2025 Volume 73 Issue 3 Pages 168-172
Two new isoflavone glucosides, irisolone 4′-O-[O-β-d-glucopyranosyl-(1→6)-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside] (1) and iriskashmirianin 4′-O-[O-β-d-glucopyranosyl-(1→6)-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside] (2) were isolated from the underground parts of Iris florentina (Iridaceae). The structures of 1 and 2 were determined based on extensive spectroscopic data analyses and hydrolytic cleavage results. The isoflavone derivatives previously isolated from this plant were evaluated for their ability to inhibit the formation of advanced glycation end products.
The genus Iris, which belongs to the family Iridaceae, comprises approximately 150 species. Iris florentina L. (Iridaceae) is distributed along the Mediterranean coast of Europe.1) The dried roots of this plant, called Orris roots, have been traditionally used as a perfume ingredient and a fixative in aromatherapy. A few norisoprenoids, including α-, β- and γ-irones, along with xanthone glucosides and flavone glucosides, have been isolated from I. florentina.2–4) Previously, 10 isoflavone derivatives (3–12) have been isolated as a result of a phytochemical study on the MeOH extract of the underground parts of I. florentina (Fig. 1). Among them, 9 and 10 showed cytotoxicity against HL-60 human promyelocytic leukemia cells with IC50 values of 30.6 and 31.7 μM, respectively.5) As part of our ongoing phytochemical study on bioactive compounds from ornamental plants, the further phytochemical investigation into the isoflavone components of I. florentina resulted in the discovery of two new isoflavone glucosides (1 and 2) (Fig. 2). This study deals with the structural determination of 1 and 2 and the inhibitory activity on the formation of advanced glycation end products (AGEs) of the isoflavone compounds isolated from I. florentina.
The underground parts of Iris florentina (6.0 kg) were extracted using MeOH. The concentrated MeOH extract was passed through a porous-polymer polystyrene resin (Diaion HP-20) column and eluted successively with 30%, 50%, and 100% MeOH; EtOH, or EtOAc. The 50% MeOH-eluted fraction was subjected to multiple chromatographic steps over silica gel and octadecylsilanized (ODS) silica gel, resulting in the isolation of 1 and 2.
Compound 1 was obtained as an amorphous solid with the molecular formula of C35H42O21 as determined from its high-resolution (HR)-electrospray ionization (ESI)-time of flight (TOF)-MS data (m/z: 821.2116 [M + Na]+, Calcd for C35H42NaO21: 821.2116). The UV spectrum exhibited an absorption maximum at 261.8 nm, indicating a conjugated system. The IR spectrum of 1 showed absorption bands for hydroxy groups (3364 cm−1) and a carbonyl group (1714 cm−1). The 1H- and 13C-NMR spectrum of 1 included signals for the characteristic resonances of the isoflavone skeleton at δH 7.99 (1H, s, H-2)/δC 151.2 (C-2), 125.7 (C-3), and 174.8 (C-4), a 1,4-disubstituted aromatic ring [δH 7.77 (2H, d, J = 8.4 Hz, H-2′ and H-6′) and 7.55 (2H, d, J = 8.4 Hz, H-3′ and H-5′)/δC 126.4, 131.1, 117.0, 158.4, 117.0, 131.1 (C-1′–6′)], three anomeric protons/carbons [δH 5.61 (1H, d, J = 7.8 Hz, Glc-I-1)/δC 102.1 (Glc-I-1), δH 5.09 (1H, d, J = 8.4 Hz, Glc-II-1)/δC 105.2 (Glc-II-1), and δH 5.06 (1H, d, J = 8.4 Hz, Glc-III-1)/δC 105.5 (Glc-III-1)], one methoxy group [δH 4.11 (3H, s, OMe)/δC 61.2 (OMe)], one methylenedioxy group [δH 6.07 (2H, s, OCH2O)/δC 102.9 (OCH2O)]. These NMR spectral features indicated that 1 is an isoflavonoid triglycoside with one methoxy and one methylenedioxy group. Acid hydrolysis of 1 with 1M HCl yielded irisolone6) and d-glucose. d-Glucose was identified by direct HPLC analysis of the hydrolysate using an optical rotation detector. Analysis of the 1D total correlation spectroscopy (TOCSY) spectra followed by interpretation of the 1H–1H shift correlation spectroscopy (COSY), the 1H-detected heteronuclear single-quantum coherence (HSQC), and HSQC-TOCSY spectra revealed that the sugar moiety of 1 comprised two C-6 substituted β-d-glucopyranosyl unit (Glc-I and Glc-II) [δH 5.77 (1H, d, J = 7.8 Hz); δC 102.1, 74.9, 78.3, 71.1, 78.2, and 69.8, and δH 5.09 (1H, d, J = 8.4 Hz); δC 105.2, 75.1, 78.4, 71.5, 77.0, and 69.9] and a terminal β-d-glucopyranosyl unit (Glc-III) [δH 5.06 (1H, d, J = 8.4 Hz); δC 105.5, 75.1, 78.4, 71.5, 77.8, and 62.6]7) (Table 1). The anomeric configurations of the glucosyl moieties were ascertained as β by the relatively large 3JH-1, H-2 values (7.8, 8.4, and 8.4 Hz).8,9) In the 1H-detected heteronuclear multiple-bond connectivity (HMBC) spectrum of 1, long-range correlations were observed between the anomeric proton (H-1) of Glc-III at δH 5.06 and C-6 of Glc-II at δC 69.9; between H-1 of Glc-II at δH 5.09 and C-6 of Glc-I at δC 69.8; and between H-1 of Glc at δH 5.61 and C-4′ of the aglycone at δC 158.4 (Fig. 3). Accordingly, the structure of 1 was assigned as irisolone 4′-O-[O-β-d-glucopyranosyl-(1→6)-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside].
| 1 | 2 | |||||||
|---|---|---|---|---|---|---|---|---|
| Position | δH | δC | Position | δH | δC | |||
| Glc-I 1 | 5.61 | d (7.8)a) | 102.1 | Glc-I 1 | 5.68 | d (8.7) | 102.1 | |
| 2 | 4.25 | dd (9.0, 7.8) | 74.9 | 2 | 4.29 | dd (9.0, 8.7) | 74.8 | |
| 3 | 4.33 | dd (9.0, 9.0) | 78.3 | 3 | 4.34 | dd (9.0, 9.0) | 78.3 | |
| 4 | 4.22 | dd (9.3, 9.0) | 71.1 | 4 | 4.23 | dd (9.0, 9.0) | 71.1 | |
| 5 | 4.33 | m | 78.2 | 5 | 4.34 | m | 78.2 | |
| 6a | 4.87 | dd (12.0, 1.8) | 69.8 | 6a | 4.85 | dd (12.6, 1.2) | 69.6 | |
| 6b | 4.36 | dd (12.0, 6.0) | 6b | 4.42 | dd (12.6, 6.0) | |||
| Glc-II 1 | 5.09 | d (8.4) | 105.2 | Glc-II 1 | 5.08 | d (8.4) | 105.2 | |
| 2 | 4.02 | dd (9.0, 8.4) | 75.1 | 2 | 4.01 | dd (9.0, 8.4) | 75.1 | |
| 3 | 4.17 | dd (9.0, 9.0) | 78.4 | 3 | 4.17 | dd (9.0, 8.7) | 78.4 | |
| 4 | 4.23 | dd (9.0, 9.0) | 71.5 | 4 | 4.23 | dd (8.7, 8.7) | 71.1 | |
| 5 | 3.94 | m | 77.0 | 5 | 3.93 | m | 76.9 | |
| 6a | 4.76 | dd (11.7, 1.8) | 69.9 | 6a | 4.76 | dd (12.0, 1.2) | 69.9 | |
| 6b | 4.32 | dd (11.7, 6.0) | 6b | 4.32 | dd (12.0, 6.0) | |||
| Glc-III 1 | 5.06 | d (8.4) | 105.5 | Glc-III 1 | 5.05 | d (8.1) | 105.5 | |
| 2 | 4.04 | dd (9.0, 8.4) | 75.1 | 2 | 4.05 | dd (9.0, 8.1) | 75.1 | |
| 3 | 4.21 | dd (9.0, 9.0) | 78.4 | 3 | 4.22 | dd (9.0, 9.0) | 78.4 | |
| 4 | 4.24 | dd (9.0, 9.0) | 71.5 | 4 | 4.26 | dd (9.0, 9.0) | 71.5 | |
| 5 | 3.88 | m | 77.8 | 5 | 3.88 | m | 77.8 | |
| 6a | 4.50 | dd (11.7, 1.8) | 62.6 | 6a | 4.49 | dd (11.4, 1.8) | 62.6 | |
| 6b | 4.37 | dd (11.7, 5.4) | 6b | 4.38 | dd (11.4, 5.4) | |||
a) δ in ppm and J values in Hz in parentheses.
Compound 2 (C36H44O22) was obtained as an amorphous solid. The UV and IR spectra exhibited an absorption maximum at 262.4 nm, indicative of a conjugate system, and absorption bands for hydroxy groups at 3391 cm−1 and a carbonyl group at 1734 cm−1. The 1H- and 13C-NMR spectral properties of 2 were analogous to those of 1, including the presence of signals for a methylenedioxy group, a methoxy group, and a triglycoside moiety attached to C-4′ of the aglycone. However, the molecular formula of 2 was larger than that of 1 by a CH2O unit, indicating the presence of an additional methoxy group. Instead of the signals corresponding to a 1,4-disubstituted aromatic ring in the 1H- and 13C-NMR spectra of 1, signals for a 1,2,4-trisubstituted aromatic ring [δH 7.83 (1H, d, J = 8.4 Hz, H-5′), 7.54 (1H, d, J = 1.8 Hz, H-2′), and 7.37 (1H, dd, J = 8.4, 1.8 Hz, H-6′)/δC 126.9, 114.4, 149.0, 147.7, 116.7, and 122.3 (C-1′–6′)] and an additional methoxy group [δH 3.77 (3H, s, OMe)/δC 55.9 (OMe)] were observed in those of 2. The acid hydrolysis of 2 yielded iriskashmirianin10) and d-glucose. The additional methoxy group was confirmed to be linked to C-3′, and the O-β-d-glucopyranosyl-(1→6)-O-β-d-glucopyranosyl-(1→6)- β-d-glucopyranosyl group to C-4′ of the aglycone, based on HMBC correlations as shown in Fig. 3. The structure of 2 was formulated as iriskashmirianin 4′-O-[O-β-d-glucopyranosyl-(1→6)-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside].
Inhibition of the Formation of AGEsAGEs are generated either through non-enzymatic glycation and oxidation of proteins and reducing sugars or as intermediates in carbohydrate metabolism. The formation of AGEs in the skin, blood vessel walls, and bones is closely related to aging.11) Furthermore, AGEs accumulation influences the pathology of diabetes, chronic inflammation, Alzheimer's disease, and cancer.12) In this study, isoflavonoid derivatives 4–10, previously isolated from I. florentina, were evaluated for their inhibitory effects on the formation of AGEs. As a result, 4–6 and 10 were found to inhibit the formation of AGEs, with IC50 values of 8.8 ± 1.5, 6.5 ± 0.08, 2.1 ± 0.05, and 8.0 ± 0.41 mM, respectively, whereas aminoguanidine used as a positive control exhibited an IC50 value of 15.6 ± 0.16 mM. The low yields of 1–3, 11, and 12 did not allow the assessment of their inhibitory activity on AGEs formation. Irigenin (6) exhibited the most potent inhibitory activity with an IC50 value of 2.1 mM. Additionally, irigenin 7-O-diglucoside 4 and irigenin 7-O-glucoside 5 also inhibited the formation of AGEs with IC50 values of 8.8 and 6.5 mM, respectively. Conversely, irisolon 4′-O-glucoside 9 and irisolon 4′-O-diglucosides 7 and 8 did not show any inhibitory activity at 20 mM. However, iriskashmirianin 4′-O-glucoside 10, in which the 5-methoxy group of 9 was replaced with a hydroxy group, exhibited inhibitory activity with an IC50 value of 8.0 mM. A number of flavones, flavanes, flavonols, flavanols, and their glycosides have been reported to exhibit inhibitory activity on AGEs formation.13) However, in the case of isoflavones, only representative compounds such as genistein and daidzein have been disclosed to inhibit AGEs formation.14,15) These results suggest that variations in the substituents on the isoflavone skeleton and the binding positions of the sugar moieties influence the inhibitory activity on AGEs formation.
In summary, two new isoflavone triglucosides containing a glucosyl-(1→6)-glucosyl-(1→6)-glucosyl group (1 and 2) were isolated from the underground parts of I. florentina. Isoflavone glycosides with a sugar chain consisting of three d-glucose units have only been reported in Parepigynum fluminense (Apocynaceae).16) This is the first isolation of triglucosyl isoflavonoids from an Iridaceae plant. Among the isolated isoflavonoids, irigenin (6), its glucosides (4 and 5), and iriskashmirianin glucoside (10) inhibited AGEs formation. Irigenin and iriskashmirianin have previously been reported to exhibit cytotoxicity against human cancer cells and anti-tumor promoting properties.17) In this study, the inhibitory activity of 4–6 and 10 on AGEs formation was revealed, suggesting that they may have new potential for anti-aging effects on vascular walls and bones.
Optical rotations (OR) were measured using a JASCO P-1030 (Tokyo, Japan) automatic digital polarimeter. UV and IR spectra were recorded on a JASCO V-630 UV-Vis spectrophotometer and a JASCO FT-IR 620 spectrophotometer, respectively. NMR spectral data were obtained using a JEOL JNM-ECZ-600R (600 MHz for 1H-NMR, JEOL, Tokyo, Japan) spectrometer using standard JEOL pulse programs at 300 K. Herein, chemical shifts are presented as δ values with reference to tetramethylsilane (TMS) as an internal standard. HR-ESI-TOF-MS data were recorded on a Waters-Micromass LCT mass spectrometer (Manchester, U.K). Diaion HP-20 (50 mesh, Mitsubishi-Chemical, Tokyo, Japan), silica gel Chromatorex BW-300 (300 mesh, Fuji-Silysia Chemical, Aichi, Japan), and COSMOSIL 75C18-OPN (75 μM particle size, Nacalai Tesque, Kyoto, Japan) were used for column chromatography (CC). TLC was conducted on precoated Si gel 60 F254 or RP18 F254S plates (0.25 mm thick, Merck, Darmstadt, Germany), and spots were detected by spraying the plates with a 10% H2SO4 aqueous solution, followed by heating. HPLC was performed using a system consisting of a DP-8020 pump (Tosoh, Tokyo, Japan), a Shodex OR-2 detector (Showa-Denko, Tokyo, Japan), and Rheodyne™ injection port (Thermo Fisher Scientific, Waltham, MA, U.S.A). HL-60 cells (JCRB0085) were obtained from the Japanese Collection of Research Bioresources (JCRB) cell bank (Osaka, Japan). All other chemicals used were of biochemical reagent grade.
Plant MaterialsThe underground parts of Iris florentina L. were purchased from Tochimoto Tenkaido Co., Ltd. (Osaka, Japan) in November 2011. A voucher specimen has been deposited in our laboratory (Voucher No. IF-2011-001, Department of Medicinal Pharmacognosy).
Extraction and IsolationThe underground parts of I. florentina (dry weight, 6.0 kg) were extracted with MeOH (45 L). After the MeOH extract was concentrated under reduced pressure, the resulting viscous concentrate (920 g) was passed through a Diaion HP-20 column and successively eluted with H2O-MeOH (7 : 3), H2O-MeOH (1 : 1), MeOH, EtOH, and EtOAc. The H2O-MeOH (1 : 1) eluate fraction (48 g) was subjected to silica gel CC (80 mm i.d. × 300 mm) eluted with stepwise gradient mixtures of EtOAc-MeOH-H2O (19 : 1 : 0.1; 9 : 1 : 0.1; 4 : 1 : 0.1; 2 : 1 : 0.1), which produced 8 fractions (A–I). Fraction H was separated by silica gel CC (50 mm i.d. × 300 mm) eluted with EtOAc-MeOH-H2O (3 : 1 : 0.1; 2 : 1 : 0.1), resulting in 6 subfractions H-1–H-6. Fraction H-5 was purified by ODS silica gel CC (40 mm i.d. × 250 mm) eluted with MeCN-H2O (2 : 9; 2 : 7; 1 : 3) and silica gel CC eluted with CHCl3-MeOH-H2O (3 : 1 : 0.1; 2 : 1 : 0.1) to yield 1 (6.6 mg) and 2 (5.9 mg).
Compound 1: Amorphous solid;
Compound 2: Amorphous solid;
Compounds 1 (1.68 mg) and 2 (1.5 mg) were each separately dissolved in 1 M HCl (dioxane-H2O, 1 : 1, 2 mL). Each solution was heated at 90 °C for 2 h under an argon atmosphere. After cooling, the reaction mixture was neutralized by passing through an Amberlite IRA-96SB (Organo, Tokyo, Japan) column and chromatographed on silica gel eluted with CHCl3-MeOH-H2O (19 : 1 : 0.1) to yield 1a (0.5 mg from 1) and 2a (0.7 mg from 2) and the sugar fractions (0.9 mg from 1; 0.7 mg from 2). Each sugar fraction was analyzed by HPLC under the following conditions. Capcell Pak NH2 UG80 column (4.6 mm i.d. × 250 mm, 5 μm, Shiseido, Tokyo, Japan); solvent: MeCN-H2O (17 : 3); detected by OR; flow rate: 1.0 mL/min. HPLC analysis of the sugar fractions revealed the presence of d-glucose (tR = 23.1 min, positive optical rotation).
Assay for Inhibition of the Production of AGEsThe inhibitory activity of the compounds on AGEs formation was evaluated during the incubation of glyceraldehyde (GA) (Sigma-Aldrich, St. Louis, MO, U.S.A.) and bovine serum albumin (BSA) (Sigma-Aldrich). The BSA was dissolved in 1/15 mol/L phosphate buffer (pH 7.2) to prepare a BSA solution (10 mg/mL). The sample solution was dissolved in 1/15 mol/L phosphate buffer (pH 7.2) to prepare a 20 mM solution. As a positive control, aminoguanidine solution (0.8, 4, 20 mM) was used. First, 50 μL of BSA solution was dispensed into each well of a 96-well black plate (Greiner Bio-one, Frickenhausen, Germany). Next, 40 μL of sample dilution buffer containing the sample or positive control was added. Then, 10 μL of GA solution (500 mM) was added to each well and mixed on a microplate shaker. After mixing, the fluorescence intensity at 0 h of reaction was measured using a Varioskan flash fluorescence microplate reader (Thermo Fisher Scientific) at the excitation wavelength (Ex) 370 nm/fluorescence wavelength (Em) 440 nm (Fluorescent intensity A). The solution was incubated at 37 °C for 24 h under the high humidity conditions. After the termination of the reaction, the fluorescence intensity was measured (Fluorescent intensity B). The inhibitory ratio of the samples was calculated using the following formula:
Inhibition against the production of AGEs (%) = {1 − [(Fluorescent intensity B of sample) − [(Fluorescent intensity A of sample)]/[(Fluorescent intensity B of control) − (Fluorescent intensity A of control)]} × 100
The data on the inhibition of AGEs formation are presented as the mean ± standard error (S.E.). The concentration resulting in 50% AGEs inhibition (IC50) was calculated from a dose-response curve.
The authors declare no conflict of interest.
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