Symplocosionosides A-C , Three Megastigmane Glycosides , a Neolignan Glucoside , and Symplocosins A and B , Two Triterpene Glycosyl Esters from the Leaves of Symplocos cochinchinensis var . Philippinensis

From the 1-BuOH-soluble fraction of a MeOH extract of the leaves of Symplocos cochinchinensis var. philippinensis, 12 compounds were isolated. Spectroscopic analyses of compounds 1 3 established their structures to be megastigmane glycosides, named symplocosionosides A-C. The absolute structure of 1 was determined by the modified Mosher’s method. Compound 4 was found to be a neolignan glucoside and named symplocosneolignan. The structures of compounds 5 and 6, named symplocosins A and B, were elucidated to be the saponins of hederagenin sugar esters. The structures of the remaining known compounds (7 12) were identified by comparison of spectroscopic data with those reported in the literature.


Introduction
Genus Symplocos comprises about 300 species, which are mainly found in tropical, except for Africa, and subtropical areas, with a small number of species in the temperate zone.Symplocos cochinchinensis (Loureiro) Spencer Le Marchant Moore var.philippinensis (Brand) Nootboom (Symplocaceae) is an evergreen tall tree, which is distributed in the Amami and Okinawa Islands, Taiwan, Southern China and Indochina.It grows up to about 15 m in height and bears white flowers in spikes [1].So far as we know, no chemical investigation has been performed on this plant.Even for its elementary species, S. cochinchinensis (Loureiro) Spencer Le Marchant Moore, only few pharmacological works have been performed on its extract [2][3][4].Thus, we were promted to invetigate the chemical constituents in the title plant.From the 1-BuOH-soluble fraction of a MeOH extract of the leaves of S. cochinchinensis var.philippinensis, three new megastigmane glycosides, named symplocosiono-sides A-C (1-3), a new neolignan glucoside, named symplocosneolignan A (4), and two new triterpene glycosyl esters, named symplocosins A and B (5,6), together with six known compounds, dendranthemoside A (7) [5], 4,5dihydroblumenol (8) [6], alangionoside B (9) [7], ampelopsisionoside (10) [8], citroside A (11) [9], and nigaichigoside F1 (12) [10] were isolated.This paper deals with structural elucidation of the new compounds.


-44.0, was isolated as an amorphous powder and its elemental composition was determined to be C 24 H 42 O 11 by high-resolution (HR)electrospray ionization (ESI)-mass spectrometry (MS).
Symplocosionosides A-C, Three Megastigmane Glycosides, a Neolignan Glucoside, and Symplocosins A and B, 610 Two Triterpene Glycosyl Esters from the Leaves of Symplocos cochinchinensis var.Philippinensis Strong absorption bands at 3394 cm -1 and 1055 cm -1 in the IR spectrum indicated that symplocosionoside A (1) was a glycoside and in the NMR spectra, two anomeric carbon and proton signals (δ C 102.9 with δ H 4.33 and δ C 110.9 with δ H 5.00) were observed.The 1 H-NMR spectrum exhibited signals for two singlet and two doublet methyls, and one trans double bond.Of the 24 carbon NMR signals, 11 were assigned to those for a β-D-(6-O-β-D- apiofuranosyl) glucopyranoside moiety (Table 1) [11].
The absolute configurations of apiose and glucose were determined to be of the D-series by HPLC analysis of a hydrolyzate of 1.The remaining 13 signals comprised of those of four methyls, two methylenes, two oxymethines, two methines, one quaternary carbon and a double bond.
From the above evidence, the aglycone of symplocosionoside A (1) was assumed to have a megastigmane skeleton, and the proton spin-spin coupling sequence from H 2 -2 through H 3 -10 was revealed by the 1 H-      the structure of the aglycone moiety.Since the H-3 proton (δ H 3.83) was coupled with the axial protons at H-2 and H-4 with a coupling constant of 12 Hz, the hydroxy substituent at the 3-position was placed in the equatorial position, and the similar coupling constants for H-4ax and H-5, and H-5 and H-6 also are favorable for placement of both of the methyl group at the 5-position and the side chain at the 6-position in the equatorial position.The site of sugar attachment was established to be to the hydroxy group at the 3-position by the HMBC correlation cross peak between H-1' (δ H 4.33) and C-3 (δ C 76.0).
The absolute configuration at the 3-position was first expected to be S from the empirical β-D-glucopyranosylation-induced shift-trend rule for glycoside (1) and the aglycone (1a) (Table 1) [12], and then the final confirmation of that of the aglycone (1a) was achieved by the modified Mosher's method (Figure 2) [13].Therefore, the structure of symplocosionoside A (1) was elucidated to be (3S, 5R, 6R, 7E, 9R)-megastigman-7-ene-  spectrum exhibited a glycosidic feature (3399 cm -1 and 1056 cm -1 ) and the presence of a ketonic functional group (1694 cm -1 ).In the 1 H-NMR spectrum, the signals for two singlet methyls, two doublet methyls, one trans double bond and two anomeric protons were observed.The 13 C-NMR spectrum exhibited 24 signals, 11 of which were assignable to those of a 9-O-β-D-(6-Oβ-D-apiofur-anosyl) glucopyranoside moiety.The absolute configurations of apiose and glucose were determined to be of the D-series by HPLC analysis of the hydrolyzate of 2. The remaining 13 signals comprised those of four methyls, two methylenes, one oxymethine, one methine, one double bond, two quaternary carbons and a ketone functional group.From the above evidence together with the information obtained from two dimensional spectra, the structure of symplocosonoside B (2) was determined to be 3-keto-6-hydro-xymegastigmane glycoside, as shown in Figure 1.From the proton-proton coupling constant, J 4ax-5 = 14 Hz, the methyl group at the 5-position was placed in an equatorial position, and then the correlation cross peak between H-5 (δ H 2.28) and H-7 (δ H 5.74) observed on phase-sensitive (PS)-nuclear Overhauser effect spectroscopy (NOESY) allowed placement of the side chain also in an equatorial position.Based on the octant rule, the absolute configuration at the 5-position was determined to be R from the positive Cotton effect at 289 nm in the CD spectrum and thus the 6-position must have the S configuration.By comparing the reported data, the absolute configuration at the 9-position was determined to be R, and the linkage of the sugar unit to the hydroxy group at the 9-positon was confirmed by the HMBC correlation cross peak between H-1' (δ H 4.34) and C-9 (δ C 78.1).Therefore, the structure of symplocosionoside B (2) was elucidated to be (5R, 6S, 7E, 9R)-megastigman-7-en-9-ol-3-


-57.9, was isolated as an amorphous powder and its elemental composition was determined to be C 24 H 38 O 12 by HR-ESI-MS.The IR spectrum exhibited a characteristic absorption band for an allenic part (1938 cm -1 ), and the presence of this func-tional group was supported by the 13 C-NMR spectrum (δ C 118.6, 197.6 and 100.9 in C 5 D 5 N).Other 13 C-NMR signals showed good similarity to those of citrosides A and B, isolated from Citrus unshiu [9], except for the presence of signals for a terminal α-arabinopyranose, and the upfield and downfield shifts of the C-5' (δ C 76. [ ]  -13.5, was isolated as an amorphous powder and its elemental composition was determined to be C 28 H 40 O 14 by HR-ESI-MS.The IR spectrum showed that symplocosneolignan (4) was a glycosidic compound (3395 cm -1 ) with aromatic ring (s) (1592 cm -1 ).A UV absorption band (269 nm) also supported the presence of the aromatic ring (s).In the 1 H-NMR spectrum, signals for two singlet aromatic protons (δ H 6.53 and 6.75), two methoxy protons (δ H 3.79 and 3.84), and an anomeric proton (δ H 4.83) were observed (Table 2).Each of the aromatic signals accounted for two protons and that of the methoxy signals six protons.The 13 C-NMR spectrum exhibited 22 signals, of which six were assignable to β-glucopyranose.As to the remaining 16 signals, two methoxy signals were expected to include those of four carbons from their peak heights and each of the four aromatic signals was obviously for two carbons.Thus, the two aromatic rings must be substituted symmetrically and the other signals comprised those of two primary alcohols, two methylenes and two oxymethines.The proton chains from H-7 to H-9 and H-7' to H-9' were assigned by inspection of the 1 H-1 H COSY spectrum, and the correlation cross peaks between H-8 (δ H 4.18), and C-1 (δ C 139.5) and C-4' (δ C 134.9) in the HMBC spectrum established the structure of symplocosneolignan (4), as shown in Figure 1.Other HMBC correlations shown in Figure 3 also supported the structure.The site of the sugar linkage was determined to be to the hydroxy group at the 4-position, judging from the cross peak between H-1' and C-4 (δ C 135.6) in the HMBC spectrum, and the mode of linkage to be β from the coupling constant (J = 8 Hz) of the anomeric proton.Judging from the coupling constant (J = 5 Hz) for H-7 and 8, symplocosneolignan (4) was concluded to possess the erythro relative stereochemistry [15].Enzymatic hydrolysis of symplocosneolignan (4) gave its aglycone (4a), whose J 7-8 remained as 5 Hz [16].Judging from the negative Cotton effect at 242 nm, the absolute configuration of 4 was determined to be 7S, 8R [16][17][18].Therefore, the structure of symplocosneolignan (4) was elucidated to be (7S, 8R)-4,7,9,9'-tetrahydroxy-3,5,3',5'-tetramethoxy-8,4'-oxyneolignan 4-O-β-D-glucopyranoside, as shown in Figure 1.
From the leaves of S. cochinchiensis var.philippinensis, three new megastigmane diglycosides, named symploco-sionosides A-C (1-3), neolignan glucoside (4) and sym-plocosins A and B, two ursane-type triterpenoid glycosyl esters (5,6) were isolated.Their structures were eluci-dated by the spectroscopic evidence.The absolute struc-ture of 1 was finally determined by the modified Mosher's method and that of 4 by the Cotton effect, observed in the CD spectrum.Three species of Symplocos were medicinally used as "lodhra" in Indian system of traditional medicine, Ayurveda.For treatment of diabetes mellitus in Ayuveda, Symplocos species were given along with the juice of cucumber [19].It is of interest for further works to define the compounds responsible for this treatment.

Plant Material
Leaves of S. cochinchinensis var.philippinensis were collected in Taketomi-cho, Yaeyama-gun, Okinawa, Japan, in November, 2003, and a voucher specimen was deposited in the Herbarium of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Hiroshima University (03-SC-Okinawa-1105).

General Experimental Procedure
Optical rotations were measured on a JASCO P-1030 digital polarimeter.IR and UV spectra were measured on Horiba FT-710 and JASCO V-520 UV/Vis spectrophotometers, respectively. 1H-and 13 C-NMR spectra were taken on JEOL JNM α-400 and ECA-600 spectrometers at 400 MHz or 600 MHz and 100 MHz or 150 MHz, respectively, with tetramethylsilane as an internal standard.CD spectra were obtained with a JASCO J-720 spectropolarimeter.Positive-ion HR-ESI-MS was performed with an Applied Biosystems QSTAR ® XL NanoSprayTM System.

Sugar Analysis
About 350 μg each of 1 and 2 was hydrolyzed with 1N HCl (0.2 ml) at 90˚C for 2 h.The reaction mixtures were washed with an equal amount of EtOAc and then passed through Amberlite MB-3.The pass-through fractions were evaporated to dryness to give residues.The residues were dissolved in 0.1 ml of dry pyridine and then 0.5 mg of L-cysteine methyl ester was added.To these mixtures, 1.4 mg of o-tolylthioisocyanate in 70 μl of pyridine was added, followed by standing at 60˚C for 1 h.(1:3), 0.8 ml/min, UV detector at 250 nm] to give peaks for thiocarbamoylthiazolidine derivatives of D-glucose and D-apiose at 18.0 min and 31.0 min, respectively [21].The peaks were identified by co-chromatography with thiocarbamoyl-thiazolidine derivatives of authentic Dglucose and D-apiose.About 500 μg of each compound, except for 1 and 2, was hydrolyzed with 1N HCl (0.1 ml) at 90˚C for 2 h.The reaction mixtures were partitioned with an equal amount of EtOAc (0.1 ml), and the water layers were analyzed with a chiral detector (JASCO OR-2090plus) on an amino column [Asahipak NH 2 P-50 4E, CH 3 CN-H 2 O (3:1), 1 ml/min].A hydrolyzate of 4 gave a peak for D-glucose at 7.9 min, and ones of 3, 5 and 6 gave peaks for L-arabinose and D-glucose at 6.1 min and 13.7 min, respectively, with positive optical rotation signs.The peaks were identified by co-chromatography with authentic samples.
one 9-O-β-D-(6'-O-β-D-apiofuranosyl) glucopyranoside.Symplocosonoside C 6 in C 5 D 5 N) and C-6' (δ C 68.8 in C 5 D 5 N) signals.A significant cross peak, observed between H-1" (δ H 5.05 in C 5 D 5 N) and C-6' in the HMBC spectrum, confirmed the sugar linkage.The absolute configurations of arabinose and glucose were determined to be of the L-and D-series, respectively, by HPLC analysis of the hydrolyzate of 3 using the chiral detector.The axis chirality of the allene part was determined to be the same as that of citroside A, tentatively R, as judged on comparison of the 13 C-NMR chemical shift of C-7 (δ C 197.6 in C 5 D 5 N) with those for citrosides A (C-7: δ C 197.6) and B (C-7: δ C 199.0).Therefore, the structure of symplocosionoside C (3) was elucidated to be citroside A 6'-O-α-L-arabinopyranoside, as shown in Figure 1.Symplocosneolignan (4), 25 D

Table 3 . 13 C-NMR data for compounds 5, 6 and 12 (C 5 D 5 N + one drop of D 2 O, 150 MHz).
Three Megastigmane Glycosides, a Neolignan Glucoside, and Symplocosins A and B, 613 Two Triterpene Glycosyl Esters from the Leaves of Symplocos cochinchinensis var.Philippinensis Copyright © 2011 SciRes.AJPS Symplocosionosides A-C, a) Data taken from ref. 10.Data for C 5 D 5 N without D 2 O at 25 MHz.b) Data for C 5 D 5 N without D 2 O at 100 MHz.<i>c</i>), <i>d</i>), <i>e</i>).The signals with the same superscripts in each column may be interchangeable.