New Quercetin Triglycoside from the Leaves of Soybean Cultivar ‘Clark’

Abstract

A new flavonol triglycoside, Quercetin 3-O-α-rhamnopyranosyl-(1→4)-[α-rhamnopyranosyl-(1→6)-β-galactopyranoside], was isolated from the leaves of soybean (Glycine max) cultivar “Clark” and identified by UV spectra, LC-ESI-MS, acid hydrolysis, and ¹H and ¹³C NMR. The compound was found together with 7 known flavonol glycosides, quercetin 3-O-robinobioside, quercetin 3-O-rutinoside, kaempferol 3-O-rhamnosyl-(1→4)-[rhamnosyl-(1→6)-galactoside], kaempferol 3-O-robinobioside, kaempferol 3-O-rutinoside, isorhamnetin 3-O-robinobioside, and isorhamnetin 3-O-rutinoside.

Soybean (Glycine max (L.) Merr.) is a major food crop and offers many compounds such as proteins, oil, and phenolic compounds including flavonoids, for the benefit of human health. ¹ Flavonoids are effective bioactive compounds produced by plants and most of them possess antioxidant properties. ² Although flavonoids of soybean have been well surveyed, many studies have been focused on isoflavonoids, which are mainly accumulated in the roots, seeds, and hypocotyls. ^3–6 On the other hand, other flavonoids, especially flavonol glycosides, were frequently reported from the leaves and flowers rather than roots and seeds. ^7–10 Recently, we revealed the leaf flavonoids and their biosynthetic genes (especially for glycosyltransferase genes) in several soybean cultivars and lines. ^11–14 While elucidating the flavonoid composition of a US soybean cultivar ‘Clark’, we found an unknown flavonol glycoside. This cultivar is used as a parent of many soybean cultivars and lines. Furthermore, its genetic background is well understood. In this paper, we describe the isolation and identification of the unknown flavonoid obtained from soybean cultivar ‘Clark.’

We preliminarily analyzed the flavonoid composition of cv. ‘Clark’ by high-performance liquid chromatography (HPLC) and recognized the presence of 8 flavonoids. Furthermore, we developed an HPLC separation method for each compound with a core–shell column.

Flavonoids 1 -8 were isolated as pale yellow powders or solutions from the leaves of cv. ‘Clark.’ The UV spectral properties of 1 indicated the presence of free 5-, 7-, 3’-, and 4’-hydroxyls and a substituted 3-hydroxyl group by the addition of NaOMe, AlCl₃, AlCl₃/HCl, NaOAc, and NaOAc/H₃BO₃. ¹⁵ Quercetin, galactose, and rhamnose were obtained by acid hydrolysis of 1. The molecular ion peak, m/z 755 [M–H]^–, obtained by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS), showed that 1 mol galactose and 2 mol rhamnose are attached to quercetin. The proton and carbon signals were assigned by heteronuclear single quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC), ¹H-¹H correlation spectroscopy (COSY), and ¹H-¹H nuclear Overhauser effect spectroscopy (NOESY). In the ¹H nuclear magnetic resonance (NMR) spectrum (Table 1), 5 aromatic protons for H-2’ at δ _H 8.42 (d, J = 2.2 Hz), H-5’ at δ _H 7.34 (d, J = 8.3 Hz), H-6’ at δ _H 8.24 (dd, J = 2.2 and 8.4 Hz), H-6 at δ _H 6.68 (d, J = 1.9 Hz), and H-8 at δ _H 6.72 (d, J = 2.0 Hz) appeared, together with a galactosyl anomeric proton at δ _H 6.47 (d, J = 7.8 Hz) and 2 rhamnosyl anomeric protons at δ _H 6.29 (brs) and δ _H 5.13 (d, J = 1.0 Hz), and 2 rhamnosyl Me protons at δ _H 1.56 (d, J = 6.2 Hz) and δ _H 1.44 (d, J = 5.8 Hz). The HMBC spectrum of 1 showed that rhamnosyl anomeric protons at δ _H 6.29 and δ _H 5.13 correlated with the carbon signals at δ _C 76.7 and δ _C 66.0 corresponding to C-4 and C-6 of galactose. The HMBC correlation between the galactosyl anomeric proton at δ _H 6.47 and C-3 of quercetin at δ _C 134.3 was also observed. Thus, 1 was identified as quercetin 3-O-α-rhamnopyranosyl-(1→4)-[α-rhamnopyranosyl-(1→6)-β-galactopyranoside] (Figure 1), which was found for the first time in nature. The chemical structure was further confirmed by referring to NMR data of kaempferol 3-O-rhamnosyl-(1→4)-[rhamnosyl-(1→6)-galactoside], ¹⁰ isorhamnetin and quercetin 3-O-rhamnosyl-(1→2)-[rhamnosyl-(1→6)-galactosides], ¹⁶ and kaempferol and quercetin 3-O-[rhamnosyl-(1→4)-rhamnosyl-(1→6)-galactosides]. ¹⁷

Table 1. ¹H and ¹³C NMR Data of 1 (Pyridine-d ₅, ¹H 600 MHz and ¹³C 150 MHz).

Position	δ H (J, Hz)	δ C
Aglycone
2		157.5
3		134.3
4		178.6
5 (OH)	13.43 s	162.8
6	6.68 d (1.9)	94.4
7		165.6
8	6.72 d (2.0)	99.6
9		157.5
10		105.4
1′		122.9
2′	8.42 d (2.2)	117.4
3′		146.8
4′		150.5
5′	7.34 d (8.3)	116.4
6′	8.24 dd (2.2, 8.4)	122.6
3-O-Galactose
1	6.47 d (7.8)	100.8
2	4.99 m	69.9
3	4.05 t (6.6)	74.7
4	5.00 m	76.7
5	4.34 m	75.8
6a	4.38 m	66.0
6b	3.80 m
4"-O-Rhamnose
1	6.29 brs	102.5
2	4.36 m	72.5
3	4.89 dd (1.2, 3.2)	72.8
4	4.30 m	74.2
5	4.13 m	69.6
6	1.56 d (6.2)	18.4
6"-O-Rhamnose
1	5.13 d (1.0)	101.7
2	4.37 m	72.0
3	4.83 dd (3.3, 9.4)	72.7
4	4.37 m	73.8
5	4.17 m	70.0
6	1.44 d (5.8)	18.2

Figure 1

Chemical structure of 1 from soybean cv. ‘Clark.’

The other flavonoids were identified as quercetin 3-O-robinobioside (2), kaempferol 3-O-rhamnopyranosyl-(1→4)-[rhamnopyranosyl-(1→6)-galactopyranoside] (3), quercetin 3-O-rutinoside (4), kaempferol 3-O-robinobioside (5), kaempferol 3-O-rutinoside (6), isorhamnetin 3-O-robinobioside (7), and isorhamnetin 3-O-rutinoside (8) by UV and mass spectrometry surveys, and HPLC comparisons with authentic samples isolated from soybean cultivars and lines, ¹⁰ and Cassytha spp (Lauraceae). ¹⁸

In the present study, we determined the chemical structures of 8 flavonol glycosides from the leaves of soybean cultivar ‘Clark.’ Though 2 -8 have previously been reported from some plant species including soybean, ^19,20 1 was found for the first time in nature. The isolated glycosides attach rhamnose, and galactose or glucose. Their glycosylation patterns are common in soybean leaves. ^7–10,21,22 Buttery and Buzzell ⁷ suggested the presence of 9 kaempferol 3-O-glycosides and 10 quercetin 3-O-glycosides in the leaves of some soybean cultivars by thin-layer chromatography (TLC). ²¹ Their chemical structures were gradually revealed by current analytical techniques. ^7–10,21,23 In addition to kaempferol and quercetin glycosides, we also found several glycosides of isorhamnetin, apigenin, luteolin, and daidzein from soybean leaves. ^4,7–10 It is probable that the flavonoid diversity of soybean leaves is higher than that reported by Buttery and Buzzell. The comprehensive profiling of leaf flavonoids in major soybean cultivars and lines is now in progress. Furthermore, some glycosyltransferase genes concerning this complex glycosylation of flavonoids in soybean leaves were successfully isolated based on such flavonoid profiling. ^11–14

Experimental

General

NMR spectra were recorded on a Bruker AV-600 in pyridine-d ₅ at 600 MHz (¹H NMR) and 150 MHz (¹³C NMR). LC-ESI-MS was measured using a Shimadzu LCMS-2010EV with an Inertsil ODS-4 column (3 µm particle material, I.D. 2.1 × 100 mm, GL sciences, Inc., Japan) at a flow rate of 0.2 mL min^–1, eluting with HCOOH/MeCN/H₂O (1:15:84), ESI⁺, 4.5 kV, ESI^– 3.5 kV, 250°C.

First, preparative HPLC was performed with an L-column2 ODS (5 µm particle material, I.D. 10 × 250 mm, Chemicals Evaluation and Research Institute, Japan) at a flow rate of 3.0 mL min^–1, detection: 350 nm, and eluent: HCOOH/MeCN/H₂O (1:12:87). Second, preparative HPLC was performed with an InertSustein AQ-C18 (5 µm particle material, I.D. 10 × 250 mm, GL Sciences, Inc.) at a flow rate of 4.0 mL min^–1, detection: 350 nm, and eluent: HCOOH/MeCN/H₂O (1:12:87). The HPLC survey of the crude extracts and isolated flavonoids was performed with a Shimadzu Prominence HPLC system using a core–shell column, SunShell C18 column (2.6 µm particle material, I.D. 4.6 × 75 mm, ChromaNik Technologies Inc., Japan) at a flow rate of 0.8 mL min^–1, detection: 350 nm, and eluent: H₃PO₄/MeCN/H₂O (0.2:12:88) (Figure 2). The TLC survey (BAW, 15% HOAc, and BEW) of 1 was carried out using Merck cellulose plastic plates. Compound 1 was hydrolyzed in 12% HCl for 30 minutes on a boiling water bath. Aglycones (organic layer) and sugars (mother liquor) were separated by the addition of diethyl ether and then shaken, and characterized by HPLC (aglycones) and TLC (sugars) comparisons with authentic samples.

Figure 2

HPLC chromatogram of crude extracts from soybean cv ‘Clark.’ 1 = Quercetin 3-O-α-rhamnopyranosyl-(1→4)-[α-rhamnopyranosyl-(1→6)-β-galactopyranoside] (retention time 5.8 min), 2 = kaempferol3-O-rhamnosyl-(1→4)-[rhamnosyl-(1→6)-galactoside] (8.9min), 3 = quercetin 3-O-robinobioside (9.8 min), 4 =quercetin 3-O-rutinoside (11.1 min), 5 = kaempferol3-O-robinobioside (15.1 min), 6 = kaempferol 3-O-rutinoside(21.1 min), 7 = isorhamnetin 3-O-robinobioside (22.5 min),and 8 = isorhamnetin 3-O-rutinoside (26.4 min).

Plant Materials

The leaves of Glycine max cultivar ‘Clark’ were used as the plant materials. The plant was cultivated in the experimental field of the Institute of Crop Science, NARO, Tsukuba, Japan.

Extraction and Separation

Fresh leaves (ca. 200 g) of soybean cultivar ‘Clark’ were extracted with MeOH. The concentrated extracts were separated by preparative paper chromatography using BAW (n-BuOH/HOAc/H₂O = 4:1:5, upper phase), 15% HOAc, and BEW (n-BuOH/EtOH/H₂O = 4:1:2.2). The compounds were purified by Sephadex LH-20 column chromatography using 70% MeOH. The isolated flavonoids were further purified by 2 steps of preparative HPLC.

Quercetin 3-O-Rhamnopyranosyl-(1→4)-[Rhamnopyranosyl-(1→6)-β-galactopyranoside] (1)

TLC (Rf): 0.30 (BAW), 0.86 (15% HOAc), 0.50 (BEW).

UV (365 nm): dark purple; UV/NH₃—dark yellow.

HPLC (tR): 5.8 minutes.

UV λ _max nm MeOH: 256, 266sh, 356. + NaOMe: 271, 327, 401 (inc.). +AlCl₃: 274, 430. +AlCl₃/HCl: 270, 298, 364, 402. + NaOAc: 272, 323, 384. + NaOAc/H₃BO₃: 261, 374.

¹H and ¹³C NMR: Table 1.

LC-ESI-MS: m/z 757 [M + H]⁺, 755 [M–H]^– (quercetin +1 mol galactose and 2 mol rhamnose), 611 [M–146 + H]⁺ (quercetin +each 1 mol of galactose and rhamnose), 465 [M–292 + H]⁺ (quercetin + 1 mol galactose), 303 [M–454 + H]⁺ (quercetin).