Three Minor Flavone Glycosides from Aeschynanthus Species and Cultivars (Gesneriaceae)

Abstract

Three minor flavonoids were isolated from the corollas and leaves of Aeschynanthus species and cultivars using MeOH, and identified as scutellarein 7-O-(6″-methylglucuronopyranoside) (1), 6-hydroxyluteolin 7-O-(6″-methylglucuronopyranoside) (2) and nepetin 4′-O-glucuronopyranoside (3) by UV and ¹H NMR spectroscopy, LC–MS, and acid hydrolysis. These flavone glycosides are reported in nature for the first time.

Introduction

The genus Aeschynanthus consists of ca. 150 species and is mainly native to subtropical and tropical zones in China and Malesia. ¹ Some cultivars are bred for ornamentals. Thirteen anthocyanins including six unreported compounds has recently been isolated from the flowers of two Aechynanthus species, A. fulgens and A. pulcher, and six cultivars. ² Moreover, 46 flavonoids including flavones, flavonol, chalcone, dihydroflavonol, and flavanones were isolated and identified from the flowers and leaves of Aeschynanthus species and cultivars. ³ We have isolated three minor flavonoids in that time. However, they were not characterized for small amount of the compounds. In this study, we identified their flavonoids through UV spectroscopy, HR–MS and/or LC–MS, acid hydrolysis, and ¹H NMR spectroscopy.

Results and Discussion

Flavonoid 1 was obtained as pale yellow powder (0.7 mg) from the mixtures of the corolla of Aeschynanthus fulgens and A. pulcher, and six cultivars, “Mahligai,” “Bravera,” “Mona Lisa,” “Redona,” “SoeKa,” and “Fresya.” Glucuronic acid and an aglycone were liberated by acid hydrolysis of 1. The aglycone was identified as scutellarein by HPLC comparison with an authentic sample. Since the molecular ion peaks at m/z 477 [M+H]⁺, 475 [M-H]⁻ appeared on LC–MS, the attachment of 1 mol methyl-glucuronic acid moiety to scutellarein was shown. The ¹H NMR spectrum of 1 showed six aromatic proton signals at δ_H 7.99 (d, J = 9.0 Hz), 6.97 (d, J = 9.0 Hz), 6.95 (s) and 6.84 (s) corresponding to H-2′,6′, H-3′,5′, H-8 and H-3, respectively. Anomeric proton signal of methyl-glucuronic acid was observed at δ_H 4.80 (d, J = 9.0 Hz) along with methylglucuronyl CH₃ proton signals at 3.95 (s), showing that methyl-glucuronic acid is β-d-form. ^{4 13}C NMR could not be measured for small amount of the sample. The attachment of methyl-glucuronic acid to 7-position of scutellarein was confirmed by no bathochromic shift of Band II (287 nm) while NaOAc was added to MeOH solution by UV spectral survey. ⁵ Since methyl group was released by acid hydrolysis, the attachment of methyl group to COOH of glucuronic acid was confirmed. Thus, 1 was identified as scutellarein 7-O-β-d-(6″-methylglucuronopyranoside) (Figure 1). The compound was reported in nature for the first time.^6-8

Figure 1.

Chemical structures of flavonoids from Aeschynanthus species and cultivars.

Flavonoid 2 was obtained as pale yellow powder (0.6 mg) from the leaves of Aeschynanthus cultivars “Mahligai” and “Mona Lisa.” HR–MS of 2 showed a molecular ion peak at m/z 491.0826 [M-H]⁻ calcd. for C₂₂H₁₉O₁₃, Found 491.0913. Molecular ion peaks at m/z 493 [M+H]⁺, 491 [M-H]⁻ appeared on LC–MS, along with a fragment ion peak at m/z 303 [M-196+H]⁺, showing the attachment of 1 mol methyl-glucuronic acid moiety to pentahydroxyflavone. Glucuronic acid and an aglycone which was characterized as 6-hydroxyluteolin by HPLC comparison with authentic sample were produced by acid hydrolysis of 2. Since methyl group was released by acid hydrolysis, the attachment of methyl group to COOH of glucuronic acid was confirmed. The ¹H NMR spectrum of 2 showed five aromatic proton signals at δ_H 7.47 (d, J = 2.4 Hz), 7.42 (brd, J = 8.4 Hz), 6.98 (s), 6.87 (d, J = 7.8 Hz) and 6.87 (s) corresponding to H-2′, H-6′, H-8, H-5′ and H-3, respectively. Anomeric proton signal of methyl-glucuronic acid was observed at δ_H 5.32 (d, J = 7.8 Hz), along with methyl proton signal at δ_H 3.71 (s), showing that methyl-glucuronic acid is β-d-form. ^{4 13}C NMR could not be measured for small amount of the sample. The attachment of methyl-glucuronic acid to 7-position of 6-hydroxyluteolin was determined by UV spectral survey according to Mabry et al, ⁵ that is, no bathochromic shift of Band II (283 nm) while NaOAc was added to MeOH solution. Thus, 2 was identified as 6-hydroxyluteolin 7-O-β-d-(6″-methylglucuronopyranoside), which was found in nature for the first time^6-8 (Figure 1).

Flavonoid 3 was obtained as pale yellow powder (0.6 mg) from the leaves of Aeschynanthus cultivars “Mahligai” and “Mona Lisa.” Glucuronic acid and an aglycone were liberated by acid hydrolysis of 3. The aglycone was identified as nepetin by HPLC comparison with authentic sample. The molecular ion peaks at m/z 493 [M+H]⁺, 491 [M-H]⁻ and fragment ion peaks at m/z 317 [M-176+H]⁺, 315 [M-176-H]⁻ appeared on LC–MS, showing that 1 mol glucuronic acid is attached to nepetin. The ¹H NMR spectrum of 3 showed five aromatic proton signals at δ_H 7.49 (brd, J = 8.4 Hz), 7.46 (brs), 7.26 (d, J = 8.4 Hz), 6.96 (s), and 6.71 (s) corresponding to H-6′, H-2′, H-5′, H-8, and H-3, respectively, along with the glucuronyl anomeric proton signal at δ_H 4.82 (d, J = 7.2 Hz) and methoxyl proton signal at δ_H 3.74 (s) corresponding to 6-OCH₃. The attachment of glucuronic acid to 4′-position of nepetin was determined by the bathochromic shift of Band I (336 nm) with remarkable decrease in intensity in the presence of NaOMe by UV spectral survey. ⁵ Thus, 3 was identified as nepetin 4′-O-β-d-glucuronopyranoside (Figure 1). The compound was found in nature for the first time.^6-8

We have reported 13 anthocyanins, 41 flavones, each one of flavonol, chalcone and dihydroflavonol, two flavanones and four phenylethanoids from the flowers and leaves of Aeschynanthus species and cultivars.^2,3 In this study, three minor flavones were identified as scutellarein 7-O-methylglucuronide (1), 6-hydroxyluteolin 7-O-methylglucuronide (2) and nepetin 4′-O-glucuronide (3). The flavone glycosides from Aeschynanthus species and cultivars were glucuronides and/or methylglucuronides in almost cases. ³ Although flavonoid information of the Aeschynanthus is limited, flavone glucuronides and/or methylglucuronides may be characteristic of the genus.

Conclusions

Three flavone glucuronides were isolated from the corollas and leaves of Aeschynanthus species and cultivars as minor compounds. They were identified as scutellarein 7-O-(6″-methylglucuronopyranoside) (1), 6-hydroxyluteolin 7-O-(6″-methylglucuronopyranoside) (2) and nepetin 4′-O-glucuronopyranoside (3). These flavone glycosides are reported in nature for the first time.

Materials and Methods

Plant Materials

Aeschynanthus flugens Wall ex R. Br. and A. pulcher (Blume) G. Don, and cv. “Freshya” are cultivated in the Bogor Botanic Gardens (BBG), Indonesia. Aeschynanthus cultivars, cv. “Bravera” and cv. “SoeKa” are cultivated in the Experiment Gardens, Ministry of Agriculture, Manoko-Lembang, Indonesia. Aeschynanthus cultivars, cv. “Mahligai,” cv. “Mona Lisa” and cv. “Redona” are growing in Sunbur Makmur, Lembang-Bandung, Indonesia. Voucher specimens and live specimens are deposited in the Bogor Botanic Gardens (BBG), Indonesia, ie, A. pulcher (B200408570), A. fulgens (SRY-A1701), A. cv. “Bravera” (PL-62009), A. cv. “Freshya” (LR-09), A. cv. “Mahligai” (LR-01), A. cv. “Mona Lisa” (SRY-A1003), A. cv. “Redona” (LR-07) and A. cv. “SoeKa” (PR-22007).

General

A preparative paper chromatography (prep. PC) was performed with solvent systems, BAW (n-BuOH/HOAc/H₂O = 4:1:5, upper phase) and 15% HOAc. Preparative high performance liquid chromatography (prep. HPLC) was performed with Shimadzu HPLC systems using an Inertsil ODS-4 column (I.D. 10 × 250 mm) at a flowrate of 1.5 mL/min, detection wavelength of 350 nm, and the eluent used was MeCN/H₂O/HCOOH (30:65:5). Analytical thin layer chromatography (TLC) was performed with BAW, BEW (n-BuOH/EtOH/H₂O = 4:1:2.2) and 15% HOAc solvent system. Analytical HPLC was performed with a Shimadzu HPLC system using an Inertsil ODS-4 column (I.D. 6.0 × 150 mm, GL Science Inc., Tokyo) at a flowrate of 1.0 mL/min. Acid hydrolysis was performed in a 12% aq. HCl, for 30 minutes at 100 °C. The aglycones and sugars were characterized by HPLC [eluent, MeCN/H₂O/H₃PO₄ (20:80:0.2)] and PC [solvent systems, BBPW = n-BuOH/benzene/pyridine/H₂O (5:1:3:3) and BTPW = n-BuOH/toluene/pyridine/H₂O (5:1:3:3)] comparisons with authentic samples, respectively. Liquid chromatography–mass spectrometry (LC–MS) was performed with a Shimadzu LC–MS system (LCMS-2010EV) using an Inertsil ODS-4 column (I.D. 2.1 × 100 mm) at a flowrate of 0.2 mL/min, electrospray ionization (ESI⁺) 4.5 kV, ESI⁻ of 3.5 kV, at 250 °C. The eluent was MeCN/H₂O/HCOOH (15:80:5). UV–visible absorption spectra were performed with a Shimadzu MPS-2000 multipurpose recording spectrophotometer. The high resolution–mass spectra (HR–MS) (ESI⁻) was performed using a JMS-T100LP mass spectrometer (JEOL Ltd, Tokyo). The nuclear magnetic resonance (NMR) spectra were measured on a Bruker AV-600 spectrometer in dimethysulfoxide-d₆ (DMSO-d₆).

Extraction and Isolation

Fresh leaves of cv. “Mahligai” (35.61 g) and “Mona Lisa” (49.79 g) were macerated in MeOH for two days. Fresh corollas of Aeschynanthus fulgens (6.4 g), A. pulcher (8.17 g), cv. “Bravera” (7.55 g), cv. “Freshya” (0.76 g), cv. “Mahligai” (92.35 g), cv. “Mona Lisa” (41.66 g), cv. “Redona” (29 g), and cv. “SoeKa” (35 g) were extracted using MeOH/HCOOH (9:1). After checking the flavonoid composition by HPLC, the corolla and leaf extracts were gathered and applied to the prep. PC, respectively. Roughly isolated flavonoids were further purified by the prep. HPLC. Flavonoids 1 (0.7 mg), 2 (0.6 mg), and 3 (0.6 mg) were isolated from the corollas and leaves, together with other flavonoids, and obtained as pale yellow powders. The other flavonoids have already been identified and reported as an article. ³

Identification of Flavonoids

Flavonoids were identified using UV–vis spectroscopy, HR–MS, and/or LC–MS, hydrolysate characterization and ¹H NMR. The authentic scutellarein and 6-hydroxyluteolin were obtained by acid hydrolysis of scutellarein 7-O-glucuronide and 6-hydroxyluteolin 7-O-glucuronide from Aeschynanthus species and cultivars. ³ The authentic nepetin was obtained by acid hydrolysis of nepetin 4′-O-glucoside from Cirsium oligophyllum (Franch. et Savat.) Matsum. ⁹ TLC, HPLC, HR–MS and/or LC–MS, UV, and ¹H NMR data of the isolated flavonoids are as follows.

Scutellarein 7-O-methylglucuronide (1).

TLC (Rf): 0.65 (BAW), 0.71 (BEW), 0.10 (15%HOAc); color (365 nm) dark purple, UV/NH₃ dark yellow. HPLC (retention time, tR): 34.98 minutes. LC–MS: m/z 477 [M+H]⁺, 475 [M-H]⁻ (scutellarein + 1 mol methyl-glucuronic acid). UV: λ_max (nm) MeOH 287, 335; +NaOMe 309sh, 372 (inc.); +AlCl₃ 234sh, 302, 363; +AlCl₃/HCl 233sh, 300, 358; +NaOAc 297sh, 325, 385sh; +NaOAc/H₃BO₃ 292, 330. ¹H NMR (600 MHz, DMSO-d₆): δ 12.69 (1H, s, 5-OH), 7.99 (2H, d, J = 9.0 Hz, H-2′, 6′), 6.97 (2H, d, J = 9.0 Hz, H-3′, 5′), 6.95 (1H, s, H-8), 6.84 (1H, s, H-3), 5.30 (1H, d, J = 8.6 Hz, methyl-glucuronyl H-1), 4.80 (1H, d, J = 9.0 Hz, methyl-glucuronyl H-5), 3.95 (3H, s, 6″-COOCH₃), 3.90-3.20 (3H, m, methyl-glucuronyl H-2, H-3, and H-4).

6-Hydroxyluteolin 7-O-methylglucuronide (2).

TLC (Rf): 0.41 (BAW), 0.45 (BEW), 0.06 (15%HOAc); color (365 nm) dark purple, UV/NH₃ dark yellow. HPLC (tR): 20.26 minutes. HR–MS: m/z 491.0826 [M-H]⁻ calcd. for C₂₂H₁₉O₁₃, Found 491.0913. LC–MS: m/z 493 [M+H]⁺, 491 [M-H]⁻ (6-hydroxyluteolin + 1 mol methyl-glucuronic acid), m/z 303 [M-190+H]⁺ (6-hydroxyluteolin). UV: λ_max (nm) MeOH 254sh, 283, 345; +NaOMe 270sh, 307sh, 395 (inc.); +AlCl₃ 276sh, 296, 382, 424sh; +AlCl₃/HCl 260sh, 296, 366; +NaOAc 288, 352, 395sh; +NaOAc/H₃BO₃ 262, 288sh, 361. ¹H NMR (600 MHz, DMSO-d₆): δ 7.47 (1H, d, J = 2.4 Hz, H-2′), 7.42 (1H, brd, J = 8.4 Hz, H-6′), 6.98 (1H, s, H-8), 6.87 (1H, d, J = 7.8 Hz, H-5′), 6.70 (1H, s, H-3), 5.32 (1H, d, J = 7.8 Hz, methyl-glucuronyl H-1), 3.71 (3H, s, 6″-COOCH₃), 3.50-3.10 (4H, m, methyl-glucuronyl H-2, H-3, H-4 and H-5).

Nepetin 4′-O-glucuronide (3).

HPLC (tR): 29.47 minutes. LC–MS: m/z 493 [M+H]⁺, 491 [M-H]⁻ (nepetin + 1 mol glucuronic acid), m/z 317 [M-176+H]⁺, 315 [M-176-H]⁻ (nepetin). UV: λ_max (nm) MeOH 273, 336; +NaOMe 265, 296sh, 374 (dec.); +AlCl₃ 256, 280, 288sh, 349, 385sh; +AlCl₃/HCl 255, 282sh, 287, 347, 385sh; +NaOAc 271, 371; +NaOAc/H₃BO₃ 273, 339. ¹H NMR (600 MHz, DMSO-d₆): δ 12.97 (1H, s, 5-OH), 7.49 (1H, brd, J = 8.4 Hz, H-6′), 7.46 (1H, brs, H-2′), 7.26 (1H, d, J = 8.4 Hz, H-5′), 6.96 (1H, s, H-8), 6.71 (1H, s, H-3), 4.82 (1H, d, J = 7.2 Hz, glucuronyl H-1), 4.08 (1H, d, J = 6.6 Hz, glucuronyl H-5), 3.74 (3H, s, 6-OCH₃), 3.55-3.20 (3H, m, glucuronyl H-2, H-3, and H-4).