Chemical Constituents of Aerial Parts and Roots of Pycnanthemum flexuosum

Abstract

An extract of whole plants of Pycnanthemum flexuosum showed an inhibitory effect on hyaluronidase activity. From an 80% acetone extract of aerial parts, 3-[(3E)-4-phenylbut-3-enoylamino]propionic acid, 3-O-β-d-glucuronopyranosyl-echinocystic acid 28-O-β-d-xylopyranosyl-(1→3)-[3,4-diacetyl-β-d-xylopyranosyl-(1→4)]-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl ester, vanillic acid 1-O-[(5-O-syringoyl)-β-d-apiofuranosyl]-(1→2)-β-d-glucopyranoside, and (4S,5R)-4-hydroxy-5-phenyl-tetrahydrofuran-2-one were isolated together with 30 known compounds. Six known compounds were isolated from an 80% acetone extract of roots, and eritrichin was revealed as a hyaluronidase inhibitor in P. flexuosum.

Pycnanthemum flexuosum (Walter) Britton, Sterns et Poggenb., a herbaceous perennial that belongs to the family Lamiaceae, grows in Japan as a naturalized plant that originated in North America.¹⁾

We have examined extracts of Lamiaceae plants for hyaluronidase inhibitory activity and isolated phenylpropanoid oligomers and flavone glucuronides.²⁾ An 80% acetone extract of whole plants of P. flexuosum also showed inhibitory activity (IC₅₀ 608 µg/mL), and 40 compounds (1–40) were isolated from 80% acetone extracts of the aerial parts or roots. Compounds 1–3 were isolated as new compounds (Fig. 1), and 4 was identified as a natural product for the first time. Chrysoeriol (6), apigenin 7-O-[6-O-(p-E-coumaroyl)-β-d-glucopyranoside] (15), apigenin 7-O-[6-O-(p-Z-coumaroyl)-β-d-glucopyranoside] (16), acteoside (23), leucosceptoside A (24), martynoside (25), plantainoside (26), darendoside B (28), eritrichin (31), dihydrosyringin (32), benzyl-O-β-d-glucopyranoside (34), chlorogenic acid (36), and caffeic acid (37) were identified as compounds isolated from other Lamiaceae plants previously.¹⁾ Genkwanin (5),³⁾ cirsilineol (7),⁴⁾ isothymusin (8),⁵⁾ acacetin 7-O-α-l-rhamnopyranosyl-(1→6)-β-d-glucuronopyranoside (9),⁶⁾ luteoline 7-O-[(6-O-acetyl)-β-d-allopyranosyl-(1→2)-β-d-glucopyranoside] (10),^7,8) 4′-O-methylhypolaetin 7-O-[6-O-acetyl-β-d-allopyranosyl-(1→2)-β-d-glucopyranoside] (11),⁸⁾ apigenin 7-O-[6-O-acetyl-β-d-allopyranosyl-(1→2)-β-d-glucopyranoside] (12),⁷⁾ isoscutellarein 7-O-[6-O-acetyl-β-d-allopyranosyl-(1→2)-β-d-glucopyranoside] (13),⁷⁾ isoscutellarein 4′-O-methylether 7-O-[(6-O-acetyl)-β-d-allopyranosyl-(1→2)-β-d-glucopyranoside] (14),⁸⁾ 3′-hydroxy-4′-O-methylisoscutellarein 7-O-[(6-O-acetyl)-β-d-allopyranosyl-(1→2)-(6-O-acetyl)-β-d-glucopyranoside] (17),⁹⁾ 4′-O-methylisoscutellarein 7-O-[(6-O-acetyl)-β-d-allopyranosyl-(1→2)-(6-O-acetyl)-β-d-glucopyranoside] (18),¹⁰⁾ luteolin 7-O-[β-d-allo-pyranosyl-(1→2)-β-d-glucopyranoside] (19),⁷⁾ hypolaetin 7-O-[β-d-allopyranosyl-(1→2)-β-d-glucopyranoside] (20),^7,9) hypolaetin 7-O-[(6-O-acetyl)-β-d-allopyranosyl-(1→2)-β-d-glucopy-ranoside] (21),⁹⁾ neoponcirin (22),¹¹⁾ artselaeroside A (27),¹²⁾ 2″,3″-di-O-acetyl martynoside (29),¹³⁾ stachysoside B (30),¹⁴⁾ albizinin (33),¹⁵⁾ (+)-pinoresinol 4-O-β-d-glucopyranoside (35),¹⁶⁾ syringic acid (38),¹⁷⁾ vanillic acid (39),¹⁸⁾ and methyl syringate 4-O-β-d-glucopyranoside (40)¹⁹⁾ were identified by comparing their spectroscopic data with those in the literature.

Fig. 1. Structures of 1–4, 4a, and 4b and the Values of ¹H-NMR Chemical Shift Differences (in ppm) for MTPA Esters of 4 [Δδ=δ(S)−δ(R)]

The molecular formula of compound 1 was established as C₁₃H₁₅NO₃ based on high resolution-electron ionization-mass spectra (HR-EI-MS) [m/z 233.1055 (Calcd for C₁₃H₁₅NO₃: 233.1053)], suggesting the presence of a nitrogen. In the ¹H-NMR spectrum, five aromatic protons at δ 7.27 (1H, t, J=7.5 Hz, H-4), 7.35 (2H, t, J=7.5, H-3, 5), and 7.44 (2H, d, J=7.5 Hz, H-2, 6), two olefinic protons at δ 6.58 (1H, br d, J=16.5 Hz, H-7) and 6.37 (1H, m, H-8), and six metylene protons at δ 3.13 (2H, d, J=7.0 Hz, H-9), 3.48 (2H, t, J=7.0 Hz, H-12), and 2.56 (2H, t, J=7.0 Hz, H-13) were observed. The corresponding carbons of these protons were assigned using a heteronuclear multiple quantum correlation (HMQC) spectrum. The aromatic protons, the spin system of the olefinic protons and the H-9 metylene protons, and a carbonyl carbon at δ 174.1 (C-10) in the ¹³C-NMR spectrum suggested the presence of a styrylacetic acid moiety. The coupling constant of the olefinic protons (16.5 Hz) showed their trans-configuration. On the other hand, the presence of nitrogen, the spin system of the four metylene protons (H-12, 13), and a carbonyl carbon at δ 175.3 (C-14) suggested the presence of a β-alanine moiety. In the heteronuclear multiple bond correlation (HMBC) spectrum, H-12 was correlated with C-10. This result showed that 1 was an amide of trans-styrylacetic acid and β-alanine, as shown in Fig. 1.

Compound 2 was obtained as a colorless powder. Its ¹H- and ¹³C-NMR spectra showed many aliphatic resonances, including seven singlet methyl and oxygenated resonances of sugars, which suggested that 2 was a triterpene saponin. In the ¹³C-NMR spectrum, 30 carbon resonances of an aglycone including two olefinic carbons (δ 123.8, C-12; 144.7, C-13), two oxygenated carbons (δ 91.2, C-3; 74.7, C-16), and a carboxylic carbon (δ 177.1, C-28) were observed with the exception of resonances of sugars. These signals were similar to echinocystic acid, and acid hydrolysis of 2 gave echinocystic acid as the aglycone of 2.²⁰⁾ In the ¹H-NMR spectrum, five anomeric (δ 4.39, 1H, d, J=7.5 Hz; 4.53, 1H, d, J=7.0 Hz; 4.88, 1H, d, J=7.5 Hz; 5.01, 1H, d, J=1.5 Hz; 5.65, 1H, d, J=3.5 Hz), a doublet methyl (δ 1.28, 3H, d, J=5.0 Hz), two singlet methyl (δ 1.99, 3H and 2.05, 3H) proton resonances, and oxygenated proton resonances corresponding to five sugar units were observed, exclusive of resonances of the aglycone moiety. The homonuclear Hartmann Hahn (HOHAHA) spectra radiated at the anomeric protons, and a doublet methyl was recorded. Then, the proton coupling systems of each sugar unit were assigned as shown in the Experimental section. Derivatized sugar analysis using HPLC and an octadodecyl silica (ODS) column after acid hydrolysis showed the presence of d-glucuronic acid, l-rhamnose, l-arabinose, and d-xylose.²¹⁾ The α or β configurations of the sugars were determined from their coupling constants as described previously.^22,23) The correlations in the HMBC spectrum suggested the partial structure of the sugar chain 3-O-β-d-glucuronopyranosyl and 28-O-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl ester; the anomeric proton resonance of the β-d-glucuronic acid moiety (δ 4.39, H-Glc A-1)²²⁾ was long-range coupled with C-3 (δ 91.2) of the aglycone, the anomeric proton resonance of α-l-arabinopyranosyl (δ 5.65, H-Ara-1)²³⁾ was coupled with the carboxylic carbon (δ 177.1, C-28) of the aglycone, and the anomeric proton resonance of α-l-rhamnopyranosyl (δ 5.01, H-Rha-1) was coupled with the resonance at δ 75.8 (C-Ara-2). The chemical shifts of C-3 (δ 82.2) and C-4 (δ 79.0) of α-l-rhamnopyranosyl were lower shifted than those of the terminal rhamnopyranosyl.²³⁾ In addition, their assigned proton and carbon resonances were similar to those of the 3,4-disubstituted rhamnopyranosyl moiety.²³⁾ The anomeric proton resonance of the terminal unsubstituted β-d-xylopyranosyl moiety (δ 4.53, H-Xyl-I-1) was coupled with C-Rha-3 (δ 82.2) in the HMBC spectrum. Another sugar unit was determined as β-d-xylopyranosyl on the basis of coupling constants of the proton spin systems (δ 5.04, t, J=9.5 Hz, H-Xyl-II-3) and the chemical shifts of their protons.²³⁾ The HMBC correlation between H-Xyl-II-1 and C-Rha-4 suggested that the second β-d-xylopyranosyl unit also bonded to the rhamnopyranosyl at its C-4. The proton and carbon resonances of Xyl-II-3 and Xyl-II-4 were lower shifted than those of the unsubstituted terminal xylopyranosyl moiety. The singlet methyl protons and carboxylic carbons at δ 171.1 and 172.4 suggested the presence of two acetyl groups. In the HMBC spectrum, H-Xyl-II-3 (δ 5.04, 1H, t, J=9.5 Hz) and H-Xyl-II-4 (δ 4.84, 1H, overlapped) were correlated with the carboxylic carbons of acetyl groups (δ 172.4, 171.1, respectively), suggesting that β-d-xylopyranosyl was a 3,4-diacetyl-β-d-xylopyranosyl moiety. The molecular formula C₆₁H₉₄O₂₈, which was established by HR-FAB-MS, supported the presence of echinocystic acid, the five sugars, and the two acetyl moieties, as mentioned above. From these data, compound 2 was identified as 3-O-β-d-glucuronopyranosyl- echinocystic acid 28-O-β-d-xylopyranosyl-(1→3)-[(3,4-di-O-acetyl)-β-d-xylopyranosyl-(1→4)]-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl ester.

The molecular formula of 3 was established as C₂₈H₃₄O₁₇ based on HR-FAB-MS [C₂₈H₃₄O₁₇Na, m/z 665.1670 (Calcd for C₂₈H₃₄O₁₇Na: 665.1692)]. In the ¹³C-NMR spectrum, two carboxylic carbons at δ 167.6 and 169.7 were observed. In the ¹H-NMR spectrum, singlet proton (δ 7.16, 2H, H-2′, 6′) and ABX system proton (δ 7.33, 1H, d, J=2.0 Hz, H-3 7.50, 1H, dd, J=8.5, 2.0 Hz, H-5; 6.97, 1H, d, J=8.5 Hz, H-6) resonances were observed in the aromatic field. A methoxy singlet proton resonance (δ 3.84, 6H) correlated with H-2′ and 6′, and another methoxy singlet resonance (δ 3.71, 3H) correlated with H-3 in the differential nuclear Overhauser effect (NOE) spectra. These data suggested that 3 had a vanillic acid and syringic acid moieties. Two anomeric proton resonances and 11 oxygenated carbon signals suggested the presence of two sugar units, which were similar to those of a 5-O-acylated apiofuranosyl-(1→2)-β-d-glucopyranosyl moiety.^{24,25) 1}H–¹H correlation spectroscopy (COSY) correlations showed the spin systems of the sugars (δ 5.05, 1H, d, J=7.5 Hz, H-Glc-1; 3.79, 1H, dd, J=9.0, 7.5 Hz, H-Glc-2; 3.65, 1H, dd, J=9.0, 8.5 Hz, H-Glc-3) and (δ 5.59, 1H, d, J=1.0 Hz, H-Api-1; 4.07, 1H, d, J=1.0 Hz, H-Api-2). In the HMBC spectrum, H-Glc-1 was long-range coupled with C-1 (δ 151.5), H-Api-1 was coupled with C-Glc-2 (δ 76.8), the carboxylic carbon of the syringic acid moiety at δ 167.6 (C-7′) was coupled with the H-Api-5 protons (δ 4.19, 1H, d, J=11.0 Hz; 4.36, 1H, d, J=11.0 Hz). The differential NOE correlation between H-Glc-1 and H-6 was demonstrated that 3 was a glycoside of vanillic acid. Although configurations of the sugar moieties were unknown, d-glucopyranosyl and d-apiofuransyl were expected, because only d-types are known for naturally occurring glucose and apiose.¹⁵⁾ The coupling constants of the anomeric protons were 7.5 Hz (glucopyranosyl) and 1.0 Hz (apiofuranosyl), suggesting that they are β-configurations.¹⁵⁾ As a result, 3 was identified as vanillic acid 1-O-[(5-O-syringoyl)-β-d-apiofuranosyl]-(1→2)-β-d-glucopyranoside, as shown in Fig. 1.

Compound 4 showed an ion peak in HR-EI-MS at m/z 178.0631 corresponding to a molecular formula of C₁₀H₁₀O₃. The ¹H- and ¹³C-NMR resonances of 4 were consistent with those of trans-4-hydroxy-5-phenyl-tetrahydrofuran-2-one.²⁶⁾ The NMR data of cis-4-hydroxy-5-phenyl-tetrahydrofuran-2-one were not inconsistent with those of 4,²⁷⁾ suggesting that 4 had a (4R,5S) or (4S,5R) configuration. The (4S,5R)-configuration of 4 was deduced from the optical rotation data ([α]_D²⁰ −51.1°), which was different from that of the synthesized (4R,5S)-isomer.²⁸⁾ The advanced Mosher method has been used to define the absolute configuration of natural products.²⁹⁾ The ¹H-NMR chemical shift difference of the (S)-2-methoxy-2-trifluoromethyl phenylacetic acid (MTPA) and (R)-MTPA ester of 4 supported the (4S,5R)-configuration (Fig. 1). The chemical structure of 4 was determined as (4S,5R)-4-hydroxy-5-phenyl-tetrahydrofuran-2-one. To the best of our knowledge, this represent the first time that this compound has been identified from a natural source.

As the first step in assessing the efficacy of plants for inflammatory and allergic diseases, some of isolated compounds had been evaluated for their hyaluronidase inhibitory activity, and only eritrichin (31) is known as an inhibitor (IC₅₀ 0.55 mm).²⁾ In this study, the inhibitory activity of compounds 1, 2, 4, 8–14, 17–21, 29, 30, 35 were also evaluated, and all of them at 1.0 mm were under 30%. A positive control was disodium cromoglicate (IC₅₀ 297 µm). Although the extract of P. flexuosum showed the highest hyaluronidase inhibitory activity among 49 Lamiaceae plant extracts, this was the only compound that showed activity. Eritrichin is a phenylpropanoid oligomer that is known as a potent hyaluronidase inhibitor.²⁾ It was isolated from the roots of the plant, and the obtained compounds from aqueous fractions of the aerial parts and the roots were different completely.

Experimental

General Procedures

Optical rotations were recorded on a JASCO P-2300 polarimeter. ¹H-NMR (400 MHz), ¹³C-NMR (100 MHz), ¹H–¹H COSY, HMQC (optimized for ¹J_C–H=145 Hz) and HMBC (optimized for ⁿJ_C–H=8 Hz) spectra were recorded on a JEOL JNM-AL400 FT-NMR spectrometer, and chemical shifts were given as δ values with TMS as an internal standard. HR-FAB- and HR-EI-MS data were obtained on a Jeol JMS700 mass spectrometer, using a m-nitrobenzyl alcohol or a glycerol matrix. A porous polymer gel (Mitsubishi Chemical, Diaion HP-20, 60×300 mm) and ODS (Cosmosil 140 C₁₈-OPN, Nacalai Tesque, 150 g) were used for column chromatography. Preparative HPLC was performed on a JASCO 2089 and detected with UV at 210 nm (columns, Capcell Pak Phenyl, Shiseido, 20×250 mm; Cosmosil AR-II, Nacalai tesque, 20×250 mm; Cosmosil 5PE-MS, Nacalai Tesque, 20×250 mm; YMC Pack ODS-AM, YMC Co., Ltd., 10×300 mm; Mightysil RP-18 GP, Kanto Chemical, 10×250 mm).

Plant Material

P. flexuosum was harvested in Medicinal Plant Garden of University of Shizuoka, Japan. The plant was identified by Prof. Akira Ueno, School of Pharmaceutical Sciences, University of Shizuoka. A voucher specimen has been deposited in the herbarium of Tohoku Pharmaceutical University, No. 20080815.

Extraction and Isolation

Powdered root parts of P. flexuosum (950 g) were extracted with acetone–water (8 : 2) at room temperature for two weeks (2×6 L). The extract was concentrated at reduced pressure (16.6 g), suspended in water (1.5 L) and subjected to extraction with diethyl ether (1.0 L) three times. The aqueous layer extract (8.0 g) was dissolved in water and passed through a porous polymer gel (Diaion HP-20, 70×180 mm) eluted with EtOH–water (95 : 5) after being washed with 5% EtOH. The 95% EtOH fraction (2.6 g) was chromatographed on a reversed-phase column using ODS and then eluted with 30%, 40%, 50%, 80% MeOH, and MeOH (frs. 1A–E). Fraction 1C (372.6 mg) was subjected to HPLC [Capcell pak Phenyl, mobile phase, methanol–0.2% trifluoroacetic acid (TFA) in water (40 : 60)→(70 : 30); ODS-AM, mobile phase, acetonitrile–0.2% TFA in water (30 : 70)], to yields compounds 6 (12.4 mg), 22 (4.7 mg), 31 (5.3 mg). Fraction 1D (388.8 mg) was subjected to HPLC [Capcell pak Phenyl, mobile phase, methanol–0.2%TFA in water (70 : 30); ODS-AM, mobile phase, acetonitrile–0.2% TFA in water (30 : 70); AR-II, mobile phases, acetonitrile–0.2% TFA in water (30 : 70) and (40 : 60)], to yields compounds 5 (3.5 mg), 7 (3.5 mg), 9 (28.5 mg).

Powdered aerial parts of P. flexuosum (2.8 kg) were extracted with acetone–water (8 : 2) at room temperature for two weeks (2×10 L). The extract was concentrated at reduced pressure (76.5 g), suspended in water (1.5 L) and subjected to extraction with diethyl ether (1.0 L) three times. The aqueous layer extract (27.4 g) was dissolved in water and passed through a porous polymer gel (Diaion HP-20, 70×180 mm) eluted with EtOH–water (95 : 5) after being washed with 5% EtOH. The 95% EtOH fraction (13.6 g) was chromatographed on a reversed-phase column using ODS and then eluted with 5%, 30%, 50%, 90% MeOH (frs. 2A–D). Fraction 2A (4.2 g) was subjected to Ultra Pack ODS-SM-50C-M, mobile phase, methanol–water (15 : 85)→(60 : 40) and HPLC [AR-II, mobile phases, acetonitrile–0.2% TFA in water (12.5 : 87.5) and (15 : 85); 5PE-MS, mobile phases, acetonitrile–0.2% TFA in water (12.5 : 87.5) and (15 : 85); Mightysil RP-18 GP, mobile phases, acetonitrile–0.2% TFA in water (10 : 90) and (12.5 : 87.5)], to yields compounds 3 (0.5 mg), 27 (12.3 mg), 28 (4.8 mg), 32 (3.9 mg), 33 (1.6 mg), 34 (6.9 mg), 36 (31.2 mg), 37 (4.3 mg), 38 (5.0 mg), 39 (4.3 mg), 40 (9.1 mg). Fraction 2B (4.0 g) was subjected to Ultra Pack ODS-SM-50C-M, mobile phase, methanol–water (15 : 85)→(50 : 50) and HPLC [AR-II, mobile phases, acetonitrile–0.2% TFA in water (15 : 85) and (20 : 80); 5PE-MS, mobile phase, acetonitrile–0.2% TFA in water (25 : 85); Capcell pak Phenyl, mobile phases, acetonitrile–0.2%TFA in water (15 : 85) and (20 : 80); Mightysil RP-18 GP, mobile phase, acetonitrile–0.2% TFA in water (20 : 80)], to yields compounds 4 (37.4 mg), 10 (37.2 mg), 23 (255.2 mg), 24 (196.8 mg), 30 (19.6 mg), 35 (5.5 mg). Fraction 2C (3.4 g) was subjected to Ultra Pack ODS-SM-50C-M, mobile phase, methanol–water (30 : 70)→(80 : 20) and HPLC [AR-II, mobile phases, acetonitrile–0.2% TFA in water 20 : 80) and (25 : 75); 5PE-MS, mobile phases, acetonitrile–0.2% TFA in water (20 : 80) and (25 : 75); Mightysil RP-18 GP, mobile phases, acetonitrile–0.2% TFA in water (25 : 75) and (30 : 70)], to yields compounds 1 (3.5 mg), 11 (102.5 mg), 12 (27.3 mg), 13 (4.7 mg), 14 (57.4 mg), 19 (3.3 mg), 20 (4.8 mg), 21 (10.5 mg), 25 (33.8 mg), 26 (2.7 mg), 29 (5.4 mg). Fraction 2D (0.89 g) was subjected to Ultra Pack ODS-SM-50C-M, mobile phase, methanol–water (50 : 50)→(80 : 20) and HPLC [AR-II, mobile phases, acetonitrile–0.2% TFA in water 35 : 65) and (40 : 60); Mightysil RP-18 GP, mobile phases, acetonitrile–0.2% TFA in water (30 : 70) and (32.5 : 67.5)], to yields compounds 3 (21.0 mg), 8 (1.6 mg), 15 (18.2 mg), 16 (3.2 mg), 17 (27.9 mg), 18 (4.8 mg).

3-[(3E)-4-Phenylbut-3-enoylamino]propionic Acid (1): Colorless amorphous solid, HR-EI-MS (positive): m/z 233.1055 [M]⁺ (Calcd for C₁₃H₁₅NO₃: 233.1053). UV (MeOH) λ_max (log ε) 203 (4.31), 237 (3.84, sh). ¹H-NMR: (CD₃OD, 400 MHz), δ: 7.44 (2H, d, J=7.5 Hz, H-2, 6), 7.35 (2H, dd, J=7.5, 7.5 Hz, H-3, 5), 7.27 (1H, t, J=7.5 Hz, H-4), 6.58 (1H, br d, J=16.5 Hz, H-7), 6.37 (1H, m, H-8), 3.13 (1H, d, J=7.0 Hz, H-9), 3.48 (2H, t, J=7.0 Hz, H-12), 2.56 (2H, t, J=7.0 Hz, H-13). ¹³C-NMR: (CD₃OD, 100 MHz), δ: 138.6 (C-1), 127.3 (C-2, 6), 129.6 (C-3, 5), 128.5 (C-4), 134.7 (C-7), 124.0 (C-8), 41.1 (C-9), 174.1 (C-10), 36.6 (C-12), 34.7 (C-13), 175.3 (C-14). Key HMBC correlations: H-2,6/C-4,7; H-4/C-2,3,5,6; H-7/C-2,6,9; H-8/C-1,7,9; H-9/C-7,8,10; H-12/C-10,13,14; H-13/C-12,14.

3-O-β-d-Glucuronopyranosyl-echinocystic Acid 28-O-β-d-Xylopyranosyl-(1→3)-[3,4-diacetyl-β-d-xylopyranosyl-(1→4)]-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl Ester (2): Colorless amorphous solid, [α]_D²⁰ −37.3° (c=2.01, MeOH), HR-FAB-MS (positive): m/z 1297.5879 [M+Na]⁺ (Calcd for C₆₁H₉₄O₂₈Na: 1297.5829). ¹H-NMR: (CD₃OD, 400 MHz), δ: 1.62 (overlapped, H-1), 3.27 (overlapped, H-3), 0.79 (overlapped, H-5), 1.65 (overlapped, H-6), 1.63 (overlapped, H-9), 1.90 (overlapped, H-11), 5.36 (1H, m, H-12), 1.40 (overlapped, H-15), 1.77 (overlapped, H-15), 4.49 (1H, m, H-16), 3.03 (1H, dd, J=14.0, 3.5 Hz, H-18), 1.04 (1H, m, H-19), 2.28 (1H, br t, J=13.5 Hz, H-19), 1.15 (overlapped, H-21), 1.92 (overlapped, H-21), 1.78 (overlapped, H-22), 1.92 (overlapped, H-22), 1.06 (3H, s, H-23), 0.86 (3H, s, H-24), 0.97 (3H, s, H-25), 0.79 (3H, s, H-26), 1.37 (3H, s, H-27), 0.88 (3H, s, H-29), 0.97 (3H, s, H-30), 4.39 (1H, d, J=7.5 Hz, H-Glc A-1), 3.25 (overlapped, H-Glc A-2), 3.38 (overlapped, H-Glc A-3), 3.50 (overlapped, H-Glc A-4), 3.78 (overlapped, H-Glc A-5), 5.65 (1H, d, J=3.5 Hz, H-Ara-1), 3.78 (overlapped, H-Ara-2), 3.88 (overlapped, H-Ara-3), 3.85 (1H, m, H-Ara-4), 3.49 (overlapped, H-Ara-5), 3.90 (overlapped, H-Ara-5), 5.01 (1H, d, J=1.5 Hz, H-Rha-1), 4.04 (1H, dd, J=3.0, 1.5 Hz, H-Rha-2), 3.89 (overlapped, H-Rha-3), 3.71 (overlapped, H-Rha-4), 3.73 (overlapped, H-Rha-5), 1.28 (3H, d, J=5.0 Hz, H-Rha-6), 4.53 (1H, J=7.0 Hz, H-Xyl-I-1), 3.25 (overlapped, H-Xyl-I-2), 3.28 (overlapped, H-Xyl-I-3), 3.49 (overlapped, H-Xyl-I-4), 3.21 (1H, m, H-Xyl-I-5), 3.83 (overlapped, H-Xyl-I-5), 4.88 (1H, d, J=7.5 Hz, H-Xyl-II-1), 3.35 (overlapped, H-Xyl-II-2), 5.04 (1H, t, J=9.5 Hz, H-Xyl-II-3), 4.84 (overlapped, H-Xyl-II-4), 3.33 (overlapped, H-Xyl-II-5), 4.00 (1H, dd, J=11.5, 6.0 Hz, H-Xyl-II-5), 2.05 (3H, s, H-Ac-Xyl-II-3), 1.99 (3H, s, H-Xyl-II-4). ¹³C-NMR: (CD₃OD, 100 MHz), δ: 39.9 (C-1), 27.1 (C-2), 91.2 (C-3), 40.9 (C-4), 57.2 (C-5), 19.5 (C-6), 34.4 (C-7), 40.3 (C-8), 48.2 (C-9), 37.9 (C-10), 24.5 (C-11), 123.8 (C-12), 144.7 (C-13), 42.8 (C-14), 36.5 (C-15), 74.7 (C-16), 50.4 (C-17), 42.2 (C-18), 47.8 (C-19), 31.4 (C-20), 36.5 (C-21), 31.9 (C-22), 28.6 (C-23), 17.1 (C-24), 16.2 (C-25), 18.1 (C-26), 27.3 (C-27), 177.1 (C-28), 33.4 (C-29), 25.2 (C-30), 107.0 (C-Glc A-1), 75.4 (C-Glc A-2), 77.7 (C-Glc A-3), 73.2 (C-Glc A-4), 76.5 (C-Glc A-5), 172.6 (C-Glc A-6), 94.1 (C-Ara-1), 75.8 (C-Ara-2), 71.0 (C-Ara-3), 67.0 (C-Ara-4), 63.7 (C-Ara-5), 101.2 (C-Rha-1), 73.2 (C-Rha-2), 82.2 (C-Rha-3), 79.0 (C-Rha-4), 69.0 (C-Rha-5), 18.4 (C-Rha-6), 106.1 (C-Xyl-I-1), 75.4 (C-Xyl-I-2), 78.2 (C-Xyl-I-3), 71.0 (C-Xyl-I-4), 67.1 (C-Xyl-I-5), 104.6 (C-Xyl-II-1), 73.8 (C-Xyl-II-2), 76.4 (C-Xyl-II-3), 71.2 (C-Xyl-II-4), 63.6 (C-Xyl-II-5), 172.4 (C-Ac-Xyl-II-3), 21.0 (C-Ac-Xyl-II-3), 171.1 (C-Ac-Xyl-II-4), 20.6 (C-Ac-Xyl-II-4). Key HMBC correlations: H-16/C-14,17; H-18/C-12,13,14,17,28; H-23/C-3,4,5,24; H-24/C-3,4,5,23; H-25/C-1,9,10; H-26/C7,8,9,14; H-27/C-8,13,14,15; H-29/C-19,20,21,30; H-30/C-19,20,21,29; H-Glc A-1/C-3; H-Ara-1/C-28, Ara-5; H-Ara-4/C-Ara-2, Ara-5; H-Rha-1/C-Ara-2, Rha-3, Rha-5; H-Rha-6/C-Rha-4; H-Xyl-I-1/C-Rha-3; H-Xyl-II-1/C-Rha-4; H-Xyl-II-3/C-Ac-Xyl-II-3; H-Xyl-II-4/C-Ac-Xyl-II-4.

Vanillic Acid 1-O-[(5-O-Syringoyl)-β-d-apiofuranosyl]-(1→2)-β-d-glucopyranoside (3): Colorless amorphous solid, [α]_D²⁰ −15.7° (c=1.07, MeOH), HR-FAB-MS (positive): m/z 665.1670 [M+Na]⁺ (Calcd for C₂₈H₃₄O₁₇: 665.1692). UV (MeOH) λ_max (log ε) 207 (4.71), 252 (4.10), 282 (4.16). ¹H-NMR: (CD₃OD, 400 MHz), δ: 7.33 (1H, d, J=2.0 Hz, H-3), 7.50 (1H, dd, J=8.5, 2.0 Hz, H-5), 6.97 (1H, d, J=8.5 Hz, H-6), 3.71 (H-OMe of C-2), 7.16 (2H, s, H-2′, 6′), 3.84 (6H, s, H-OMe of C-3′,5′), 5.05 (1H, d, J=7.5 Hz, H-Glc-1), 3.79 (1H, dd, J=9.0, 7.5 Hz, H-Glc-2), 3.65 (1H, dd, J=9.0, 8.5 Hz, H-3), 3.40 (1H, overlapped, H-4), 3.40 (1H, overlapped, H-5), 3.66 (1H, overlapped, H-Glc-6), 3.88 (1H, overlapped, H-Glc-6), 5.59 (1H, d, J=1.0 Hz H-Api-1), 4.07 (1H, d, J=1.0 Hz, H-Api-2), 3.85 (1H, d, J=10.0 Hz, H-Api-4), 4.47 (1H, d, J=10.0 Hz, H-Api-4), 4.19 (1H, d, J=11.0 Hz, H-Api-5), 4.36 (1H, d, J=11.0 Hz, H-Api-5). ¹³C-NMR: (CD₃OD, 100 MHz), δ: 151.5 (C-1), 150.0 (C-2), 114.0 (C-3), 125.0 (C-4), 124.5 (C-5), 115.4 (C-6), 169.7 (C-7), 56.2 (C-OMe of C-2), 121.0 (C-1′), 108.6 (C-2′,6′), 148.8 (C-3′,5′), 142.2 (C-4′), 167.6 (C-7′), 57.0 (C-OMe of C-3′,5′), 99.7 (C-Glc-1), 76.8 (C-Glc-2), 79.2 (C-Glc-3), 71.6 (C-Glc-4), 78.3 (C-Glc-5), 62.5 (C-Glc-6), 110.0 (C-Api-1), 78.8 (C-Api-2), 79.3 (C-Api-3), 75.4 (C-Api-4), 67.9 (C-Api-5). Key HMBC correlations: H-3/C-1,7; H-5/C-1,7; H-6/C-2; H-OMe of C-2/C-2; H-Glc-1/C-1; H-Glc-2/C-Api-1; H-Api-1/C-Api-3, Api-4; H-Api-5/C-7′; H-2′/C-1′,3′,4′,7′; H-6′/C-1′,4′,5′,7′; H-OMe of C-3′/C-3′; H-OMe of C-5′/C-5′. Key differential NOE correlations: H-3/H-OMe of C-2; H-6/H-Glc-1; H-Api-1/H-Api-2, Api-4; H-2′/H-OMe of C-2′; H-5′/H-OMe of C-5′.

(4S,5R)-4-Hydroxy-5-phenyl-tetrahydrofuran-2-one (4): Colorless amorphous solid, [α]_D²⁰ −51.1° (c=0.19, MeOH), HR-EI-MS (positive): m/z 178.0631 [M]⁺ (Calcd for C₁₀H₁₀O₃: 178.0631). UV (MeOH) λ_max (log ε) 207 (4.26), 257 (2.91). ¹H-NMR: (CDCl₃, 400 MHz), δ: 2.61 (1H, dd, J=18.0, 5.0 Hz, H-3), 2.87 (1H, dd, J=18.0, 6.5 Hz, H-3), 4.89 (1H, m, J=H-4), 5.37 (1H, d, J=3.5 Hz, H-5), 7.25–7.43 (5H, overlapped, H-7,8,9,10,11). ¹³C-NMR: (CDCl₃, 100 MHz), δ: 174.7 (C-2), 37.0 (C-3), 74.5 (C-4), 87.7 (C-5), 136.8 (C-6), 125.1, 128.7, 128.9 (C-7,8,9,10,11). Key HMBC correlations: H-3/C-2,4; H-5/C-2,3,4,6. A key differential NOE correlation: H-4/H-7,11.

(S)- and (R)-MTPA Esterifications of Compound 4

To a solution of 4 (3.0 mg each) in pyridine (100 µL) was added (R)-(−)-MTPA chloride or (S)-(+)-MTPA chloride (5 µL), and the mixture was stirred for 1 h at 0°C. The solvent was subjected to preparative HPLC (column, Mightysil RP-18 GP, 10×250 mm) to give the (S)-(−)-MTPA ester of 4 (4a, 2.7 mg) and the (R)-(+)-MTPA ester of 4 (4b, 2.5 mg). 4a: ¹H-NMR (in CDCl₃, 400 MHz, 30°C), δ: 2.62 (1H, br dd, J=18.0, 4.0 Hz, H-3), 2.83 (1H, dd, J=18.0, 6.5 Hz, H-3), 4.49 (1H, ddd, J=6.5, 4.0, 3.5 Hz H-4), 5.46 (1H, d, J=3.5 Hz, H-5), 7.25–7.45 (4H, m), 7.61 (1H, m). 4b: ¹H-NMR (in CDCl₃, 400 MHz, 30°C), δ: 2.62 (1H, dd, J=18.0, 3.5 Hz, H-3), 2.86 (1H, dd, J=18.0, 6.5 Hz, H-3), 4.49 (1H, ddd, J=6.5, 3.5, 3.5 Hz H-4), 5.41 (1H, d, J=3.5 Hz, H-5), 7.25–7.45 (4H, m), 7.61 (1H, m).

Acid Hydrolysis and Sugar Identification

Compound 2 (4.3 mg) was hydrolyzed using 7% HCl (1 mL) at 60°C for 2 h. The reaction mixture was neutralized using an Amberlite IRA400 column, and the eluate was concentrated. The residues were stirred with l-cysteine methyl ester (10 mg) and o-tolyl isothiocyanate (20 µL) in pyridine (0.5 mL) by using the procedure reported by Tanaka et al.²¹⁾ The reaction mixture was analyzed by HPLC (column, Cosmosil 5C₁₈–AR II column, 4.6×250 mm; mobile phase, CH₃CN–0.2% TFA in H₂O (25 : 75), 1.0 mL/min; detector, UV at 210 nm) at 20°C. d-Glucuronic acid derivative (t_R 16.2 min), d-xylose derivative (t_R 19.1 min), l-rhamnose derivative (t_R 27.7 min), and l-arabinose derivative (t_R 18.3 min) were used for identification of the sugar moieties of 2 based on comparisons with derivatives of authentic samples [d-glucuronic acid derivative (t_R 16.2 min), l-glucuronic acid derivative (using d-cystein methyl ester and d-glucuronic acid, t_R 15.1 min), d-xylose derivative (t_R 19.1 min), l-xylose derivative (t_R 17.7 min), l-rhamnose derivative (t_R 27.7 min), d-rhamnose derivative (using d-cystein methyl ester and l-rhamnose, t_R 15.4 min), l-arabinose derivative (t_R 18.3 min), and d-arabinose derivative (using d-cystein methyl ester and l-arabinose, t_R 20.0 min)].

Assay of Hyaluronidase Inhibition

Hyaluronidase activity was measured as described previously.³⁰⁾ Each compound (final concentration: 1, 0.3, 0.1, 0.03 mm) was dissolved in 0.1 m acetate buffer as the sample solution. Each acetone extract of Lamiaceae plant (final concentration: 2, 0.6, 0.2, 0.06 mg/mL) was dissolved in 0.1 m acetate buffer as the sample solution. Disodium cromoglicate was used as a positive control (IC₅₀ 297 µm). The final concentration of hyaluronidase was 400 unit/mL.

Acknowledgment

We thank Mr. S. Sato and Mr. T. Matsuki of Tohoku Pharmaceutical University for assisting with the MS measurements.

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