2-Aminobenzoyl and megastigmane glycosides from Wrightia antidysenterica

Phytochemistry Letters 29 (2019) 61–64

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2-Aminobenzoyl and megastigmane glycosides from Wrightia antidysenterica a

a

a

a

Chutima Srinroch , Poolsak Sahakitpichan , Supanna Techasakul , Nitirat Chimnoi , ⁎ Somsak Ruchirawata, Tripetch Kanchanapooma,b, a b

T

Chulabhorn Research Institute, Kamphaeng Phet 6, Talat Bang Khen, Lak Si, Bangkok 10210, Thailand Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand

ARTICLE INFO

ABSTRACT

Keywords: Wrightia antidysenterica Apocynaceae 2-Aminobenzoyl glycoside Benzoxazinoid Megastigmane glycoside Wrightiaionosides A-B

A new aminobenzoyl glycoside, 2-aminobenzoyl O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (1) and two new megastigmane glycosides, wrightiaionosides A (10), and B (11), were isolated along with 17 known compounds, including four benzoxazinoid glycosides (2-5), one indole diglycoside (6), three simple aromatic glycosides (7-9), three megastigmane glycosides (12-14), five flavonoid glycosides (15-19) and one lignan (20). The structure elucidation of these compounds was based on analyses of physical and spectroscopical data including 1D and 2D NMR experiments.

1. Introduction Wrightia antidysenterica (L.) R.Br. (syn. Nerium antidysentericum L., Nerium zelaynicum L., Walidda antidysenterica (L.) Pichon, Wrightia zelaynica (L.) R.Br.; Thai name: Put-Pit-Cha-Ya; Family Apocynaceae) is an evergreen shrub up to 2 m high. It is native to Sri Lanka, but is widely cultivated as an ornamental plant in many parts of Thailand. This species has been used in Sri Lankan traditional medicine for the treatment of blood disorders, chronic fever, jaundice and hemorrhoids (Wickramaratne et al., 2015). In the biological activity, the antioxidant and antibacterial investigation of plant extracts were reported (Wickramaratne et al., 2015). The present paper reports the isolation of a new 2-aminobenzoyl diglycoside (anthranillic acid diglycoside) (1) and two new megastigmane glycosides (10, 11) together with 17 known compounds from the water soluble fraction of the MeOH extract of the aerial parts and roots of this plant. 2. Results and discussion The methanolic extract of W. antidysenterica was partitioned between Et2O and H2O. The aqueous soluble fraction was separated by combination of chromatographic methods to afford three new compounds (1, 10 and 11) (Fig. 1) together with 17 known compounds. The known compounds were identified as (2R)-2-O-β-D-glucopyranosyl-2H1,4-benzoxazin-3(4H)-one (HBOA-Glc or blepharin, 2), (2R)-2-O-β-Dglucopyranosyl-5-hydroxy-2H-1,4-benzoxazin-3(4H)-one (3), (2R)-2-Oβ-D-glucopyranosyl-7-hydroxy-2H-1,4-benzoxazin-3(4H)-one (DHBOA-

⁎

Glc, 4), (2R)-2-O-β-D-glucopyranosyl-4-hydroxy-2H-1,4-benzoxazin3(4H)-one (DIBOA-Glc, 5) (Kanchanapoom et al., 2001), indoxyl 3-O-βD-apiofuranosyl-(1→6)-β-D-glucopyranoside (6) (Sahakitpichan et al., 2018), benzyl β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (icariside F2, 7) (Miyase et al., 1988), 3,4,5-trimethoxyphenyl-β-D-apiofuranosyl(1→6)-β-D-glucopyranoside (8) (Fuchino et al., 1995), icariside D1 (9) (Miyase et al., 1987), ampelopsisionoside (12) (De Marino et al., 2004), symplocosionoside B (13) (Cai et al., 2011), (6R,9R)-3-oxo-α-ionol β-Dapiofuranosyl-(1→6)-β-D-glucopyranoside (14) (De Tommasi et al., 1992), rutin (15), kaempferol 3-O-rutinoside (16), kaempferol 3-O-α-Lrhamnopyanosyl-(1→6)-β-D-galactopyranoside (17), kaempferol 3-Oα-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside (18), schaftoside (19) (Agrawal and Bansal, 1989; Kazuma et al., 2003) and (+)-isolariciresinol (20) (Okuyama et al., 1995). All known compounds were identified by comparison of physical data with literature values and from spectroscopic evidence. Compound 1 was isolated as an amorphous powder. The molecular formula was determined to be C18H25NO11 by high resolution electrospray time-of-flight (HR-ESI-TOF) mass spectrometric analysis. The 1H NMR spectrum showed the characteristic pattern of 1,2-disubstituted aromatic ring system from the chemical shifts at δH 6.58 (ddd, J = 8.1, 7.1, 1.1 Hz), 6.75 (dd, J = 8.4, 1.1 Hz), 7.26 (ddd, J = 8.4, 7.1, 1.6 Hz) and 7.90 (dd, J = 8.1, 1.6 Hz) in addition to two anomeric protons at δH 4.68 (d, J = 8.0) and 4.98 (d, J = 2.4 Hz) for the sugar moieties. The chemical shifts of the aglycone part were coincidence with those of 2aminobenzoyl moiety (Parada et al., 1996; Syahrani et al., 1999), and the disaccharide moiety was assigned as a β-D-apiofuranosyl-(1→6)-O-

Corresponding author at: Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand. E-mail address: [email protected] (T. Kanchanapoom).

https://doi.org/10.1016/j.phytol.2018.11.006 Received 29 August 2018; Received in revised form 19 October 2018; Accepted 8 November 2018 1874-3900/ © 2018 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

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Fig. 3. COSY and HMBC correlations of compound 10.

and H-1" (δH 4.98) to C-6' (δC 68.4) as shown in Fig. 2. Therefore, this compound was identified to be 2-aminobenzoyl O-β-D-apiofuranosyl(1→6)-β-D-glucopyranoside. Wrightiaionoside A (10) was obtained as an amorphous powder. Its molecular formula was determined to be C19H32NO8 by HR-ESI-TOF mass spectrometric analysis. Positive ESI-MS of this compound displayed a significant fragment ion at m/z 249 [M+H-Glc]+. Inspection of the 13C NMR spectrum revealed the presence of a β-glucopyranosyl unit from six carbon signals in addition to 13 signals for the aglycone moiety, corresponding to a megastigmane skeleton. These signals were indicated to be three methyls (δC 21.2, 21.3, 31.0), three methylenes (δC 44.6, 56.5, 65.3), five methines (δC 42.1, 51.9, 77.4, 130.6, 137.9) and two quaternary carbons (δC 39.2 and 214.4) by DEPT spectra. The partial structure of C-4 to C-10 [CH2 (δC 44.6, C-4)-CH (δC 42.1, C-5)CH (δC 51.9, C-6)-CH (δC 130.6, C-7)-CH (δC 137.5, C-8)-CH (δC 77.4, C9)-CH3 (δC 21.2, C-10)]; and C-5 to C-13 [CH (δC 42.1, C-5)-CH2 (δC 65.3, C-13)] was deduced by COSY and HMQC analysis as shown in Fig. 3. The presence of the chemical shifts of C-9 at δC 77.4 and C-13 at δC 65.3 indicated that these carbon atoms were oxygenated. The quaternary carbon signals at δC 39.2 and 214.4 were characteristic for C-1 and C-3, respectively, as well as, two tertiary methyl signals at δC 21.3 and 31.0 were assignable to C-11 and C-12, respectively, connected to C-1. The remaining methylene signal at δC 56.5 belonged to C-2. Therefore, the aglycone of 10 was suggested to be a 9,13-dihydroxymegastigm-7-en-3-one. The structure was confirmed by HMBC spectrum (Fig. 3), in which the significant correlations were observed from i) H3-11, H3-12 to C-1, C-2 and C-6; ii) H-6 to C-1, C-2, C-4, C-5, C7, C-8, C-11 and C-12; and iii) H2-13 to C-4, C-5 and C-6. The glucopyranosyl moiety was located at C-9 due to the appearance of HMBC correlation from H-1' to C-9. The NOESY correlations from H-5 (δH 1.95) to H3-11 (δH 0.82); and from H-6 (δH 2.22) to H3-12 (δH 1.01), as well as the large coupling constant between H-5 and H-6 with J = 11.4 Hz indicated that H-5 and H-6 protons had axial orientations, while the side chains at C-5 and C-6 were in the equatorial orientations. The configuration of C-6 was determined to be S from the CD spectrum, in which the extreme value was observed at Δε (nm): +60.2 (235) (Pabst et al., 1992; Yean et al., 2014); and in turn, that of C-5 to be R. The chemical shifts of C-9 at δC 77.4 and C-10 at δC 21.2 were indicative of a 9Rconfiguration, being similar to report in the literatures (Pabst et al., 1992; Yamano and Ito, 2005; Yean et al., 2014). Consequently, compound 10 was elucidated as (5R,6S,7E,9R)-9,13-dihydroxymegastigm7-en-3-one 9-O-β-D-glucopyanoside. Wrightiaionoside B (11) was isolated as an amorphous powder. The molecular formula was determined to be C24H40O12 by HR-ESI-TOF mass spectrometric analysis. The 1H and 13C NMR indicated that compound 11 was a megastigmane diglycoside. The chemical shifts (Table 2) were closely related to those of 10 except for a set of additional signals arising from a β-D-apiofuranosyl unit in 11. Positive ESIMS of compound 11 exhibited significant fragment ions at m/z 411 [M +H-Api]+, 249 [M+H-Api-Glc]+. The apiofuranosyl moiety was connected to C-6' of the glucopyranosyl moiety due to the downfield shift of this carbon atom, and was confirmed by the HMBC correlation from H-1" (δH 5.00) to C-6' (δC 68.6). The sugar part was identified to be

Fig. 1. Structures of compounds 1, 10 and 11. Table 1 NMR spectroscopic data of compound. (400 MHz for 1H NMR and 100 MHz for 13 C NMR, in CD3OD). Position

1 δC

1 2 3 4 5 6 7

110.2 153.4 117.7 135.7 116.4 132.4 168.0

Glc-1' 2' 3' 4' 5' 6'

δH

6.75 7.26 6.58 7.90

(1H, (1H, (1H, (1H,

dd, J = 8.4, 1.1 Hz) ddd, J = 8.4, 7.1, 1.6 Hz) ddd, J = 8.1, 7.1, 1.1 Hz) dd, J = 8.1, 1.6 Hz)

95.4 74.0 78.1 71.4 77.7 68.4

4.68 3.48 3.46 3.37 3.58 3.64 4.00

(1H, (1H, (1H, (1H, (1H, (1H, (1H,

d, J = 8.0 Hz) dd, J = 8.4, 8.0 Hz) dd, J = 9.1, 8.4 Hz) dd, J = 9.2, 9.1 Hz) m) dd, J = 11.0, 5.8 Hz) br d, J = 11.0 Hz)

Api-1" 2" 3" 4"

110.2 77.9 80.6 75.0

4.98 (1H, d, J = 2.4 Hz) 3.90 (1H, d, J = 2.4 Hz)

5"

65.6

3.74 (1H, d, J = 9.7 Hz) 3.97 (1H, d, J = 9.7 Hz) 3.57 (2H, s)

Fig. 2. HMBC correlations of compound 1.

β-D-glucopyranosyl unit, compared to compounds 6-9. The sugar part was confirmed by the detailed analysis of COSY and HMQC experiments as well as the splitting patterns of each proton (Table 1). Positive ESIMS of compound 1 exhibited significant fragment ions at m/z 300 [M +H-Api]+, 138 [M+H-Api-Glc]+. From HMBC spectrum, the long range correlations were detected from H-1' (δH 4.68) to C-7 (δC 168.0), 62

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Micro TOF-LC mass spectrometer. Optical rotations were measured with a Jasco P-1020 digital polarimeter. Circular dichroism spectra were recorded on a Jasco J-815 spectropolarimeter. For column chromatography, Diaion HP-20 (Mitsubishi Chemical Industries Co. Ltd.), silica gel 60 (70−230 mesh, Merck), and RP-18 (50 μm, YMC) were used. HPLC (Jasco PU-980 pump) was carried out on ODS columns (column 20 mm i.d. x 250 mm length, YMC ODS-AQ) with a Jasco UV970 detector at 220 nm. The flow rates were 6 mL/min. The spraying reagent used for TLC was 10% H2SO4 in H2O-EtOH (1:1, v/v).

Table 2 NMR spectroscopic data of compounds 10 and 11 (400 MHz for 1H NMR and 100 MHz for 13C NMR, in CD3OD). Position

10 δC

1 2

39.2 56.5

3 4

214.4 44.6

5 6

42.1 51.9

7

130.6

8

137.9

9

77.4

10 11 12 13

21.2 21.3 31.0 65.3

Glc-1' 2'

102.6 75.3

3' 4'

78.1 71.5

5' 6'

78.0 62.6

11 δC

δH 2.04 (1H, d, J = 13.3 Hz) 2.41 (1H, d, J = 13.3 Hz) 1.92a 2.38a 1.95 (1H, 2.22 (1H, 9.8 Hz) 5.48 (1H, 9.8 Hz) 5.73 (1H, 6.4 Hz) 4.38 (1H, 6.4 Hz) 1.31 (3H, 0.82 (3H, 1.01 (3H, 3.46 (1H, 5.9 Hz) 3.54 (1H, 3.2 Hz)

39.2 56.5 214.5 44.6

m) dd, J = 11.4,

42.1 51.9

dd, J = 15.5,

130.9

dd, J = 15.5,

137.8

qd, J = 6.4,

77.7

d, J = 6.4 Hz) s) s) dd, J = 11.0,

21.3 21.4 31.2 65.3

dd, J = 11.0,

4.36 (1H, d, J = 7.7 Hz) 3.18 (1H, dd, J = 9.8, 7.7 Hz) 3.32a 3.31a

102.6 75.3

3.22 (1H, m) 3.62 (1H, dd, J = 11.9, 5.2 Hz) 3.82 (1H, dd, J = 11.9, 2.3 Hz)

76.9 68.6

78.0 71.6

Api-1" 2" 3" 4"

111.0 78.0 80.5 74.9

5"

65.4

a

δH 2.04 (1H, d, J = 13.2 Hz) 2.44 (1H, d, J = 13.2 Hz) 1.92a 2.40a 1.94 (1H, m) 2.25 (1H, dd, J = 11.4, 10.0 Hz) 5.50 (1H, dd, J = 15.4, 10.0 Hz) 5.74 (1H, dd, J = 15.4, 6.5 Hz) 4.37 (1H, qd, J = 6.5, 6.5 Hz) 1.31,(3H, d, J = 6.5 Hz) 0.83 (3H, s) 1.02 (3H, s) 3.47 (1H, dd, J = 10.8, 5.6 Hz) 3.56a 4.35 (1H, 3.17 (1H, 7.7 Hz) 3.32a 3.28 (1H, 8.7 Hz) 3.39 (1H, 3.59a 3.95 (1H, 1.8 Hz)

3.2. Plant material Wrightia antidysenterica (L.) R. Br. was cultivated and collected from Khon Kaen Province, Thailand, in August 2017. Plant specimen was identified by one of us (TK). Voucher specimens (TK-PSKKU-0083) are on files in the Herbarium of the Faculty of Pharmaceutical Sciences, Khon Kaen University. 3.3. Extraction and isolation The aerial parts and roots of W. antidysenterica (1.4 kg) were extracted three times with MeOH (8.0 L for each extraction, 2 h under reflux) and concentrated to dryness. The residue (96.8 g) was suspended in H2O and partitioned with Et2O. The water soluble fraction (53.6 g) was subjected to a Diaion HP-20 column, and eluted with H2O, and MeOH, successively. The fraction eluted with MeOH (13.1 g) was applied to a silica gel column (36 mm i.d.) using solvent systems EtOAcMeOH (9:1, 8.0 L), EtOAc-MeOH-H2O (40:10:1, 6.0 L), EtOAc-MeOHH2O (70:30:3, 4.0 L) and EtOAc-MeOH-H2O (6:4:1, 2.0 L), respectively to provide five fractions (A to E). Fraction B (2.1 g) was applied to a RP-18 column using a gradient solvent system, H2O-MeOH (90:10 → 20:80, v/v) to provide nine subfractions. Compound 4 (2.5 mg) was crystallized from sub-fraction B-2. Sub-fraction B-3 was purified by preparative HPLC-ODS (YMC ODS-AQ) with solvent system H2O-MeCN (90:10, v/v) to provide compounds 1 (3.9 mg), 3 (2.3 mg), 6 (36.3 mg) and 8 (15.3 mg). Sub-fraction B-4 was purified by preparative HPLC-ODS with solvent system H2O-MeCN (85:15, v/v) to afford compounds 2 (20.0 mg), 7 (29.1 mg), 10 (8.3 mg) and 13 (10.9 mg). Sub-fraction B-5 was purified by preparative HPLCODS with solvent system H2O-MeCN (93:17, v/v) to yield compounds 9 (3.0 mg) and 12 (4.0 mg). Sub-fraction B-6 was purified by preparative HPLC-ODS with solvent system H2O-MeCN (83:17, v/v) to provide compound 14 (7.4 mg). Sub-fraction B-7 was purified by preparative HPLC-ODS with solvent system H2O-MeCN (80:20, v/v) to give compounds 16 (62.6 mg) and 17 (38.9 mg). Fraction C (2.6 g) was separated on a RP-18 column using solvent system, H2O-MeOH (90:10 → 20:80, v/v) to provide ten sub-fractions. Sub-fraction C-3 was purified by preparative HPLC-ODS with solvent system H2O-MeCN (90:10, v/v) to give compound 5 (13.8 mg). Subfraction C-4 was purified by preparative HPLC-ODS with solvent system H2O-MeCN (83:17, v/v) to afford compounds 11 (1.6 mg) and 20 (2.6 mg). Sub-fraction C-5 was purified by preparative HPLC-ODS with solvent system H2O-MeCN (80:20, v/v) to obtain compound 18 (273.8 mg). Sub-fraction C-6 was purified by preparative HPLC-ODS with solvent system H2O-MeCN (80:20, v/v) to provide compound 15 (39.8 mg). Fraction D (4.8 g) was applied to a RP-18 column using solvent system, H2O-MeOH (90:10 → 20:80, v/v) to give six sub-fractions. Subfraction D-4 was purified by preparative HPLC-ODS with solvent system H2O-MeCN (85:15, v/v) to provide compound 19 (37.3 mg).

d, J = 7.7 Hz) dd, J = 9.8, dd, J = 9.0, m) dd, J = 10.5,

5.00 (1H, d, J = 2.5 Hz) 3.92 (1H, d, J = 2.5 Hz) 3.76 (1H, d, J = 9.6 Hz) 3.98 (1H, d, J = 9.6 Hz) 3.57 (2H, s)

Chemical shifts were assigned by COSY and HMQC.

the same as that of compounds 1 and 6-9. The NOESY correlations were also observed from H-5 (δH 1.94) to H3-11 (δH 0.83); and from H-6 (δH 2.25) to H3-12 (δH 1.02). The configuration of C-5, C-6 and C-9 was concluded to be 5R, 6S, and 9R, respectively, since its CD spectrum [Δε (nm): +8.42 (244)], and the chemical shifts of C-9 (δC 77.7) and C-10 (δC 21.3) were consistent with those of 10. Thus, compound 11 was established as (5R,6S,7E,9R)-9,13-dihydroxymegastigm-7-en-3-one 9O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside. In conclusion, the present work reported nineteen compounds from the water soluble part of the MeOH extract of W. antidysenterica, including one 2-aminobenzoyl glycoside (1), four benzoxazinoid glycosides (2-5), one indole diglycoside (6), three simple aromatic glycosides (7-9), five megastigmane glycosides (10-14), five flavonoid glycosides (15-19) and one lignan (20). The occurrence of these compounds from W. antidysenterica was related to our previous investigation on the same genus, W. religiosa (Sahakitpichan etal., 2018). Also, it was the second time to report benzoxazinoid glycosides from the family Apocynaceae. 3. Experimental 3.1. General procedures

3.4. 2-Aminobenzoyl O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (1)

NMR spectra were recorded in CD3OD using Bruker AV-300 or Bruker AV-400 spectrometers. The MS data was obtained on a Bruker

Amorphous powder, [α]D26 −77.3 (MeOH, c 0.37); 1H and 13C NMR (CD3OD): Table 1; Positive HRESITOF-MS, m/z: 454.1316 [M 63

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+Na]+ (C18H25NNaO11 required 454.1320).

De Tommasi, N., Aquino, R., De Simone, F., Pizza, C., 1992. Plant metabolites. New sesquiterpene and ionone glycosides from Eriobotrya japonica. J. Nat. Prod. 55, 1025–1032. Fuchino, H., Satoh, T., Tanaka, N., 1995. Chemical evaluation of Betula species in Japan. I. Constituents of Betula ermanii. Chem. Pharm. Bull. 43, 1937–1942. Kanchanapoom, T., Kamel, M.S., Kasai, R., Picheansoonthon, C., Hiraga, Y., Yamasaki, K., 2001. Benzoxazinoid glucosides from Acanthus ilicifolius. Phytochemistry 58, 637–640. Kazuma, K., Noda, N., Suzuki, M., 2003. Malonylated flavonol glycosides from the petals of Clitoria ternatea. Phytochemistry 62, 229–237. Miyase, T., Ueno, A., Takizawa, N., Kobayashi, H., Oguchi, H., 1987. Studies on the glycosides of Epimedium grandiflorumMorr. var.thunbergianum (Miq.) Nakai. II. Chem. Pharm. Bull. 35, 3713–3719. Miyase, T., Ueno, A., Takizawa, N., Kobayashi, H., Oguchi, H., 1988. Studies on the glycosides of Epimedium grandiflorumMorr. var.thunbergianum (Miq.) Nakai. III. Chem. Pharm. Bull. 36, 2475–2484. Okuyama, E., Suzumurz, K., Yamazaki, M., 1995. Pharmacologically active components of todopon puok (Fagraea racemosa), a medicinal plant from Borneo. Chem. Pharm. Bull. 43, 2200–2204. Pabst, A., Barron, D., Semon, E., Schreier, P., 1992. Two diastereomeric 3-oxo-α-ionol βD-glucosides from raspberry fruit. Phytochemistry 31, 1649–1652. Parada, F., Krajewski, D., Duque, C., Jäger, E., Herderich, M., Schreier, P., 1996. 1-O-β-DGlucopyranosyl anthranilate from piñuela (Bromelia plumier) fruit. Phytochemistry 42, 871–873. Sahakitpichan, P., Chimnoi, N., Srinroch, C., Ruchirawat, S., Kanchanapoom, T., 2018. Benzoxazinoid and indoxyl glycosides from Wrightia religiosa. Phytochemistry Lett. 26, 30–32. Syahrani, A., Ratnasari, E., Indrayanto, G., Wilkins, A.L., 1999. Biotransformation of oand p-aminobenzoic acids and N-acetyl p-aminobenzoic acid by cell suspension cultures of Solanum mammosum. Phytochemistry 51, 615–620. Wickramaratne, M.N., Gunatilake, L.P., Anuradha, N.G.D., Godavillathanna, A.N., Perera, M.G.N., Nicholas, I., 2015. Antioxidant activity and antibacterial activity of Walidda antidysenterica. J. Pharmacogn. Phytochem. 2, 121–126. Yamano, Y., Ito, M., 2005. Synthesis of optically active vomifoliol and roseoside stereoisomers. Chem. Pharm. Bull. 53, 541–546. Yean, M.H., Kim, J.S., Kang, S.S., Kim, Y.S., 2014. A new megastigmane glucoside and three new flavonoid glycosides fromSpiraea prunifolia var. simpliciflora. Helv. Chim. Acta 97, 1123–1131.

3.5. Wrightiaionoside A (10) Amorphous powder, [α]D27 +10.7 (MeOH, c 0.80); CD (MeOH, c 1.0 × 10−3) Δε (nm) +60.2 (235); 1H and 13C NMR (CD3OD): Table 2; Positive HRESITOF-MS, m/z: 411.1979 [M+Na]+ (C19H32NaO8 required 411.1989). 3.6. Wrightiaionoside B (11) Amorphous powder, [α]D27 −4.8 (MeOH, c 0.13); CD (MeOH, c 1.3 × 10−5) Δε (nm) +8.42 (244); 1H and 13C NMR (CD3OD): Table 2; Positive HRESITOF-MS, m/z: 543.2412 [M+Na]+ (C24H40NaO12 required 543.2404). Acknowledgements This research work was supported by the grant from Chulabhorn Research Institute, and Khon Kaen University. References Agrawal, P.K., Bansal, M.C., 1989. Flavonoid glycosides. In: Agrawal, P.K. (Ed.), Carbon13 NMR of Flavonoids. Elsevier, Amsterdam, pp. 283–364. Cai, W.-H., Matsunami, K., Otsuka, H., Takeda, Y., 2011. 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. Am. J. Plant Sci. 2, 609–618. De Marino, S., Borbone, N., Zollo, F., Ianaro, A., Di Meglio, P., Iorizzi, M., 2004. Megastigmane and phenolic components from Laurus nobilis L. Leaves and their inhibitory effects on nitric oxide production. J. Agric. Food Chem. 52, 7525–7531.

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