2′-Hydroxylated 3-Deoxyanthocyanin from the Flowers of Cosmos sulphureus Cultivars

Abstract

A new 3-deoxyanthocyanin was isolated from the orange flowers of Cosmos sulphureus cultivar “Diabolo,” together with known aurone, chalcone, flavone, flavonol, and flavanone. The chemical structure of 3-deoxyanthocyanin was established as 5,7,2′,4′-tetrahydroxyflavylium 4′-O-β-d-glucopyranoside by chemical and spectroscopic methods including UV, FAB MS, and ¹H and ¹³C nuclear magnetic resonance spectroscopy (NMR), and named as cosmonidin 4′-O-glucoside (1a). Other known flavonoids were characterized as sulfuretin 6-O-glucoside (2), butein 4′-O-glucoside (3), quercetin 3-O-glucoside (4), luteolin 7-O-glucuronide (5), and eriodictyol 7-O-glucuronide (6) by UV, liquid chromatograph - mass spectra (LC-MS), NMR, and high performance liquid chromatography comparisons with authentic samples. Anthocyanin, which 2′-position is hydroxylated, has not been reported until now. Compound (1a) was also found in other 10 C. sulphureus cultivars “Lamala Lemon,” “Sunrise,” “Dwarf Yellow,” “Cosmic Yellow,” “Carpet Gold,” “Mandarin,” “Cosmic Orange,” “Orange Flare,” “Cosmic Red,” and “Sunset.”

The genus Cosmos (Asteraceae) consists of circa 35 species and is distributed to Central and South America of which Cosmos bipinnatus Cav., Cosmos sulphureus Cav., and Cosmos atrosanguineus (Hook.) A. Voss are widely cultivated as the ornamentals. As flavonoids in the flowers of Cosmos species, anthocyanin, cyanidin 3-O-glucoside, was reported from C. bipinnatus, ^1,2 together with flavones, apigenin 7-O-glucoside, luteolin 7-O-glucuronide, and chrysoeriol 7-O-glucuronide. ^3,4 Recently, 5 anthocyanins, cyanidin 3-O-glucoside, 3-O-rutinoside and 3-O-(malonylglucoside), and pelargonidin 3-O-glucoside and 3-O-rutinoside, were reported from the black flowers of C. atrosanguineus and the hybrids with C. sulphureus, together with a chalcone, butein 4′-O-glucoside, and 2 flavanones, eriodictyol 7-O-glucoside and 7-O-glucuronide. ⁵ On the other hand, aurone, sulfuretin 6-O-glucoside, and chalcone, butein 4′-O-glucoside, were found in the flowers of C. sulphureus, together with flavonol, quercetin 3-O-glucoside. ^6,7 Samata et al ⁸ also surveyed the flower pigments of C. sulphureus and found 2 anthocyanins, cyanidin 3-O-glucoside and 3-O-rhamnosylglucoside, together with each one of aurone and chalcone which were described above. Although they noticed the presence of an unknown orange pigment by two-dimensional paper chromatography, isolation and identification of this pigment were not performed.

In this survey, an anthocyanin (1a) was isolated from the flowers of C. sulphureus cultivar “Diabolo,” together with 5 other flavonoids (2-6). Pigment 1a was obtained as orange red amorphous powder from the flowers of C. sulphureus. UV-Vis absorption maxima of 1a in 0.01%HCl-MeOH were 290 and 483 nm, which presumed that the compound is a 3-deoxyanthocyanin. Its molecular formula was assigned as tetrahydroxyflavylium monohexoside such as pelargonidin 3-O-glucoside and luteolinidin 5-O-glucoside based on the [M]⁺ ion peak at m/z 433.1230 (calcd. for C₂₁H₂₁O₁₀, 433.1213) in the high resolution fast atom bombardment mass spectra (HRFAB MS). On the other hand, that of anthocyanidin 1b (10 mg) which was obtained by acid hydrolysis of 1a was assigned as tetrahydroxyflavylium based on the [M]⁺ ion peak at m/z 271.0648 (calcd. for C₁₅H₁₁O₅, 271.0606). However, high performance liquid chromatography (HPLC) behavior and UV-Vis spectral properties of 1b did not agree with those of authentic pelargonidin and luteolinidin. Moreover, since UV-Vis absorption maxima of 1a and 1b did not shift by the addition of AlCl₃, the absence of ortho-dihydroxyl group in the B-ring was shown. Final estimation of 1b was performed by ¹H and ¹³C NMR. Proton and carbon signals of ¹H and ¹³C NMR were assigned by hetero-nuclear single quamtum coherence (HSQC), hetero-nuclear multiple-bond connectivity (HMBC), ¹H-¹H correlation spectroscopy (COSY), and ¹H-¹H nuclear overhauser effect spectroscopy (NOESY). ¹H NMR data of 1b showed the presence of 7 aromatic protons. Of their protons, δ_H 7.46 (brs) and 7.53 (brs) were assigned to H-6 and H-8 by COSY correlation between their protons and NOESY correlation with H-4. Two protons, δ_H 8.58 (d, J = 9.0 Hz) and 8.87 (d, J = 9.1 Hz), were assigned to H-3 and H-4 by comparison with the data of apigeninidin 5-O-glucoside ⁹ and luteolinidin 5-O-glucoside. ¹⁰ Other 3 protons, δ_H 6.69 (d, J = 2.3 Hz), 8.20 (d, J = 9.0 Hz), and 6.64 (dd, J = 2.2 and 9.1 Hz), which were derived from the B-ring were similar to those of H-2′, H-5′, and H-6′ of luteolinidin 5-O-glucoside. ^9,10 However, the absence of ortho-dihydroxyl group in the B-ring and NOESY correlation between δ_H 6.69 (H-3′) and 6.64 (H-5′) showed that they are H-3′, H-6′, and H-5′. In ¹³C NMR of 1b, 7 carbon signals, ie, C-3 (δ_C 116.3), C-4 (δ_C 150.5), C-6 (δ_C 111.2), C-8 (δ_C 103.3), C-3′ (δ_C 104.0), C-5′ (δ_C 111.5), and C-6′ (δ_C 132.6), were assigned by HSQC. On the other hand, other 8 carbon signals were assigned by HMBC, ie, both H-6 and H-8 correlated to C-7 and C-9 at δ_C 159.6 and 149.3; H-3 and H-4 with C-2 at δ_C 168.4; H-4, H-6, and H-8 with C-5 at δ_C 154.0; and H-3 and H-8 with C-10 at δ_C 119.4. Moreover, H-3′ and H-5′, and H-3′ and H-6′ correlated with C-1′ at δ_C 108.8, and C-2′ and C-4′ at δ_C 163.7 and 167.3 with equivalent intensity, respectively. Such a fact was followed by comparison with the data of norartocarpetin (5,7,2′,4′-tetrahydroxyflavone). ¹¹ Moreover, it was confirmed by COSY and NOESY correlations between H-3 (δ_H 8.58) and H-4 (δ_H 8.87), and H-5′ (δ_H 6.64) and H-6′ (δ_H 8.20), NOESY correlation between H-3′ (δ_H 6.69) and H-6′, and H-4 and H-6. Thus, anthocyanidin 1b was identified as 5,7,2′,4′-tetrahydroxyflavylium (Figure 1(b)). Thirty-one kinds of anthocyanidins have been reported in plants. ¹² However, since 2′-hydroxylated anthocyanidin was found in nature for the first time, we named this compound 1b as cosmonidin.

Figure 1

Chemical structures of cosmonidin 4′-O-glucoside (1a) and its anthocyanidin (1b) from the flowers of Cosmos sulphureus cultivar “Diabolo.” Solid arrows: COSY correlations; dotted arrows: NOESY correlations; bold arrows: both COSY and NOESY correlations.

The chemical structure of original glycoside 1a was determined by ¹H and ¹³C NMR. Cosmonidin and d-glucose were liberated by acid hydrolysis of 1a. In ¹H NMR, glucosyl anomeric proton δ_H 5.10 (d, J = 7.4 Hz) appeared with 7 aromatic protons which are derived from cosmonidin. Other glucosyl protons were assigned by HSQC correlation with carbon signals C-2″-6″ of ¹³C NMR. The attachment of glucose to 2′- or 4′-position of cosmonidin was shown by no shift of H-6 (δ_H 7.49) and H-8 (δ_H 7.58) to lower magnetic field in comparison with those of 1b. The attachment of glucose to 4′-position was confirmed by the larger shift of lower magnetic field of both H-3′ (δ_H 6.91) and H-5′ (δ_H 6.88) in comparison with those of 1b than the shift of H-2′. Moreover, it was followed by NOESY correlation between both H-3′ and H-5′, and between their protons and anomeric proton (δ_H 5.10) of 1a. Thus, 1a was identified as 5,7,2′,4′-tetrahydroxyflavylium 4′-O-β-d-glucopyranoside (cosmonidin 4′-O-glucoside) (Figure 1(a)).

As other flavonoids, sulfuretin 6-O-glucoside (2) (aurone), ⁶ butein 4′-O-glucoside (3) (chalcone), ¹³ quercetin 3-O-glucoside (4) (flavonol), ¹⁴ luteolin 7-O-glucuronide (5) (flavone), ¹⁵ and eriodictyol 7-O-glucuronide (6) (flavanone) ¹⁶ were isolated and identified by UV, LC-MS, acid hydrolysis, NMR, and/or HPLC comparisons with authentic samples. Although 2 and 3 have already been reported from the flowers of C. sulphureus, ⁶ 4 to 6 were found in the species for the first time. Cosmonidin 4′-O-glucoside (1a) was also found in the flowers of other 10 cultivars, “Limala Lemon,” “Sunrise,” “Dwarf Yellow,” “Cosmic Yellow,” “Carpet Gold,” “Mandarin,” “Cosmic Orange,” “Orange Flare,” “Cosmic Red,” and “Sunset” by HPLC survey.

Experimental

General

Analytical HPLC was performed with a Shimadzu HPLC systems (Shimadzu Co. Ltd., Tokyo) using Inertsil octadecyl-silica (ODS)-4 column (GL Science Co. Ltd., Tokyo) (I.D. 6.0 × 150 mm) for anthocyanin, and L-Column 2 ODS column (CERI Co. Ltd., Tokyo) for other flavonoids at 40°C with flow-rate of 1.0 mL/min, and eluents MeCN/HOAc/H₂O/H₃PO₄ (10:8:79:3) for anthocyanin and MeCN/H₂O/H₃PO₄ (20:80:0.2) for other flavonoids. Thin layer chromatography (TLC) was performed on cellulose-coated plastic plates (Merck, Darmstadt) using solvent systems, BAW (n-BuOH/HOAc/H₂O = 4:1:5, upper phase), BuH (n-BuOH/2 N HCl = 1:1, upper phase), Forestal (HOAc/HCl/H₂O = 30:3:10) and 1%HCl for anthocyanin, and BAW, BEW (n-BuOH/EtOH/H₂O = 4:1:2.2) and 15%HOAc for other flavonoids. Acid hydrolysis was performed in 12%HCl, 100°C, 30 minutes. After shaking with isoamyl alcohol, anthocyanidin was migrated to the organic layer, and sugar was left in the aqueous layer. Sugar was identified by paper chromatographic comparisons with authentic sugars using solvent systems, BBPW (n-BuOH/benzene/pyridine/H₂O = 5:1:3:3) and BTPW (n-BuOH/toluene/pyridine/H₂O = 5:1:3:3). Sugar spots were visualized by spraying 1% methanolic hydrochloride on the chromatograms and heating. UV-Vis spectra were measured on Shimadzu MPS-2000 and Shimadzu UV-2600 (Shimadzu) in 0.01%HCl-MeOH (anthocyanin) and MeOH (other flavonoids). Prep. HPLC was performed with Tosoh HPLC systems (Tosoh Co. Ltd., Tokyo) using Inertsil ODS-4 column (I.D. 10 × 250 mm), flow-rate 1.5 to 3.0 mL/min, eluents MeCN/H₂O/HCOOH (12-20:83-75:5). HRFAB MS were performed using JEOL HX-110 spectrometer. ¹H (600 MHz) and ¹³C NMR (150 MHz) were measured on a Bruker AV-600 using solvents, DMSO-d ₆ + CF₃COOD (3:1) for 1a and (9:1) for 1b at room temperature and 60°C.

Plant Materials

The petals of C. sulphureus Cav. cultivars, which were cultivated in the nursery of Tamagawa University, Machida, Tokyo, were collected in September 2015 and used as plant materials for isolation of the pigments.

Extraction and Isolation

Fresh petals (circa 250 g) were extracted with MeOH/HCOOH (92:8) and concentrated to dryness. The extracts were dissolved in 5%HCOOH, applied to Amberlight XAD 7HP (Organo, Tokyo), and eluted with 5%HCOOH-MeOH for removal of polysaccharides. After concentration, solution was applied to preparative paper chromatography using solvent systems, BAW and then 15%HOAc. The isolated anthocyanin and other flavonoids were purified by Sephadex LH-20 column chromatography using solvent system, MeOH/H₂O/HCOOH (70:25:5) for anthocyanin or 70%MeOH for other flavonoids and then prep. HPLC. Anthocyanin 1a (19 mg) and other flavonoids 2 (circa 20 mg) and 3 (circa 5 mg) were obtained as orange red solid or yellow powders. Other flavonoids were obtained as pure solutions.

Cosmonidin 4′-O-glucoside (1a)

TLC: Rf 0.30 (BAW), 0.07 (BuH), 0.03 (1%HCl), 0.54 (Forestal); color visible orange, UV (365 nm) orange yellow fluorescent, HPLC: MeCN/H₂O/HOAc/H₃PO₄ (10:79:8:3): t _R 6.10 minutes. UV-Vis: λ_max (nm) 0.01%HCl-MeOH 290, 483; E ₄₄₀/E _max (%) 43; +AlCl₃ no shift. ¹H NMR (600 MHz, DMSO-d ₆ + CF₃COOD = 3:1) δ_H 8.63 (1H, d, J = 8.8 Hz, H-3), 8.94 (1H, d, J = 8.9 Hz, H-4), 7.49 (1H, brs, H-6), 7.58 (1H, brs, H-8), 6.91 (1H, d, J = 2.3 Hz, H-3′), 6.88 (1H, dd, J = 2.3 and 9.1 Hz, H-5′), 8.27 (1H, d, J = 8.6 Hz, H-6′), 5.10 (1H, d, J = 7.3 Hz, 4′-glucosyl H-1), 3.45 (1H, dd, J = 7.5 and 8.7 Hz, 4′-glucosyl H-2), 3.48 (1H, t, J = 8.6 Hz, 4′-glucosyl H-3), 3.39 (1H, t, J = 9.1 Hz, 4′-glucosyl H-4), 3.51 (1H, m, 4′-glucosyl H-5), 3.85 (1H, dd, J = 2.3 and 12.1 Hz, 4′-glucosyl H-6a), 3.68 (1H, dd, J = 5.3 and 12.1 Hz, 4′-glucosyl H-6b). ¹³C NMR (150 MHz, DMSO-d ₆ + CF₃COOD = 3:1) (cosmonidin) δ_C 167.5 (C-2), 116.5 (C-3), 151.2 (C-4), 154.8 (C-5), 111.0 (C-6), 160.7 (C-7), 103.3 (C-8), 149.8 (C-9), 120.3 (C-10), 111.0 (C-1′), 162.7 (C-2′), 104.9 (C-3′),165.0 (C-4′), 110.7 (C-5′), 132.2 (C-6′); (4′-O-glucose) δ_C 100.6 (C-1), 73.7 (C-2), 77.1 (C-3), 70.1 (C-4), 78.0 (C-5), 61.1 (C-6).

Cosmonidin (1b)

TLC: Rf 0.38 (BAW), 0.31 (BuH), 0.00 (1%HCl), 0.52 (Forestal); color visible orange, UV (365 nm) orange yellow fluorescence. HPLC: MeCN/H₂O/HOAc/H₃PO₄ (12:77:8:3): t _R 11.58 minutes. ¹H NMR (600 MHz, DMSO-d ₆+ CF₃COOD = 9:1) δ_H 8.58 (1H, d, J = 9.0 Hz, H-3), 8.87 (1H, d, J = 9.1 Hz, H-4), 7.46 (1H, brs, H-6), 7.53 (1H, brs, H-8), 6.69 (1H, d, J = 2.3 Hz, H-3′), 6.64 (1H, dd, J = 2.2 and 9.1 Hz, H-5′), 8.20 (1H, d, J = 9.0 Hz, H-6′). ¹³C NMR (150 MHz, DMSO-d ₆+ CF₃COOD = 9:1) δ_C 168.4 (C-2), 116.3 (C-3), 150.5 (C-4), 154.0 (C-5), 111.2 (C-6), 159.6 (C-7), 103.3 (C-8), 149.3 (C-9), 119.4 (C-10), 108.8 (C-1′), 163.7 (C-2′), 104.0 (C-3′), 167.3 (C-4′), 111.5 (C-5′), 132.6 (C-6′)

Supplemental Material

Supplemental material - Supplemental material for 2′-Hydroxylated 3-Deoxyanthocyanin from the Flowers of Cosmos sulphureus Cultivars

Supplemental material, Supplemental material, for 2′-Hydroxylated 3-Deoxyanthocyanin from the Flowers of Cosmos sulphureus Cultivars by Tsukasa Iwashina, Kotarou Amamiya, Tsunashi Kamo, Junichi Kitajima, Takayuki Mizuno, Ayumi Uehara and Nobuya Koizuka in Natural Product Communications