Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs

Abstract

Two iridoid glycosides (1 and 2), 3 phenolic glycosides (3–5), 1 flavone glycoside (6), 3 biflavonoids (7–9), 1 flavone (10), 2 triterpenes (11 and 12), 1 sterol (13), and 1 naphthoquinone derivative (14) were isolated from the whole plant of Verbena hastata (Verbenaceae). Compounds 3-13 were isolated from V. hastata for the first time. Compound 14 is undescribed in the literature. Incubation of glyceraldehyde and collagen either with phenolic glycosides (3), (4), or (5) or with biflavonoid (8) inhibited the production of advanced glycation end products, with IC₅₀ values of 6.3, 6.4, 6.2, and 6.8 mM, respectively. Aminoguanidine, which was used as a positive control, had an IC₅₀ value of 10.2 mM.

The genus Verbena belongs to Verbenaceae, and is mainly found in the subtropical regions of North America and South America and partially distributed in Europe and Asia. ¹ Although most of the Verbena species have been cultivated for horticulture, a few Verbena species have been used for medicinal purposes. For instance, Verbena officinalis L., also known as ‘Vervain,’ is used as a diuretic, an astringent for poorly healing wounds, a galactagogue, an expectorant, and an antirheumatic. ² Iridoids, triterpenes, and flavonoids are the characteristic components isolated from V. officinalis. ³ Verbena hastata L., the species of interest in this study, is native to North America. V. hastata, also known as ‘Blue Vervain,’ is used as a folk medicine for the treatment of headaches, externally wounds, ulcers, and acne. Although a few iridoid glucosides such as verbenalin and hastatoside were isolated from V. hastata, ^4,5 there has been no systematic study of its chemical components. As a part of our ongoing study on traditional medicinal plants, a phytochemical examination was carried out on the whole plant of V. hastata. This study involves the structural identification of the isolated compounds and their inhibitory activity against the production of advanced glycation end products (AGEs).

Results and Discussion

The whole plant of V. hastata was extracted with MeOH. After removing the solvent, the concentrated MeOH extract was passed through a column packed with porous polymer polystyrene resin (Diaion HP-20). The samples were eluted with 30% MeOH, 50% MeOH, MeOH, EtOH, and EtOAc. The eluates with 50% MeOH and MeOH were subjected to silica gel (Si) column chromatography (CC) and octadecylsilanized (ODS) Si CC to yield compounds 1-6 and 7-14, respectively.

Based on the physicochemical and spectroscopic data, compounds 1-13 were identified as verbenalin (1), ⁶ hastatoside (2), ⁷ verbascoside (3), ⁸ leucosceptoside A (4), ⁹ martynoside (5), ¹⁰ apigenin 7-O-β-D-glucuronide methyl ether (6), ¹¹ lophirone B (7), ¹² lophirone C (8), ¹³ luxenchalcone (9), ¹⁴ luteorin (10), ¹⁵ oleanolic acid (11), ¹⁶ ursolic acid (12), ¹⁷ and (22R)-stigmast-5-ene-3β,4β,7α,22-tetrol (13) ¹⁸ (Figures 1 and 2). Among these, compounds 3-13 were isolated from V. hastata for the first time.

Figure 1

Structures of 1-6.

Figure 2

Structures of 7-14.

Compound 14 was isolated as a red amorphous powder ([α]_D ²⁵ –21.4 in MeOH). Its molecular formula was determined to be C₁₅H₁₄O₅ from the combined high resolution mass spectrometry-electrospray ionization time-of-flight data (HRMS-ESITOF) and ¹³C NMR (nuclear magnetic resonance) data. UV spectrum of 14 showed the absorption maxima at 407.4, 285.5, and 245.6 nm, indicating a highly conjugated system. The IR spectrum suggested the presence of hydroxy groups at 3501 cm^-1 and carbonyl groups at 1643 cm^-1. Analysis of the ¹H and ¹³C NMR spectra, along with the distortionless enhancement by polarization transfer (DEPT) and heteronuclear single quantum coherence (HSQC) spectra, indicated the presence of 2 carbonyl carbons [δ_C 184.8 (C-5) and 186.4 (C-10)], 5 sp ² quaternary carbons [δ_C 122.3 (C-4a), 184.8 (C-5), 133.6 (C-5a), 162.7 (C-9), 115.6 (C-9a), 186.4 (C-10), and 155.2 (C-10a)], and 3 sp ² methine groups [δ_H 7.58 (1H, dd, J = 7.5, 1.1 Hz, H-6)/δ_C 119.6 (C-6), δ_H 7.65 (1H, dd, J = 8.4, 7.5 Hz, H-7)/δ_C 138.1 (C-7), and δ_H 7.20 (1H, dd, J = 8.4, 1.1 Hz, H-8)/δ_C 124.4 (C-8)], which were characteristic of the 1,4-naphthoquinone structure (please see online supplemental Figures S1–S9). In addition, signals corresponding to 1 sp ³ oxygen-bearing quaternary carbon [δ_C 80.5 (C-2)], 1 sp ³ oxygen-bearing methine group [δ_H 4.90 (1H, dd, J = 5.3, 4.3 Hz, H-4)/δ_C 59.6 (C-4)], 1 methylene group [δ_H 2.07 (1H, dd, J = 14.6, 4.3 Hz, H-3a) and 2.03 (1H, dd, J = 14.6, 5.3 Hz, H-3b)/δ_C 41.0 (C-3)], and 2 methyl groups [δ_H 1.51 (3H, s, H₃-11)/δ_C 26.5 (C-11), 1.47 (3H, s, H₃-12)/δ_C 24.4 (C-12)] were observed. In the ¹H-¹H correlation spectroscopy (COSY) spectrum, the oxygen-bearing methine proton at δ_H 4.90 (H-4) showed spin-coupling correlations with a pair of methylene protons at δ_H 2.07 and 2.03 (H₂-3), and the aromatic proton at δ_H 7.65 (H-7) exhibited correlation peaks with both δ_H 7.58 (H-6) and 7.20 (H-8) aromatic protons. These signals were consistent with the presence of a 1,2,3-trisubstituted aromatic ring. In the heteronuclear multiple bond correlation (HMBC) spectrum, long-range correlations from the aromatic protons at δ_H 7.58 (H-6) to δ_C 184.8 (C-5)/124.4 (C-8)/115.6 (C-9a), δ_H 7.20 (H-8) to δ_C 119.6 (C-6)/162.7 (C-9)/C-9a, and δ_H 7.65 (H-7) to δ_C 133.6 (C-5a)/C-9 were observed. Long-range correlations were also observed from the oxygen-bearing methine proton at δ_H 4.90 (H-4) to δ_C 80.5 (C-2)/41.0 (C-3)/122.3 (C-4a)/184.8 (C-5)/155.2 (C-10a), and from a pair of geminal dimethyl protons at δ_H 2.07 (H₃-11) and 2.03 (H₃-12) to δ_C 80.5 (C-2)/41.0 (C-3). These correlations indicated that 14 was 4,9-dihydroxy-α-lapachone (Figure 3). The absolute configuration of the C-4 position was determined to be R. This was because the specific optical rotation of 14 is –21.4 in MeOH while those of (4S)−4,9-dihydroxy-α-lapachone isolated from Catalpa ovata (Bignoniaceae) and (4S)−4-hydroxy-9-methoxy-α-lapachone isolated from Mansoa alliacea (Bignoniaceae) are +17.9 and +26.8, respectively, in MeOH. ^19,20 Thus, 14 was identified to be (4R)−4,9-dihydroxy-α-lapachone.

Figure 3

Key HMBC correlations of 14.

AGEs are either produced by the nonenzymatic glycation and oxidation of proteins and reducing sugars or as sugar metabolism intermediates. The formation of AGEs in the skin, blood vessel walls, and bones is closely related to their aging. ²¹ Moreover, the accumulation of AGEs has implications in the pathology of diabetes, chronic inflammation, Alzheimer’s disease, and cancer. ²² Therefore, the inhibitory activities of the isolated compounds (1, 6, 8, and 11-13) on the production of AGEs were evaluated. Compounds 3, 4, 5, and 8 were found to inhibit the production of AGEs, with IC₅₀ values of 6.3, 6.4, 6.2, and 6.8 mM, respectively. Aminoguanidine, which was used as a positive control, exhibited an IC₅₀ value of 10.2 mM. Compounds 1, 2, 6, and 11-13 did not show any inhibitory activity at a concentration of 20 mM.

In summary, 14 compounds were isolated from the whole plant of V. hastata. Compounds 3-13 were isolated from V. hastata for the first time. Compound 14 is undescribed in the literature. The isolated compounds were evaluated for their inhibitory activity against the production of AGEs. Phenolic glycosides 3-5 and a biflavonoid 8 could inhibit the production of AGEs. These phenolic compounds may be effective in preventing the aging of the blood vessel walls and bones.

Experimental

General Experimental Procedures

Optical rotations were measured using a JASCO P-1030 (Tokyo, Japan) automatic digital polarimeter. IR spectra were recorded on a JASCO FT-IR 620 spectrophotometer. UV spectra were recorded on a JASCO V-630 spectrophotometer. NMR spectra were recorded on a Bruker DRX-500 (500 MHz for ¹H NMR, Karlsruhe, Germany) spectrometer using standard Bruker pulse programs. Chemical shifts are given as δ values in reference to tetramethylsilane (TMS) as an internal standard. MS-ESITOF data were recorded on a Waters-Micromass LCT mass spectrometer (Manchester, UK). Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan), silica gel (Fuji Silysia Chemical, Aichi, Japan), and ODS silica gel (Nacalai Tesque, Kyoto, Japan) were used for column chromatography. TLC was carried out on silica gel 60 F₂₅₄ (thickness: 0.25 mm, Merck, Darmstadt, Germany) and RP₁₈ F_254S (thickness: 0.25 mm, Merck) plates, and spots were visualized by spraying the plates with 10% H₂SO₄ aqueous solution, followed by heating. HPLC was performed using a system composed of a DP-8020 pump (Tosoh, Tokyo, Japan), a Shodex OR-2 (Showa-Denko, Tokyo, Japan) detector, and a Rheodyne injection port. All other chemicals used were of biochemical reagent grade.

Plant Material

Verbena hastata L. was purchased from a wholesale firm in Lichters (Ontario Canada) in November 2013. A voucher specimen has been deposited in our laboratory (voucher no. KS-2013‐004, Department of Medicinal Pharmacognosy).

Extraction and Isolation

The whole plants of V. hastata (dry weight, 2.0 kg) were extracted with MeOH. The MeOH extract was concentrated under reduced pressure, and the viscous concentrate (235 g) was passed through a porous-polymer polystyrene resin (Diaion HP-20), successively eluted with 30% MeOH, 50% MeOH, MeOH, EtOH, and EtOAc. The 50%MeOH-eluate portion (25 g) was subjected to Si CC and eluted with stepwise gradient mixtures of EtOAc-MeOH-H₂O (190:10:1; 90:10:1; 40:10:1; 20:10:1), and finally with MeOH alone, to give 12 fractions (A–L). Fraction C was purified by ODS Si CC eluted with MeOH-H₂O (1:1; 6:4; 7:3) to give 6 (12.6 mg). Fraction D was purified by ODS Si CC eluted with MeCN-H₂O (1:4) and MeOH-H₂O (1:2; 2:3) to give 5 (17.7 mg). Fraction E was separated by ODS Si CC eluted with MeCN-H₂O (1:4) to give subfractions E-1–E-8. Fraction E-2 was purified by Si CC eluted with EtOAc-MeOH-H₂O (90:10:1) and by ODS Si CC eluted with MeOH-H₂O (2:5) to give 1 (79.8 mg) and 3 (28.0 mg). Fraction E-3 was purified by preparative HPLC using MeOH-H₂O (1:1) as the mobile phase to give 4 (16.7 mg). Fraction H was purified by Si CC eluted with EtOAc-MeOH-H₂O (70:10:1; 40:10:1; 20:10:1) and by ODS Si CC eluted with MeOH-H₂O (2:5) and MeCN-H₂O (1:4) to give 2 (13.9 mg). The MeOH-eluate portion (27 g) was subjected to Si CC and eluted with stepwise gradient mixtures of hexane-acetone (4:1; 3:1; 2:1; 1:1) to produce 10 fractions (a–j). Fraction c was purified by Si CC eluted with hexane-EtOAc (3:1; 2:1) and by ODS Si CC eluted with MeOH-H₂O (6:4; 7:3; 8:2) to give 14 (11.9 mg). Fraction f was purified by Si CC eluted with hexane-EtOAc (3:1) and by ODS Si CC eluted with MeOH-H₂O (9:1) to give 11 (163.8 mg). Fraction d was purified by Si CC eluted with hexane-EtOAc (3:1) and by ODS Si CC eluted with MeCN-H₂O (1:1) to give 12 (16.2 mg). Fraction g was separated by Si CC eluted with hexane-EtOAc (1:1) to give subfractions g-1–g-8. Fraction g-4 was purified by Si CC eluted with hexane-Me₂CO (6:4) and CHCl₃-MeOH-H₂O (190:10:1) and by ODS Si CC eluted with MeCN-H₂O (1:1) to give 7 (2.7 mg), 8 (52.1 mg), and 9 (1.8 mg). Fraction g-6 was purified by Si CC eluted with hexane-Me₂CO (2:1; 1:1) and by ODS Si CC eluted with MeOH-H₂O (1:1) to give 10 (5.1 mg). Fraction g-8 was purified by Si CC eluted with hexane-EtOAc (1:2) and by ODS Si CC eluted with MeOH-H₂O (6:4; 7:3; 8:2) to give 13 (19.6 mg).

(4R)-4,9-Dihydroxy-α-lapachone (14)

Red amorphous powder. $[\alpha {]}_{\mathrm{D}}^{25}$ : −21.4 (c 0.10, MeOH). UV (MeOH) λ_max: 407.4 (log ε 3.63), 285.5 (log ε 4.11), 245.6 (log ε 4.16). IR ν_max (film): 3501, 2955, 2925, 1697, 1643, 1606, 1577 cm^-1. HRMS-ESITOF: m/z 297.0702 [M + Na]⁺, calcd for C₁₅H₁₄NaO₅: 297.0739. ¹H NMR (500 MHz, CD₃OD): δ_H 7.65 (1H, dd, J = 8.4, 7.5 Hz, H-7), 7.58 (1H, dd, J = 7.5, 1.1 Hz, H-6), 7.20 (1H, dd, J = 8.4, 1.1 Hz, H-8), 4.90 (1H, dd, J = 5.3, 4.3 Hz, H-4), 2.07 (1H, dd, J = 14.6, 4.3 Hz, H-3a), 2.03 (1H, dd, J = 14.6, 5.3 Hz, H-3b), 1.51 (3H, s, H-11), 1.47 (3H, s, H-12). ¹³C NMR (125 MHz, CD₃OD): δ_C 80.5 (C-2), 41.0 (C-3), 59.6 (C-4), 122.3 (C-4a), 184.8 (C-5), 133.6 (C-5a), 119.6 (C-6), 138.1 (C-7), 124.4 (C-8), 162.7 (C-9), 115.6 (C-9a), 186.4 (C-10), 155.2 (C-10a), 26.5 (C-11), 24.4 (C-12).

Assay for Determining the Inhibitory Activity Against the Production of AGEs

The inhibitory activity of the compounds against the production of AGEs upon the incubation of glyceraldehyde and collagen was measured using a commercially available kit (Collagen Glycation Assay Kit, Glyceraldehyde, CosmoBio, Tokyo, Japan). Briefly, collagen solution was stored in ice, and 50 µL of this solution was placed in each well of a 96-well black plate (Greiner Bio-one, Frickenhausen, Germany). The solution was incubated for 24 hours at 37 °C under the high humidity conditions. After incubation, 40 µL of sample dilution buffer containing the sample or positive control was added to obtain a final concentration of 2‐20 mM. Then, 10 µL of 500 mM glyceraldehyde solution was added to each well. The plate was mixed on a microplate shaker and read on Varioskan flash fluorescence microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at Ex/Em  =  360/475  nm (Fluorescent intensity A). The solution was further incubated for 24 hours at 37 °C. After termination of the reaction, the fluorescence intensity was measured using a fluorescence microplate reader at Ex/Em  =  360/475  nm (Fluorescent intensity B). Inhibitory ratio of the samples was calculated using the following formula:

$$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{a}\mathrm{g}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{s}\mathrm{t}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{A}\mathrm{G}\mathrm{E}\mathrm{s}\left(\mathrm{\%}\right)=\{1-[(\mathrm{F}\mathrm{l}\mathrm{u}\mathrm{o}\mathrm{r}\mathrm{s}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{B}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}-\mathrm{F}\mathrm{l}\mathrm{u}\mathrm{o}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{A}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e})\left]∕\right[\left(\mathrm{F}\mathrm{l}\mathrm{u}\mathrm{o}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{B}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)-\left(\mathrm{F}\mathrm{l}\mathrm{u}\mathrm{o}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{A}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)\left]\right\}\times 100$$

Data of the inhibition against the production of AGEs were present as the mean ± SE. The concentration resulting in 50% inhibition (IC₅₀) was calculated from a dose-response curve.

Statistical Analysis

The results are present as the mean ± SE. The significance of the differences was examined by one-way ANOVA followed by Dunnett’s test. Differences of P < 0.05 were considered significant.

Supplemental Material

Online supplementary file 1 - Supplemental material for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs

Supplemental material, Online supplementary file 1, for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs by Akihito Yokosuka, Misaki Honda, Hitoshi Kondo and Yoshihiro Mimaki in Natural Product Communications

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Online supplementary file 2 - Supplemental material for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs

Supplemental material, Online supplementary file 2, for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs by Akihito Yokosuka, Misaki Honda, Hitoshi Kondo and Yoshihiro Mimaki in Natural Product Communications

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Online supplementary file 3 - Supplemental material for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs

Supplemental material, Online supplementary file 3, for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs by Akihito Yokosuka, Misaki Honda, Hitoshi Kondo and Yoshihiro Mimaki in Natural Product Communications

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Online supplementary file 4 - Supplemental material for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs

Supplemental material, Online supplementary file 4, for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs by Akihito Yokosuka, Misaki Honda, Hitoshi Kondo and Yoshihiro Mimaki in Natural Product Communications

Supplemental Material

Online supplementary file 5 - Supplemental material for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs

Supplemental material, Online supplementary file 5, for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs by Akihito Yokosuka, Misaki Honda, Hitoshi Kondo and Yoshihiro Mimaki in Natural Product Communications

Supplemental Material

Online supplementary file 6 - Supplemental material for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs

Supplemental material, Online supplementary file 6, for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs by Akihito Yokosuka, Misaki Honda, Hitoshi Kondo and Yoshihiro Mimaki in Natural Product Communications

Supplemental Material

Online supplementary file 7 - Supplemental material for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs

Supplemental material, Online supplementary file 7, for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs by Akihito Yokosuka, Misaki Honda, Hitoshi Kondo and Yoshihiro Mimaki in Natural Product Communications

Supplemental Material

Online supplementary file 8 - Supplemental material for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs

Supplemental material, Online supplementary file 8, for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs by Akihito Yokosuka, Misaki Honda, Hitoshi Kondo and Yoshihiro Mimaki in Natural Product Communications

Supplemental Material

Online supplementary file 9 - Supplemental material for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs

Supplemental material, Online supplementary file 9, for Chemical Constituents of the Whole Plant of Verbena hastata and Their Inhibitory Activity Against the Production of AGEs by Akihito Yokosuka, Misaki Honda, Hitoshi Kondo and Yoshihiro Mimaki in Natural Product Communications