Characterization of New Ent-kaurane Diterpenoids of Yunnan Arabica Coffee Beans

Nat. Prod. Bioprospect. () 6:217 DI 10.1007/s59-0-0099-1 RIGINAL ARTILE haracterization of New Ent-kaurane Diterpenoids of Yunnan Arabica offee Beans Rui hu. Luo-Sheng Wan. Xing-Rong Peng. Mu-Yuan Yu. Zhi-Run Zhang. Lin Zhou. Zhong-Rong Li. Ming-ua Qiu Received: March / Accepted: 10 April / Published online: 10 May The Author(s). This article is published with open access at Springerlink.com Abstract Five new ent-kaurane diterpenoids, named mascaroside IIIV (1), and -nor-cofaryloside III (5), together with seven known diterpenoids, were isolated from methanol extracts of the green coffee beans of Yunnan Arabica offee. Their chemical structures were elucidated by extensive spectroscopic analyses. anwhile, cytotoxicity assay against L- 60, A-59, SMM-7721, MF-7 and SW80 cell lines showed that they have not evident inhibition of cytotoxicity. Graphical Abstract 1 R=A 2 R=B R A B 5 Keywords offea arabica L. Green coffee beans Diterpenoids Structural elucidation 1 Introduction Electronic supplementary material The online version of this article (doi:10.1007/s59-0-0099-1) contains supplementary material, which is available to authorized users. R. hu L.-S. Wan X.-R. Peng M.-Y. Yu Z.-R. Zhang L. Zhou Z.-R. Li M.-. Qiu (&) State Key Laboratory of Phytochemistry and Plant Resources in West hina, Kunming Institute of Botany, hinese Academy of Sciences, Kunming 6501, hina e-mail: mhchiu@mail.kib.ac.cn R. hu M.-Y. Yu M.-. Qiu University of hinese Academy of Sciences, Beijing 10009, hina offea arabica L., commonly known as coffee and widely distributed throughout the world, involving Africa, Latin America and Asia, is a very popular hot drink around the world because of its attractive aroma and unique taste [1, 2]. Previous studies have shown that coffee beans are consisted of caffeine, chlorogenic acids, saccharides [5], as well as diterpenoids, although, taking a minor proportion in the chemical constituents of coffee. owever, due to their broad spectrum of biological activities, such as cytotoxicity, antioxidant, anti-inflammatory, researchers have carried out work on diterpenoids components from

218 R. hu et al. 2 1 19 18 10 5 12 11 6 7 9 1 8 1 R=A 2 R=B 1 15 17 1' 21 6' 5' ' 2' ' 2 2 R 1 10 5 5" 6" 7" 8" 19 18 5 11 6 9 10" 12 8 7 1 A 1 15 " " 9" 17 2" 1" 10" 1" " 5" 2" " 6" 9" 7" 8" 11" B 21 2 6 7 8 9 R 2 R 1 10 R 1 = α - R 2 = 11 R 1 = β - R 2 = 12 R 1 = β - R 2 = Glc Fig. 1 Structures of compounds 112 coffee beans, and found nearly 90 diterpenoids [69]. Yunnan Arabica offee was the offea arabica which were planted in Yunnan province. Along with the planting scale expanded to 1 thousand hectares, Yunnan province became a well-known cultivation base of. arabica in the world. In order to investigate the chemical constituents of Yunnan Arabica offee and find bioactivity diterpenoids, we took Yunnan Arabica offee green beans collected in Dehong as the subject, and discovered five new ent-kaurane diterpenoids, along with seven known diterpenoids (Fig. 1). erein, the isolation, structural elucidation, and their relevant bioactivities were also described. 2 Results and Discussion Mascaroside III (1) was isolated as a white amorphous powder. The molecular formula 6 1 was deduced from the molecular ion peak at m/z [M? Na]? 707.2670 (calcd for 6 1 Na, 707.267) in REIMS. The IR spectrum indicated that 1 possessed hydroxyl (0 cm -1 )anda,bunsaturated ketone (50 cm -1 ) groups. The 1 NMR (DEPT) data (Tables 2, ) showed 6 carbon resonances, attributed to a monosaccharide, a cinnamic acids group and an aglycone moiety, and the aglycone moiety were classified as one methyl (d 15.7), 7 methylenes (including one

haracterization of New Ent-kaurane Diterpenoids of Yunnan Arabica offee Beans 219 Table 1 1 NMR spectral data of compounds 15 [d in ppm, J in z] Position 1 a 2 a b a 5 b 1 2.9 (d,.) 2.2 (d,.) 2. (d,.1) 1.28 (dd, 1.6,.) 0.9 (m) 2.7 (d,.) 2.77 (d,.) 2.8 (d,.1) 1.81 (m) 2.01 (dd, 1.8, 5.9) 2 1.19 (m) 1.52 (m) 1.72 (m) 1.6 (m) 1.58 (m) 1.01 (m) 1.65 (m) 2.10 (m) 5 2.8 (br d, 1.2) 2.85 (br d, 12.6) 2.77 (dd, 12., 2.) 1.9 (dd, 1.0,.8) 1.5 (m) 6 1.61 (m) 1.6 (m) 1.62 (m) 1.57 (m) 1.8 (m) 2.01 (m) 2.0 (m) 1.99 (m) 1.86 (m) 1.89 (m) 7 1.72 (m) 1.7 (m) 1.78 (m, 2) 1.77 (m) 1.6 (m) 2.0 (m) 2.06 (m) 1.90 (m) 2. (d, 12.1) 9 1.61 (m) 1.6 (m) 1.61 (m) 11.86 (m).89 (m).9 (m) 1.72 (m) 1. (m) 2. (dd, 1.8, 5.8) 1.75 (m) 12 1.72 (m) 1.7 (m) 1.81 (m) 1.61 (m) 1.5 (m) 1.78 (m) 1.78 (m) 1.95 (m) 1.6 (m) 1.51 (m) 1 2.10 (m) 2.1 (m) 2.15 (m) 1.90 (m) 2.11 (m) 1 1.7 (m) 1.7 (m) 1.57 (m) 1.77 (m) 1.50 (m) 1.77 (m) 1.76 (m) 1.85 (m) 1.91 (m) 1.65 (m) 15 1.7 (d, 1.) 1.0 (d, 1.) 1.85 (m) 1.9 (d, 1.7) 1.5 (m) 2.12 (m) 2.15 (d, 1.) 2.0 (m) 2.02 (d, 1.7) 2. (d, 15.0) 17.59 (d, 10.2).5 (d, 10.2).10 (d, 9.).6 (d, 11.5).89 (d, 8.6).71 (d, 10.2).7 (d, 10.2).27 (d, 9.).70 (d, 11.5).0 (d, 8.6) 18 6.58 (s) 6.61 (s) 6.2 (d, 1.) 1.06 (s) 1.2 (s) 19 7.78 (s) 7.81 (s) 7.60 (d, 1.) 0.78 (s) 0.81 (s) 0.87 (s) 1. (s) 1.08 (s) 2 a Data were measured at 600 Mz in D D b Data were measured at 600 Mz in Dl 1.7 (s) 1.6 (s) 1.9 (s) 1.6 (s) oxygenated), 6 methines (including one oxygenated, two olefinic), and 6 quaternary carbons (including one oxygenated, two olefinic, and one carbonyl). These data (Tables 1, 2, ) were similar to those of mascaroside I [10, 11] except for 10 additional signals for a cinnamic acids group. The coupling constant (J 2 00 00, = 15.9 z) suggested the double bond of the cinnamic acid group was trans.besides,d 6.80(d, J = 8.2 z), 7.10(d, J = 8.2 z) were due to orthoaromatic hydrogen suggested the two oxygenated sp 2 quaternary carbons were at -6 00 and -7 00, along with -10 00 linked to -7 00 confirmed by the MB correlations from -10 00 (d.88) to -7 00. Further, the -6 0 in the glucose and -1 00 in trans-cinnamic acids group formed into ester were confirmed by MB correlations of -6 0 (d.29,.69) to -1 00 (d 9.5). The relative configuration of glucose anomeric proton was confirmed as b on the basis of the coupling constant (J 1 0 0, 2= 7.8 z). Furthermore, the glucose was identified as D-form by G analysis comparing with a standard after acid hydrolysis [12, 1].Therelativeconfiguration of 1 was same with mascaroside I by comparison of the NMR data. Furthermore, owing to the greatly predominant occurrence of this enantiomeric form in nature world, and until now, all the kaurene skeleton diterpenoids have been isolated from offea arabica were ent-kaurene series. Therefore, compound 1 was confirmed as an ent-kauranoid with the negative specific rotation value (-126.67) confirmed it further. ent-kauranoid with the configuration of - being a-orientated and -5, -9 being b-orientated. The key RESY correlations of -11 (d.86)/- (d 0.78), and -5 (d 2.8)/-9 (d 1.61), -9/-15b (d 2.12), and -15b/ 2-17 (d.59,.71) allowed to assign -11 as a-orientated, and 2-17 as b-orientated, separately [1]. ence, the structure of 1 was determined and named as mascaroside III (Fig. 2). Mascaroside IV (2) had the molecular formula of 7 6 1 according to the RESIMS analysis at m/z

0 R. hu et al. Table 2 1 NMR spectral data of compounds 15 [d in ppm] Position 1 a 2 a b a 5 b 1 5. (t) 5. (t) 5. (t) 1.8 (t) 29.7 (t) 2 187.7 (s) 187.6 (s) 185.2 (s) 19.8 (t) 28.1 (t) 18.0 (s) 17.9 (s).6 (s) 5.8 (t) 7.6 (t) 1.8 (s) 1.8 (s) 12.1 (s) 50.1 (s) 9.5 (s) 5 5.8 (d) 5.8 (d).8 (d) 52.2 (d) 50.1 (d) 6 2. (t) 2. (t). (t) 21.6 (t) 21.8 (t) 7 7.1 (t) 7.0 (t) 9.6 (t) 1. (t) 9. (t) 8 5.2 (s) 5.2 (s) 2. (s).5 (s).8 (s) 9 62.1 (d) 62.1 (d) 61.0 (d) 5.9 (s).8 (s) 10.8 (s).8 (s).9 (s) 90.6 (s) 77.1 (s) 11 66.1 (d) 66.1 (d) 65.2 (d). (t) 6.2 (t) 12 7.5 (t) 7.5 (t) 6.7 (t) 2.9 (t) 19.0 (t) 1 6.5 (d) 6.5(d).7 (d).8 (d).2 (d) 1 1. (t) 1.2 (t) 8.0 (t).5 (t) 2. (t) 15 52.0 (t) 52.0 (t) 55.1 (t) 50.9 (t) 50.2 (t) 82. (s) 82. (s) 89. (s) 8.9 (s) 89.2 (s) 17 75. (t) 75. (t) 70.5 (t) 66.2 (t) 69.7 (t) 18 111. (d) 111. (d) 110.0 (d) 17.5 (q) 29.1 (q) 19 150. (d) 150. (d) 18.2 (d) 18.0 (s) 18.2 (s) 15.7 (q) 15.7 (q) 15. (q) 19. (q) 17.5 (q) 21 2 108.1 (s) 26.8 (q) 26.6 (q) a Data were measured at 150 Mz in D D 10.2 (s) 26.9 (q) 26.8 (q) Table 1 NMR and 1 NMR of the glucose and cinnamic acids group [d in ppm, J in z] 1 NMR 1 NMR 1 2 1 2 1 0.2 (d, 7.8). (d, 7.5) 10.8 (d) 10.8 (d) 2 0. (m).26 (t, 8.) 75. (d) 75. (d) 0.0 (m). (m) 77.6 (d) 77.6 (d) 0.0 (m). (m) 71.5 (d) 71.5 (d) 5 0.50 (m).61 (d, 10.1) 75.5 (d) 75.5 (d) 6 0.29 (m).2 (d,.7) 6.9 (t) 6.8 (t).69 (m).7 (m) 1 00 9.5 (s) 9.5 (s) 2 00 6.2 (d, 15.9) 6.9 (d, 15.9) 115.2 (d) 115.7 (d) 00 7.65 (d, 15.9) 7.67 (d, 15.9) 17. (d) 17.6 (d) 00 127.6 (s) 126.9 (s) 5 00 7. (s) 6.95 (s) 111.8 (d) 107.0 (d) 6 00 150.8 (s) 19.5 (s) 7 00 19. (s) 19.8 (s) 8 00 6.80 (d, 8.2) 1.5 (d) 19.5 (s) 9 00 7.10 (d, 8.2) 6.95 (s) 12. (d) 107.0 (d) 10 00.88 (s).89 (s) 56.5 (q) 56.9 (q) 11 00.89 (s) 56.9 (q) d Data were measured at 600 Mz in D D d Data were measured at 150 Mz in D D b Data were measured at 150 Mz in Dl [M? Na]? 77.2786 (calcd for 7 6 1 Na, 77.2780). The 1D NMR data (Tables 1, 2, ) of2 was identical to that of 1, except that the cinnamic acids group in 2 was substituted by one more oxygenated methyl, which was further verified by the MB correlations from -10 00, and -11 00 (d.89) to -6 00, and -8 00 (d 19.5). The chiral centers of 2 were same with those of compound 1. Therefore, the structure of 2 was elucidated as shown and given the name mascaroside IV. Mascaroside V () was isolated as white powder. The RESIMS of showed an ion peak at m/z [M? Na]? 09.1987 (calcd for 09.1985) suggesting a molecular formula 2 0 5. The 1D NMR spectroscopic features showed it similar to the aglycone moiety of 1 with the differences being that three more carbon atoms (d 108.1 s, 26.8 q, 26.6 q). SQ together with MB spectral signals, showed that - (d 26.8), -2 (d 26.6) were located at the same sp quaternary carbon (-21 d 108.1 s) which was confirmed by correlations from -, -2 to -21. This three carbons group was substituted at -, 17- to form a ketal ring [15] based on MB correlations from 2-17 (d.10,.27) to 19 18-21.The RESY correlations were similar to compound 1, consequently, the structure of was confirmed as mascaroside V. -nor-cofaryloside I () a white amorphous powder, displayed a [M? Na]? 57.2 (calcd for 57.6) in REIMS, consistent with the molecular formula of 0, indicating 6 degrees of unsaturation. The compound displayed similar characteristic signals to 10a,a,17-trihydroxy-9a-methyl-15-oxo--nor-kauran- 2 1 11 10 5 9 7 6 8 15 12 1 1 1-1 SY MB RESY R Fig. 2 Key correlations in 2D NMR spectra of compound 1

haracterization of New Ent-kaurane Diterpenoids of Yunnan Arabica offee Beans 1 18 2 5 19 1 10 11 9 6 7 8 1 12 15 17 1 2 1 18 10 11 5 9 19 6 7 8 12 15 17 21 1 1 2 1-1 SY MB RESY Fig. Key correlations in 2D NMR spectra of compound 19-oic acid c-lactone (A) [, 17] except for the absence of the carbonyl groups at -15. This was confirmed by the chemical shift at -15 (d 50.9) in which was upfield shifted comparing to that in A (d.0). Besides, the MB correlations from 2-15 (d 1.9, 2.02) to - (d 8.9) and 8 (d.5) also supported these change. n biogenetic grounds, compound as an ent-kaurane with the configuration of -5 and -9 being b-orientated, while -10 being a-orientated. Its RESY correlations showed cross peaks of -5 (d 1.9)/-18 (d 1.06), -5/- (d 1.), -/-15b (d 2.02), -15b/ 2-17 (d.6,.70), revealing that the orientation of -18, - and -17 was b-orientated (Fig. ). Thereupon, the structure of was identified as 10a,a,17-trihydroxy-9b-methyl--norent-kauran-19-oic acid c-lactone, and named -nor-cofaryloside I. -nor-cofaryloside II (5) possessed the molecular formula of 2 6 5, according to the RESIMS analysis at m/z [M - ] - 91.288 (calcd for 91.290). Analyses of 5 0 s 1 and 1 NMR data indicated the existence of 2 carbon resonances, and -21 (d 10.2 s), - (d 26.9 q) and -2 (d 26.9 q) suggesting that the structure of compound 5 had the same ketal ring as compound and this deduction was further supported by MB correlations from 2-17, - and -2 to -21. The other carbon signals showed that compound 5 was similar to while the chemical shift of the oxygenated sp quaternary carbon -10 (d 77.1) of 5 was upfield shifted comparing to that of (d 90.6), and the molecular weight of 5 was 18 units more than that of along with the degrees of unsaturation decrease by one. Therefore, the lactone linkage which assigned between -19 and -10 was ring opened in 5. The relative configuration of 5 (Fig. ) was same with. Thus, the structure was defined as 10a-hydroxy- a,17-[(1- methylethylidene)bis(oxy)]-9b-methyl--nor-ent-kauran- 19-oic acid, and named -nor- cofaryloside II. Seven known diterpenoids were also obtained from this genus, bengalensol [18], villanovane [10], tricalysione A [1], 2b,a,17-trihydroxy-ent-kauran-19a-oic acid [19], 2--(2- -isovaleryl-b- D-gluco-pyranosyl)-a-atractyligenin [], 2--(2--isovaleryl-b-D-gluco-pyranosyl)-b-atrac- tyligenin [], --b-d-glucopyranosyl-2--(2--isovaleryl-b-dgluco-pyranosyl)-b-atracty ligenin []. Their structures were identified by comparison of their NMR data with literature data. ompounds 15, 7, 8 were evaluated for cytotoxicity against L-60, A-59, SMM-7721, MF-7 and SW80 cell lines. Unfortunately, they were inactive against all test cells (Electronic supplementary material, Table S1). Experimental Section.1 General Experimental Procedures 1D and 2D NMR spectra were obtained on a Bruker Avance III 600 Mz spectrometer (Bruker Biospin Gmb, Karlsruhe, Germany). REIMS was measured on Waters Xevo TQ-S and Waters Autospec Premier P776 mass spectrometers (Waters, Milford, MA, USA). RESIMS were recorded on an Agilent 60 Q-TF MS system (Agilent Technologies, Santa lara, A, USA). UV spectra were recorded on a Shimadzu UV- 1P (Shimadzu, Kyoto, Japan). ptical rotations were obtained on a JAS P-10 digital polarimeter (oriba, Kyoto, Japan). IR spectra were detected on Bruker Tensor 27 FTIR (KBr pellets) spectrometers. Sephadex L- (Amersham Biosciences, Upssala, Sweden) and silica gel (Qingdao aiyang hemical o., Ltd) were used for column chromatography (). Preparative high performance liquid chromatography (prep-pl) was performed on an Agilent 1100 liquid chromatography system equipped with Zorbax SB-18 columns (9. mm 9 250 mm) and a DAD detector (Agilent Technologies, Santa lara, A, USA). Thin-layer chromatography was performed on precoated TL plates (0250 lm thickness, silica gel 60 F25, Qingdao Marine hemical, Inc.), and spots were visualized by heating after spraying..2 Plant Material 1-1 SY MB RESY Fig. Key correlations in 2D NMR spectra of compound 5 The green coffee beans of offea arabica L. were harvested in December 1 and identified by ong-bo Zhang, Dehong Institute of Tropical Agriculture. A voucher specimen of. arabica was deposited in the erbarium of Kunming Institute of Botany, hinese Academy of Sciences (No. KF 112).

2 R. hu et al.. Extraction and Isolation The air-dried and powdered green Dehong coffee beans (18 kg) were extracted with 95 % methanol three times, and then the combined filtrates were concentrated under reduced pressure to give a crude extract (5 kg). The crude extract was suspended in 2 and extracted with petroleum ether (PE), ethyl acetate (EtAc), respectively. The EtAc layer (0 g) was got rid of saccharides by D101 and then subjected to RP-18 column chromatography which eluted with 2 (gradient from 15:85 to 100:0 v/v) to yield four fractions: fraction 1 ( g), fraction 2 (19 g), fraction (0 g), and fraction (2 g). Fraction was separated on silica gel using a l gradient solvent system (80:0? 1:1, v/v) to obtain eight subfractions (fraction 1 to 8). Then fraction 6 (6.5 g) was chromatographed on RP-18 ( 2 10:90100:0 v/v), Sephadex L- () and then purified by semipreparative PL (elute with N 2 1575 %, 0 min) to afford 1 (5 mg), 2 (7 mg), 10 (2 mg), 11 (8 mg), 12 (0 mg). In the same way, 5 (1 mg), 6 ( mg), 7 (17 mg) were isolated from fraction -2 (70 mg) and (2 mg), (5 mg), 8 ( mg), 9 (6 mg) were isolated from fraction - (2.7 g).. Mascaroside III (1) White amorphous powder; ½a 19 D -126.67 (c 0.150, ); UV () k max (log e) 29 (.87), 281 (.1), (.9), 2 (.97), 2 (.05) nm; IR (KBr) m max 9, 292, 2878,170, 57, 1515, 17, 1270, 15, 1126, 10 cm -1 ; 1 (600 Mz, D D) and 1 NMR (150 Mz, D D) data, Tables 1, 2 and ; ESIMS m/z 707 [M? Na]? ; REIMS m/z [M? Na]? 707.2670 (calcd for 6 1 Na, 707.267)..5 Mascaroside IV (2) White amorphous powder; ½a 19 D -107. (c 0.21, ); UV () k max (log e) 2 (.00), 279 (.0), 21 (.0), 2 (.12) nm; IR (KBr) m max 9, 292, 2855, 1706, 6, 151,, 1258, 1155, 111, 102 cm -1 ; 1 (600 Mz, D D) and 1 NMR (150 Mz, D D) data, Tables 1, 2, and ; ESIMS m/z 77 [M? Na]? ; REIMS m/z [M? Na]? 77.2786 (calcd for 7 6 1 Na, 77.2780)..6 Mascaroside V () White amorphous powder; ½a 19 D -12.81 (c 0.2, l ); UV (l ) k max (log e) 276 (.05), (.71), 8 (.67), 198 (.6) nm; IR (KBr) m max 92, 2925, 2866, 65,, 8, 18, 1128, 109 cm -1 ; 1 (600 Mz, Dl ) and 1 NMR (150 Mz, Dl ) data, Tables 1,2; ESIMS m/z 09 [M? Na]? ; REIMS m/z [M? Na]? 09.1987 (calcd for 2 0 5 Na, 09.1985)..7 -Nor-cofaryloside I () white amorphous powder; a -7.77 (c 0.6, ); UV () k max (log e) 27 (1.85), 6 (2.59) nm; IR (KBr) m max 9, 291, 2870, 1759, 2, 1, 182, 117, 1, 118, 95 cm -1 ; 1 (600 Mz, D D) and 1 NMR (150 Mz, D D) data, Tables 1,2; ESIMS m/ z 77 [M? Na]? ; REIMS m/z [M? Na]? 57.2 (calcd for 0 Na, 57.6). ½ 19 D.8 -Nor-cofaryloside II (5) White amorphous powder; a D -51.01 (c 0.105, l ); UV (l ) k max (log e) 280 (2.85), 21 (2.96), 9 (2.8), 192 (1.77) nm; IR (KBr) m max 2, 296, 2872, 1718, 99,, 17, 1252, 121, 1052, 970 cm -1 ; 1 NMR (600 Mz, Dl ) and 1 NMR (150 Mz, Dl ) data, Tables 1 and 2; ESIMS m/z 91 [M - ] - ; REIMS m/z [M ] - 91.288 (calcd for 2 5 5, 91.290)..9 ytotoxicity Assay ½ 18 The cytotoxicity against L-60, A-59, SMM-7721, MF-7 and SW80 cell lines of compounds 15, 7, 8 were tested by using MTS method. MTS [-(,5-dimethylthia- zol-2-yl)-5(-carboxymethoxyphenyl)-2-(-sulfopheny)- 2-tetrazolium] is an analogue of MTT [21], which can be reduced into soluble formazan by succinate dehydrogenase in mitochondria of living cells. Moreover, the optical density value of formazan (90 nm) is proportional to the number of living cells. Acknowledgments This work was supported financially by Programme of Key New Productions of Yunnan Province, entre of INA (No. 15BB002), The STS Programme of hinese Academy of Sciences (KFJ-SW-STS-1-8), as well as Foundation of State Key Laboratory of Phytochemistry and Plant Resources in West hina (P15-ZZ09). ompliance with Ethical Standards onflict of Interest All authors declare no conflict of interest. pen Access This article is distributed under the terms of the reative ommons Attribution.0 International License (http:// creativecommons.org/licenses/by/.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the reative ommons license, and indicate if changes were made.

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