Phenolic Compounds from the Leaves of Castanopsis Fargesii

molecules

Article Phenolic Compounds from the Leaves of Castanopsis fargesii

Yong-Lin Huang 1,*, Ya-Feng Wang 1, Jin-Lei Liu 1, Lei Wang 1, Takashi Tanaka 2, Yue-Yuan Chen 1, Feng-Lai Lu 1 and Dian-Peng Li 1

1 Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization, Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin 541006, China; [email protected] (Y.-F.W.); [email protected] (J.-L.L.); [email protected] (L.W.); [email protected] (Y.-Y.C.); [email protected] (F.-L.L.); [email protected] (D.-P.L.) 2 Department of Natural Product Chemistry, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan; [email protected] * Correspondence: [email protected]; Tel.: +86-773-355-0829; Fax: +86-773-355-0067

Academic Editor: Derek J. McPhee Received: 6 October 2016; Accepted: 13 January 2017; Published: 19 January 2017

Abstract: In the course of a phytochemical and chemotaxonomical investigation of Castanopsis species 0 0 00 (Fagaceae), three new phenolic compounds, (3R,1 S)-[1 -(6 -O-galloyl-β-D-gluco-pyranosyl)oxyethyl]- 3-hydroxy-dihydrofuran-2(3H)-one (1), (2R,3S)-2-[20-(galloyl)oxyethyl]-dihydroxybutanoic acid (2), and (3S,4S)-3-hydroxymethyl-3,4-dihydro-5,6,7-trihydroxy-4-(40-hydroxy-30-methoxyphenyl)-1H-[2]- benzopyran-1-one (3) were isolated from the fresh leaves of Castanopsis fargesii. In addition, a known phenolic glycoside, gentisic acid 5-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (4) was also isolated and identiﬁed. Their structures were elucidated by means of spectroscopic methods including one- and two-dimensional NMR techniques.

Keywords: Castanopsis fargesii; Castanopsis; phenolic; cytotoxicity

1. Introduction The genus Castanopsis belongs to the Fagaceae family and is commonly found in the evergreen forests of East Asia. There are about 120 species of Castanopsis, however, the classical plant taxonomy of the species is very complicated and sometimes confusing [1], thus, the application of other auxiliary methods and technologies, such as chemotaxonomy and cytotaxonomy, is necessary to identify species within this genus [2]. Previous phytochemical investigations on the plants of this genus C. fissa, C. cuspidata var. seiboldii, and C. hystrix have led to the isolation of triterpene hexahydroxydiphenoyl (HHDP) esters, HHDP glucoses, galloyl, acylated quinic acids, phenol glucosides, condensed tannins, and flavonol glycosides [3–8]. In this study, we investigated C. fargesii, which is widely distributed in southern China, where it is usually used as a traditional medicine for the treatment of diarrhea, hemorrhage, and chronic ulcers [1]. Our preliminary analysis by HPLC and TLC indicated that the leaves are rich in tannins. Subsequent chromatographic separation of the extract yielded two metabolites 1 and 2, which were identified as 2,3-dihydroxy-2-(2-hydroxyethyl)-butanoic acid derivatives, and two phenolic compounds 3 and 4. This paper reports the isolation and structural characterization of the new compounds 1–3 and an assessment of the cytotoxicity of these molecules.

2. Results and Discussion The fresh leaves of C. fargesii were extracted with 80% aqueous ethanol, and the extract was partitioned between Et2O and water. The Et2O and aqueous fractions were separated by a combination

Molecules 2017, 22, 162; doi:10.3390/molecules22010162 www.mdpi.com/journal/molecules Molecules 2017, 22, 162 2 of 8

Molecules 2017, 22, 162 2 of 8 of Sephadexof Sephadex LH-20, LH-20, MCI MCI gel gel CHP CHP 20P, 20P,Toyopearl Toyopearl Butyl-650C, Chromatorex Chromatorex ODS, ODS, and and Diaion Diaion HP20SS HP20SS columncolumn chromatography chromatography and and semi-preparative semi-preparative reverse-phase reverse-phase HPLC, HPLC, to to yield yield three three new new compounds compounds1 – 3 and1– one3 and known one known phenolic phenolic compound compound gentisic gentisic acid acid 5-O -5-αO-L--rhamnopyranosyl-(1α-L-rhamnopyranosyl-(1→→2)-2)-β-βD--glucosideD-glucoside [ 9] (4)[9] (Figure (4) (Figure1). 1).

OH HO O O O O 2 OH 2' 6" HO O 1 O OH HO O O O 3 HO 2 1' HO 1' 1" HO OH HO HO OH 2' 3

1 2 OH O HOOC HO 9 1 1 O A B OH 5 HO 10 5 3a HO O O OH HO 1' C HO O 3' OCH 3 O 1" OH HO HO OH 3 4

FigureFigure 1.1. StructuresStructures of compounds 11––44. .

Compound 1 was isolated as a brown amorphous powder and gave a positive FeCl3 test (dark blue), Compound 1 was isolated as a brown amorphous powder and gave a positive FeCl test (dark blue), which suggested the presence of phenol moieties in the molecule. The molecular formula3 C19H24O13 whichwas suggesteddetermined the based presence on the of phenolliquid chromatograp moieties in thehy-mass molecule. spectrometry The molecular IT-TOF formula (LC-MS/IT-TOF), C19H24O13 was determinedwhich showed based [M on − theH]− liquidand [M chromatography-mass + Na]+ ion peaks at m/ spectrometryz 459.1143 (calcd. IT-TOF for C (LC-MS/IT-TOF),19H23O13, 459.1144) whichand − + showed483.1118 [M (calcd.− H] for Cand19H24 [MO13Na, + Na] 483.1109),ion peaks respectively. at m/ zIn459.1143 the 1H- and (calcd. 13C-NMR for spectra C19H23 (TableO13, 459.1144)1), two 1 13 andproton 483.1118 singlets (calcd. at δH for7.13 C (2H,19H24 s)O and13Na, four 483.1109), aromatic respectively.carbon signals Inat δ theC 109.1H- and and 145.2,C-NMR along spectrawith (Tablean ester1), two carbonyl proton signal singlets at δC at166.4δH suggested7.13 (2H, the s) presence and four of aromatic a galloyl carbongroup [10].The signals hydrolysis at δC 109.1 of and1 145.2,produced along D with-glucose, an ester which carbonyl was identified signal by at GCδC 166.4 analysis. suggested The coupling the presence constant of the a galloyl anomeric group sugar [10 ]. Theproton hydrolysis was 7.8 of Hz,1 produced indicatingD -glucose,that the sugar which moiety was identifiedwas in the byβ configuration. GC analysis. TheThe couplinglarge downfield constant of theshift anomeric of the glucose sugar H-6” proton protons was ( 7.8δH 4.32 Hz, indicatingand 4.58) suggested that the sugaresterifica moietytion with was inthe the galloylβ configuration. moiety at this position. This was confirmed by the HMBC00 correlation between H-6” and the carboxy carbon The large downfield shift of the glucose H-6 protons (δH 4.32 and 4.58) suggested esterification with(δC the166.4) galloyl (Figure moiety 2). A second at this HMBC position. correlation This was between confirmed H-1” and by C-1’ the HMBCsuggested correlation that the glycosyl between group00 is linked to C-1′. HSQC experiment showed that the remaining moiety was composed of six 00 H-6 and the carboxy carbon (δC 166.4) (Figure2). A second HMBC correlation between H-1 andcarbons: C-10 suggested a carboxy carbon that the (δC glycosyl178.6, C-2), group an oxygenated is linked methine to C-1 (0δ.C HSQC77.8, C-1’), experiment an oxygenated showed quaternary that the carbon (δC 76.6, C-3), an oxygenated methylene (δC 65.8, C-5), a methylene (δC 29.9, C-4), and a methyl remaining moiety was composed of six carbons: a carboxy carbon (δC 178.6, C-2), an oxygenated (δC 15.6, C-2’) carbon.0 The presence of a γ-lactone structure in the remaining moiety was suggested methine (δC 77.8, C-1 ), an oxygenated quaternary carbon (δC 76.6, C-3), an oxygenated methylene by the lower field shift of the carboxy carbon (δC 178.6) and the unsaturation index (eight). This was (δ 65.8, C-5), a methylene (δ 29.9, C-4), and a methyl (δ 15.6, C-20) carbon. The presence of a Calso confirmed by HMBC correlationsC shown in Figure 2, in whichC the oxygenated methylene protons γ-lactone structure in the remaining moiety was suggested by the lower field shift of the carboxy (δH 4.31–4.34, H-5) correlated with C-2. Furthermore, the 1H-1H COSY correlations of H-4 (δH 2.10) with carbon (δ 178.6) and the unsaturation index (eight). This was also confirmed by HMBC correlations H-5 (δHC 4.32) and H-1′ with H-2′ and the HMBC correlations of H-1’ with C-3 and C-4 and H-4 with shownC-2, C-3, in Figure and C-52, indicated in which that the oxygenatedthe remaining methylene moiety is 3-hydroxy-3-(1’- protons ( δH 4.31–4.34,hydroxyethyl)dihydrofuran- H-5) correlated with 1 1 0 C-2.2(3 Furthermore,H)-one. Consequently, the H- Hthe COSY structure correlations of compound of H-4 1 was (δH 2.10)established with H-5 as 3-[1’-(6”- (δH 4.32)O-galloyl- and H-1β-withD- 0 0 H-2glucopyranosyl)oxyethyl]-3-hydroxy-dihydrofuran-2(3and the HMBC correlations of H-1 with C-3 and C-4H)-one. and H-4 with C-2, C-3, and C-5 indicated 0 that theCompound remaining 2 moietywas isolated is 3-hydroxy-3-(1 as a yellow amorphous-hydroxyethyl)dihydrofuran-2(3 powder and gave a positiveH FeCl)-one.3 test Consequently, (dark blue). 0 00 theThe structure presence of of compound a galloyl group1 was was established deduced from as 3-[1 the-(6 13C-NMR-O-galloyl- signalsβ- D(Table-glucopyranosyl)oxyethyl]- 1) [6]. The molecular 3-hydroxy-dihydrofuran-2(3formula of C13H16O9 was establishedH)-one. based on the LC-MS/IT-TOF (m/z 339.0672 [M + Na]+, calcd. for 13 13 C13CompoundH16O9Na, 339.0687)2 was and isolated C-NMR as adata. yellow The amorphousC-NMR and DEPT powder spectra and show gaveed a signals positive attributable FeCl3 test 13 (darkto ablue). methyl The (δC presence16.9), a methylene of a galloyl (δC group34.2), an was oxymethine deduced ( fromδC 71.4), the an C-NMRoxymethylene signals (δ (TableC 60.2),1 )[an 6]. Theoxy molecular quaternary formula (δC 77.7), of and C13 aH carboxyl16O9 was (δC established 175.4) carbons. based The on NMR the data LC-MS/IT-TOF of 2 (Table 1) (werem/z similar339.0672 Molecules 2017, 22, 162 3 of 8

Molecules 2017, 22, 162 3 of 8 + 13 13 [M + Na] , calcd. for C13H16O9Na, 339.0687) and C-NMR data. The C-NMR and DEPT spectra to those of 1, except for the absence of the signals for one glucosyl moiety, which was supported by its showed signals attributable to a methyl (δC 16.9), a methylene (δC 34.2), an oxymethine (δC 71.4), an MS data. A 2,3-dihydroxy-2-(2’-oxyethyl)-butanoic acid moiety could be constructed by 1H-1H COSY oxymethylene (δ 60.2), an oxy quaternary (δ 77.7), and a carboxyl (δ 175.4) carbons. The NMR correlations (FigureC 2) of H-4 with H-3 and H-1C′a with H-2′a and the HMBCC correlations of H-4 with data of 2 (Table1) were similar to those of 1, except for the absence of the signals for one glucosyl C-3 (δC 71.4), H-3 with C-1, H-1′ with C-1, and H-2′ with C-2 (Figure 2). The HMBC correlation of H-2’ moiety, which was supported by its MS data. A 2,3-dihydroxy-2-(20-oxyethyl)-butanoic acid moiety with the carboxy carbon (δC 165.9) indicated that the galloyl group was attached to C-2’. Based on these 1 1 0 0 couldresults, be constructedthe structure byof 2H- wasH determined COSY correlations to be 2-[2’-(galloyl)-oxyethyl]-2,3-dihydroxybutanoic (Figure2) of H-4 with H-3 and H-1 a with acid. H-2 a 0 0 and the HMBC correlations of H-4 with C-3 (δC 71.4), H-3 with C-1, H-1 with C-1, and H-2 with 0 C-2 (Figure2Table). The 1. HMBC1H (500 MHz) correlation and 13C (125 of H-2 MHz)with NMR the data carboxy of compounds carbon 1 and (δC 2165.9) in acetone- indicatedd6. that the galloyl group was attached to C-20. Based on these results, the structure of 2 was determined to be 1 2 2-[20-(galloyl)-oxyethyl]-2,3-dihydroxybutanoicPositions acid. 1H 13C 1H 13C 1 1 13 175.4 Table 1. H (500 MHz) and C (125 MHz) NMR data of compounds 1 and 2 in acetone-d6. 2 178.6 77.7 3 176.6 3.88 (1H, q, J = 2 6.5 Hz) 71.4 Positions 4 2.10 (1H,1H m) 29.913C 1.19 (3H,1 Hd, J = 6.4 Hz) 13C 16.9 2.58 (1H, m) 1 175.4 5 2 4.31–4.34 (2H, m) 65.8178.6 77.7 1′ 3 4.01 (1H, d, J = 6.4 Hz) 77.8 76.6 2.31 3.88 (1H, (1H, m), q, J =2.36 6.5 Hz)(1H, m) 71.4 34.2 2′ 4 1.26 (1H, 2.10 d, (1H,J = 6.4 m) Hz) 15.6 29.9 4.31 1.19 (1H, (3H, m), d, J =4.33 6.4 Hz)(1H, m) 16.9 60.2 2.58 (1H, m) 1″ 5 4.41 (1H, 4.31–4.34 d, J = (2H, 7.8 m)Hz) 104.1 65.8 2″ 10 3.19 (1H,4.01 dd, (1H, J d,= J7.8,= 6.4 8.9 Hz) Hz) 73.8 77.8 2.31 (1H, m), 2.36 (1H, m) 34.2 0 3″ 2 3.431.26 (1H, (1H, t, d,J =J =8.9 6.4 Hz) Hz) 76.4 15.6 4.31 (1H, m), 4.33 (1H, m) 60.2 1” 4.41 (1H, d, J = 7.8 Hz) 104.1 4″ 2” 3.46 3.19 (1H, dd,t, JJ == 8.9 7.8, Hz) 8.9 Hz) 70.4 73.8 3” 3.433.62 (1H, (1H, t, J =m) 8.9 Hz) 76.4 5″ 73.9 4” 3.464.32 (1H, (1H, t, J =m) 8.9 Hz) 70.4 3.62 (1H, m) 5” 73.9 6″ 4.58 (1H, dd,4.32 J = (1H, 2.1, m) 11.8 Hz) 63.8 Galloyl 6” 4.58 (1H, dd, J = 2.1, 11.8 Hz) 63.8 1 Galloyl 120.6 121.0 2,6 1 7.13 (2H, s) 109.1 120.6 7.10 (2H, s) 121.0 109.1 2,6 7.13 (2H, s) 109.1 7.10 (2H, s) 109.1 3,5 3,5 145.2 145.2 145.0 145.0 4 4 138.1 138.1 137.7 137.7 7 7 166.4 166.4 165.9 165.9

1 1 FigureFigure 2. 2.Key Key HMBC HMBC andand 1H-1HH COSY COSY correlations correlations of of 1 1andand 2. 2.

The absolute configurations at C-2 and C-3 of 2 were established using the modified Mosher’s The absolute configurations at C-2 and C-3 of 2 were established using the modified Mosher’s method [11,12]. Treatment of 2 with CH3I, then with (R)-(−)- and (S)-(+)-2-methoxy-2-trifluoromethyl- method2-phenylacetyl [11,12]. Treatment (MTPA) chloride of 2 with to CHget 3theI, then C-3 with(S)- and (R)-( (R−)-MTPA)- and ( Sester)-(+)-2-methoxy-2-trifluoromethyl- derivatives, respectively. Δδ 2-phenylacetylvalues obtained (MTPA) from the chloride 1H-NMR to data get of the the C-3 ( (RS)-)- and and (S ()-MTPAR)-MTPA ester ester derivative derivatives, indicated respectively. that the 1 ∆δabsolutevalues obtainedconfiguration from at the C-3H-NMR of 2 was data S (Figure of the 3). C-3 Compound (R)- and ( S2)-MTPA reacted esterwith 2,2-dimethoxypropane derivative indicated that the(DMP) absolute and configuration pyridinium p-toluene at C-3 of sulfonate2 was S (Figure (PPTS) 3to). form Compound the 2,3-O2-isopropylidenereacted with 2,2-dimethoxypropane derivative. The C-2 (DMP)and C-3 and relative pyridinium configurationp-toluene of sulfonate 2,3-O-isopropylidene (PPTS) to form derivative the 2,3-O was-isopropylidene determined based derivative. on the TheNOE C-2 andcorrelation C-3 relative of H-1 configuration′ with H3-4 of(Figure 2,3-O 4).-isopropylidene Thus, the absolute derivative configurations was determined at C-2 and based C-3 onof 2 the were NOE 0 correlationassigned as of R H-1 andwith S, respectively. H3-4 (Figure 4). Thus, the absolute configurations at C-2 and C-3 of 2 were assigned as R and S, respectively. Molecules 2017, 22, 162 4 of 8 Molecules 2017, 22, 162 4 of 8 Molecules 2017, 22, 162 4 of 8

Figure 3. Δδ(S-R) values of MTPA ester derivative of 2. Figure 3. ∆δ - values of MTPA ester derivative of 2. Figure 3. Δδ(S(SR-R)) values of MTPA ester derivative of 2.

Figure 4. Key NOE correlations of 2,3-O-isopropylidene derivative of 2. Figure 4. Key NOE correlations of 2,3-O-isopropylidene derivative of 2. Figure 4. Key NOE correlations of 2,3-O-isopropylidene derivative of 2. Compounds 1 and 2 both contain a 2,3-dihydroxy-2-(2-hydroxyethyl)-butanoic acid moiety. The hydrolysisCompounds of 1 in 1 M1 and HCl 2 bothyielded contain 3-hydroxy-3- a 2,3-dihydroxy-2-(2-hydroxyethyl)-butanoic(1-hydroxyethyl)dihydrofuran-2(3H)-one acid moiety.that was The hydrolysisCompounds of 1 in1 and1 M HCl2 both yielded contain 3-hydroxy-3- a 2,3-dihydroxy-2-(2-hydroxyethyl)-butanoic(1-hydroxyethyl)dihydrofuran-2(325 H)-one acid that moiety. was identified to have the same absolute configuration as 2 by comparing their [α]D and CD data. Hence, The hydrolysis of 1 in 1 M HCl yielded 3-hydroxy-3-(1-hydroxyethyl)dihydrofuran-2(325 H)-one that was theidentified absolute configurationsto have the same of absolute1 were assigned configuration as 3R,1’ asS 2. by comparing their [α]D and CD data. Hence, identified to have the same absolute configuration as 2 by comparing their [α]25 and CD data. Hence, theCompound absolute configurations 3 was obtained of as 1 a werebrown assigned amorphous as 3 Rpowder,1’S. , which gave a dark blueD color with FeCl3. 0 the absolute configurations of 1 were assigned as 3R,1 S. − 3 The molecularCompound formula 3 was C 17obtainedH16O8 was as adeduced brown amorphous from the [M powder − H] peak, which at mgave/z 347.0768 a dark blue in the color LC-MS/IT- with FeCl . − TOFThe Compound(calcd. molecular for C17 formula3Hwas15O8, obtained347.0772). C17H16O8 aswasComparison a browndeduced amorphous of from the 1theH- and[M powder, − 13 H]C-NMR peak which dataat m /of gavez 347.07683 (Table a dark 2)in andthe blue LC-MS/IT-(3 colorS,4S)- with 1 13 − FeCl3-[(TOFβ-3D.-glucopyranosyl)oxymethyl]-3,4-d The(calcd. molecular for C17H15 formulaO8, 347.0772). C17H Comparison16ihydro-5,6,7-trihydroxy-4-(4O8 was deduced of the H- from and theC-NMR’-hydroxy-3’-methoxyphenyl)-1 [M − dataH] ofpeak 3 (Table at m 2)/ andz 347.0768 (3HS-,4S)- in 1 13 the[2]-benzopyran-1-one3-[( LC-MS/IT-TOFβ-D-glucopyranosyl)oxymethyl]-3,4-d (calcd. [13] revealed for C17 thatH15 Othe8ihydro-5,6,7-trihydroxy-4-(4, 347.0772).methyl at C-3 Comparison in the known’-hydroxy-3’-methoxyphenyl)-1 of thecompoundH- and wasC-NMR replaced data byH - of 3 0 (Tablea hydroxymethyl[2]-benzopyran-1-one2) and (3 S,4 inS )-3-[(3. This [13]β -wasD -glucopyranosyl)oxymethyl]-3,4-dihydro-5,6,7-trihydroxy-4-(4revealed confirmed that bythe the methyl MS data at C-3 an din the the correlations known compound of the methine was replaced proton-hydroxy- by 0 3(δ-methoxyphenyl)-1Ha 4.67) hydroxymethyl with the methylene inH 3-[2]-benzopyran-1-one. This carbon was confirmed (δC 62.4) in by the the [ 13HMBC MS] revealed data spectr andum that the (Figure correlations the methyl 6), as well of at the as C-3 methine1H- in1H the COSY proton known 1 1 compoundcorrelations(δH 4.67) with wasof H-3 the replaced (methyleneδH 4.67) by with acarbon hydroxymethyl H-3a ( δ(δC H62.4) 3.54) in and the in HMBCH-43. This(δH spectr 4.50) was um(Figure confirmed (Figure 6). Comparison6), by as thewell MS as ofH- data theH COSYCD and the correlations of H-3 (δH 4.67)25 with H-3a (δH 3.54) and H-4 (δH 4.50) (Figure 6). Comparison of the CD correlationsand the optical of rotation the methine ([α]D proton+18.3°) (dataδ 4.67) of 3 with with those the of methylene similar co carbonmpounds (δ suggested62.4) in that the the HMBC 25 H C and the optical rotation ([α]D 1+18.3°)1 data of 3 with those of similar compounds suggested that the spectrumabsolute configuration (Figure5), as is well 3S,4S as [14].H- BasedH COSY on the correlations above evidence of H-3s, the (δ structureH 4.67) with of compound H-3a (δH 33.54) was and concludedabsolute to configuration be (3S,4S)-3-hydroxymethyl-3,4-dihydro-5,6,7-trihydroxy-4-(4’-hydroxy-3’-methoxyphenyl)- is 3S,4S [14]. Based on the above evidences, the structure of25 compound◦ 3 was H-4 (δH 4.50) (Figure5). Comparison of the CD and the optical rotation ( [α] +18.3 ) data of 3 concluded to be (3S,4S)-3-hydroxymethyl-3,4-dihydro-5,6,7-trihydroxy-4-(4’-hydroxy-3’-methoxyphenyl)-D with1H-[2]-benzopyran-1-one. those of similar compounds suggested that the absolute configuration is 3S,4S [14]. Based on 1H-[2]-benzopyran-1-one. the above evidences, the structure of compound 3 was concluded to be (3S,4S)-3-hydroxymethyl- Table 2. 1H-NMR (500 MHz) and 13C-NMR (125 MHz) data of compound 3 in DMSO-d6. 3,4-dihydro-5,6,7-trihydroxy-4-(40-hydroxy-30-methoxyphenyl)-1H-[2]-benzopyran-1-one. Table 2. 1H-NMR (500 MHz) and 13C-NMR (125 MHz) data of compound 3 in DMSO-d6. Positions 1H 13C 1 13 1 13 Table 2. H-NMRPositions1 (500 MHz) and C-NMR H (125 MHz) data of compound164.6 C3 in DMSO-d6. 2 1 164.6 3 2 Positions 4.67 (1H, td, J = 1.1, 1H 6.5 Hz) 13 84.7C 3 3.54 4.67(1H, (1H, dd, td,J = 7.6,J = 1.1, 11.2 6.5 Hz) Hz) 84.7 3a 1 164.662.4 3.683.54 (1H, (1H, dd, dd, J = J6.5, = 7.6, 11.2 11.2 Hz) Hz) 3a 2 62.4 4 33.68 4.67 4.50(1H, (1H, (1H,dd, td, Jbr =J =6.5,s)1.1, 11.2 6.5 Hz) 84.7 36.7 4 3.54 (1H, 4.50 dd,(1H,J = br 7.6, s) 11.2 Hz) 36.7 5 3a 62.4144.7 6 5 3.68 (1H, dd, J = 6.5, 11.2 Hz) 139.7144.7 4 4.50 (1H, br s) 36.7 6 139.7 7 5 144.7145.3 8 7 67.15 (1H, s) 139.7107.7145.3 9 8 77.15 (1H, s) 145.3115.3107.7 10 9 8 7.15 (1H, s) 107.7 119.9115.3 1’ 10 9 115.3133.5 119.9 10 119.9 1’ 133.5 2’ 10 6.83 (1H, d, J = 2.1 Hz) 133.5 111.8 3’ 2’ 20 6.836.83 (1H, (1H, d, d, J J= =2.1 2.1 Hz) Hz) 111.8147.6 111.8 4’ 3’ 30 147.6144.8147.6 0 5’ 4’ 4 6.67 (1H, d, J = 8.1 Hz) 144.8 114.9144.8 50 6.67 (1H, d, J = 8.1 Hz) 114.9 6’ 5’ 6.64 (1H,6.67 dd, (1H, J =d, 2.1, J = 8.18.1 Hz)Hz) 119.6 114.9 60 6.64 (1H, dd, J = 2.1, 8.1 Hz) 119.6 OCH6’3 6.64 (1H,3.72 dd,(3H, J s)= 2.1, 8.1 Hz) 55.4 119.6 OCH3 3.72 (3H, s) 55.4 OCH3 3.72 (3H, s) 55.4 Molecules 2017, 22, 162 5 of 8 Molecules 2017, 22, 162 5 of 8

Figure 5.6. Key HMBC and COSY correlations ofof 33..

All isolates were subjected to a cytotoxicity assay in vitro against human lung epithelial A549, All isolates were subjected to a cytotoxicity assay in vitro against human lung epithelial A549, human hepatocellular carcinoma SMMC-7721 cell, human gastric carcinoma MGC-803 cell, liver human hepatocellular carcinoma SMMC-7721 cell, human gastric carcinoma MGC-803 cell, liver hepatocellular HepG2 cell, and human breast adenocarcinoma MCF-7 tumour cell. Unfortunately, hepatocellular HepG2 cell, and human breast adenocarcinoma MCF-7 tumour cell. Unfortunately, none of the isolates showed inhibitions of those tumour cells at the highest concentration tested (IC50 none of the isolates showed inhibitions of those tumour cells at the highest concentration tested value > 10 μM). (IC50 value > 10 µM).

3. Experimental Section

3.1. Materials The leaves of C. fargesii were collected at Guangxi Institute of Botany, Guangxi, China, in August 2014, and were identiﬁedidentified byby Prof.Prof. Shi-Hong Lu. A voucher specimen (20140627) was deposited in the Guangxi KeyKey LaboratoryLaboratory ofof Functional Functional Phytochemicals Phytochemicals Research Research and and Utilization, Utilization, Guangxi Guangxi Institute Institute of Botany,of Botany, China. China.

3.2. General Experimental Procedures Optical rotations were measured with a 341 digital polarimeter (Perkin-Elmer Corp., Corp., Waltham, Waltham, MA, USA). 11H- and 13C-NMRC-NMR spectra spectra were were me measuredasured in in acetone acetone at at 27 °C,◦C, using using an Avance 500 spectrometer (500(500 MHzMHz forfor 11H and 125125 MHzMHz forfor 1313C, Bruker Biospin AG, Fällanden,Fällanden, Switzerland).Switzerland). Coupling constantsconstants and chemical shifts were given in Hz andand onon aa δδ (ppm)(ppm) scale,scale, respectively.respectively. GCGC waswas performedperformed onon aa 6890N instrument equipped withwith a FID detector (Ag (Agilentilent Technologies, SantaSanta Clara,Clara, CA, USA) operated at 280 °C◦C( (column:column: 28 28 m m ×× 0.320.32 mm mm i.d. i.d. HP-5, HP-5 column, column temp. temp. 160 160 °C).◦ C).LC-MS/IT-TOF LC-MS/IT-TOF was wasrecorded recorded on a onLCMS-IT-TOF a LCMS-IT-TOF spectrometer spectrometer (Shimadzu, (Shimadzu, Kyoto, Kyoto, Japan). Japan). Semi-preparative Semi-preparative HPLC HPLC was was performed performed on onan Agilent an Agilent 1200 1200 apparatus apparatus equipped equipped with with a UV adetect UV detectoror and a Zorbax and a Zorbax SB-C-18 SB-C-18 (9.4 × 250 (9.4 mm)× 250 column mm) column(Agilent). (Agilent). Column Column chromatography chromatography (CC) was (CC) performed was performed using using Sephadex Sephadex LH-20 LH-20 (25–100 (25–100 μm; µGEm; GEHealthcare Healthcare Bio-Science Bio-Science AB, AB, Uppsala, Uppsala, Sweden), Sweden), MCI MCI gel gel CHP CHP 20P 20P (75–150 (75–150 μµm;m; Mitsubishi Mitsubishi Chemical, Chemical, Tokyo,Tokyo, Japan),Japan), DiaionDiaion HP20SSHP20SS (Mitsubishi(Mitsubishi Chemical),Chemical), Chromatorex Chromatorex ODS (100–200(100–200 mesh;mesh; FujiFuji SilysiaSilysia Chemical, Aichi, Aichi, Japan), Japan), and and Toyopearl Toyopearl Butyl-650C Butyl-650C (TOSOH, (TOSOH, Tokyo, Tokyo,Japan) columns. Japan) columns.TLC was performed TLC was performedon precoated on Kieselgel precoated 60 F Kieselgel254 plates (0.260 F mm254 plates thick; Merck, (0.2 mm Darmstadt, thick; Merck, Germany) Darmstadt, with toluene–HCO Germany) with2Et– toluene–HCOHCO2H (1:7:1,2 Et–HCOv/v) as the2H solvent, (1:7:1, v and/v) asspots the were solvent, detected and spots by spraying were detected with a by2% spraying ethanolic with FeCl a3.2% ethanolic FeCl3. 3.3. Extraction and Separation 3.3. Extraction and Separation The fresh leaves of C. fargesii (5.20 kg) were cut into small pieces and extracted three times with EtOH/HThe2 freshO (8:2, leaves v/v, 36 of L)C. by fargesii maceration(5.20 kg) at room were cuttemperature into small for pieces 7 days. and The extracted extracts three were times combined with EtOH/Hand concentrated2O (8:2, v /underv, 36 L)reduced by maceration pressure at to room give temperature an aqueous forsolution. 7 days. The The solution extracts was were partitioned combined andwith concentrated Et2O four times under to give reduced the Et pressure2O fraction to give (32.4 an g). aqueous The aqueous solution. layer The was solution subjected was to partitioned Sephadex withLH-20 Et CC2O four(8 cm times i.d. × to 40 give cm) the with Et 20%–100%O fraction MeOH–H (32.4 g).2 TheO (20% aqueous stepwise layer elution, was subjected each 1.5 to L) Sephadex to give 9 LH-20fractions: CC frs (8 cm1 (15.6 i.d. g),× 402 (84.5 cm) withg), 3 0%–100%(26.6 g), 4 MeOH–H (27.0 g), 52 (130.0O (20% g), stepwise 6 (12.9 g), elution, 7 (3.3 eachg), 8 1.5(2.3 L) g), to and give 9 9(2.2 fractions: g). Fraction frs 1fr. (15.6 2 (84.5 g), g) 2 (84.5was separated g), 3 (26.6 by g), MCI 4 (27.0 gel CHP g), 5 20PCC (130.0 g),(6 cm 6 (12.9 i.d. × g), 40 7 cm) (3.3 with g),8 MeOH– (2.3 g), H2O (10% stepwise elution, each 1.0 L) to yield seven fractions, and fraction fr. 2-2 (5.3 g) was further fractionated by Diaion HP20SS CC (4 cm i.d. × 30 cm) with H2O containing increasing proportions of Molecules 2017, 22, 162 6 of 8 and 9 (2.2 g). Fraction fr. 2 (84.5 g) was separated by MCI gel CHP 20PCC (6 cm i.d. × 40 cm) with MeOH–H2O (10% stepwise elution, each 1.0 L) to yield seven fractions, and fraction fr. 2-2 (5.3 g) was further fractionated by Diaion HP20SS CC (4 cm i.d. × 30 cm) with H2O containing increasing proportions of MeOH (0%–100%, 10% stepwise elution, each 0.5 L) to give 4 (105 mg). The Et2O fraction was subjected to MCI gel CHP 20PCC (5 cm i.d. × 50 cm) with 0%–100% MeOH in H2O (10% stepwise elution, each 0.5 L) to yield 10 fractions: frs E-1 (5.3 g), 2 (7.5 g), 3 (1.6 g), 4 (3.2 g), 5 (4.3 g), 6(1.5 g), 7 (2.3 g), 8 (1.0 g), 9 (9.9 g) and 10 (3.5 g). Fr. E-3 was fractionated by Toyopearl Butyl-650C CC (3 cm i.d. × 30 cm) with 0%–100% MeOH–H2O containing 0.1% CF3CO2H (TFA) (10% stepwise elution, each 0.3 L) to give fr. E-31 (1.3 g) and fr. E-32 (122 mg). Fr. E-32 was further puriﬁed by Chromatorex ODS CC (3 cm i.d. × 30 cm) with 0%–80% MeOH in H2O (5% stepwise elution, each 0.2 L) to give 3 (12 mg). Fraction E-4 was separated by Sephadex LH-20 CC (4 cm i.d. × 40 cm) with H2O containing increasing amounts of MeOH (0%–100%, 10% stepwise elution, each 0.5 L) to yield Fr. E-41 (250 mg), Fr. E-42 (150 mg), Fr. E-43 (296 mg) and Fr. E-44 (1.7 g). The Fr. E-42 and Fr. E-43 were further puriﬁed by semi-preparative HPLC (MeCN/H2O, 20:80, 2.5 mL/min) to give 1 (46 mg, tR 14.5 min) and 2 (68 mg, tR 13.2 min), respectively.

3.4. Spectroscopic Data

(3R,1'S)-[1'-(6"-O-Galloyl-β-D-glucopyranosyl)oxyethyl]-3-hydroxy-dihydrofuran-2(3H)-one (1): Brown 25 ◦ amorphous powder; [α]D +52.1 (c = 0.12, MeOH); UV (MeOH) λmax nm (log ε): 272 (4.32); 1 13 CD (MeOH) λmax (∆ε) 278 (8.4), 254 (4.5), 209 (2.7). H- and C-NMR data, see Table1; LC-MS/IT-TOF − + m/z [M − H] 459.1143 (calcd. for C19H23O13, 459.1144) and [M + Na] 483.1118 (calcd. for C19H24O13Na, 483.1109). 25 ◦ (3R,1'S)-3-Hydroxy-1'-hydroxyethyl-dihydrofuran-2(3H)-one (Hydrochloride of 1): [α]D −17.0 (c = 0.15, MeOH); UV (MeOH) λmax nm (log ε): 265 (3.16); CD (MeOH) λmax (∆ε) 267 (5.7), 251 (3.2), 211 (1.2). 1 H-NMR (MeOH-d4, 500 MHz) δ 4.36 (1H, m, H-5a), 4.25 (1H, m, H-5b), 2.11 (1H, m, H-4a), 2.33 (1H, m, H-4b), 1.21 (3H, d, J = 6.5 Hz, H-4), 4.12 (1H, q, J = 6.5 Hz, H-10), 1.21 (3H, d, J = 6.5 Hz, H-20); + LC-MS/IT-TOF m/z 169.0475 [M + Na] (calcd. for C6H10O4Na, 169.0477). 25 ◦ (2R,3S)-2-[2'-(Galloyl)oxyethyl]-dihydroxybutanoic acid (2): Yellow amorphous powder; [α]D −16.7 (c = 0.12, MeOH); UV (MeOH) λmax nm (log ε): 267 (4.26); CD (MeOH) λmax (∆ε) 268 (10.6), 252 (4.6), 212 (1.4). 1H- and 13C-NMR data, see Table1; LC-MS/IT-TOF m/z 339.0672 [M + Na]+ (calcd. for C13H16O9Na, 339.0687). (3S,4S)-3-Hydroxymethyl-3,4-dihydro-5,6,7-trihydroxy-4-(4'-hydroxy-3'-methoxyphenyl)-1H-[2]-benzopyran- 25 ◦ 1-one (3): Brown amorphous powder; [α]D +18.3 (c = 0.11, MeOH); UV (MeOH) λmax nm (log ε): 1 13 220 (4.26), 278 (2.35); CD (MeOH) λmax (∆ε) 288 (11.4), 242 (6.5), 218 (2.7). H- and C-NMR data, − see Table2; LC-MS/IT-TOF m/z 347.0768 [M − H] (calcd. for C17H15O8, 347.0772).

3.5. Preparation of MTPA Esters Derivatives

CH3I (30 mg) and K2CO3 (15 mg) were added to a solution of 2 (10 mg) in DMF (5 mL). After stirring for 24 h at room temperature (r.t.), the reaction mixture was suspended in H2O and extracted with CHCl3. The CHCl3 layer was vacuum dried to afford a residue (6.2 mg). Then, DMAP (3.8 mg), Et3N (4.0 µL), and (R)-(−)-MTPACl (3.0 µL) were added to a solution of the residue (3.1 mg) in CH2Cl2 (1.0 mL) and stirred for 4 h at r.t. The reaction mixture was dried under a stream of N2. Separation of the residue was done by a silica gel column (hexane/EtOAc, 4:1) to afford the (S)-MTPA ester derivative (2.1 mg). The (R)-MTPA ester derivative (2.3 mg) was obtained according to the same procedure using (S)-(+) MTPACl.

1 (3S)-MTPA Ester derivative of 2: Colorless oil; H-NMR (MeOH-d4, 500 MHz) δ 7.0512–7.3901 (7H), 4.8212 (1H, q, J = 6.6 Hz, H-3), 1.3648 (3H, d, J = 6.6 Hz, H-4), 2.2511 (1H, m, H-10a), 2.3004 (1H, m, Molecules 2017, 22, 162 7 of 8

0 0 0 H-1 b), 4.2526 (1H, m, H-2 a), 4.2812 (1H, m, H-2 b), 3.3206–3.8516 (-OCH3 × 5). LC-MS/IT-TOF m/z + 611.17147 [M + Na] (calcd. for C27H31F3O11Na, 611.17162). 1 (3R)-MTPA Ester derivative of 2: Colorless oil; H-NMR (MeOH-d4, 500 MHz) δ 7.0510–7.3902 (7H), 4.8210 (1H, q, J = 6.6 Hz, H-3), 1.3603 (3H, d, J = 6.6 Hz, H-4), 2.2572 (1H, m, H-10a), 2.3088 (1H, m, 0 0 0 H-1 b), 4.2548 (1H, m, H-2 a), 4.2858 (1H, m, H-2 b), 3.3312–3.8716 (-OCH3 × 5). LC-MS/IT-TOF m/z + 611.1710 [M + Na] (calcd. for C27H31F3O11Na, 611.1716).

3.6. Preparation of Acetonide Derivative of 2 Compound 2 (10.2 mg) was dissolved in acetone (1.0 mL) and treated with DMP (0.2 mL) and PPTS (6.5 mg) at r.t. After 4 h, Et3N (7.5 µL) was added and the mixture was concentrated by N2 blowing. The residue was separated on a silica gel column (CH2Cl2/EtOAc, 4:1–2:1) to afford the 2,3-O-isopropylidene derivative (3.2 mg) of 2.

1 2,3-O-Isopropylidene Derivative of 2: Colorless oil; H-NMR (MeOH-d4, 500 MHz) δ 6.98 (2H, s), 4.36 (1H, q, J = 6.5 Hz, H-3), 1.21 (3H, d, J = 6.5 Hz, H-4), 2.11 (1H, m, H-10a), 2.23 (1H, m, H-10b), 4.25 (1H, m, 0 0 H-2 a), 4.29 (1H, m, H-2 b), 1.27 (3H, s, acetonide-CH3), 1.31 (3H, s, acetonide-CH3); LC-MS/IT-TOF + m/z 379.1007 [M + Na] (calcd. for C18H20O9Na, 379.1005).

3.7. Acid Hydrolysis and Sugar Analysis by GC

Compound 1 (6 mg) was dissolved in MeOH (4.0 mL) and 1 M H2SO4 (2.0 mL) and reﬂuxed for 2 h on a H2O bath. After the hydrolysate was cool, H2O (8.0 mL) was added, then extracted with EtOAc (3 × 10.0 mL). The EtOAc layer was vacuum dried and chromatographed on semi-preparative HPLC eluting with a gradient of MeOH–H2O (5:95–25:75, v/v) to afford 3-hydroxy-10-hydrooxyethyl-dihydrofuran-2(3H)-one. The aqueous layer was neutralized with aqueous Ba(OH)2 and evaporated under reduced pressure to give a residue. The residue was dissolved in pyridine (100 µL), subsequent treated with 0.1 mL cysteine methyl ester hydrochloride (150 µL; Sigma, St. Louis, MO, USA) and warmed at 60 ◦C for 1 h, then the trimethysilylation reagent HMDS/TMCS (hexamethyldisilazane/trimethylchlorosilane/pyridine 2:1:10; Acros Organics, Geel, Belgium) was added and warmed at 60 ◦C for 30 min. The reaction mixture was partitioned between water and hexane. The hexane extract was analyzed by GC [15] (detector temperature: 280 ◦C; injector temperature: 250 ◦C; temperature gradient: start at 160 ◦C, hold for 5 min, increase to 280 ◦C at ◦ 5 C/min, hold for 10 min). The authentic samples were analyzed in the same way. The tR values of D-glucose and L-glucose were 13.25 min and 15.32 min, respectively. The thiazolidine derivatives of the samples were conﬁrmed by comparison with authentic standards.

3.8. Cytotoxicity Assay All isolates were tested for cytotoxicity in vitro against A549, SMMC-7721, MGC-803, HepG2, and MCF-7 tumour cells via the MTT assay [16,17] with hydroxycamptothecine as a positive control.

4. Conclusions In this study, we separated and identiﬁed three new compounds 1–3 and a known compound 4 from the leaves of C. fargesii. The 2,3-dihydroxy-2-(2-hydroxyethyl)-butanoic acid moiety in 1 and 2 is an unusual carboxylic acid in Nature, thus, these compounds might be recognized as chemotaxonomic markers. Our preliminary examination also suggested this plant contains triterpene HHDP esters, which are important chemotaxonomical markers of Castanopsis sp.; therefore, further phytochemical investigations of the leaves of C. fargesii are in progress.

Acknowledgments: We gratefully acknowledge support from the Guangxi Natural Science Foundation (2014GXNSFCB118001), National Natural Science Foundation China (21562008), “Western Light” Talent Training Plan of Chinese Academy of Sciences (No. [2013]165), Science Research Foundation of Guangxi Academy of Sciences (15YJ22ZWS22) and Program for Bagui Scholars of Guangxi Institute of Botany (The third batch). Molecules 2017, 22, 162 8 of 8

Author Contributions: In this work, Yong-Lin Huang designed the experiments and the manuscript writing; Ya-Feng Wang, Jin-Lei Liu, Yue-Yuan Chen, and Feng-Lai Lu performed the extraction, isolation; Lei Wang and Dian-Peng Li carried out the structural elucidations; Takashi Tanak are viewed the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. CAS. Flora Republicae Popularis Sinicae; China Science Publishing & Media Ltd.: Beijing, China, 1998; Volume 13–14, p. 55. 2. Suranto. Application of modern experimental technique to solve morphological complexity in plants taxonomy. Biodiversitas 2000, 10, 80–84. 3. Ageta, M.; Nonaka, G.; Nishioka, I. Tannins and related compounds. LXVII. Isolation and characterization of castanopsinins A–H, novel ellagitannins containing a triterpenoid core, from Castanopsis cuspidata var. sieboldii NAKAI (3). Chem. Pharm. Bull. 1988, 36, 1646–1663. [CrossRef] 4. Chen, H.F.; Tanaka, T.; Nonaka, G.; Fujoka, T.; Mihsahi, K. Hydrolysable tannins based on a triterpenoid glycoside core, from Castanopsis hystrix. Phytochemistry 1993, 32, 1457–1460. [CrossRef] 5. Huang, Y.L.; Tsujita, T.; Tanaka, T.; Matsuo, Y.; Kouno, I.; Li, D.P.; Nonaka, G.I. Triterpene hexahydroxydiphenoyl esters and a quinic acid purpurogallin carbonyl ester from the leaves of Castanopsis fissa. Phytochemistry 2011, 72, 2006–2014. [CrossRef][PubMed] 6. Huang, Y.L.; Matsuo, Y.; Tanaka, T.; Kouno, I.; Li, D.P.; Nonaka, G.I. New phenylpropanoid-substituted flavan-3-ols from the leaves of Castanopsis sclerophylla. Heterocycles 2011, 83, 2321–2328. 7. Huang, Y.L.; Tanaka, T.; Matsuo, Y.; Kouno, I.; Li, D.P.; Nonaka, G.I. Two new phenolic glucosides and an ellagitannin from the leaves of Castanopsis sclerophylla. Phytochem. Lett. 2012, 5, 158–161. [CrossRef] 8. Huang, Y.L.; Tanaka, T.; Matsuo, Y.; Kouno, I.; Li, D.P.; Nonaka, G.I. Isolation of ellagitannin monomer and macrocyclic dimer from Castanopsis carlesii leaves. Heterocycles 2012, 86, 381–389. 9. Fujii, S.; Aoki, H.; Kômoto, M. Fluorescent glycosides accumulated in Taphrina weisneri-infected Cherry stems. Agric. Biol. Chem. 1971, 35, 1526–1534. [CrossRef] 10. Yoshi, T.; Fenga, W.S.; Okuda, T. Two polyphenol glycosides and tannins from Rosa cymosa. Phytochemistry 1993, 32, 1033–1036. [CrossRef] 11. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. A new aspect of the high-field NMR application of Mosher’s method. The absolute configuration of marine triterpene sipholenol A. J. Am. Chem. Soc. 1991, 113, 4092–4095. [CrossRef] 12. Kusumi, T.; Ooi, T.; Ohkubo, Y.; Yabuuchi, T. The Modified Mosher’s Method and the Sulfoximine Method. ChemInform 2006, 37, 965–980. [CrossRef] 13. Duan, W.J.; Jin, X.; Chen, L.X. Four new compounds from Paeonia albiflora. J. Asian Nat. Prod. Res. 2009, 11, 299–305. [CrossRef][PubMed] 14. Magid, A.A.; Voutquenne-Nazabadioko, L.; Moroy, G.; Moretti, C.; Lavaud, C. Dihydroisocoumarin glucosides from stem bark of Caryocar glabrum. Phytochemistry 2007, 68, 2439–2443. [CrossRef][PubMed] 15. Chen, Y.Y.; Pan, Q.D.; Li, D.P.; Liu, J. L.; Wen, Y.X.; Huang, Y.L.; Lu, F.L. New pregnane glycosides from Brucea javanica and their antifeedant activity. Chem. Biodivers. 2011, 8, 460–466. [CrossRef][PubMed] 16. Alley, M.C.; Scudiero, D.A.; Monks, A.; Hursey, M.L.; Czerwinski, M.J.; Fine, D.L.; Abbott, B.J.; Mayo, J.G.; Shoemaker, R.H.; Boyd, M.R. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 1988, 48, 589–601. [PubMed] 17. Ge, Y.Z.; Zhang, H.; Liu, H.C.; Dong, L.; Ding, J.; Yue, J.M. Cytotoxic dinorditer penoids from Drypetes perreticulata. Phytochemistry 2014, 100, 120–125. [CrossRef][PubMed]

Sample Availability: Samples of the compounds 1–4 are available from the authors.

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