Glycosides from the Aerial Parts of Patrinia villosa

Abstract

An investigation of the Korean medicinal plant Patrinia villosa (THUNB.) JUSS. (Valerianaceae) led to the isolation of two new flavonoid glycosides, patrivilosides 1 (1) and 2 (2), a new iridoid glycoside, patrinovalerosidate (3), and two new saponins, patrinovilosides A (4) and B (5), along with six known compounds including three flavonoid glycosides and three iridoid glycosides. The structures of the new compounds were elucidated based on analysis of their one dimensional (1D)- and 2D-NMR spectra along with their mass spectrometric data and the results of acid hydrolysis.

The genus Patrinia (Valerianaceae) consists of about 20 species, four of which, P. scabiosaefolia FISCH., P. saniculaefolia HEMSL., P. rupestris JUSS. and P. villosa (THUNB.) JUSS. are distributed throughout Korea.¹⁾ The whole plants have traditionally been used for inflammation, wound healing, ascetics, and abdominal pain after childbirth.^2,3) Previous phytochemical research on Patrinia villosa showed dominant presence of various types of iridoids,^4–6) and flavonoids^7–18) as well as steroids and triterpenoids,^8,18,19) and saponins.⁸⁾ Our previous phytochemical research on the genus mainly focused on P. scabiosaefolia and P. saniculaefolia and led to the isolation of a series of iridoids²⁾ and saponins.³⁾ Modern pharmacological research has demonstrated that P. villosa has broad pharmacological activity, including antitumor, anti-inflammatory, and antioxidant effects.^7,20–22) Therefore, we conducted phytochemical studies on P. villosa and resulted in the isolation of two new flavonoid glycosides, patrivilosides 1 (1) and 2 (2), a new iridoid glycoside, patrinovalerosidate (3), and two new saponins, patrinovilosides A (4) and B (5), along with six known compounds. The structures were determined using spectroscopic methods including one dimensional (1D)- and 2D-NMR spectra along with their mass spectroscopic data and the results of acid hydrolysis. Here, we describe the structure elucidation of the new compounds 1–5.

The dried aerial parts of P. villosa were crushed and extracted with MeOH. The concentrated extract was suspended in H₂O and successively extracted with hexane, EtOAc and BuOH. The BuOH extract was subjected to sequential column chromatography over silica gel, Sephadex LH-20 and RP-18 gel to yield two new flavonoid glycosides, patrivilosides 1 (1) and 2 (2), a new iridoid glycoside, patrinovalerosidate (3), and two new saponins, patrinovilosides A (4) and B (5), along with six known compounds that included three iridoid glycosides, loganin (6),²³⁾ valerosidate (7),^24,25) and loganic acid (11),²³⁾ and three flavonoid glycosides, kaempferol 3-O-4‴-acetylrhamninoside (8),²⁶⁾ catharticin (9),²⁶⁾ and kaempferol 3-O-rhamninoside (10).^8,26) Known compounds were identified by detailed 1D- and 2D-NMR analyses, comparison of their FAB-MS and spectral data with those reported in literature.

Fig. 1. Structures of Compounds 1–12

Patriviloside 1 (1) was isolated as a yellow amorphous powder and its molecular formula was established as C₃₆H₄₄O₂₀ from the [M+H]⁺ peak at m/z 797.2505 (Calcd for C₃₆H₄₅O₂₀: 797.2504) in the high resolution (HR) (+)-FAB-MS. The IR spectrum showed typical absorption bands for hydroxy (3390 cm⁻¹), carbonyl (1739 cm⁻¹), conjugated carbonyl (1657 cm⁻¹), aromatic C=C (1598, 1588, 1497 cm⁻¹), and glycosidic C–O groups (1072, 1039 cm⁻¹). The UV spectrum of 1 showed two major absorption bands of 350 nm and 266 nm, suggesting a flavonol skeleton.^{8,26) 1}H- and ¹³C-NMR data also exhibited signals characteristic of 3,5,7,4′-tetrasubstituted flavonol glycosides, with a methoxyl [δ_H 3.86; δ_C 56.1], an acetoxyl [δ_H 1.99; δ_C 20.6, 169.7], two secondary methyl groups [δ_H 0.95 (d, J=6.2 Hz, H-6‴), δ_C 17.4 (C-6‴); δ_H 0.98 (d, J=6.2 Hz, H-6″″), δ_C 17.6 (C-6″″)], and three sugar units evidenced by signals of three anomeric carbons [δ_C 102.0 (C-1″), 100.5 (C-1‴), 102.5 (C-1″″)] and their corresponding anomeric protons [δ_H 5.35 (d, J=7.6 Hz, H-1″), 4.47 (br s, H-1‴), 4.51 (br s, H-1″″)]. Acid hydrolysis of 1 yielded rhamnocitrin,²⁶⁾ galactose (Gal) and rhamnose (Rha) as sugar components identified by TLC comparison with authentic samples. The absolute configuration of the sugars were determined to be L-form for Rha and D-form for Gal, which were identified by GC analysis of the thiazolidine derivatives.²⁷⁾ (+)-FAB-MS exhibited fragment ions at m/z 797 [M+H]⁺, 651 [(M+H)−146]⁺, 609 [(M+H)−146−42]⁺, 463 [(M+H)−(146×2)−42]⁺, and 301 [(M+H)−(146×2)−42−162]⁺, which showed the presence of a linear sugar chain, and the sugar sequence appeared to be that of Rha−Rha−Gal.³⁾ A correlation between H-1″ (δ_H 5.35) of Gal and C-3 (δ_C 133.5) in the heteronuclear multiple bond connectivity (HMBC) spectrum of 1 indicated C-3 as the site of O-glycosylation. The interglycosidic linkages of the trisaccharide moiety were also determined from the HMBC data. Correlations from the inner Rha H-1‴ at δ_H 4.47 to C-6″ (δ_C 66.6) of Gal, and the terminal Rha H-1″″ at δ_H 4.51 to the inner Rha of C-3‴ at δ_C 76.3 indicated that the trisaccharide moiety was a rhamninose. The acetoxyl moiety was attached at C-4‴ by HMBC correlations between δ_H 4.82 (t, J=9.8 Hz, H-4‴) and δ_C 169.7 (CH₃CO). Thus, 1 was identified unambiguously as rhamnocitrin 3-O-α-L-rhamnopyranosyl-(1→3)-(4‴-O-acetyl-α-L-rhamnopyranosyl)-(1→6)-β-D-galactopyranoside, and named patriviloside 1.

Patriviloside 2 (2) had a molecular formula of C₄₂H₅₄O₂₅, as deduced from HR-FAB-MS. The ¹H- and ¹³C-NMR data were closely similar to those of 1, except for the presence of an additional β-D-glucopyranosyl (Glc) unit, confirmed by the acid hydrolysis experiment.²⁷⁾ The location of the additional β-D-Glc unit in 2 was deduced to be at C-5 hydroxy group of 1 by the HMBC correlation between H-1″‴ of Glc at δ_H 4.92 (d, J=7.5 Hz) and C-5 at δ_C 159.6.²⁸⁾ From the above results, 2 was characterized as rhamnocitrin 3-O-4‴-acetylrhamninoside 5-O-β-D-glucopyranoside, and named patriviloside 2.

Patrinovalerosidate (3) was obtained as a white amorphous powder. The IR spectrum showed typical absorption bands for hydroxy (3413 cm⁻¹), carbonyl (1715 cm⁻¹), conjugated C=C groups (1651 cm⁻¹), and glycosidic C–O (1077, 1032 cm⁻¹). The HR-(+)-FAB-MS showed a protonated molecular ion peak at m/z 449.2388 [M+H]⁺, suggestive of a molecular formula of C₂₁H₃₆O₁₀ (Calcd for C₂₁H₃₇O₁₀: 449.2387) and four degrees of unsaturation. The ¹H- and ¹³C-NMR spectroscopic data were closely similar to those of jatamanin J (12)²⁹⁾ except for the presence of one hexose and isovaleryl moiety in 3. These observations, coupled with analysis of the ¹H–¹H correlation spectroscopy (COSY), the heteronuclear multiple quantum correlation (HMQC) and HMBC spectra, disclosed that 3 was a derivative of 12 with an isovaleroxyl group at C-1 and sugar moiety at C-11. The presence of a Glc unit was supported by the ¹³C-NMR and ¹H–¹H COSY spectra. The absolute configuration of the Glc was determined to be D-form in the above manner.²⁷⁾ The HMBC correlations between the Glc anomeric proton at δ_H 4.27 (d, J=7.8 Hz) and C-11 (δ_C 73.5), and between the oxymethylene protons at C-1 [δ_H 4.05 (1H, dd, J=7.3, 11.2 Hz), 4.17 (1H, dd, J=6.0, 11.2 Hz)] and the ester carbonyl of the isovaleroxyl moiety at δ_C 175.0 (C-1″) clearly established the full structure of 3 for assignment. The nuclear Overhauser effect (NOE) correlations of H-9 with H-5 and 10-CH₃, and H-7 with H-6α, and H-5 with 10-CH₃ and H-6β showed that the H-5 and H-9 hydrogens possess a cis-configuration and indicated 7β,8α-orientations for the 7,8-OH groups. The above data strongly suggest that 3 is a monocyclic iridoid glycoside ring-opened between C-1 and C-2 as seen in the patrinioside which was already isolated from the same genus P. scabra.³⁰⁾ Thus, the structure of 3 was established as 1-O-isovaleroxyl-11-O-β-D-glucopyranosyl jatamanin J and named patrinovalerosidate. Iridoids with an isovaleroyl group at C-1 and a sugar moiety at C-11 have been previously isolated from several species within the genera Penstemon, Viburnum and Valeriana.^31–34) The occurrence of monocyclic iridoid glycoside produced by the cleavage of the pyran ring isolated from natural sources is relatively rare.^30,32–34) Although the positions of glycosidation and acylation are different from those of patrinioside, this is the first positional isomer of patrinioside isolated from the same genus Patrinia.

Patrinoviloside A (4) was obtained as a white amorphous powder. HR-(−)-FAB-MS gave the molecular formula C₄₆H₇₄O₁₇. The inspection of the ¹H- and ¹³C-NMR spectra of the compound readily indicated the presence of three monosaccharide units through easily identifiable signals for anomeric protons and carbons. Acid hydrolysis of 4 yielded Glc, xylose (Xyl), and arabinose (Ara) as the sugar components identified by TLC analysis with comparison of authentic samples. The absolute configurations of sugars were determined as the D-form for Glc, L-form for Ara and D-form for Xyl, respectively, in the above manner.²⁷⁾ The (−)-FAB-MS of 4 exhibited an [M−H]⁻ ion at m/z 897, which is consistent with a trisaccharide glycoside carrying one mol each of Glc, Xyl and Ara, and an aglycon with a molecular mass 472. The daughter ions at m/z 765 [(M−H)−132]⁻, 603 [(M−H)−132−162]⁻, and 471 [(M−H)−132−162−132]⁻ showed the presence of a linear sugar chain, and the sugar sequence appeared to be that of pentose-Glc-pentose.³⁾ The ¹H- and ¹³C-NMR data of the aglycon moiety in 4 were closely related to those of reported compound, mesembryanthemoidigenic acid,³⁵⁾ except for the presence of the three sugar units described above. An unambiguous determination of the sequence of the sugars and linkage sites at the aglycon was determined from the HMBC spectrum (Fig. 2), which showed key correlation peaks between the proton signal at δ_H 4.72 (H-1′) and the carbon resonance at δ_C 88.6 (C-3), δ_H 5.34 (H-1″) and δ_C 84.3 (C-3′), and the proton signal at δ_H 5.11 (H-1‴) and the carbon resonance at δ_C 80.7 (C-4″). The large J values of the anomeric protons established the sugar units as α-L-arabinopyranosyl (J=7.5 Hz), β-D-glucopyranosyl (J=7.9 Hz) and β-D-xylopyranosyl (J=7.7 Hz) in 4.³⁾ Thus, the structure of 4 was established as mesembryanthemoidigenic acid 3-O-β-D-xylopyranosyl-(1→4)-β-D-glucopyranosyl-(1→3)-α-L-arabinopyranoside, named patrinoviloside A.

Fig. 2. Key HMBC Correlations of Patrinoviloside A (4)

Patrinoviloside B (5) was isolated as a white amorphous powder and the molecular formula was established as C₆₀H₉₆O₂₇ based on the HR-(−)-FAB-MS. Its IR spectrum showed absorptions at 3397, 1737, 1644 and 1067 cm⁻¹, compatible with the presence of hydroxyl (OH), carbonyl (C=O), double bond (C=C), and glycosidic C–O functionalities. Acid hydrolysis of 5 yielded an aglycon identified as ursolic acid by direct comparison with an authentic sample and sugars identified as L-Ara, D-Glc, and apiose as described in 4. The stereochemistry of the apiose could be assigned as the D-form by the coupling of H-5 at δ_H 4.16, which showed the magnetic equivalent.^36,37) The (−)-FAB-MS of 5 displayed a quasimolecular ion peak at m/z 1247 [M−H]⁻ ion, which is consistent with a pentasaccharide glycoside carrying one mol each of Ara and apiose and three moles of Glc with an acetoxyl group, and an aglycon with a molecular mass 456. Further fragment ion peaks were observed at m/z 1115 [(M−H)−132]⁻, 1073 [(M−H)−132−42]⁻, 953 [(M−H)−132−162]⁻, 911 [(M−H)−132−162−42]⁻, 791 [(M−H)−132−162−162]⁻, 749 [(M−H)−132−162−42−162]⁻, 731 [(M−H)−132−162−42−162−18]⁻, 629 [(M−H)−132−162−162−162]⁻, 587 [(M−H)−132−162−162−162−42]⁻ and 455 [(M−H)−132−162−162−162−42−132]⁻, corresponding to the successive losses of one pentosyl, three glucosyl, and again one pentosyl moieties, respectively.³⁾ The presence of five monosaccharides for 5 was confirmed by the presence of five anomeric proton signals at δ_H 4.70 (d, J=7.9 Hz), 5.06 (d, J=7.9 Hz), 6.19 (d, J=8.0 Hz), 4.98 (d, J=7.8 Hz) and 6.00 (d, J=2.7 Hz) correlated in the HMQC spectrum with the carbon signals at δ_C 104.7, 106.5, 95.6, 105.0, and 111.0, respectively. Complete assignments of each sugar by extensive 1D- and 2D-NMR spectroscopic analyses allowed for the identification of three β-D-glucopyranosyl, one α-L-arabinopyranosyl, and one β-D-apiofuranosyl moiety. From the downfield ¹³C-NMR chemical shift of C-3 at δ_C 89.1 and the upfield chemical shift value observed for C-28 at δ_C 176.3 compared with those of ursolic acid, it was established that 5 is a bisdesmosidic glycoside of ursolic acid with sugar linkages at C-3 through an ether bond and at C-28 through an ester bond.³⁾ The sequence of the sugars and binding sites at the aglycon of 5 were determined based on HMBC experiments (Fig. 3). HMBC correlations between the proton at δ_H 5.06 (d, J=7.9 Hz, H-1″) and the carbon at δ_C 81.2 (C-3′), and between the proton at δ_H 4.70 (d, J=7.9 Hz, H-1′) and the carbon at δ_C 89.1 (C-3), and between the proton at δ_H 6.02 (br t, J=9.0 Hz, H-2′) and the acetyl carbonyl carbon at δ_C 169.9 (CH₃CO) proved the sugar chain sequence at C-3 to be β-D-glucopyranosyl-(1→3)-(2-O-acetyl-α-L-arabinopyranosyl). Other HMBC correlations between the proton at δ_H 6.00 (d, J=2.7 Hz, H-1″‴) and the carbon at δ_C 78.9 (C-4″″), and between the proton at δ_H 4.98 (d, J=7.8 Hz, H-1″″) and the carbon at δ_C 71.0 (C-4‴), and between the proton at δ_H 6.19 (d, J=8.0 Hz, H-1‴) and the carbon at δ_C 176.3 (C-28) suggested the sequence at C-28 to be β-D-apiofuranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl. Thus, the structure of 5 was established as 3-O-β-D-glucopyranosyl-(1→3)-(2′-O-acetyl-α-L-arabinopyranosyl)ursolic acid 28-O-β-D-apiofuranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, and named patrinoviloside B. Although many saponins have been isolated from the genus Patrinia,³⁸⁾ only two saponins such as sulfapatrinosides I and II have been isolated from this plant.⁸⁾ Saponins of the Patrinia plants, especially those with sugars acylated with acetic acid, have only been isolated from P. scabiosaefolia.^39,40) To the best of our knowledge, patrinoviloside B (5) is the first example of a bisdesmosidic triterpenoid saponin bearing the apiofuranose sugar moiety in the Patrinia species.

Fig. 3. Key HMBC Correlations of Patrinoviloside B (5)

Experimental

General

UV spectra were obtained with the Hitachi JP/U3010 spectrometer and IR spectra were obtained on a JASCO FT/IR-5300 spectrometer. Optical rotations were determined on a JASCO P-1020 polarimeter. The FAB mass spectra were obtained on a JEOL JMS-700 spectrometer. The NMR spectra were determined on a Bruker Avance-400 (400 MHz) or a Bruker Avance-500 (500 MHz), and the chemical shifts were referenced to tetramethylsilane (TMS). Column chromatography (CC) was run on silica gel 60 (70–230 mesh or 230–400 mesh, Merck), LiChroprep RP₁₈ (40–63 µm, Merck) and Sephadex LH-20 (Amersham Biosciences AB). TLC was performed on silica gel 60 F₂₅₄ plates (Merck) and RP18_254S plates (Merck). GC analysis was performed with a Younglin YL 6100 gas chromatograph equipped with an H₂ flame ionization detector. The column was an HP-5 capillary column (30 m×0.32 mm×0.25 mm); column temperature, 230°C; injector and detector temperature, 200°C; and He flow rate, 1 mL/min.

Plant Material

The aerial parts of P. villosa were collected at Chuyeop Mt., Yangpyung, Gyunggido, Korea, in September 2010. The botanical identification was made by Prof. Y.-B. Suh. A voucher specimen (KSS2010-3) was deposited in the Natural Products Research Institute, College of Pharmacy, Seoul National University.

Extraction and Isolation

The dried aerial parts (1.5 kg) were extracted seven times with MeOH under reflux at 80°C. The MeOH extract were evaporated to dryness under reduced pressure. The MeOH extract was suspended in H₂O and successively partitioned with hexane, EtOAc and BuOH. The BuOH fraction (51 g) was purified by silica gel CC and eluted with CH₂Cl₂–MeOH–H₂O (7 : 1 : 0.5→6 : 4 : 1) to yield 13 fractions (B-1–B-13). Fraction B-4 (6 g) was purified by silica gel CC with EtOAc–MeOH–H₂O (100 : 8 : 6→100 : 24 : 18) to yield 6 fractions. Fraction B-4-2 (250 mg) was purified by silica gel CC with CHCl₃–MeOH–H₂O (7 : 1 : 0.5→7 : 2 : 0.5) to yield 5 fractions. Fraction B-4-2-3 was purified by silica gel CC with EtOAc–MeOH–H₂O (100 : 1 : 1→100 : 8 : 6) to afford 6 (5 mg) and 4 fractions. Fraction B-4-2-3-3 was purified by RP-18 with MeOH–H₂O (4.5 : 5.5) to afford 3 (8 mg). Fraction B-4-3 (2.8 g) was purified by silica gel CC with CHCl₃–MeOH–H₂O (7 : 1.5 : 0.5→7 : 3 : 1) to yield 6 fractions. Fraction B-4-3-5 (1.5 g) was purified by silica gel CC with EtOAc–MeOH–H₂O (100 : 1 : 1→100 : 8 : 6) to yield 8 fractions. Fraction B-4-3-5-5 (800 mg) was purified by RP-18 with MeOH–H₂O (1 : 1→7 : 3), respectively to afford 7 (410 mg) and 1 (190 mg). Fraction B-6 (5.3 g) was purified by silica gel CC with EtOAc–MeOH–H₂O (100 : 16.5 : 13.5) to yield 7 fractions. Fraction B-6-2 (2.5 g) was purified by RP-18 with MeOH–H₂O (6 : 4→8 : 2) to afford 8 (166 mg) and 4 fractions. Fraction B-6-2-2 was purified by RP-18 with MeOH–H₂O (1 : 1) to afford 9 (138 mg). Fraction B-6-3 (1.3 g) was purified by silica gel CC with CHCl₃–MeOH–H₂O (7 : 2 : 0.5→520 : 280 : 80) to yield 7 fractions. Fraction B-6-3-3 was recrystallized by MeOH and CH₂Cl₂ to afford 4 (4 mg). Fraction B-8 (7.4 g) was purified by silica gel CC with EtOAc–MeOH–H₂O (100 : 16.5 : 13.5→100 : 40 : 20) to yield 6 fractions. B-8-2 (1 g) was purified by Sephadex LH-20 with MeOH to afford 10 (985 mg). Fraction B-8-5 (300 mg) was purified by RP-18 with MeOH–H₂O (9 : 2) to yield 3 fractions. Fraction B-8-5-2 (150 mg) was purified by RP-18 with MeOH–H₂O (8 : 2) to afford 5 (10 mg). Fraction B-8-6 (3.9 g) was purified by RP-18 with MeOH–H₂O (9 : 2) to 3 fractions. B-8-6-2 (1 g) was purified by RP-18 with MeOH–H₂O (10 : 0.5) to yield 4 fractions. Fraction B-8-6-2-3 (100 mg) was purified by RP-18 with MeOH–H₂O (1 : 1) to yield 4 fractions. Fraction B-8-6-2-3-3 (50 mg) was purified by RP-18 with MeOH–H₂O (6 : 4) to afford 2 (8 mg). Fraction B-9 (5.2 g) was purified by silica gel CC with EtOAc–MeOH–H₂O (100 : 16.5 : 13.5→100 : 40 : 20) to yield 5 fractions. Fraction B-9-5 (2.7 g) was purified by RP-18 with MeOH–H₂O (9 : 2) to yield 4 fractions. Fraction B-9-5-2 (210 mg) was purified by RP-18 with MeOH–H₂O (10 : 10→10 : 3) to yield 3 fractions. Fraction B-9-5-2-2 (130 mg) was purified by RP-18 with MeOH–H₂O (5 : 7) to yield 3 fractions. Fraction B-9-5-2-2-2 (10 mg) was purified by Sephadex LH-20 with MeOH to afford 11 (4 mg).

Patriviloside 1 (1): Yellow amorphous powder. [α]_D²⁰ −34.6 (c=0.2, MeOH); IR (KBr) ν_max cm⁻¹: 3390 (OH), 1739, 1215 (OAc), 1657 (conjugated C=O), 1598, 1588, 1497 (aromatic C=C), 1453, 1350, 1283, 1168, 1072, 1039 (glycosidic C–O), 889, 838; UV λ_max (MeOH) nm (log ε): 350 (4.57), 266 (4.63); (NaOAc): 398 (4.19), 265 (4.22); (NaOAc+H₃BO₃): 354 (4.21), 266 (4.25); (NaOMe): 395 (4.69), 269 (4.57); (AlCl₃): 398 (4.52), 353 (4.50), 304 (4.37), 274 (4.64); (AlCl₃+HCl): 396 (4.45), 350 (4.47), 302 (4.33), 275 (4.59); ¹H-NMR (400 MHz, DMSO-d₆) and ¹³C-NMR (100 MHz, DMSO-d₆) δ: Table 1; FAB-MS m/z: 819 [M+Na]⁺, 797 [M+H]⁺, 651 [(M+H)−146]⁺, 609 [(M+H)−146−42]⁺, 463 [(M+H)−146−42−146]⁺, 301 [rhamnocitrin+H]⁺; HR-(+)-FAB-MS m/z: 797.2505 (Calcd for C₃₆H₄₅O₂₀: 797.2504).

Table 1. NMR Data of 1 and 2 (δ in ppm, J in Hz)

No.	1a)		2b)
	δHc)	δCd)	δHc)	δCd)
C-2	—	157.0	—	158.0
C-3	—	133.5	—	138.0
C-4	—	177.6	—	176.1
C-5	—	160.9	—	159.6
C-6	6.38 (d, 1.8)	97.9	6.91 (d, 1.8)	103.8
C-7	—	165.2	—	166.2
C-8	6.72 (d, 1.8)	92.4	6.84 (d, 1.8)	97.0
C-9	—	156.3	—	159.8
C-10	—	104.9	—	110.3
OCH3	3.86 (s)	56.1	3.92 (s)	56.8
C-1′	—	120.7	—	122.3
C-2′, 6′	8.10 (d, 8.7)	131.0	8.16 (d, 8.7)	132.5
C-3′, 5′	6.87 (d, 8.7)	115.1	6.89 (d, 8.7)	116.2
C-4′	—	160.2	—	161.7
5-OH	12.57
Galactose
C-1″	5.35 (d, 7.6)	102.0	5.00 (d, 7.8)	105.5
C-2″	3.58 (br t, 7.9)	71.1	3.82 (br t, 9.2)	73.0
C-3″	3.45e)	72.9	3.60e)	75.1
C-4″	3.62e)	68.2	3.78 (d, 3.3)	70.4
C-5″	3.61e)	73.4	3.69e)	75.3
C-6″	3.55e)	66.6	3.54e)	68.8
	3.62e)		3.79e)
Rhamnose
C-1‴	4.47 (br s)	100.5	4.56 (br s)	102.2
C-2‴	3.58e)	69.8	3.70 (dd, 1.8, 3.5)	71.8
C-3‴	3.53 (dd, 3.0, 9.8)	76.3	3.71 (dd, 3.5, 10.0)	77.9
C-4‴	4.82 (t, 9.8)	72.4	4.93 (t, 10.0)	74.2
C-5‴	3.62e)	66.0	3.71e)	67.8
C-6‴	0.95 (d, 6.2)	17.4	0.99 (d, 6.2)	17.7
COCH3	—	169.7	—	172.0
COCH3	1.99 (s)	20.6	2.02 (s)	21.0
Rhamnose
C-1″″	4.51 (br s)	102.5	4.66 (br s)	103.8
C-2″″	3.54 (br d, 2.8)	70.3	3.64 (br d, 3.3)	72.0
C-3″″	3.43e)	70.7	3.65e)	72.3
C-4″″	3.13 (t, 9.2)	71.9	3.32 (t, 10.5)	73.8
C-5″″	3.55e)	68.6	3.63e)	70.2
C-6″″	0.98 (d, 6.2)	17.6	1.11 (d, 6.2)	18.0
Glucose
C-1″‴			4.92 (d, 7.5)	104.9
C-2″‴			3.59e)	74.8
C-3″‴			3.51 (t, 9.1)	78.7
C-4″‴			3.42 (t, 9.1)	71.3
C-5″‴			3.53 (m)	77.2
C-6″‴			3.72 (dd, 5.2, 11.2)	62.6
			3.93 (br d, 11.2)

a) DMSO-d₆. b) CD₃OD. c) Recorded at 400 MHz. d) Recorded at 100 MHz. e) Overlapping signal.

Patriviloside 2 (2): Yellow amorphous powder. [α]_D²⁰ −83.8 (c=0.6, MeOH); IR (KBr) ν_max cm⁻¹: 3374 (OH), 1732, 1222 (OAc), 1626 (conjugated C=O), 1603, 1511 (aromatic C=C), 1447, 1358, 1285, 1222, 1178, 1073, 1041 (glycosidic C–O), 843; UV λ_max (MeOH) nm (log ε): 341 (4.40), 259 (4.39); (NaOAc): 395 (4.02), 345 (4.33), 259 (4.39); (NaOAc+H₃BO₃): 342 (4.42), 259 (4.41); (NaOMe): 396 (4.51), 260 (4.30); (AlCl₃): 409 (4.35), 350 (sh, 4.07), 305 (sh, 4.05), 271 (4.43); (AlCl₃+HCl): 410 (4.32), 340 (sh, 4.07), 305 (sh, 4.05), 270 (4.41); ¹H-NMR (400 MHz, CD₃OD) and ¹³C-NMR (100 MHz, CD₃OD) δ: Table 1; FAB-MS m/z: 981 [M+Na]⁺, 651 [(M+H)−146−162]⁺, 609 [(M+H)−146−162−42]⁺, 301 [rhamnocitrin+H]⁺; HR-(+)-FAB-MS m/z: 981.2863 (Calcd for C₄₂H₅₄O₂₅Na: 981.2852).

Patrinovalerosidate (3): White amorphous powder. [α]_D²¹ −12.9 (c=0.5, MeOH); IR (KBr) ν_max cm⁻¹: 3413 (OH), 2960, 2931, 1715 (carbonyl), 1651 (C=C), 1077, 1032 (glycosidic C–O), 827; ¹H-NMR (400 MHz, CD₃OD) and ¹³C-NMR (100 MHz, CD₃OD) δ: Table 2; FAB-MS m/z: 471 [M+Na]⁺, 449 [M+H]⁺, 287 [(M+H)−162]⁺. HR-(+)-FAB-MS m/z: 449.2388 (Calcd for C₂₁H₃₇O₁₀: 449.2387).

Table 2. NMR Data of 3 in CD₃OD (δ in ppm, J in Hz)

No.	δHa)	δCb)
C-1	4.05 (dd, 7.3, 11.2), 4.17 (dd, 6.0, 11.2)	64.1
C-3	5.13 (br s), 5.31 (br s)	115.3
C-4	—	147.1
C-5	3.19 (m)	41.4
C-6	2.19 (ddd, 4.9, 10.6, 13.4), 1.76 (br dd, 8.2, 13.4)	39.0
C-7	3.78 (br d, 4.3)	81.2
C-8	—	83.7
C-9	2.34 (br td, 6.8, 10.3)	47.9
C-10	1.35 (s)	23.0
C-11	4.14 (d, 12.6), 4.31 (d, 12.6)	73.5
Glucose
C-1′	4.27 (d, 7.8)	102.9
C-2′	3.21 (t, 8.4)	75.1
C-3′	3.33 (dd, 8.4, 9.2)	78.1
C-4′	3.32 (t, 9.2)	71.7
C-5′	3.23 (m)	77.9
C-6′	3.66 (dd, 5.5, 11.9), 3.85 (dd, 1.7, 11.9)	62.8
Isovaleric acid
C-1″	—	175.0
C-2″	2.15 (d, 6.6)	44.4
C-3″	2.04 (dq, 6.6, 13.8)	26.8
C-4″	0.94 (d, 6.6)	22.8
C-5″	0.94 (d, 6.6)	22.8

a) Recorded at 400 MHz. b) Recorded at 100 MHz.

Patrinoviloside A (4): White amorphous powder. [α]_D²² +31.0 (c=0.5, MeOH); IR (KBr) ν_max cm⁻¹: 3378 (OH), 1690 (COOH), 1643 (C=C), 1376, 1246, 1157, 1077, 1043 (glycosidic C–O), 871; ¹H-NMR (500 MHz, pyridine-d₅) and ¹³C-NMR (125.8 MHz, pyridine-d₅) δ: Table 3; (−)-FAB-MS m/z: 897 [M−H]⁻, 765 [(M−H)−132]⁻, 603 [(M−H)−132−162]⁻ and 471 [(M−H)−132−162−132]⁻; HR-(−)-FAB-MS m/z: 897.4837 (Calcd for C₄₆H₇₃O₁₇: 897.4848).

Table 3. NMR Data of 4 and 5 in Pyridine-d₅ (δ in ppm, J in Hz)

No.	4		5
	δHa)	δCb)	δHc)	δCd)
C-1	0.92 (br t, 13.1), 1.48e)	38.7	0.83,e) 1.48e)	38.7
C-2	1.85,e) 2.11e)	26.7	1.78 (br t, 13.0), 2.05e)	26.5
C-3	3.31 (dd, 4.2, 11.7)	88.6	3.22 (dd, 3.9, 11.5)	89.1
C-4	—	39.7	—	39.2
C-5	0.78 (br d, 11.7)	55.8	0.77 (br d, 11.4)	55.7
C-6	1.26,e) 1.47e)	18.4	1.30,e) 1.48e)	18.5
C-7	1.26,e) 1.45e)	33.2	1.33,e) 1.48e)	33.5
C-8	—	39.6	—	40.1
C-9	1.63 (t, 8.7)	48.0	1.55 (dd, 7.5, 11.6)	48.0
C-10	—	37.0	—	36.9
C-11	1.87e)	23.8	1.91 (m)	23.7
C-12	5.49 (br s)	122.5	5.43 (br s)	126.1
C-13	—	144.9	—	138.4
C-14	—	42.1	—	42.5
C-15	2.19e)	28.3	2.42 (m)	28.7
C-16	1.99,e) 2.19e)	23.8	1.91,e) 2.05e)	24.6
C-17	—	47.1	—	48.4
C-18	3.41 (br dd, 3.6, 13.7)	41.2	2.51 (br d, 11.2)	53.2
C-19	1.49,e) 2.16 (t, 13.1)	41.4	1.45e)	39.3
C-20	—	36.6	1.48e)	39.0
C-21	1.86e)	29.1	1.27,e) 1.36e)	30.8
C-22	1.93,e) 2.15e)	32.7	1.78 (br t, 13.0), 1.97 (br d, 13.0)	36.8
C-23	1.28 (s)	28.1	1.12 (s)	28.0
C-24	0.96 (s)	16.9	0.89 (s)	16.8
C-25	0.81 (s)	15.5	0.83 (s)	15.7
C-26	0.99 (s)	17.4	1.13 (s)	17.6
C-27	1.30 (s)	26.1	1.19 (s)	23.8
C-28	—	180.3	—	176.3
C-29	3.58e)	73.9	0.92 (d, 6.6)	17.4
C-30	1.20 (s)	19.7	0.89 (d, 6.6)	21.3
Arabinose
C-1′	4.72 (d, 7.5)	107.4	4.70 (d, 7.9)	104.7
C-2′	4.58 (br t, 8.9)	71.9	6.02 (br t, 9.0)	72.2
C-3′	4.17 (dd, 3.4, 9.5)	84.3	4.23e)	81.2
C-4′	4.39 (br s)	69.4	4.48 (br s)	69.5
C-5′	3.75 (br d, 11.5)	67.1	3.69 (br d, 11.8)	67.1
	4.21 (br d, 11.5)		4.16 (br d, 11.8)
COCH3	—		—	169.9
COCH3	—		2.26 (s)	21.4
Glucose
C-1″	5.34 (d, 7.9)	106.2	5.06 (d, 7.9)	106.5
C-2″	4.04 (t, 8.2)	75.4	3.86 (t, 8.2)	74.6
C-3″	4.25 (t, 8.9)	76.2	4.24 (t, 8.4)	78.5
C-4″	4.32 (t, 9.3)	80.7	4.16e)	71.6
C-5″	3.90 (br dt, 3.3, 9.3)	76.7	4.00e)	78.4
C-6″	4.45 (dd, 1.8, 11.8)	61.5	4.35 (dd, 5.1, 11.9)	62.8
	4.57 (dd, 3.7, 11.8)		4.55 (br d, 11.9)
Xylose			Glucose
C-1‴	5.11 (d, 7.7)	105.6	6.19 (d, 8.0)	95.6
C-2‴	4.00 (t, 8.4)	74.9	4.14e)	73.8
C-3‴	4.09 (t, 8.7)	78.3	4.00e)	78.7
C-4‴	4.13 (dd, 5.2, 9.2)	70.8	4.31e)	71.0
C-5‴	3.64 (t, 10.5)	67.4	4.10 (m)	77.8
	4.20 (dd, 1.8, 10.5)
C-6‴	—	—	4.31e)	69.6
			4.66 (br d, 10.4)
Glucose
C-1″″			4.98 (d, 7.8)	105.0
C-2″″			3.96 (t, 8.4)	74.9
C-3″″			4.17e)	76.4
C-4″″			4.23e)	78.9
C-5″″			3.76 (m)	76.9
C-6″″			4.29e)	61.3
Apiofuranose
C-1″‴			6.00 (d, 2.7)	111.0
C-2″‴			4.76 (d, 2.7)	77.5
C-3″‴			—	80.1
C-4″‴			4.30 (d, 9.4)	75.1
			4.74 (d, 9.4)
C-5″‴			4.16 (s)	65.0

a) Recorded at 500 MHz. b) Recorded at 125.8 MHz. c) Recorded at 400 MHz. d) Recorded at 100 MHz. e) Overlapping signal.

Patrinoviloside B (5): White amorphous powder. [α]_D²⁰ −4.8 (c=0.6, MeOH); IR (KBr) ν_max cm⁻¹: 3397 (OH), 1737, 1251 (OAc), 1644 (C=C), 1455, 1373, 1165, 1067, 1034 (glycosidic C–O), 829; ¹H-NMR (400 MHz, pyridine-d₅) and ¹³C-NMR (100 MHz, pyridine-d₅) δ: Table 3; (−)-FAB-MS m/z: 1247 [M−H]⁻, 1115 [(M−H)−132]⁻, 1073 [(M−H)−132−42]⁻, 953 [(M−H)−132−162]⁻, 911 [(M−H)−132−162−42]⁻, 791 [(M−H)−132−162−162]⁻, 749 [(M−H)−132−162−42−162]⁻, 731 [(M−H)−132−162−42−162−18]⁻, 629 [(M−H)−132−162−162−162]⁻, 587 [(M−H)−132−162−162−162−42]⁻ and 455 [(M−H)−132−162−162−162−42−132]⁻; HR-(−)-FAB-MS m/z: 1247.6086 (Calcd for C₆₀H₉₅O₂₇: 1247.6061).

Acid Hydrolysis and Determination of the Absolute Configuration of Sugars of 1–5

Each compound (1 mg each) was refluxed separately with 5% HCl in 60% dioxane (0.1 mL) for 3 h. The reaction solution was evaporated under reduced pressure, and the hydrolysate was extracted with CHCl₃. The aqueous layer was neutralized with Ag₂CO₃, and filtered, and the filtrate was concentrated under reduced pressure. The dried sugar mixture was treated with pyridine (0.1 mL), and then the solution was added to a pyridine solution (0.1 mL) of L-cysteine methyl ester hydrochloride (2 mg) and warmed at 60°C for 1 h. The solvent was evaporated under a N₂ stream and dried in vacuo. The residue was trimethylsilylated with trimethylchlorosilane and hexamethyldisilazane in anhydrous pyridine (0.1 mL) at 60°C for 30 min. After the addition of hexane and water, the hexane layer was removed and checked by GC. The retention times (t_R) of the peaks were 7.97 and 12.68 (1), and 7.95, 11.67 and 12.67 min (2), 11.63 min (3), 6.76, 6.82 and 11.68 (4), and 6.81, 11.65 and 5.80 min (5). The t_R of the peaks of the authentic samples were 11.67 (D-glucose), 7.95 (L-rhamnose), 12.69 min (D-galactose) 6.81 (L-arabinose), 6.78 min (D-xylose), and 5.81 (D-apiose), respectively.

Acknowledgment

This work was financially supported by the third phase of the Brain Korea 21 Program in 2012. The authors are grateful to Prof. Y.-B. Suh, College of Pharmacy, Seoul National University, for the identification of the plant materials.

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