6β,7β,8α-Tetrahydroxy-2-[2-(4-methoxyphenyl)ethyl]- S3 HO OH O OCH3 13.0 349 331, 137 α β β α S3 HOHO OH OO 13.0 349 331, 137 5α,6β,7β,8α-Tetrahydroxy5 ,6-2-[2,7-(4,8-methoxyphenyl)ethyl]-Tetrahydroxy-2-[2-(4-methoxyphenyl)ethyl]-- S3S3 HO O 13.013.0 349 349331, 137 331, 1375α,6β,7β,8α-Tetrahydroxy5,6,7,8-tetarahydrochromone-2-[2-(4-methoxyphenyl)ethyl] - S3S3 HO O 13.013.0 349349 331331,, 137137 5α,6β,7β,8α-Tetrahydroxy5,6,7,8-tetarahydrochromone-2-[2-(4-methoxyphenyl)ethyl]5,6,7,8-tetarahydrochromone - S3 HO OH O 13.0 349 331, 137 5α,6β,7β,8α-Tetrahydroxy5,6,7,8-tetarahydrochromone-2-[2-(4-methoxyphenyl)ethyl] - S3 HOHO OH O 13.0 349 331, 137 5,6,7,8-tetarahydrochromone HO OH O 5,6,7,8-tetarahydrochromone HO OHOH OO 5,6,7,8-tetarahydrochromone OH O OH OHO OH HO OH O HO OHOH O 5α,6β,7β,8α-Tetrahydroxy-2-[2-(4-methoxyphenyl)ethyl)]- S4 HO OH O 14.1 319 301, 283 5α,6β,7β,8α-Tetrahydroxy-2-[2-(4-methoxyphenyl)ethyl)]- S4 HOHO OH OO 14.1 319 301, 283 5α,6β,7β,8α-Tetrahydroxyα-2β-[2-β(4-methoxyphenyl)ethyl)]α - S4 HO O 14.1 319 301, 283 5α,6β,7β,8α5α,6β,7β,8α5,6,7,8--Tetrahydroxy-tetrahydrochromone5 ,6--22--[2[2,7--(4(4,8--methoxyphenyl)ethyl)]-Tetrahydroxy-2-[2-(4-methoxyphenyl)ethyl)]- (Agaroretrol) -- S4 S4S4 HO O 14.114.114.1 319319 319301301,, 283283 301,5α,6β,7β,8α 283 5,6,7,8-Tetrahydroxy-tetrahydrochromone-2-[2-(4-methoxyphenyl)ethyl)] (Agaroretrol) - S4 HO OH O 14.1 319 301, 283 5α,6β,7β,8α5,6,7,8-Tetrahydroxy-tetrahydrochromone-2-[2-(45,6,7,8-tetrahydrochromone-methoxyphenyl)ethyl)] (Agaroretrol) - (Agaroretrol) HOHO OH O 5,6,7,85,6,7,8--tetetrahydrochromonetrahydrochromone ((Agaroretrol)) S4 HO OH O 14.1 319 301, 283 5,6,7,8-tetrahydrochromone (Agaroretrol) HO OHOH OO 5,6,7,8-tetrahydrochromone (Agaroretrol) OH O OH O OCH 3 OH OCH 3 OH OCH3 HO OH O OCH3 HO OH OCH3 5:6-Epoxy-7β,8α-dihydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]- OH O OHOCH3 5:6-Epoxy-7β,8α-dihydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]- S5 HO OH O OHOCH3 18.4 347 329, 301 S5 HOHO OH OO OH 18.4 347 329, 301 5:6-Epoxy-7β,8α-dihydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]- S5 HO O OHOH 18.4 347 329, 301 5:6-Epoxy-7β,8α-dihydroxy5,6,7,85:6-Epoxy-7-tetarahydrochromone-2-[2-(3-βhydroxy,8α-dihydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]--4-methoxyphenyl)ethyl] - S5S5 HO O O OH 18.418.4 347347 329,329, 301301 5:6-Epoxy-7β,8α-dihydroxy5,6,7,8-tetarahydrochromone-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl] - S5 S5 O O OH 18.418.4 347 347329, 301 5:6 329,-Epoxy 301-7β,8α-dihydroxy5,6,7,8-tetarahydrochromone-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl] - S5 O O 18.4 347 329, 301 5,6,7,8-tetarahydrochromone5,6,7,8-tetarahydrochromone OO O 5,6,7,8-tetarahydrochromone O OO 5,6,7,8-tetarahydrochromone O O O OCH3 OCH3 O OCH3 O O OCH33 O O OHOCH3 (5R,6R,7R,8R)-5,6 :7,8-Diepoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]- S6 OO O OHOCH3 23.4 329 301, 137 (5R,6R,7R,8R)-5,6 :7,8-Diepoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]- S6 O OO OH 23.4 329 301, 137 (5R,6R,7R,8R)-5,6 :7,8-Diepoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]- S6 O O OHOH 23.4 329 301, 137 (5R,6R,7R5,6,7,8,8R)-5,6-tetrahydrochromone :7,8-Diepoxy-2-[2-(3 (-Oxidoagarochromonehydroxy-4-methoxyphenyl)ethyl] C) - S6 O OH 23.4 329 301, 137 (5R,6R,7R5,6,7,8,8R)-5,6-tetrahydrochromone :7,8-Diepoxy(5R,6R,7-2R-,8[2R-(3)-5,6 (-Oxidoagarochromonehydroxy :7,8-Diepoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]--4-methoxyphenyl)ethyl] C) - S6 O OH 23.4 329 301, 137 (5R,6R,7R5,6,7,8,8R)-5,6-tetrahydrochromone :7,8-Diepoxy-2-[2-(3 (-Oxidoagarochromonehydroxy-4-methoxyphenyl)ethyl] C) - S6 S6 O O 23.423.4 329 329301, 137 301, 1375,6,7,85,6,7,8--tetrahydrochromonetetrahydrochromone ((Oxidoagarochromone C)) S6 O O 23.4 329 301, 137 5,6,7,8-tetrahydrochromone5,6,7,8-tetrahydrochromone (Oxidoagarochromone (OxidoagarochromoneC) C) OO O 5,6,7,8-tetrahydrochromone (Oxidoagarochromone C) O OO O O O OH OH OH H CO O OHOH H 3CO O OH H3 OH S7 H3CO O 30.0 313 207, 107 6-Hydroxy-7-methoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone S7 H33CO O 30.0 313 207, 107 6-Hydroxy-7-methoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone S7 H3HOCO O 30.0 313 207, 107 6-Hydroxy-7-methoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone S7S7 H3HOCO O 30.030.0 313313 207207,, 107107 66--Hydroxy--77--methoxy--22--[2[2--(4(4--hydroxyphenyl)ethyl]chromonehydroxyphenyl)ethyl]chromone S7 HO O 30.0 313 207, 107 6-Hydroxy-7-methoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone S7 S7 HOHO O 30.030.0 313 313207, 107 207,6-Hydroxy 107-7-methoxy-2 6-Hydroxy-7-methoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone-[2-(4-hydroxyphenyl)ethyl]chromone HO O HO OO O O Molecules 2018, 23, 1261 12 of 29

Table 3. Cont. Molecules 2018, 23, x FOR PEER REVIEW 12 of 28 MoleculesMolecules 20182018,,, 2323,,, xxx FORFORFOR PEERPEERPEER REVIEWREVIEWREVIEW 1212 ofof 2828 Molecules 2018, 23, x FOR PEER REVIEW + 12 of 28 MoleculesMolecules 20182018No.,, 2323,, xx FORFOR PEERPEER StructureREVIEWREVIEW tR (min) [M + H] (m/z) Fragment Ions (m/z) Name 1212 ofof 2828

OCH OCH333 OHOH OCH3 OH OCH3 HO OH O OCHOCH3 HOHO OHOH OO 3 HO O β α S8S8S8 HO 32.732.732.7 331331 331313313,,, 121121 5:65:6 313,--EpoxyEpoxy 121--77ββ,8,8,8αα--dihydroxydihydroxy 5:6-Epoxy-7--22--[2[2--(4(4--methoxyphenyl)methoxyphenyl),8 -dihydroxy-2-[2-(4-methoxyphenyl) ethyl]chromoneethyl]chromone ethyl]chromone S8S8 HO OO 32.732.7 331331 313313,, 121121 5:65:6--EpoxyEpoxy--77ββ,8,8αα--dihydroxydihydroxy--22--[2[2--(4(4--methoxyphenyl)methoxyphenyl) ethyl]chromoneethyl]chromone S8S8 O 32.732.7 331331 313313,, 121121 5:65:6--EpoxyEpoxy--77ββ,8,8αα--dihydroxydihydroxy--22--[2[2--(4(4--methoxyphenyl)methoxyphenyl) ethyl]chromoneethyl]chromone OO O O OO OO O OO OCH3 OCH33 OCH3 OCH3 HOHO OO OCHOCH3 6-Methoxy-7-hydroxy-2-[2-(3-hydroxy-4-methoxyphenyl) HO O OHOH 3 6-Methoxy-7-hydroxy-2-[2-(3-hydroxy-4-methoxyphenyl) S9S9S9 HO O OHOH 32.832.832.8 343343 343137137 13766--MethoxyMethoxy--77--hydroxyhydroxy6-Methoxy-7-hydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)--22--[2[2--(3(3--hydroxyhydroxy--44--methoxyphenyl)methoxyphenyl) ethyl]chromone S9 HOHO OO OH 32.8 343 137 6-Methoxy-7-hydroxyethyl]chromone-2-[2-(3-hydroxy -4-methoxyphenyl) S9 HH3COCO OH 32.8 343 137 6-Methoxy-7-hydroxyethyl]chromone-2-[2-(3-hydroxy -4-methoxyphenyl) S9 H33COCO OH 32.8 343 137 ethyl]chromone S9 3 O 32.8 343 137 ethyl]chromone H3CO O ethyl]chromone H3CO O ethyl]chromone H3CO O OO OH OHOH HOHO OH O HOHO OH OO HO OH O S10S10S10 HO O 33.433.433.4 301301 301283,283, 255255 283, 2555:65:6--EpoxyEpoxy--77ββ,8,8,8αα--dihydroxydihydroxy 5:6-Epoxy-7--22--(2(2--phenylethyl)chromonephenylethyl)chromoneβ,8α-dihydroxy-2-(2-phenylethyl)chromone S10S10 HO O 33.433.4 301301 283,283, 255255 5:65:6--EpoxyEpoxy--77ββ,8,8αα--dihydroxydihydroxy--22--(2(2--phenylethyl)chromonephenylethyl)chromone S10S10 OO 33.433.4 301301 283,283, 255255 5:65:6--EpoxyEpoxy--77ββ,8,8αα--dihydroxydihydroxy--22--(2(2--phenylethyl)chromonephenylethyl)chromone O OO O O OO O OO OCH3 OCH33 OCH3 OCH3 OO OCHOCH3 O OHOH 3 S11S11 O OHOH 36.636.6 313313 137137 66--HydroxyHydroxy--22--[2[2--(3(3--hydroxyhydroxy--44--methoxyphenyl)ethyl]chromonemethoxyphenyl)ethyl]chromone S11S11 OO OH 36.636.6 313 313137 6 137-Hydroxy-2-[2-(3-hydroxy 6-Hydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone-4-methoxyphenyl)ethyl]chromone S11 HOHO OH 36.6 313 137 6-Hydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone S11 HOHO OH 36.6 313 137 6-Hydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone S11 HO OO 36.6 313 137 6-Hydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone HO OO HO O O O OCH3 OCH33 OHOH OCH3 OH OCH3 OH OO OCH3 OH OO OCH3 S12S12 OH O 38.238.2 313313 121121 6,86,8--DihydroxyDihydroxy--22--[2[2--(4(4--methoxyphneyl)ethyl]chromonemethoxyphneyl)ethyl]chromone S12 OO 38.2 313 121 6,8-Dihydroxy-2-[2-(4-methoxyphneyl)ethyl]chromone S12S12 HOHO 38.238.2 313 313121 1216,8-Dihydroxy-2-[2-(4-methoxyphneyl)ethyl]chromone 6,8-Dihydroxy-2-[2-(4-methoxyphneyl)ethyl]chromone S12 HOHO 38.2 313 121 6,8-Dihydroxy-2-[2-(4-methoxyphneyl)ethyl]chromone S12 HO OO 38.2 313 121 6,8-Dihydroxy-2-[2-(4-methoxyphneyl)ethyl]chromone HO OO HO O O OHO OHOH OH O OH OO S13 OH O 39.3 283 192, 91 6,8-Dihydroxy-2-(phenylethyl)chromone S13S13 OO 39.339.3 283283 192192,,, 9191 6,86,8--DihydroxyDihydroxy--22--(phenylethyl)chromone(phenylethyl)chromone S13 HOHO 39.3 283 192, 91 6,8-Dihydroxy-2-(phenylethyl)chromone S13 HOHO 39.3 283 192, 91 6,8-Dihydroxy-2-(phenylethyl)chromone S13S13 HO O 39.339.3 283 283192, 91 192, 916,8-Dihydroxy-2-(phenylethyl)chromone 6,8-Dihydroxy-2-(phenylethyl)chromone HO OO HO O OO OHOH OHOH OH H3CO O OH H33CO O OH H3CO O S14S14 H3CO O 40.540.5 327327 221,221, 107107 6,76,7--DimethoxyDimethoxy--22--[2[2--(4(4--hydroxyphenyl)ethyl]chromonehydroxyphenyl)ethyl]chromone S14 HH3COCO OO 40.5 327 221, 107 6,7-Dimethoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone S14 HH33COCO 40.5 327 221, 107 6,7-Dimethoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone S14 H33COCO 40.5 327 221, 107 6,7-Dimethoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone S14S14 3 O 40.540.5 327 327221, 107 221, 1076,7-Dimethoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone 6,7-Dimethoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone H3CO O H3CO O H3CO O OO

O OO S15 O 41.0 283 177, 107 6-Hydroxy-2-[2-(2-hydroxyphenyl)ethyl]chromone S15S15 OO OHOH 41.041.0 283283 177,177, 107107 66--HydroxyHydroxy--22--[2[2--(2(2--hydroxyphenyl)ethyl]chromonehydroxyphenyl)ethyl]chromone S15 HOHO OH 41.0 283 177, 107 6-Hydroxy-2-[2-(2-hydroxyphenyl)ethyl]chromone S15 HOHO OH 41.0 283 177, 107 6-Hydroxy-2-[2-(2-hydroxyphenyl)ethyl]chromone S15 HO O OHOH 41.0 283 177, 107 6-Hydroxy-2-[2-(2-hydroxyphenyl)ethyl]chromone HO OO HO O OO Molecules 2018, 23, x FOR PEER REVIEW 12 of 28

OCH OH 3 HO O S8 32.7 331 313, 121 5:6-Epoxy-7β,8α-dihydroxy-2-[2-(4-methoxyphenyl) ethyl]chromone

O O

OCH3 HO O 6-Methoxy-7-hydroxy-2-[2-(3-hydroxy-4-methoxyphenyl) S9 OH 32.8 343 137 H3CO ethyl]chromone O

OH HO O S10 33.4 301 283, 255 5:6-Epoxy-7β,8α-dihydroxy-2-(2-phenylethyl)chromone O O

OCH3 O S11 OH 36.6 313 137 6-Hydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone HO O OCH OH 3 O S12 38.2 313 121 6,8-Dihydroxy-2-[2-(4-methoxyphneyl)ethyl]chromone HO O

OH O S13 39.3 283 192, 91 6,8-Dihydroxy-2-(phenylethyl)chromone Molecules 2018, 23, 1261HO 13 of 29 O

H3CO O Table 3. Cont. S14 40.5 327 221, 107 6,7-Dimethoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone H3CO O + No. Structure tR (min) [M + H] (m/z) Fragment Ions (m/z) Name

S15S15 OH 41.041.0 283 283177, 107 177, 1076-Hydroxy-2-[2-(2-hydroxyphenyl)ethyl]chromone 6-Hydroxy-2-[2-(2-hydroxyphenyl)ethyl]chromone HO MoleculesMolecules 20182018,,, 2323,,, xxx FORFORFOR PEERPEERPEER REVIEWREVIEWREVIEW 1313 ofof 2828 MoleculesMolecules 2018 20182018,, , 23 2323,, , x xx FOR FORFOR PEER PEERPEER REVIEW REVIEWREVIEWO 131313 ofofof 282828

OCH OCHOCH3 OCHOCH33 O OCH33 OO O OO OO (5(5RR,6,6RR,7,7RR,8,8RR))--5,65,6 :7,8:7,8--DiepoxyDiepoxy(5R,6R,7--22R--[2[2,8-R-(4(4)-5,6--methoxyphenyl)ethyl]methoxyphenyl)ethyl] :7,8-Diepoxy-2-[2-(4-methoxyphenyl)ethyl]--- S16S16S16 OO 42.342.342.3 313313 313285,285, 121121 285,(5(5(5RR 121,6,6,6RR,7,7,7RR,8,8,8RR)))---5,65,65,6 :7,8 :7,8:7,8---DiepoxyDiepoxy---222---[2[2[2---(4(4(4---methoxyphenyl)ethyl]methoxyphenyl)ethyl]--- S16S16S16 42.342.342.3 313313313 285,285,285, 121 121121 5,6,7,85,6,7,8--tetrahydrochromonetetrahydrochromone5,6,7,8-tetrahydrochromone ((OxidoagarochromoneOxidoagarochromone B (OxidoagarochromoneB)) B) O 5,6,7,85,6,7,8--tetrahydrochromonetetrahydrochromone ( (OxidoagarochromoneOxidoagarochromone B B)) OO O 5,6,7,8-tetrahydrochromone (Oxidoagarochromone B) OO OO OO

O O O OO OO O OO (5(5RR,6,6RR,7,7RR,8,8RR))--5,65,6 :7,8:7,8--DiepoxyDiepoxy(5R,6R,7--22R--(2(2,8-R-phenylethyl)phenylethyl))-5,6 :7,8-Diepoxy-2-(2-phenylethyl)--- S17S17S17 O 42.742.742.7 283283 283255255 255 (5(5(5RR,6,6,6RR,7,7,7RR,8,8,8RR)))---5,65,65,6 :7,8 :7,8:7,8---DiepoxyDiepoxy---222---(2(2(2---phenylethyl)phenylethyl)phenylethyl)--- S17S17S17 42.742.742.7 283283283 255255255 5,6,7,85,6,7,8--tetrahydrochromonetetrahydrochromone5,6,7,8-tetrahydrochromone ((OxidoagarochromoneOxidoagarochromone A (OxidoagarochromoneA)) A) O 5,6,7,85,6,7,8--tetrahydrochromonetetrahydrochromone ( (OxidoagarochromoneOxidoagarochromone A A)) OO O 5,6,7,8-tetrahydrochromone (Oxidoagarochromone A) OO OO OO OCH OCHOCH3 OCHOCH33 OCH33 H3CO O H3CO O OH HH3COCO OO OHOH S18 H33CO O OH 42.9 357 137 6,7-Dimethoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl] chromone S18S18 OHOH 42.942.9 357 357137 6,7 137-Dimethoxy-2-[2-(3-hydroxy 6,7-Dimethoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]-4-methoxyphenyl)ethyl] chromone chromone S18S18 H CO 42.942.9 357357 137137 6,76,7--DimethoxyDimethoxy--22--[2[2--(3(3--hydroxyhydroxy--44--methoxyphenyl)ethyl]methoxyphenyl)ethyl] chromone chromone S18 HH3COCO 42.9 357 137 6,7-Dimethoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl] chromone HH33COCO H33CO O OO OO OH OHOH OHOH O OO OO OCH3 S19 O OCHOCH3 47.7 297 161, 137 2-[2-(4-hydroxy 3-methoxyphenyl)ethyl]chromone S19 OCH33 47.7 297 161, 137 2-[2-(4-hydroxy 3-methoxyphenyl)ethyl]chromone S19S19S19S19 OCH3 47.747.747.747.7 297297297 297161,161,161, 137 137137 161, 137222---[2[2[2---(4(4(4---hydroxyhydroxyhydroxy 3 33---mmethoxyphenyl)ethyl]chromoneethoxyphenyl)ethyl]chromoneethoxyphenyl)ethyl]chromone 2-[2-(4-hydroxy 3-methoxyphenyl)ethyl]chromone O OO OO OH OHOH OHOH O OO OO S20S20 O 49.249.2 267267 9191,, 176176 88--HydroxyHydroxy--22--(2(2--phenylethyl)chromonephenylethyl)chromone S20S20S20S20 49.249.249.249.2 267267267 267919191,, , 176 176176 91, 176888---HydroxyHydroxy---222---(2(2(2---phenylethyl)chromonephenylethyl)chromonephenylethyl)chromone 8-Hydroxy-2-(2-phenylethyl)chromone O OO OO OCH OCHOCH3 OCHOCH33 OCH33 HO O HOHO OO S21S21 HOHO OO 51.451.4 327327 121121 77--HydroxyHydroxy--66--mmethoxy2ethoxy2--[2[2--(4(4--methoxyphenyl)ethyl]chromonemethoxyphenyl)ethyl]chromone S21S21 H CO 51.451.4 327327 121121 77--HydroxyHydroxy--66--mmethoxy2ethoxy2--[2[2--(4(4--methoxyphenyl)ethyl]chromonemethoxyphenyl)ethyl]chromone S21S21 HH3COCO 51.451.4 327 327121 7 121-Hydroxy-6-methoxy2- 7-Hydroxy-6-methoxy2-[2-(4-methoxyphenyl)ethyl]chromone[2-(4-methoxyphenyl)ethyl]chromone HH33COCO H33CO O OO OO

O OO S22 OO 54.1 267 161 2-[2-(2-Hydroxyphenyl)ethyl]chromone S22S22 OH 54.154.1 267267 161161 22--[2[2--(2(2--Hydroxyphenyl)ethyl]chromone S22S22 OHOH 54.154.1 267267 161161 22--[2[2--(2(2--Hydroxyphenyl)ethyl]chromoneHydroxyphenyl)ethyl]chromone S22 OHOH 54.1 267 161 2-[2-(2-Hydroxyphenyl)ethyl]chromone O OO OO OCH OCHOCH3 OCHOCH33 OCH33 O OO OH S23 OO OHOH 54.4 327 137 6-Methoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone S23 OHOH 54.4 327 137 6-Methoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone S23S23 H CO 54.454.4 327327 137137 66--MethoxyMethoxy--22--[2[2--(3(3--hydroxyhydroxy--44--methoxyphenyl)ethyl]chromonemethoxyphenyl)ethyl]chromone S23 HH3COCO 54.4 327 137 6-Methoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone HH33COCO H33CO O OO OO Molecules 2018, 23, x FOR PEER REVIEW 13 of 28

OCH OCH3 O O (5R,6R,7R,8R)-5,6 :7,8-Diepoxy-2-[2-(4-methoxyphenyl)ethyl]- S16 42.3 313 285, 121 5,6,7,8-tetrahydrochromone (Oxidoagarochromone B) O O

O O (5R,6R,7R,8R)-5,6 :7,8-Diepoxy-2-(2-phenylethyl)- S17 42.7 283 255 5,6,7,8-tetrahydrochromone (Oxidoagarochromone A) O O

OCH OCH3 H CO O H3CO O OH S18 OH 42.9 357 137 6,7-Dimethoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl] chromone H CO H3CO O

OH O O OCH S19 OCH3 47.7 297 161, 137 2-[2-(4-hydroxy 3-methoxyphenyl)ethyl]chromone

OH O S20 49.2 267 91, 176 8-Hydroxy-2-(2-phenylethyl)chromone Molecules 2018, 23, 1261 14 of 29 O

OCH OCH3 HO O Table 3. Cont. S21 51.4 327 121 7-Hydroxy-6-methoxy2-[2-(4-methoxyphenyl)ethyl]chromone H CO H3CO O + No. Structure tR (min) [M + H] (m/z) Fragment Ions (m/z) Name

O S22S22 54.154.1 267 267161 1612-[2-(2-Hydroxyphenyl)ethyl]chromone 2-[2-(2-Hydroxyphenyl)ethyl]chromone S22 OH 54.1 267 161 2-[2-(2-Hydroxyphenyl)ethyl]chromone O

OCH OCH3 O O OH S23S23 OH 54.454.4 327 327137 6 137-Methoxy-2-[2-(3-hydroxy 6-Methoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone-4-methoxyphenyl)ethyl]chromone H CO MoleculesMolecules 2018 2018, ,23 23, ,x x FOR FORH3CO PEER PEER REVIEW REVIEW 1414 ofof 2828 MoleculesMolecules 20182018,, 2323,, xx FORFOR PEERPEER REVIEWREVIEWO 1414 ofof 2828 Molecules 2018, 23, x FOR PEER REVIEWO 14 of 28

OCHOCH OCH33 OCH33 OCH3 OO OO S24S24S24S24 O 55.655.655.655.6 297297297 297121121121 12166-6-Hydroxy-HydroxyHydroxy--2-2-2-[2-[2[2--(4-(4(4--methoxyphenyl)ethyl]chromone-methoxyphenyl)ethyl]chromonemethoxyphenyl)ethyl]chromone 6-Hydroxy-2-[2-(4-methoxyphenyl)ethyl]chromone S24 HOHO 55.6 297 121 6-Hydroxy-2-[2-(4-methoxyphenyl)ethyl]chromone HOHO HO OO OO O

OO OO S25S25S25S25 O 57.357.357.357.3 267267267 267919191 9166-6-Hydroxy-HydroxyHydroxy--2-2-2-(2-(2(2--phenylethyl)chromone-phenylethyl)chromonephenylethyl)chromone 6-Hydroxy-2-(2-phenylethyl)chromone S25 HOHO 57.3 267 91 6-Hydroxy-2-(2-phenylethyl)chromone HOHO HO OO OO O OCHOCH OCH33 OCH33 OCH3 OO O OHOH S26S26 O OH 58.858.8 343343 137137 55--HydroxyHydroxy--66--methoxymethoxy--22--[2[2--(3(3--hydroxyhydroxy--44--methoxyphenyl)ethyl]chromonemethoxyphenyl)ethyl]chromone S26S26S26 OH 58.858.858.8 343343 343137137 55--HydroxyHydroxy 137 --66--methoxymethoxy--225-Hydroxy-6-methoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone--[2[2--(3(3--hydroxyhydroxy--44--methoxyphenyl)ethyl]chromonemethoxyphenyl)ethyl]chromone S26 HHCOCO 58.8 343 137 5-Hydroxy-6-methoxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethyl]chromone 3H3 CO H33CO H3CO OHOH OO OHOH OO OH O

HHCOCO OO 3H3 CO O H33CO O S27S27S27 H3CO O 64.464.464.4 311311311 220220220 6,76,76,7--Dimethoxy-DimethoxyDimethoxy--2-2-2-(2-(2(2--phenylethyl)chromone-phenylethyl)chromonephenylethyl)chromone S27S27 HHCOCO 64.464.4 311 311220 2206,7-Dimethoxy-2-(2-phenylethyl)chromone 6,7-Dimethoxy-2-(2-phenylethyl)chromone S27 3H3 CO 64.4 311 220 6,7-Dimethoxy-2-(2-phenylethyl)chromone H33CO H3CO OO OO O OCHOCH OCH33 OCH33 OCH3 OO O S28S28 O 71.271.2 281281 121121 22--[2[2--(4(4--Methoxyphenyl)ethyl]chromoneMethoxyphenyl)ethyl]chromone S28S28S28 71.271.271.2 281281 281121121 12122--[2[2--(4(4--Methoxyphenyl)ethyl]chromoneMethoxyphenyl)ethyl]chromone 2-[2-(4-Methoxyphenyl)ethyl]chromone OO OO O

OO O S29S29 O 73.073.0 251251 160,160, 91 91 22--(2(2--Phenylethyl)chromonePhenylethyl)chromone S29S29 73.073.0 251251 160,160, 9191 22--(2(2--Phenylethyl)chromonePhenylethyl)chromone OO OO O OCHOCH OCH33 OCH33 OCH3 OO O S30S30 O 78.478.4 327327 206,206, 121 121 55--HydroxyHydroxy--66--methoxymethoxy--22--[2[2--(4(4--methoxyphenyl)ethyl]chromonemethoxyphenyl)ethyl]chromone S30S30 78.478.4 327327 206,206, 121121 55--HydroxyHydroxy--66--methoxymethoxy--22--[2[2--(4(4--methoxyphenyl)ethyl]chromonemethoxyphenyl)ethyl]chromone S30 HHCOCO 78.4 327 206, 121 5-Hydroxy-6-methoxy-2-[2-(4-methoxyphenyl)ethyl]chromone 3H3 CO H33CO H3CO OHOH OO OHOH OO OH O MoleculesMolecules 20182018,, 2323,, xx FORFOR PEERPEER REVIEWREVIEW 1414 ofof 2828

OCH OCH33 OO S24S24 55.655.6 297297 121121 66--HydroxyHydroxy--22--[2[2--(4(4--methoxyphenyl)ethyl]chromonemethoxyphenyl)ethyl]chromone HOHO OO

OO S25S25 57.357.3 267267 9191 66--HydroxyHydroxy--22--(2(2--phenylethyl)chromonephenylethyl)chromone HOHO OO

OCH OCH33 OO S26S26 OHOH 58.858.8 343343 137137 55--HydroxyHydroxy--66--methoxymethoxy--22--[2[2--(3(3--hydroxyhydroxy--44--methoxyphenyl)ethyl]chromonemethoxyphenyl)ethyl]chromone H CO H33CO OHOH OO

H CO O H33CO O S27S27 64.464.4 311311 220220 6,76,7--DimethoxyDimethoxy--22--(2(2--phenylethyl)chromonephenylethyl)chromone H CO Molecules 2018, 23, 1261H33CO 15 of 29 OO

OCH OCH33 OO Table 3. Cont. S28S28 71.271.2 281281 121121 22--[2[2--(4(4--Methoxyphenyl)ethyl]chromoneMethoxyphenyl)ethyl]chromone

OO + No. Structure tR (min) [M + H] (m/z) Fragment Ions (m/z) Name

OO S29S29S29 73.073.073.0 251251 251160,160, 9191 160, 9122--(2(2--Phenylethyl)chromonePhenylethyl)chromone 2-(2-Phenylethyl)chromone

OCH OCH33 OO S30S30S30 78.478.478.4 327327 327206,206, 121121 206,55--HydroxyHydroxy 121--66--methoxymethoxy--22 5-Hydroxy-6-methoxy-2-[2-(4-methoxyphenyl)ethyl]chromone--[2[2--(4(4--methoxyphenyl)ethyl]chromonemethoxyphenyl)ethyl]chromone H CO H33CO OHOH OO Molecules 2018, 23, 1261 16 of 29

2.2.1. Discussion of Substitution Pattern of Chromone and Phenylethyl Moiety of FTPECs, Respectively Although studies on chemical constituents of agarwood did not began until 1978, the uncommon structure of 2-(2-phenylethyl)chromones intrigued many research groups, which led to the rapid exploitation of PECs. Until now, more than 100 PECs have been isolated and identiﬁed from agarwood of different origin. Numerous PECs were differentiated from each other by different substituents or substituted position. Substituents attached at PECs are commonly hydroxy and methoxy, and chlorinated PECs and glycosylated PECs also existed in agarwood but with relatively rare frequency. The substituents were most frequently linked at C6-only or both C6- and C7-position of FTPECs at chromone moiety, while C40-only or both C30- and C40-position of PECs at phenylethyl moiety. The basic structure of FTPECs is a chromone skeleton bearing an uncommon phenylethyl group at the C-2 position, thus distinguishing FTPECs from other normal chromones (Figure2). However, unlike nonaromatic PECs, FTPECs are not unique in agarwood, and a few members of FTPECs, as concluded in Figure3, have also been identiﬁed in other plant species such as Flindersia laevicarpa, Bothriochloa ischaemum, Imperata cylindrica, and Cucumis melo (Table4)[ 21–26]. Although either FTPECs from agarwood or from other plants all share the same skeleton, there were differences in substitution patterns of their chromone core. Among all FTPECs in agarwood, those isolated most frequently or existed with comparatively higher relative content were basically FTPECs with a nonsubstituted A-ring, or FTPECs with 6-hydroxy, or 6-methoxy, or 6,7-dimethoxy at chromone moiety (Figure3), while several FTPECs isolated from other plant species were rarely found in agarwood, such as FTPECs bearing 5,7-dihydroxy isolated from C. melo or FTPECs bearing 5-hydroxy isolated from I. cylindrica or B. ischuemurn (Figure3). The different substitution patterns between FTPECs of different origin suggested their different biosynthetic pathway.

Table 4. Summarizing PECs of nonagarwood origin.

No. Coumpund Resources Literature Flindersia laevicarpa (Rutaceae) [21] I 2-(2-phenylethyl)chromone (ﬂindersiachromone) Imperata cylindrica (Gramineae) [23,25] 5-hydroxy-6-methoxy-2-[2-(2-hydroxyphenyl)- II Bothriochloa ischaemum (Gramineae) [22] ethyl]-chromone III 2,3-dihydro-2-(2-phenylethyl)chromone-3-one Flindersia laevicarpa (Rutaceae) [21] IV 1,5-diphenylpentane-1,3-diol Flindersia laevicarpa (Rutaceae) [21] V 5,7-dihydroxy-2-[2-(4-hydroxyphenyl)ethyl]chromone Cucumis melo (Cucurbitaceae) [24] VI 5,7-dihydroxy-2-[2-(3,4-dihydroxyphenyl)ethyl]chromone Cucumis melo (Cucurbitaceae) [24] 5,7-dihydroxy-2-[2-(3-methoxy-4- VII Cucumis melo (Cucurbitaceae) [26] hydroxyphenyl)ethyl]chromone 7-glucosyloxy-5-hydroxy-2-[2-(4- VIII Cucumis melo (Cucurbitaceae) [24] hydroxyphenyl)ethyl]chromone IX 5-hydroxy-2-(2-phenylethyl)chromone Imperata cylindrica (Gramineae) [23] 5-hydroxy-2-[2-(2-hydroxypheny1)- Bothriochloa ischaemum (Gramineae) [22] X ethyl]-chromone Imperata cylindrica (Gramineae) [23] XI 5-hydroxy-2-styrylchromone Imperata cylindrica (Gramineae) [23] XII 8-hydroxy-2-(2-phenylethyl)chromone Imperata cylindrica (Gramineae) [25] XIII 8-methoxy-ﬂindersiachromone Flindersia laevicarpa (Rutaceae) [21]

XIV 2-(2-phenylethyl)chromone-8-O-β-D-glucopyranoside Imperata cylindrica (Gramineae) [25] MoleculesMolecules 20182018, 23, 23, x, 1261FOR PEER REVIEW 1716 ofof 29 28

Major PECs in agarwood Reported PECs in other plant species

R Compound I (59) and II (64) existed OR1 O in both agarwood and other plants O O R R R = H 3 2 HO 50 49 R = OCH V. R1 = H, R2 = R3 = H O 3 VI. R = R = OH, R = H OH O 1 H, 2 3 VII. R = R = OCH , R = H O 1 H, 2 3 3 VIII. R = R = R = β O 1 2 H, 3 -glucose R I. (59) O R O III. O O 8' = H3CO 61 R H 7' O 60 R = OCH3 O IX. R = H X. R = OH OH O ∆7', 8', H3CO HO XI. R = H II. (64) OH O R R O IV. O H3CO O OH

54 R = H XII. R = H H3CO 53 R = OCH XIII. R = CH3 O 3 O - XIV. R = β D-glucopyranosyl

FigFigureure 3 3.. Comparison of major PECs derived from agarwood and other plant species. Molecules 2018, 23, 1261 18 of 29 Molecules 2018, 23, x FOR PEER REVIEW 17 of 28

As Asfor forthe the substitution substitution pattern pattern at phenylethyl at phenylethyl moiety moiety of PECs of PECs from from agarwood agarwood,, most most of them of 0 0 frequentlythem frequently appeared as appeared a nonsubstituted as a nonsubstituted C-ring, or 4′-OCH C-ring,3-only or, or 4 -OCH both 3′3-only,-OH- and or both4′-OCH 3 -OH-3-substituted and 0 C-ring4 -OCH. As 3shown-substituted in Figure C-ring. 4 and As Table shown 5, infrom Figure the4 1st and to Table 12th5 month,, from the the 1st total to 12threlative month, content the totalof PECs withrelative nonsubstitution content of PECs at phenylethyl with nonsubstitution moiety was at phenylethylgenerally much moiety higher was generallythan PECs much with higher 4′-OCH than3 at 0 0 phenylethylPECs with moiety, 4 -OCH 3andat phenylethyl the latter moiety,was also and much the latter higher was than also muchPECs higher with than3′-OH PECs and with 4′-OCH 3 -OH3 at 0 phenylethyland 4 -OCH moiety3 at phenylethyl. The general moiety. trend The of general relative trend contents of relative of PECs contents with of a PECs nonsubstituted with a nonsubstituted C-ring was decreasedC-ring was gradually decreased, for gradually, more and for more more PECs and morewith PECsdiverse with substituted diverse substituted C-rings appeared C-rings appeared from the from 1st to 0 12ththe month. 1st to 12th The month. general The tendency general tendencyof relative of relativecontent content of PECs of PECswith witha 4′- aOCH 4 -OCH3-substituted3-substituted C-ring C-ring was increasedwas increased gradually gradually from the from 1st the to 1st6th to month 6th month but dropped but dropped down down between between the the6th 6thand and 9th 9th month, month, and 0 0 thenand went then up went again up from again the from 9th the to 12th 9th to month, 12th month, while whilePECs PECswith a with 3′-OH a 3--OH- and 4 and′-OCH 4 -OCH3-substituted3-substituted C-ring appearC-ringed appearedonly from only the 5th from month the 5th and month showed and an showed uptrend an uptrendfrom thefrom 6th to the 9th 6th month, to 9th and month, then and decreased then 0 fromdecreased the 9th fromto 12th the month 9th to. 12thThe abovementioned month. The abovementioned results suggested results that suggested PECs with that PECs3′-OH with and 34′-OH-OCH3 0 0 areand hydroxylated 4 -OCH3 are products hydroxylated from 4′- productsOCH3-substituted from 4 -OCH PECs.3-substituted Apart from PECs. above Apart typical from substitution above typical pattern forsubstitution phenylethyl pattern moiety, for there phenylethyl were also moiety, FTPECs there detected were also in some FTPECs samples detected bearing in some 3′-OCH samples3 and bearing 4′-OH at 0 0 phenylethyl3 -OCH3 and moiety 4 -OH such at phenylethyl as 2-[2-(4 moiety-hydroxy such-3-methoxyphenyl)ethyl]chromone as 2-[2-(4-hydroxy-3-methoxyphenyl)ethyl]chromone (40), 4′-hydroxylated 0 FTPECs(40), 4such-hydroxylated as 6,7-dimethoxy FTPECs-2-[2 such-(4-hydroxyphenyl)ethyl]chromone as 6,7-dimethoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone (33), and 6-hydroxy-7-methoxy (33),-2- 0 [2-(4and-hydroxyphenyl)ethyl]chromone 6-hydroxy-7-methoxy-2-[2-(4-hydroxyphenyl)ethyl]chromone (19), as well as 2′-hydroxylated (FTPECs19), as wellsuch asas 6 2--hydroxylatedhydroxy-2-[2-(2- hydroxyphenyl)ethyl]chromoneFTPECs such as 6-hydroxy-2-[2-(2-hydroxyphenyl)ethyl]chromone (34) and 2-[2-(2-hydroxyphenyl)ethyl] (34) and chromone 2-[2-(2-hydroxyphenyl)ethyl] (47) (Table 2). chromone (47) (Table2).

90 Ⅰ Ⅱ Ⅲ 80 70 60 50 40 30 20

Relative Content (%) Content Relative 10 0 1 2 3 4 5 6 9 12 Months

Figure 4. The changing trend of the total relative content from the 1st to 12th month of three groups of FigPECsure 4. which The changing were subdivided trend of according the total torelative the substitution content from pattern the of1st phenylethyl to 12th month moiety. of three Group groups I were of 0 PECsPECs which with were no substitution subdivided ataccording phenylethyl to th moiety,e substitution group IIpattern were PECsof phenylethyl with 4 -OCH moiet3 aty. phenylethyl Group I were 0 0 PECsmoiety, with groupno substitution III were PECsat phenylethyl with 3 -OH moiety, and 4 group-OCH3 IIat were phenylethyl PECs with moiety. 4′-OCH3 at phenylethyl moiety, group III were PECs with 3′-OH and 4′-OCH3 at phenylethyl moiety. Table 5. The statistic results of the total relative content (%) from the 1st to 12th month of three Tablegroups 5. The of 2-(2-phenylethyl)chromonesstatistic results of the total relative which werecontent subdivided (%) from according the 1st to to12th the month substitution of three pattern groups of of 2-(2phenylethyl-phenylethyl)chromones moiety. which were subdivided according to the substitution pattern of phenylethyl moiety. 1 2 3 4 5 6 9 12 1 2 3 4 5 6 9 12 GroupGroup I І 77.6377.63 75.5675.56 66.5366.53 71.62 57.52 46.27 46.27 39.56 39.56 55.0 55.0 Group II 15.50 15.67 18.69 22.01 29.70 32.90 19.13 24.73 GroupGroup III II 15.5\\\\0 15.67 18.69 22.01 29.71.960 32.9 3.630 19.13 9.93 24.73 3.69 Group Ш \ \ \ \ 1.96 3.63 9.93 3.69 Molecules 2018, 23, 1261 19 of 29

2.2.2. Discussion of Possible Biosynthetic Pathway of 2-(2-Phenylethyl)Chromones PECs are principal components responsible for the quality of agarwood. However, the molecular basis of PECs biosynthesis remains almost unknown. The most recent progress is about a new type III polyketide synthase (PKS), AsPKS1, which is isolated and characterized in A. sinensis calli [27], and may contribute to the biosynthesis of PECs. Type III PKSs are a group of enzymes traditionally existing in plants to produce divergent natural polyketide scaffolds, such as flavonoids, stilbenes, phloroglucinol, curcuminoids, and so on. The enzyme assay experiment of AsPKS1 carried out in vitro suggested it not only exhibited the same function as curcumin synthase (CURS) or benzalacetone synthase (BAS) to yield curcuminoids or benzylacetone analogues, but also showed the ability to condensate 4-coumaroyl-CoA with malonyl-CoA and benzoyl-CoA to produce 5-(4-hydroxyphenyl)-1-phenyl-4-pentene-1,3-dione, or condensate dihydro-4-coumaroyl-CoA with malonyl-CoA and benzoyl-CoA to produce 5-(4-hydroxyphenyl)-1-phenylpentane-1,3-dione [28]. Thus, it is possible that some PKS similar to AsPKS1 may also condensate dihydro-cinnamoyl-CoA with malonyl-CoA and benzoyl-CoA to produce 1,5-diphenylpentan-1,3-dione, which possesses the same C6–C5–C6 scaffold as products of AsPKS1 (Figure5). Although 1,5-diphenylpentan-1,3-dione has not yet been reported from nature, one of its analogues, 1-hydroxy-1,5-diphenylpentan-3-one, has been found from agarwood, and another, 3-hydroxy-1,5-diphenylpentan-1-one, has been found from the medicinal plant Daphne acutiloba Rehd. (Thymelaeceae) [29], which possess obvious structural similarities with PECs except for a γ-pyrone ring. Referring to the chemoenzymatic results of HsPKS3 carried out in vitro by Wang et.al [30], coincubation of HsPKS3 with 2-hydroxybenzoyl-CoA and 3-oxo-5-phenylpentanoic acid led to the formation of a small amount of 2-(2-phenylethyl)chromone, while condensation of benzoyl-CoA, malonyl-CoA and p-hydroxyphenylpropinoyl-CoA only produced a diarylpentanoid, which suggests a hydroxy group at the C-2 position may be a prerequisite for the ring closure of the diarylpentanoid scaffold. Therefore, the formation of 2-(2-phenylethyl)chromone in vivo was possibly catalyzed by PKSs that condensate 2-hydroxybenzoyl-CoA, malonyl-CoA, and phenylpropinoyl-CoA, while 1,5-diphenylpentan-1,3-dione analogues were produced by the condensation of benzoyl-CoA with malonyl-CoA and phenylpropinoyl-CoA. As the biosynthesis of the nonsubstituted FTPEC, 2-(2-phenylethyl)chromone (59), has been described as above, from which we could know that the substitution pattern at phenylethyl moiety of PECs is in accordance with the “first” starter, phenylpropinoyl-CoA, while the nonsubstituted A-ring is derived from the “second” starter 2-hydroxybenzoyl-CoA, and malonyl-CoA acts as an unchangeable “bridge” to connect the above two parts together. Thus, the biosynthesis pathway of FTPECs with diverse substitution patterns could be deduced in a similar way, that is, the chromone moiety of FTPECs was derived from the 2-hydroxybenzoyl-CoA analogues, while the phenylethyl moiety came from the phenylpropinoyl-CoA analogues (Figure5). When considering the substitution patterns of the chromone core of FTPECs, as mentioned before, those isolated most frequently or existed with comparatively higher relative content in agarwood were basically FTPECs with a nonsubstituted A-ring, or FTPECs with a 6-hydroxy-, 6-methoxy-, or 6,7-dimethoxy-substituted chromone moiety. According to the aforementioned biosynthetic pathway of PECs with a nonsubstituted A-ring, which was derived from 2-hydroxybenzoyl-CoA substrate, 6-hydroxy-substituted FTPECs were consequently derived from 2,5-dihydroxybenzoyl-CoA. At the same time, FTPECs with a substituted A-ring, including 6-hydroxy-substituted FTPECs, also may be modified products of FTPECs with a nonsubstituted A-ring by further reaction catalyzed by hydroxylase or O-methyltransferase (OMT) (Figure5). It is difficult to deduce whether both of the two branches of pathway exist or if one of them has an advantage over the other. However, we could get some clues by considering the formation time of the identified FTPECs at least. Among all the A-ring-substituted PECs identified in this study, most of the C6-substituted PECs existed from the 1st to the 12th month with a relatively high amount, such as compounds 49 and 50 with 6-hydroxy, compounds 60 and 61 with 6-methoxy, compounds 53 and 54 with 6,7-dimethoxy, or compounds 63 and 64 with 5-hydroxy, 6-methoxy groups. Therefore, it is most Molecules 2018, 23, 1261 20 of 29 likely that 2,5-dihydroxybenzoyl-CoA was involved in the biosynthetic pathway of FTPECs and produced 6-hydroxy substituted FTPECs as a precursor for further modifications. For example, 6-hydroxy-2-[2-(4-methoxyphenyl)ethyl]chromone (49) and 6-methoxy-2-[2-(4-methoxyphenyl)ethyl] chromone (60) were detected from the 1st to the 12th month, while their possible hydroxylated products, 6,8-dihydroxy-2-[2-(4-methoxyphenyl)ethyl]chromone (31) and 7-hydroxy-6-methoxy-2-[2-(4-methoxy phenyl)ethyl]chromone (44), were only detected from the 4th to the 12th month. The relatively late time formation of 31 and 44 supported that they were hydroxylated products of 49 and 60, respectively, rather than directly formed by condensation reactions with other potential substrates beside 2-hydroxybenzoyl-CoA and 2,5-dihydroxybenzoyl-CoA. However, this study did not cover whole range of chromone compounds occurring in agarwood formation, but only the major PECs. Thus, it cannot be excluded that some other benzoyl CoA analogues may also be found to contribute to the construction of chromone moiety of FTPECs as potential substrates in further research. As mentioned before, most of the substitution patterns at the C-ring were nonsubstituted, 0 0 0 0 4 -OCH3-only, or 3 -OH- and 4 -OCH3-substituted, and in rare cases, also 2 -OH-substituted 0 0 or 3 -OCH3- and 4 -OH-substituted, which suggests the first starter substrates of PECs biosynthesis are most likely to correspond with 2,3-dihydro derivatives of cinnamoyl-CoA and 4-methoxycinnamoyl-CoA, and sometimes also feruloyl-CoA and 20-hydroxycinnamoyl-CoA. Utilization of above aromatic substrates as starters was proven by the existence of their possible BAS-like enzyme catalyzed products, e.g., (E)-4-phenylbut-3-en-2-one (benzylidene acetone), 4-phenylbutan-2-one (benzylacetone), 1-(4-methoxyphenyl)propan-2-one (anisylacetone), as well as 4-(4-hydroxy-3-methoxyphenyl) butan-2-one in agarwood [1]. Further, the latter three compounds have also been detected in our volatile constituents produced in agarwood samples from the 2nd to the 12th month (Table1). BAS is a plant-specific type III polyketide synthase (PKS) that has been isolated from raspberries and Rheum palmatum and catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6–C4 skeleton of phenylbutanoids [31–33]. However, apart from the substrates mentioned above, it cannot be excluded that 4-coumaroyl-CoA, caffeoyl-CoA, or other potential substrates may contribute to 0 0 the biosynthesis of PECs. Those PECs bearing 3 -OH and 4 -OCH3 at phenylethyl moiety are 0 0 most likely to be 3 -hydroxylated products from 4 -OCH3-substituted PECs, for the reason that 0 0 the corresponding C6–C3 substrates bearing 3 -OH and 4 -OCH3 groups have not been found 0 0 in plants, and 3 -OH and 4 -OCH3 substituted PECs were not detected from the beginning but have only been found since the 5th month or even later. Here, we took compounds 49, 50, and 30 as an example for detailed elucidation. Both 6-hydroxy-2-(2-phenylethyl)chromone) (50) and 6-hydroxy-2-[2-(4-methoxyphenyl) ethyl]chromone (49) were detected from the 1st to the 12th month, while 6-hydroxy-2[2-(3-hdroxy-4-methoxyphenyl)ethyl]chromone (30) only has been detected since the 5th month. As previously discussed, the phenylethyl moiety of compound 50 was suggested to derive from dihydro-cinnamoyl-CoA, and the 4-methoxylpenylethyl part of compound 49 was came from 4-methoxyphenylpropionyl-CoA. Thus, it can be explained that FTPECs with a nonsubstituted or 4-OCH3-substituted C-ring were produced by the condensation reactions with their corresponding 0 0 substrates, while the late formation time for 30 or other 3 -OH- and 4 -OCH3-substituted PECs, such as 25, 37, 48, and 51, were for the reason that they were not directly formed by PKS but 30-hydroxylated 0 products from 4 -OCH3 substituted PECs. MoleculesMolecules 20182018, ,2323, ,x 1261 FOR PEER REVIEW 2120of of 29 28

R1 O O + R CoAS HO SCoA 1 R2 PKS BAS-like enzyme O R3 CoAS malonyl-CoA R2 R =R =R =H O cinnamoyl-CoA: 1 2 3 O O R3 acetone 4-methoxycinnamoyl-CoA: R1=OCH3,R2=R3=H Benzylidene 2-hydroxycinnamoyl-CoA: R1=R2=H, R3=OH feruloyl-CoA: R1=OH, R2=OCH3,R3=H reductase reductase

reductase R1 R1 R1 O O BAS-like enzyme PKS CoAS R2 R CoAS + HO SCoA 2 O R3 O O R 3 Benzylacetone:R1=R2=H O R3 malonyl-CoA p-Methoxy-benzylacetone=Anisyl acetone:R1=OCH3,R2=H 4-(4-Hydroxy-3-methoxyphenyl)butan-2-one: R1=OH, R2=OCH3 phenylpropionyl-CoA: R1=R2=R3=H 4-methoxyphenylpropionyl-CoA: R1=OCH3,R2=R3=H 2-hydroxyphenylpropionyl-CoA: R1=R2=H, R3=OH 3-methoxy-4-hydroxyphenylpropionyl-CoA: R1=OH, R2=OCH3,R3=H

O O O HO SCoA PKS SCoA PKS SCoA PKS OH OH

R OH 1 R1 R1 hydroxylase O O O hydroxylase O R2 R2 R2 R R HO 3 HO 3 31:R =OCH =H O 1 3,R2 O O O 32:R1=R2=H 58: R =OCH ,R =R =H OCH 1 3 2 3 3 49: R =OCH ,R =R =H 59 R =R =R =H 1 3 2 3 (I): 1 2 3 50: R =R =R =H 47: R =R =H, R =OH O 1 2 3 1 2 3 OH hydroxylase 34: R =R =H, R =OH reductase 1 2 3 HO 30 O OCH hydroxylase OMT R1 3 HO R O OMT 1 HO HO O O hydroxylase OH OH OCH3 hydroxylase O hydroxylase R2 R2 O H3CO O O H3CO 25 OH OH 60: R =OCH ,R =H 44: R =OCH ,R =H H3CO 1 3 2 O 1 3 2 O R =R =H =R =H 1-hydroxy-1,5-diphenylpentan-3-one 3-hydroxy-1,5-diphenylpentan-1-one O 61: 1 2 46: R1 2 42(XII) H3CO 48 O O OMT hydroxylase OMT OMT hydroxylase OCH R1 3 R1 reductase OCH OCH3 3 hydroxylase hydroxylase H3CO O O H CO O OH O R2 3 R UGT O 2 OH H CO 63: R =OCH ,R =H =H H3CO 37 3 1 3 2 H3CO 53: R1=OCH3,R2 64: R =R =H =R =H O XIII H3CO 51 OH O 1 2 O 54: R1 2 O HO OH O OGlc O

OH XIV IV:1,5-diphenylpentane-1,3-diol O

FFigureigure 5. Hypothetical scheme for the biosynthetic pathway of PECs. OMT, O-methyltransferase,O-methyltransferase, UGT, glycosyltransferase.glycosyltransferase. Molecules 2018, 23, 1261 22 of 29

The possible biosynthesis pathway of major FTPECs has been elucidated in Figure5, and other known PECs which were not detected or identified in this study, either from agarwood origin or from other plants, may also be produced by a similar biosynthesis pathway, except for FTPECs bearing 5,7-dihydroxyl groups. So far, 5,7-dihydroxylated FTPECs have only been isolated from C. melo, and their biosynthetic pathway may be partly different from above mentioned agarwood-type PECs. As we know, flavonoids with 5,7-dihydroxyl or other 5,7-dioxygen groups commonly exist in the stem of healthy A. sinensis plants, while only one flavone, 5-hydroxy-7,40-dimethoxyflavone, has been reported in agarwood induced by artificial holing [34]. Instead, FTPECs with diverse substitution patterns other than 5,7-dihydroxylated FTPECs emerged in a large number and accumulated gradually during agarwood formation. It is known that the typical biosynthesis of flavonoids produces chromones with 5,7-dihydroxyl groups, and their biosynthetic pathway has been well-studied and elucidated as the involvement of CHS superfamily, which typically select 4-coumaroyl-CoA as a starter and perform sequential condensations with three C2 units derived from malonyl-CoA to produce an enzyme-bound tetraketide intermediate, following with Claisen-type cyclization to form a C6–C3–C6 scaffold of narigenin chalcone [33,35] (Figure6). It is reasonable to deduce that the biosynthesis of FTPECs with 5,7-dihydroxylated chromone moiety may be different from agarwood-type FTPECs, the A-ring of which is derived from 2-hydroxy-benzoyl-CoA substrates and its analogues, but similar to typical flavonoids with 5,7-dihydroxyl groups. Thus, we proposed another biosynthetic pathway for 5,7-dihydroxylated FTPECs (V–VIII), which is isolated from C. melo as similar to the flavonoids biosynthetic pathway, but sequentially condensing four C2 units from malonyl-CoA to form a C6–C5–C6 scaffold of chalcone analogue, following cyclization of the linear C6–C5–C6 intermediate to yield 5,7-dihydroxylated FTPECs, which may serve as skeletons for further modifications (Figure6). For the reason that the substitution pattern at phenyethyl moiety of PECs is in accordance with the starter units, the starters of the flavonoids-like pathway are proposed to be 4-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA, or their 2,3-dihydro derivatives, according to the structures of compounds V–VIII, which all bear 40-hydroxy group. It is noteworthy that the substrates involved in this biosynthetic pathway are different from those substrates of agarwood-type PECs except for feruloyl-CoA. Phenylpropanoid derived metabolites comprise and contribute to multiple biosynthetic branches other than lignin and flavonoid biosynthesis only [36]. The origin of numerous CoA derivatives involved in the biosynthesis of PECs was also suggested to be derived from the phenylpropanoid pathway (Figure7). The plant shikimate pathway is the entry to the biosynthesis of phenylpropanoids and results in the biosynthesis of phenylalanine and tyrosine. The elimination of the amino group of phenylalanine and tyrosine is catalyzed by corresponding phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL), thus yielding cinnamic acid and coumaric acid, respectively [36]. Starting from them, numerous organic acids are produced by reduction of double bond or hydroxylation at different positions, and provide diverse aromatic CoA-esters to different biosynthetic pathways by the function of CoA ligase. Among them, 4-coumaroyl CoA is the most representative one which contributes to the biosynthesis of not only flavonoids but also other important polyketides such as stilbenes, lignin, and coumarins [36]. In the case of PECs, we suggest that cinnamoyl-CoA, 4-methoxycinnamoyl-CoA, 2-hydroxycinnamoyl-CoA, and feruloyl-CoA or their corresponding dihydro analogues serve as the C6–C3 carbon skeleton to act as the first starters and contribute to the construction of phenylethyl moiety of PECs. Detection of phenylpropionic acid, phenylpropionic acid methyl ester, and 4-methoxyphenylpropionic acid methyl ester by GC-MS analysis partly proved the above deduction. Although not as popular as 4-coumaroyl CoA, at least some of them, such as cinnamoyl-CoA [36], 4-methoxycinnamoyl-CoA [37,38], feruloyl-CoA [35], and 3-hydroxyphenylpropionyl-CoA [39], have been reported as substrates for type III PKSs in different plants. However, unlike the well-established biosynthesis of phenylpropanoid CoA thioesters, the formation of phenolic acids with a C6–C1 skeleton still remain obscure, and two possible pathways are proposed, either by a β-oxidative pathway as described for Petunia hybrida [40] or by an alternative Molecules 2018, 23, 1261 23 of 29 nonoxidative pathway as identified in Anthirrhinum majus [41]. It was reported that several pathways for benzoic acid biosynthesis may coexist in a single plant. The β-oxidative pathway produced a C6–C1 skeleton from cinnamic acid acts firstly by the addition reaction between the double bond and a molecule of water to introduce hydroxy group, then by oxidation of the hydroxy into a carbonyl group, and finally by the elimination of the β-acetyl group by a reverse Claisen reaction to produce benzoyl CoA. Further reaction catalyzed by 2-hydroxylase upon benzoic acid produced 2-hydroxybenoic acid, which is also known as salicylic acid (SA). SA plays a very important role in plant defense and serves as a critical signal for the establishment of plant resistance against pathogen attack. On the basis of SA, a number of biologically relevant chemical modifications, including glucosylation, methylation, amino acid conjugation, and hydroxylation, produced diverse phenolic derivatives. Gentisic acid (GA), also named 2,5-dihydroxybenzoic acid, is the C5-hydroxylated derivative of SA, and it has been proposed to be a signal molecule for plant defense response in compatible, non-necrotizing interactions [42]. Therefore, SA and GA, as the plant-defense-related signals, may be related to the formation of PECs in the injured Aquilaria plants by contributing their corresponding CoA derivatives to the construction of chromones moiety of PECs. It is known that the selection of substrates by different type III PKSs is one of the reasons for the production of diverse polyketides [43,44]. For example, CURS1~3 catalyzed the in vivo formation of curcuminoids which were isolated from Curcuma longa [44,45]. Then, in vitro analysis revealed that CURS2 preferred feruloyl-CoA as the starter and CURS3 preferred both feruloyl-CoA and p-coumaroyl-CoA, suggesting that the availability of different substrates and different expression levels of the CURS 1~3 involved in curcuminoid synthesis could account for the variety of curcuminoids in different cultivars of turmeric (Curcuma longa). It is possible that the PKSs catalyzed the formation of PECs in Aquilaria or other plants may have different substrate specificity, and therefore, result in the different compositions of PECs. In general, 2-(2-phenylethyl)chromones, as important secondary metabolites in agarwood, are the product of complex physiological and biochemical reactions in injured A. sinensis. So far, the process of agarwood accumulation has not been fully understood, and the biological origin of PECs still remains unknown. In this study, dynamic changes of chemical constituents, especially 2-(2-phenylethyl)chromones, in agarwood samples collected in different periods were studied by GC-MS and UPLC-MS analysis. Based on the observed results, as well as by comparison and analysis of structural characteristics of reported PECs, the possible biosynthetic pathway of PECs, either of agarwood or nonagarwood origin, were elucidated by comparison with biosynthetic pathways of other structurally similar secondary metabolites such as curcuminoids and flavonoids. This is the first reports that fully discussed the biosynthesis of PECs, and it may serve as a basis, from the perspective of chemical constituents, for further studies on the mechanism of agarwood formation and discovery of biosynthetic genes of PECs. Molecules 2018, 23, x FOR PEER REVIEW 23 of 28 Molecules 2018, 23, 1261 24 of 29

a Biosynthetic pathway of flavonoids

OH OH OH OH HO OH HO O HO O CoAS CHS CHI FNS O O O + Chalcone OH O Flavanones 4-coumaroyl-CoA 3 HO SCoA OH O OH O Flavones

b Hypothetic biosynthesis of PECs isolated from C. melo OR1 OR1 CHS-like enzyme HO OH OR1 HO O CoAS cyclase R2 R2 O O O R2 + 4 HO SCoA OH O O OH O 4-coumaroyl-CoA: R1=H, R2=H caffeoyl-CoA: R1=H; R2=OH feruloyl-CoA: R1=H, R2=OCH3 reductase reductase OR1 OR reductase 1 HO O R O O CHS-like enzyme HO OH OR 3 1 R2 R OR1 cyclase UGT 2 O O V:R1=H,R2=H CoAS VIII:R1=H,R2=H,R3=glycosyl R2 OH O VI:R1=H,R2=OH R2 + 4 OH O HO SCoA OH O O VII:R =H,R =OCH O 1 2 3

2,3-dihydro-4-coumaroyl-CoA: R1=H, R2=H 2,3-dihydro-caffeoyl-CoA: R1=H, R2=OH 2,3-dihydro-feruloyl-CoA: R1=H, R2=OCH3

FFigureigure 6. Hypothetic biosynthesis of PECs isolated from C. melo and comparison of the biosynthesis of ﬂavonoids.flavonoids. MoleculesMolecules 20182018, 23, 23, x, 1261FOR PEER REVIEW 2524 ofof 2928

O O

OH OH

NH2 Phe HO NH2 Tyr

PAL TAL

O O O O O C4H OH C2H OH OH ODD OH OH acid OH cinnamic HO 4-coumaric acid HO caffeic acid HO dihydrocaffeic acid O acid 2-hydroxycinnamic HO HO S-CoA 4CL O OH O HCT,C3'H,HCT O O 2-hydroxy- O OH O cinnamoyl-CoA OH S-CoA S-CoA OH O S-CoA phenylpropionic acid HO HO HO OH S-CoA dihydro-4-coumaric acid HO 4-coumaroyl-CoA caffeoyl-CoA dihydrocaffeoyl-CoA 2-hydroxyphenyl- HO HO propionic acid cinnamoyl- O CoA OMT CCoAOMT O S-CoA O O O O H O 2 S-CoA S-CoA S-CoA S-CoA S-CoA phenylpropionyl-CoA HO HO OH H3CO HO OH O dihydro-4-coumaroyl-CoA feruloyl-CoA 4-methoxycinnamoyl-CoA H3CO H3CO 2-hydroxyphenyl- S-CoA propionyl-CoA 3-methoxy-4-hydroxyphenylpropionyl-CoA NAD+

O O O O O reverse Claisen reaction HO S-CoA S-CoA S-CoA S-CoA

OH OH phenyldiketide- HSCoA CH COSCoA benzoyl-CoA 2-hydroxybenzoyl-CoA 2,5-dihydroxybenzoyl-CoA CoA 3

O O O HO BA2H OH hydroxylase OH OH OH OH benzoic acid (BA) 2-hydroxybenzoic acid 2,5-dihydroxybenzoic acid (salicylic acid, SA) (gentisic acid, GA)

FigureFigure 7 7.. PlantPlant phenolics phenolics based based on on phenylpropanoid phenylpropanoid pathway pathway and their production as substratessubstrates forfor biosynthesisbiosynthesis ofof polyketidepolyketide class. class. PAL, PAL, phenylalanine phenylalanine ammonia ammonia lyase (MIO); TAL, tyrosine ammonia lyase; C4H, cinnamate-4-hydroxylase (O2, cytochrome P450, NADPH); C2H, cinnamate-2-hydroxylase (O2, cytochrome P450, lyase (MIO); TAL, tyrosine ammonia lyase; C4H, cinnamate-4-hydroxylase (O2, cytochrome P450, NADPH); C2H, cinnamate-2-hydroxylase (O2, cytochrome NADPH);P450, NADPH); 4CL, 4-hydroxycinnamoyl 4CL, 4-hydroxycinnamoyl-CoA-CoA ligase (ATP, ligase coenzyme (ATP, coenzyme A [CoASH]); A [CoASH]); HCT, hydroxycinnamoyl HCT, hydroxycinnamoyl-CoA:shikimate-CoA:shikimate hydroxycinnamoyl hydroxycinnamoyl transferase transferase(shikimate); C3′H, p-coumaroyl0 -5-O-shikimate 3′-hydroxylase0 (O2, cytochrome P450, NADPH); CCoAOMT, caffeoyl-CoA O-methyl transferase (AdoMet); ODD, (shikimate); C3 H, p-coumaroyl-5-O-shikimate 3 -hydroxylase (O2, cytochrome P450, NADPH); CCoAOMT, caffeoyl-CoA O-methyl transferase (AdoMet); ODD, . 2-2-oxoglutarate-dependentoxoglutarate-dependent dioxygenase dioxygenase (α (α-ketoglutarate,-ketoglutarate, Fe(II), Fe(II), ascorbate), ascorbate), BA2H, BA2H, benzoic benzoic acid acid-2-hydroxylase.-2-hydroxylase Molecules 2018, 23, 1261 26 of 29

3. Experimental

3.1. General All the reference compounds were isolated and identified from agarwood in our previous study, and the purity was determined to be more than 98% by HPLC-UV analysis. The structures of 30 reference compounds are listed in Table3. Acetonitrile and methanol of HPLC grade were purchased from Tedia (Fairfield, AL, USA). Chromatographic-grade absolute formic acid was purchased from ROE SCIENTIFIC INC (Shanxi, China). Ultrapure water was prepared with a Milli-Q Plus 185 purification system (Millipore, Bedford, MA, USA).

3.2. Material Treatment The wood samples used in this study were collected during a fixed period in an A.sinensis plantation at Dingan Town, Haikou City, Hainan province, China. The agarwood inducer invented in our lab was used for stimulating the formation of resin in A. sinensis trees. Seven-year-old A. sinensis trees were chosen for the experiment of agarwood formation, and the experiment was carried out by us from 28 July 2014 to 28 July 2015. Voucher specimens were deposited at the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences. To conduct the experiment, first of all, we needed to hold an electric drill at a 45-degree angle from the trunk and drill a hole at the depth of about 4 cm in the trunk above 45 cm off the ground. Then, 500 mL of prepared agarwood inducer was transfused into each tree. After that, the hole was blocked by rubber in case of rainwash and to prevent outflow of the inducer.

3.3. Sample Preparation The trunk initially started to decay around the hole we drilled for the transfusion of agarwood inducer. A month later, we chose three trees and chopped off a wood block about 15-cm long, 10-cm wide, and 2-cm thick at the position around the hole for each tree. Then, rotten woods were collected from three trees in the planation every month within half a year, then at the 9th and the 12th month. The dry woods were whittled into chips after the removal of whitewood, followed by subjecting them to ultrasonic extraction with ethyl ether for 30 min under the condition of ice-cold water. After another 3 min’ standing, the suspension was ﬁltered. The extraction was performed totally three times, and ethyl ether extracts were obtained after vaporizing.

3.4. GC-MS Analysis GC-MS analysis was performed with an Agilent gas chromatography instrument (GC 7820) equipped with a ZB-5MSI 5% Phenyl-95% Dimethylpolysiloxane column (30 m × 0.25 mm, 0.25 µm) and a mass selective detector (5977) (Agilent Technologies Co., Ltd., Santa Clara, CA, USA) with an ion trap detector in full scan mode under election impact ionization (70 eV). The carrier gas was helium, at a ﬂow rate of 1.0 mL/min with split ratio of 50:1. 1 µL essential oil solution in methanol (HPLC grade) was injected into the front inlet of the gas chromatograph operating at 250 ◦C in the splitless mode. The operating parameters were that the oven program commenced at 50 ◦C (2 min) and increased at a rate of 5 ◦C/min to 310 ◦C where it was held for 10 min. The ion source temperature was set at 230 ◦C and the scan range was from 50 to 500 amu under full scan.

3.5. UPLC-MS Analysis A Dionex UltiMate 3000 series HPLC system (Dionex Softron GmbH, Germering, Germany), equipped with a diode array detector, a vacuum degasser, a quaternary pump, and an autosampler, and electrospray ionization tandem mass spectrometry (Amazon SL, Bruker Daltonik GmbH, Bremen, Germany) were used for sample analysis. The data acquisition was supported by Bruker Compass Data Analysis 4.0 software. The separations were performed on a Dionex-Acclaim 120 C18 column Molecules 2018, 23, 1261 27 of 29

(250 mm × 4.6 mm, 5 µm) at 26 ◦C. The mobile phase system was acetonitrile (A) and water with 0.5% acetic acid (B) at flow rate of 0.4 mL/min. The gradient elution program was as follows: 0–60 min, 25–55% A, 60–80 min, 55–80% A, 80–90 min, 80–100% A, and 90–95 min, 100% A. The sample injection volume for analysis was 20 µL. The detection wavelength was set at 254 nm for all the tested compounds. The ESI-MS analysis was acquired in the positive ion mode. Helium gas was used as the collision gas at the flow rate of 0.4 L/min and high-purity nitrogen gas was used as the nebulizer and drying gas at 6.0 L/min. The conditions for ESI-MS analysis were as follows: capillary voltage, −4000 V; end plate voltage, −500 V; drying gas temperature, 250 ◦C; and nebulizer pressure, 15 psi. The MS spectra were scanned from 70 to 2200 m/z. The MS/MS analysis was used to obtain the mass fractions of target ions. The extracts and standards were diluted in methanol to 1 mg/mL, respectively, and filtered through 0.45 µm membranes.

Author Contributions: The list authors contributed to this work as follows: G.L. and W.-H.D. performed the process of data, collection of the agarwood samples, and preparation of the manuscript. J.-L.Y. contributed to the operation of instruments, W.L. and J.W. contributed to the revision of this manuscript. The whole research was performed based on the planning of H.-F.D. and W.-L.M. All authors approved the final version of the manuscript. Funding: This research was funded by Innovative Research Team Grant of the Natural Science Foundation of Hainan Province (No.2017CXTD020), China Agriculture Research System (CARS-21), Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (No.17CXTD-15, No.1630052016008). Conflicts of Interest: The authors declare no conflict of interest.

References

1. Naef, R. The volatile and semi-volatile constituents of agarwood, the infected heartwood of Aquilaria species: a review. Flav. Fragr. J. 2011, 26, 73–87. [CrossRef] 2. Blanchette, R.; Heuveling, V.B.H. Cultivated Agarwood. U.S. Patent 7638145B2, 29 December 2009. 3. Dai, H.F.; Liu, J.; Zeng, Y.B.; Han, Z.; Wang, H.; Mei, W.L. Two new 2-(2-phenylethyl) chromones from Chinese eaglewood. J. Asian. Nat. Prod. Res. 2010, 12, 134–137. [CrossRef][PubMed] 4. Paoli, G.D.; Peart, D.R.; Leighton, M.; Samsoedin, I. An ecological and economic assessment of the nontimber forest product gaharu wood in Gunung Palung National Park, West Kalimantan, Indonesia. Cons. Biol. 2001, 15, 1721–1732. [CrossRef] 5. Pojanagaroon, S.; Kaewrak, C. Mechanical methods to stimulate aloes wood formation in Aquilaria crassna Pierre ex H.Lec (kritsana) trees. ISHS. Acta. Hort. 2005, 676, 161–166. [CrossRef] 6. Rahman, M.A.; Basak, A.C. Agar production in agar tree by artificial inoculation and wounding. Bano. Biggyan. Patrika. 1980, 9, 87–93. 7. Sitepu, I.R.; Santoso, E.; Siran, S.A.; Turjaman, M. Fragrant Wood Gaharu.: When the Wild Can. No Longer Provide; R&D Centre for Forest Conservation and Rehabilitation: Bogor, Indonesia, 2011; pp. 23–30. 8. Mei, W.L.; Zuo, W.J.; Yang, D.L.; Dong, W.H.; Dai, H.F. Advances in the mechanism, artificial agarwood-induction techniques and chemical constituents of artificial agarwood production. Chin. J. Trop. Crop. 2013, 34, 2513–2520. 9. Tamuli, P.; Boruah, P.; Samanta, R. Biochemical changes in agarwood tree (Aquilaria malaccensis Lank.) during pathogenesis. J. Spices. Aromat. Crop. 2014, 13, 87–91. 10. Zhang, X.L. Studies on Relationships between Wound-Induced Defense Response and Agarwood Formation from Aquilaria sinensis. Ph.D. Thesis, Beijing Forestry University, Beijing, China, 2013. 11. Mei, W.L.; Dai, H.F.; Wang, H.; Cai, Z.J. Method to cultivate agarwood. China Patent ZL 201310150138.5, 14 May 2014. 12. Mei, W.L.; Yang, D.L.; Wang, H.; Yang, J.L.; Zeng, Y.B.; Guo, Z.K.; Dong, W.H.; Li, W.; Dai, H.F. Characterization and determination of 2-(2-phenylethyl) chromones in agarwood by GC-MS. Molecules 2013, 18, 12324–12345. [CrossRef][PubMed] 13. Ismail, N.; Ali, N.A.M.; Jamil, M.; Rahiman, M.H.F.; Tajuddin, S.N.; Taib, M.N. A review study of agarwood oil and its analysis. J. Teknologi. 2014, 68, 37–42. [CrossRef] Molecules 2018, 23, 1261 28 of 29

14. Lin, F.; Dai, H.F.; Wang, H.; Mei, W.L. GC-MS analysis of the chemical constituents of essential oils from two kinds of Chinese eaglewood produced by artificial fungus inoculation. Lishizhen Med. Mater. Med. Res. 2010, 21, 1901–1902. 15. Mei, W.L.; Zeng, Y.B.; Liu, J.; Dai, H.F. GC-MS analysis of volatile constituents from five different kinds of Chinese eaglewood. J. Chin. Med. Mater. 2007, 30, 554–558. 16. Li, W.; Cai, C.H.; Dong, W.H.; Guo, Z.K.; Wang, H.; Mei, W.L.; Dai, H.F. 2-(2-Phenylethyl)chromone derivatives from Chinese agarwood induced by artificial holing. Fitoterapia. 2014, 98, 117–123. [CrossRef] [PubMed] 17. Wu, B.; Kwon, S.W.; Hwang, G.S.; Park, J.H. Eight new 2-(2-phenylethyl)chromone (=2-(2-phenyl ethyl)-4H-1-benzopyran-4-one) derivatives from Aquilaria malaccensis agarwood. Helv. Chim. Acta. 2012, 95, 1657–1665. [CrossRef] 18. Yagura, T.; Shibayama, N.; Ito, M.; Kiuchi, F.; Honda, G. Three novel diepoxy tetrahydro chromones from agarwood artificially produced by intentional wounding. Tetrahedron Lett. 2005, 46, 4395–4398. [CrossRef] 19. Liao, G.; Mei, W.L.; Dong, W.H.; Li, W.; Wang, P.; Kong, F.D.; Gai, C.J.; Song, X.Q.; Dai, H.F. 2-(2-Phenylethyl) chromone derivatives in artificial agarwood from Aquilaria sinensis. Fitoterapia 2016, 110, 38–43. [CrossRef] [PubMed] 20. Okudera, Y.; Ito, M. Production of agarwood fragrant constituents in Aquilaria calli and cell suspension cultures. Plant. Biotech. 2009, 26, 307–315. [CrossRef] 21. Picker, K.; Ritchie, E.; Taylor, W. The chemical constituents of australian Flindersia. species. XXI an examination of the bark and the leaves of F. laevicarpa. Aust. J. Chem. 1976, 29, 2023–2036. [CrossRef] 22. Wang, T.; Li, L.F.; Zhang, K.; Zhang, W.Y.; Pei, Y.H. New 2-(2-phenylethyl)chromones from Bothriochloa. Ischaemum. J. Asian. Nat. Prod. Res. 2001, 3, 145–149. [CrossRef][PubMed] 23. Yoon, J.S.; Lee, M.K.; Sung, S.H.; Kim, Y.C. Neuroprotective 2-(2-phenylethyl)chromones of Imperata. cylindrica. J. Nat. Prod. 2006, 69, 290–291. [CrossRef][PubMed] 24. Ibrahim, S.R.M. New 2-(2-phenylethyl)chromone derivatives from the seeds of Cucumis melo L var. reticulatus. Nat. Prod. Commun. 2010, 5, 403–406. [PubMed] 25. Liu, X.; Zhang, B.F.; Yang, L.; Chou, G.X.; Wang, Z.T. Two new chromones and a new flavone glycoside from Imperata. cylindrica. Chin. J. Nat. Med. 2013, 11, 77–80. [CrossRef] 26. Ibrahim, S.R.M. New Chromone and Triglyceride from Cucumis melo Seeds. Nat. Prod. Commun. 2014, 9, 205–208. [PubMed] 27. Wang, X.H.; Zhang, Z.X.; Dong, X.J.; Feng, Y.Y; Liu, X.; Gao, B.W.; Wang, J.L; Zhang, L.; Wang, J.; Shi, S.P.; et al. Identification and functional characterization of three type III polyketide synthases from Aquilatia. sinensis calli. BioChem. Biophy. Res. Commun. 2017, 486, 1040–1047. [CrossRef][PubMed] 28. Tu, P.F.; Shi, S.P.; Wang, X.H.; Gao, W.B.; Li, J.; Liu, X.; Zhao, Y.F.; Zhang, Z.X.; Zhang, L.; Dong, X.J.; et al. Type III PKS with Its Coding Gene and Usage. China Patent CN 201610037246, 28 July 2017. 29. Huang, S.H.; Zhang, X.J.; Li, X.Y.; Jiang, H.Z.; Ma, Q.Y.; Wang, P.C; Liu, Y.Q.; Hu, J.M.; Zheng, Y.T.; Zhou, J.; et al. Phenols with anti-HIV activity from Daphne acutiloba. Planta. Med. 2011, 77, 1–4. 30. Wang, J.; Wang, X.H.; Liu, X.; Li, J.; Shi, X.P.; Song, Y.L.; Zeng, K.W.; Zhang, L.; Tu, P.F.; Shi, S.P. Synthesis of unnatural 2-substituted quinolones and 1,3-diketones by a member of Type III polyketide synthases from Huperzia. serrata. Org. Lett. 2016, 8, 3550–3553. [CrossRef][PubMed] 31. Abe, I.; Takahashi, Y.; Morita, H.; Noguchi, H. Benzalacetone synthase: A novel polyketide synthase that plays a crucial role in the biosynthesis of phenylbutanones in Rheum palmatum. Eur. J. Biochem. 2001, 268, 3354–3359. [CrossRef][PubMed] 32. Zheng, D.; Hrazdina, G. Molecular and biochemical characterization of benzalacetone synthase and chalcone synthase genes and their proteins from raspberry (Rubus. idaeus L.). Arch. Biochem. Biophys. 2008, 470, 139–145. [CrossRef][PubMed] 33. Abe, I.; Morita, H. Structural and function of the chalcone synthase superfamily of plant type III polyketide synthases. Nat. Prod. Rep. 2010, 27, 809–838. [CrossRef][PubMed] 34. Li, W.; Mei, W.L.; Zuo, W.J.; Cai, C.H.; Dong, W.H.; Dai, H.F. Chemical constituents of Chinese agarwood induced by artificial holing. J. Trop. Subtrop. Botany. 2016, 24, 342–347. 35. Austin, M.B.; Noel, J.P. The chalcone synthase superfamily of type III polyketide synthases. Nat. Prod. Rep. 2003, 20, 79–110. [CrossRef][PubMed] 36. Vogt, T. Phenylpropanoid biosynthesis. Mol. plant 2010, 3, 2–20. [CrossRef][PubMed] Molecules 2018, 23, 1261 29 of 29

37. Wanibuchi, K.; Zhang, P.; Abe, T.; Morita, H.; Kohno, T.; Chen, G.S.; Noguchi, H.; Abe, I. An aridone-producing novel multifunctional type III polyketide synthase from Huperzia. serrata. FEBS J. 2007, 274, 1073–1082. [CrossRef][PubMed] 38. Abe, I.; Morita, H.; Nomura, A.; Noguchi, H. Substrate speciﬁcity of chalcone synthase: Enzymatic formation of unnatural polyketides from synthetic cinnamoyl-CoA analogues. J. Am. Chem. Soc. 2000, 122, 11242–11243. [CrossRef] 39. Schröder, J. The chalcone/stilbene synthase-type family of condensing enzymes. Comprehen. Nat. Prod. Chem. 1999, 1, 749–771. 40. Van, M.A.; Schauvinold, I.; Pichersky, E.; Haring, M.A.; Schuurink, R. A plant thiolase involved in benzoic acid biosynthesis and volatile benzenoid production in plants. Plant J. 2009, 60, 292–302. 41. Long, M.C.; Nagegowda, D.A.; Kaminaga, Y.; Ho, K.K.; Kish, C.M.; Schnepp, J.; Sherman, D.; Weiner, H.; Rhodes, D.; Dudareva, N. Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis. Plant J. 2009, 59, 256–265. [CrossRef][PubMed] 42. Bellés, J.M.; Garro, R.; Fayos, J.; Navarro, P.; Primo, J.; Conejero, V. Gentisic acid as a pathogen-inducible signal, additional to salicylic acid for activation of plant defenses in tomato. Mol. Plant-Microbe Interact. 1999, 12, 227–235. [CrossRef] 43. Mizuuchi, Y.; Shi, S.P.; Wanibuchi, K.; Kojima, A.; Morita, H.; Noguchi, H.; Abe, I. Novel type III polyketide synthases from Aloe arborescens. FEBS J. 2009, 276, 2391–2401. [CrossRef][PubMed] 44. Katsuyama, Y.; Kita, T.; Funa, N.; Horinouchi, S. Curcuminoid biosynthesis by two type III polyketide synthases in the herb Curcuma longa. J. Biol. Chem. 2009, 284, 11160–11170. [CrossRef][PubMed] 45. Katsuyama, Y.; Kita, T.; Horinouchi, S. Identiﬁcation and characterization of multiple curcumin synthases from the herb Curcuma. longa. FEBS Lett. 2009, 583, 2799–2803. [CrossRef][PubMed]

Sample Availability: Samples of the compounds are not available from the authors.

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