nutrients Nutrients Nutrients Nutrients 2072-6643 Molecular Diversity Preservation International 10.3390/nu2020171 nutrients-02-00171 Article Production of Hesperetin Glycosides by Xanthomonas campestris and Cyclodextrin Glucanotransferase and Their Anti-allergic Activities Shimoda Kei 1 * Hamada Hiroki 2 * Department of Chemistry, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan Department of Life Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan Author to whom correspondence should be addressed: Email: shimoda@med.oita-u.ac.jp (K.S.); Tel.: +81-97-586-5606; Fax: +81-97-586-5619 (K.S.); Email: hamada@dls.ous.ac.jp (H.H.); Tel.:+81-86-256-9473; Fax:+81-86-256-8468 (H.H.) 09 02 2010 022010 2 2 171 180 07 01 2010 01 02 2010 © 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. 2010

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

The production of hesperetin glycosides was investigated using glycosylation with Xanthomonas campestris and cyclodextrin glucanotransferase (CGTase). X. campestris glucosylated hesperetin to its 3'-, 5-, and 7-O-glucosides, and CGTase converted hesperetin glucosides into the corresponding maltosides. The resulting 7-O-glucoside and 7-O-maltoside of hesperetin showed inhibitory effects on IgE antibody production and on O2- generation from rat neutrophils.

 Hesperetin Xanthomonas campestris cyclodextrin glucanotransferase glycoside anti-allergic activity 1. Introduction

Vitamin P is the term used for bioactive flavonoids. Hesperetin (5,7,3'-trihydroxyl-4'-methoxyl-flavanone), which occurs in Citrus species, tomatoes, and apples, is a kind of vitamin P and has been well documented for its medicinal properties as an important Chinese traditional medicine [1,2]. Hesperetin has been studied for its effects on the blood-brain barrier, signal transduction pathway, and certain kinds of cancer [3,4,5,6]. However, hesperetin barely dissolves in aqueous solution, and this low water-solubility limits its further pharmacological exploitation.

Glycosylation is considered to be an important method for the conversion of water-insoluble and unstable organic compounds into the corresponding water-soluble and chemically stable derivatives. Recently, Mizukami et al. reported that glucosyl conjugation was far more effective than cyclodextrin complexation at enhancing the water solubility of hydrophobic compounds such as curcumin [7]. Hesperidin is a hesperetin glycoside, i.e., a 7-O-β-rutinoside of hesperetin. The values of the partition coefficient (logP) of hesperetin and hesperidin are 2.6 and 1.1, respectively [8]. Hesperidin is widely used as vitamin P2, which has biologically beneficial effects such as anticancer, antimutagenic, anticarcinogenic, and cholesterol-lowering activities [9,10,11]. Furthermore, the glycosides of hesperidin have been utilized in food industries as food additives (Hesperidin S (Hayashibara Biochemical Laboratories Ltd.)). Therefore, the glycosides of hesperetin are important from a physiological point of view. One-step biocatalytic glycosylation is useful for synthesis of glycosides rather than chemical glycosylation that requires tedious protection-deprotection procedures [7]. Herein we report the glycosylation of hesperetin into the corresponding glucosides and maltosides by Xanthomonas campestris and cyclodextrin glucanotransferase (CGTase), which has been used for production of Hesperidin S. We also report the inhibitory effects of hesperetin glycosides on IgE antibody formation and on O2- generation from rat neutrophils. Hesperetin glycosides synthesized here may be useful food ingredients which have potent anti-allergic activities.

 2. Results and Discussion 2.1. Production of Hesperetin α-glycosides

The structure of hesperetin and the six newly synthesized vitamin P compounds are shown in Figure 1. The glucosylation of hesperetin (1) was investigated using the lyophilized cells of X. campestris. Incubation of lyophilized X. campestris cells with hesperetin (1) and a sugar donor, i.e., maltose, for five days gave three new compounds, 3'-, 5-, and 7-O-α-glucosides of hesperetin (2-4), which were detected in HEPES-NaOH buffer (10 mM) by HPLC. The glucoside products 2-4 were isolated from n-butanol extracts of the buffer by preparative HPLC (2, 12% yield; 3, 10%; 4, 15%). The compounds 2-4 were individually glycosylated by CGTase to give three new compounds 5-7 in 49, 62, and 50% yield, respectively. The chemical structures of products 5-7 were determined as hesperetin 3'-O-α-maltoside, hesperetin 5-O-α-maltoside, and hesperetin 7-O-α-maltoside, which have not been identified before, by HRFABMS, 1H and 13C NMR, H-H COSY, C-H COSY, and HMBC spectra.

The HRFABMS spectra of 5-7 included pseudomolecular ion [M+Na]+ peaks at 649.1751 for 5, 649.1755 for 6, and 649.1750 for 7 (calculated for C28H34O16Na, 649.1745), indicating that each product consisted of one substrate and two hexoses. The sugar components in these products were determined to be glucose on the basis of the chemical shifts of their carbon signals. The 1H NMR spectra showed two proton signals at δ 5.01 (1H, d, J = 3.6 Hz) and 5.06 (1H, d, J = 3.2 Hz) for 5, δ 5.03 (1H, d, J = 3.0 Hz) and 5.40 (1H, d, J = 3.2 Hz) for 6, and δ 5.05 (1H, d, J = 3.2 Hz) and 5.17 (1H, d, J = 3.0 Hz) for 7, indicating that the glucoside linkage in these three compounds had α-orientations. The HMBC spectra of 5-7 included correlations between the proton signal at δ 5.01 (H-1''') and the carbon signal at δ 80.7 (C-4'') and between the proton signal at δ 5.06 (H-1'') and the carbon signal at δ 146.2 (C-3') for 5, between the proton signal at δ 5.03 (H-1''') and the carbon signal at δ 80.5 (C-4'') and between the proton signal at δ 5.40 (H-1'') and the carbon signal at δ 165.1 (C-5) for 6, and between the proton signal at δ 5.05 (H-1''') and the carbon signal at δ 80.5 (C-4'') and between the proton signal at δ 5.17 (H-1'') and the carbon signal at δ 166.9 (C-7) for 7. These data indicated that 5-7 were α-maltosyl analogues of 1, the sugar moiety of which attached at their 3'-position (5), 5-position (6), and 7-position (7). Thus, products 5-7 were identified as hesperetin 3'-O-α-maltoside (5), hesperetin 5-O-α-maltoside (6), and hesperetin 7-O-α-maltoside (7), respectively. The substrate hesperetin used for this experiment was a chiral compound, the stereochemistry of which was 2S [12,13,14,15,16,17,18,19] (Sigma-Aldrich Co.). On the other hand, it has been reported that hesperidin epimerizes in C2 to form the 2S and 2R epimers [20]. The absolute configuration at C-2 of the aglycone of hesperetin α-glycosides synthesized here is not clarified.

Synthetic route of α-glucosides and α-maltosides of hesperetin-------sequential biocatalytic glycosylation of hesperetin by X. campestris and CGTase.

It has been reported that kojic acid α-glucoside was synthesized by lyophilized X. campestris in the presence of maltose as a sugar donor [21]. This report led us to produce new vitamin P compounds, that is, hesperetin α-glycosides, by lyophilized X. campestris. Six new vitamin P compounds, i.e., hesperetin α-glucosides and hesperetin α-maltosides, were successfully synthesized through sequential biocatalytic α-glycosylation by X. campestris and CGTase. Earlier, some kinds of hesperetin β-glycosides, such as β-glucosides, β-diglucosides, and β-rutinoside, had been isolated from fruits and flowers of Citrus species [22,23,24]. On the other hand, it has been reported that cultured cells of Citrus paradisi glycosylated hesperetin at its 7-position to give 7-O-β-glucoside and 7-O-β-rutinoside [25,26]. Compared to the β-glucosylation with C. paradisi, X. campestris showed high potential for the α-glucosylation at each phenolic hydroxyl groups of hesperetin to give three α-glucosides. This is the first report on the synthesis of hesperetin α-glycosides.

 2.2. Anti-allergic Activity of Hesperetin α-glycosides

The effects of hesperetin α-glucosides 2-4 and hesperetin α-maltosides 5-7 on IgE antibody formation were investigated by in vivo bioassay using ovalbumin as an antigen [27]. The average rat plasma IgE level after treatment of ovalbumin with or without test compounds was examined and the results are summarized in Table 1. Hesperetin 7-O-α-glucoside (4) and 7-O-α-maltoside (7) showed inhibitory action on IgE antibody generation. On the other hand, 3'-O-α-glycosides 2 and 5 and 5-O-α-glycosides 3 and 6 did not inhibit IgE antibody generation.

There is evidence that superoxide anion (O2-), which is generated primarily through the activation of the plasma membrane-bound NADPH-oxidase system, is responsible for allergic reactions, and anti-allergic drugs such as mequitazine have been reported to inhibit O2- generation [28].

nutrients-02-00171-t001_Table 1

Suppressive action of hesperetin α-glycosides 2-7 on IgE antibody formation.

Compound IgE levela
None 393.1 ± 188.7
2 356.6 ± 186.9
3 338.3 ± 163.0
4 155.4 ± 67.2*
5 365.7 ± 126.7
6 374.9 ± 169.1
7 164.6 ± 57.8*

aThe results were expressed as average plasma IgE level of seven rats administered a total of 10 mg/kg of each test compound. Data are presented as mean ± SE. Asterisk indicates significant differences from controls (*P < 0.05).

 nutrients-02-00171-t002_Table 2

Effects of hesperetinα-glycosides 2-7 on O2- generation from rat neutrophils.

Compound %Inhibition a
2 12
3 17
4 59
5 5
6 7
7 46
Mequitazine 62

aThe percentage reduction of the O2- generation was calculated as follows: %Inhibition = [(Pc- Pt)/Pc]x100, where Pt and Pc are the photon count of sample solutions with and without the test compound.

The effects of hesperetin α-glucosides 2-4 and hesperetin α-maltosides 5-7 on O2- generation from rat neutrophils using cypridina luciferin analog-dependent chemiluminescence as a probe for O2- generation were examined according to the previously reported methods [29]. The results are expressed in terms of the percentage reduction of the O2- generation. Percent inhibition was calculated as follows: %Inhibition = [(Pc- Pt)/Pc]x100, where Pt and Pc are the photon count of sample solutions with and without the test compound. As a result, inhibitory effects of hesperetin 7-O-α-glycosides 4 and 7 and an authentic anti-allergic drug on fMLP-induced O2- generation from rat neutrophils are as follows: 4, 59%; 7, 46%; mequitazine, 62% (Table 2). Particularly, hesperetin 7-O-α-glucoside (4) exhibited significant inhibitory activity for O2- generation, to the same level as mequitazine. This is the first time that hesperetin α-glycosides have been shown to have a strong inhibitory effect on O2- generation. Hesperetin 3'-O-α-glycosides 2 and 5 and hesperetin 5-O-α-glycosides 3 and 6 showed no inhibitory action on O2- generation. Previously, we reported that 7-O-β-glycosides of polyphenols, i.e., genistein and quercetin, showed anti-allergic activities, whereas polyphenol glycosides, where the is attached at other phenolic hydroxyl groups, exhibited no anti-allergic actions [29]. These findings suggest that α-glycosides at C-7 of hesperetin did not attenuate the anti-allergic activity of the aglycone, i.e., hesperetin, and that phenolic hydroxyl groups at 3'- and 5-positions of hesperetin might be necessary for hesperetin and its glycosides to act as anti-allergic formulations. Recently, it has been reported that hesperetin is more bioactive than its glycoside hesperidin, and efforts to formulate hesperetin are in progress to improve absorption and its disposition [30,31,32,33]. The hesperetin α-glycosides might act as pro-drugs that release hesperetin by hydrolysis in the living body.

Recently, some β-glycosides of tocopherol (vitamin E) have been reported to act as potential anti-allergic agents [34]. In addition, the β-glycosides of vitamin E showed inhibitory effects on O2- generation from rat neutrophils. It was postulated that inhibition of O2- generation by vitamin E β-glycosides was responsible for their anti-allergic activity [28,34]. Studies on the mechanism by which the hesperetin α-glycosides synthesized here act as anti-allergic compounds are now in progress.

 3. Experimental Section 3.1. General

Substrate, hesperetin, was purchased from Sigma-Aldrich Co. CGTase was purchased from Amano Pharmaceutical Co. Ltd. The NMR spectra were recorded in CD3OD using a Varian XL-400 spectrometer. The chemical shifts were expressed in δ (ppm) referring to tetramethylsilane. The HRFABMS spectra were measured using a JEOL MStation JMS-700 spectrometer. HPLC was carried out on a YMC-Pack R&D ODS column (150 x 30 mm) [solvent: CH3CN:H2O (3:17, v/v); detection: UV (280 nm); flow rate: 1.0 mL/min].

3.2. Bacterial Strain and Culture Conditions

X. campestris was a gift from the Department of Chemistry of Okayama University of Science. Culture medium used for growth of X. campestris had the following composition (in grams per liter): 5 g of maltose, 3 g of peptone, 0.4 g of yeast extract, 0.2 g of MgSO4. The cells were grown in the culture medium with continuous shaking on a rotary shaker (120 rpm) at 30°C.

 3.3. Production of Hesperetin α-glucosides by X. Campestris

The cultures of X. campestris were grown in 500 mL conical flasks containing 200 mL of culture medium at 30 °C. Prior to use for the experiments, the cells were harvested by centrifugation at 8,000 g for 15 min and were lyophilized. Hesperetin α-glucosides were prepared as follows. A total of 2 mmol of hesperetin was added to ten 300-ml conical flasks (0.2 mmol/flask), each of which contained 5 g of lyophilized X. campestris cells and 1 mmol of maltose in 100 ml of 10 mM HEPES-NaOH buffer (pH 7.5). The mixture was incubated continuous shaking on a rotary shaker (120 rpm) for 5 days at 30 °C. The reaction mixture was centrifuged at 8,000 g for 15 min and the supernatant was extracted with n-butanol. The butanol fraction was purified by preparative HPLC on YMC-Pack R&D ODS column to give hesperetin α-glucosides.

 3.4. Production of Hesperetin α-maltosides by CGTase

To a solution containing 0.1 mmol of hesperetin α-glucoside and 5 g of starch in 25 mM of sodium phosphate buffer (pH 7.0) was added 100 U of CGTase. The reaction mixture was stirred at 40 °C for 24 h, and then the mixture was centrifuged at 3,000 g for 10 min. The supernatant was subjected on to a Sephadex G-25 column equilibrated with water to remove CGTase. The fractions containing glycosides were purified by preparative HPLC on YMC-Pack R&D ODS column to give hesperetin α-maltosides.

Spectral data of selected new compounds are as follows.

Hesperetin 3'-O-α-maltoside (5): FABMS m/z: 649.1751 [M+Na]+; 1H NMR (400 MHz, DMSO-d6, δ in ppm): δ 2.77 (1H, dd, J = 17.8, 2.8 Hz, H-3a), 3.08-3.88 (12H, m, H-2'', 2''', 3'', 3''', 4'', 4''', 5'', 5''', 6'', 6'''), 3.33 (1H, dd, J = 17.8, 12.8 Hz, H-3b), 3.78 (3H, s, OCH3), 5.01 (1H, d, J = 3.6 Hz, H-1'''), 5.06 (1H, d, J = 3.2 Hz, H-1''), 5.50 (1H, dd, J = 12.6, 2.8 Hz, H-2), 5.89 (1H, d, J = 2.8 Hz, H-6), 5.90 (1H, d, J = 2.8 Hz, H-8), 7.03 (1H, dd, J = 8.4, 2.0 Hz, H-6'), 7.08 (1H, d, J = 8.4 Hz, H-5'), 7.25 (1H, d, J = 2.0 Hz, H-2'); 13C NMR (100 MHz, DMSO-d6, δ in ppm): δ 43.3 (C-3), 55.5 (OCH3), 60.5 (C-6'''), 60.7 (C-6''), 69.5 (C-4'''), 72.2 (C-5''), 72.4 (C-2''), 73.5 (C-2''', C-3''), 73.9 (C-3''', C-5'''), 78.0 (C-2), 80.7 (C-4''), 94.7 (C-8), 95.7 (C-6), 98.0 (C-1''), 100.5 (C-1'''), 101.4 (C-10), 111.5 (C-5'), 113.6 (C-2'), 120.5 (C-6'), 130.2 (C-1'), 146.2 (C-3'), 149.0 (C-4'), 162.4 (C-9), 163.2 (C-5), 166.3 (C-7), 196.9 (C-4).

Hesperetin 5-O-α-maltoside (6): FABMS m/z: 649.1755 [M+Na]+; 1H NMR (DMSO-d6): δ 2.76 (1H, dd, J = 17.8, 2.8 Hz, H-3a), 3.05-3.89 (12H, m, H-2'', 2''', 3'', 3''', 4'', 4''', 5'', 5''', 6'', 6'''), 3.33 (1H, dd, J = 17.8, 12.8 Hz, H-3b), 3.78 (3H, s, OCH3), 5.03 (1H, d, J = 3.0 Hz, H-1'''), 5.40 (1H, d, J = 3.2 Hz, H-1''), 5.51 (1H, dd, J = 12.6, 2.8 Hz, H-2), 6.08 (1H, d, J = 2.8 Hz, H-6), 6.12 (1H, d, J = 2.8 Hz, H-8), 6.90 (1H, dd, J = 8.4, 2.0 Hz, H-6'), 7.00 (2H, m, H-2', 5'); 13C NMR (DMSO-d6): δ 43.0 (C-3), 55.5 (OCH3), 60.3 (C-6'''), 60.6 (C-6''), 69.5 (C-4'''), 72.2 (C-5''), 72.5 (C-2''), 73.5 (C-2''', C-3''), 74.1 (C-3''', C-5'''), 78.0 (C-2), 80.5 (C-4''), 93.7 (C-8), 94.8 (C-6), 99.7 (C-1''), 100.6 (C-1'''), 103.5 (C-10), 111.7 (C-5'), 113.8 (C-2'), 117.7 (C-6'), 130.8 (C-1'), 146.0 (C-3'), 147.7 (C-4'), 162.5 (C-9), 165.1 (C-5), 165.0 (C-7), 198.9 (C-4).

Hesperetin 7-O-α-maltoside (7): FABMS m/z: 649.1750 [M+Na]+; 1H NMR (DMSO-d6): δ 2.76 (1H, dd, J = 17.8, 2.8 Hz, H-3a), 3.07-3.83 (12H, m, H-2'', 2''', 3'', 3''', 4'', 4''', 5'', 5''', 6'', 6'''), 3.32 (1H, dd, J = 17.8, 12.8 Hz, H-3b), 3.78 (3H, s, OCH3), 5.05 (1H, d, J = 3.2 Hz, H-1'''), 5.17 (1H, d, J = 3.0 Hz, H-1''), 5.49 (1H, dd, J = 12.6, 2.8 Hz, H-2), 6.16 (1H, d, J = 2.8 Hz, H-6), 6.22 (1H, d, J = 2.8 Hz, H-8), 6.88 (1H, dd, J = 8.4, 2.0 Hz, H-6'), 6.92 (2H, m, H-2', 5'); 13C NMR (DMSO-d6): δ 42.2 (C-3), 55.5 (OCH3), 60.4 (C-6'''), 60.6 (C-6''), 69.5 (C-4'''), 72.2 (C-5''), 72.5 (C-2''), 73.8 (C-2''', C-3''), 74.1 (C-3''', C-5'''), 78.2 (C-2), 80.5 (C-4''), 95.4 (C-6), 95.9 (C-8), 100.1 (C-1''), 100.7 (C-1'''), 103.2 (C-10), 111.5 (C-5'), 113.9 (C-2'), 117.6 (C-6'), 130.7 (C-1'), 146.0 (C-3'), 147.8 (C-4'), 162.2 (C-9), 162.7 (C-5), 166.9 (C-7), 196.7 (C-4).

 3.5. Suppressive Action on IgE Antibody Formation

The inhibitory action of hesperetin α-glycosides on IgE antibody formation was examined as follows. Ovalbumin was used as the antigen (1 mg/rat), and Al(OH)3 and pertussis vaccine were used as the adjuvants (20 mg and 0.6 mL/rat, respectively). Sensitization was made by injecting a mixture (0.6 mL) of the antigen and the adjuvant into the paws of each rat (male, ca. 200 g). Paw edema was measured 24 h after injection and the treated rats were divided in groups with an equal average swelling volume. Each sample was dissolved in physiological saline containing 10% Nikkol and the solution was injected daily into the rat for 11 days starting on the day of grouping. The amount of IgE was measured by the passive cutaneous anaphylaxis method on the 15th day [27]. The results were expressed as average of plasma IgE level of seven rats administered a total of 10 mg/kg of each test compound.

 3.6. Inhibitory Action on O2- Generation from Rat Neutrophils

Effects of hesperetin α-glycosides on O2- generation from rat neutrophils were examined as follows. Male Wistar rats, each weighing 250 to 300 g, were used. Under ether anesthesia, whole blood was collected from the carotid artery and diluted twice with Hanks' balanced salt solution (HBSS) (pH 7.4). Neutrophils were purified by Percoll density gradient centrifugation. O2- generation from rat neutrophils was measured by the cypridina luciferin analog-dependent chemiluminescence method [28]. An authentic antiallergic agent, mequitazine, was used as a positive control. Neutrophil suspensions (106 cells/mL) were incubated for 3 min in HBSS containing 0.4 mM of cypridina luciferin analog and 50 μM of hesperetin α-glycoside or authentic antiallergic agent at 37 °C in the dark. Five seconds later, N-formyl-Met-Leu-Phe (fMLP) (2.5 μM) was added into the assay mixture. Cypridina luciferin analog-dependent chemiluminescence was monitored with a Lumicounter ATP-237 (Advantec Co. Ltd.). The results are expressed in terms of the percentage reduction of the O2- generation from rat neutrophils at 5 min after the administration of fMLP by test compounds.

 4. Conclusions

Hesperetin α-glycosides, i.e., α-glucosides and α-maltosides, were successfully synthesized through sequential biocatalytic glycosylation by X. campestris and CGTase for the first time. It should be emphasized that new vitamin P glycosides, i.e., hesperetin α-maltosides, which are glycosylated at each phenolic hydroxyl group of hesperetin, can be prepared by the present two-step glycosylation system. Hesperetin 7-O-α-glucoside and hesperetin 7-O-α-maltoside showed suppressive action on IgE antibody formation and exhibited inhibitory effects on O2- generation from rat neutrophils. These new vitamin P compounds would be useful food ingredients with potent anti-allergic activities. Further studies on the physiological activity of hesperetin α-glycosides, such as cytotoxicity and anticancer activity, are now in progress.

 References and Notes Formica J.V. Regelson W. Review of the biology of quercetin and related bioflavonoids Food Chem. Toxicol. 1995 33 1061 1080 10.1016/0278-6915(95)00077-18847003 Yáñez J.A. Remsberg C.M. Miranda N.D. Vega-Villa K.R. Andrews P.K. Davies N.M. Pharmacokinetics of selected chiral flavonoids: hesperetin, naringenin and eriodictyol in rats and their content in fruit juices Biopharm. Drug Dispos. 2008 29 63 82 10.1002/bdd.58818058792 So F.V. Guthrie N. Chambers A.F. Carroll K.K. Inhibition of proliferation of estrogen receptor-positive MCF-7 human breast cancer cells by flavonoids in the presence and absence of excess estrogen Cancer lett. 1997 112 127 133 10.1016/S0304-3835(96)04557-09066718 Mitsunaga Y. Takanaga H. Matsuo H. Naito M. Tsuruo T. Ohtani H. Sawada Y. Effect of bioflavonoids on vinicristine transport across blood-brain barrier Eur. J. Pharmacol. 2000 395 193 201 10.1016/S0014-2999(00)00180-110812049 O’Prey J. Brown J. Fleming J. Harrison P.R. Effects of dietary flavonoids on major signal transduction pathways in human epithelial cells Biochem. Pharmacol. 2003 66 2075 2088 10.1016/j.bcp.2003.07.00714609732 Youdim K.A. Dobbie M.S. Kuhnle G. Proteggente A.R. Abbott N.J. Rice-Evans C. Interaction between flavonoids and the blood-brain barrier: in vitro study J. Neurochem. 2003 85 180 192 10.1046/j.1471-4159.2003.01652.x12641740 Kaminaga Y. Nagatsu A. Akiyama T. Sugimoto N. Yamazaki T. Maitani T. Mizukami H. Production of unnaturalglycosides of curcumin with drastically enhanced water solubility by cell suspension cultures of Catharanthus roseus. FEBS Lett. 2003 555 311 316 14644434 10.1016/S0014-5793(03)01265-1 Perrissoud D. Testa B. Inhibiting or potentiating effects of flavonoids on carbon tetrachloride-induced toxicity in isolated rat hepatocytes Arzneimittelforschung. 1986 36 1249 1253 3778562 Zhang M. Zhang J.P. Ji H.T. Wang J.S. Qian D.H. Effect of six flavonoids on proliferation of hepatic stellate cells in Vitro Acta Pharmacol. Sin. 2000 21 253 256 11324426 Garg A. Garg S. Zaneveld L.J.D. Singla A.K. Chemistry and pharmacology of the Citrus bioflavonoid hesperidin Phytother.Res. 2001 15 655 669 10.1002/ptr.107411746857 Chiba H. Uehara M. Wu J. Wang X. Masuyama R. Suzuki K. Kanazawa K. Ishimi Y. Hesperidin, a citrus flavonoid, inhibits bone loss and decreases serum and hepatic lipids in ovariectomized mice J. Nutr. 2003 133 1892 1897 12771335 Ng S.C. Ong T.T. Fu P. Ching C.B. Enantiomer separation of flavour and fragrance compounds by liquid chromatography using novel urea-covalent bonded methylated beta-cyclodextrins on silica J. Chromatogr. A 2002 968 31 40 10.1016/S0021-9673(02)00840-312236512 Asztemborska M. Miśkiewicz M. Sybilska D. Separation of some chiral flavanones by micellar electrokinetic chromatography Electrophoresis 2003 24 2527 2531 10.1002/elps.20030550812900864 Caccamese S. Caruso C. Parrinello N. Savarino A. High-performance liquid chromatographic separation and chiroptical properties of the enantiomers of naringenin and other flavanones J. Chromatogr. A 2005 1076 155 162 10.1016/j.chroma.2005.04.02415974082 Kwon C. Park H. Jung S. Enantioseparation of some chiral flavanones using microbial cyclic beta-(1-->3),(1-->6)-glucans as novel chiral additives in capillary electrophoresis Carbohydr. Res. 2007 342 762 766 10.1016/j.carres.2006.12.01817234165 Yáñez J.A. Remsberg C.M. Miranda N.D. Vega-Villa K.R. Andrews P.K. Davies N.M. Pharmacokinetics of selected chiral flavonoids: hesperetin, naringenin and eriodictyol in rats and their content in fruit juices Biopharm. Drug Dispos. 2008 29 63 82 10.1002/bdd.58818058792 Kwon C. Paik S.R. Jung S. Enantiomeric separation of some flavanones using shinorhizobial linear octasaccharides in CE Electrophoresis 2008 29 4284 4290 10.1002/elps.20080012718924103 Si-Ahmed K. Tazerouti F. Badjah-Hadj-Ahmed A.Y. Aturki Z. D'Orazio G. Rocco A. Fanali S. Optical isomer separation of flavanones and flavanone glycosides by nano-liquid chromatography using a phenyl-carbamate-propyl-beta-cyclodextrin chiral stationary phase J. Chromatogr. A 2009 (in press) Si-Ahmed K. Tazerouti F. Badjah-Hadj-Ahmed A.Y. Aturki Z. D'Orazio G. Rocco A. Fanali S. Analysis of hesperetin enantiomers in human urine after ingestion of blood orange juice by using nano-liquid chromatography J. Pharm. Biomed. Anal. 2010 51 225 229 10.1016/j.jpba.2009.08.01519740618 Yáñez J.A. Andrews P.K. Davies N.M. Methods of analysis and separation of chiral flavonoids J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007 848 159 181 17113835 10.1016/j.jchromb.2006.10.052 Hsieh H.-J. Giridhar R. Wu W.-T. Regioselective formation of kojic acid-7-o-alpha-D-glucopyranoside by whole cells of mutated Xanthomonas campestris Enz. Microb. Technol. 2007 40 324 328 10.1016/j.enzmictec.2006.04.014 Sadykov A.A. Isaev K.I. Ismailov A.I. Polyphenols in the bark of Persica vulgaris roots and flowers Khim. Prirod. Soed. 1975 11 281 282 Rousseff R.L. Martin S.F. Youtsey C.O. Quantitative survey of narirutin, naringin, hesperidin and neohesperidin in citrus J. Agric. Food Chem. 1987 35 1027 1030 10.1021/jf00078a040 Del Rio J.A. Fuster M.D. Sabater F. Porras I. Garcia-Lidon A. Ortuno A. Effect of benzylaminopurine on the flavanones hesperidin, hesperetin 7-O-glucoside and pruning in tangelo nova fruits J. Agric. Food Chem. 1995 43 2030 2034 10.1021/jf00056a014 Lewinsohn E. Berman E. Mazur Y. Gressel J. Glucosylation of exogenous flavanones by grapefruit (Citrus paradisi) cell cultures Phytochemistry 1986 25 2531 2535 10.1016/S0031-9422(00)84502-1 Lewinsohn E. Berman E. Mazur Y. Gressel J. Glucosylation and (1-6) rhamnosylation of exogenous flavanones by undifferentiated Citrus cell cultures Plant Science 1989 61 23 28 10.1016/0168-9452(89)90114-3 Koda A. Miura T. Inagaki N. Sakamoto O. Arimura A. Nagai H. Mori H. A method for evaluating anti-allergic drugs by simultaneously induced passive cutaneous anaphylaxis and mediator cutaneous reactions Int. Arch. Allergy. Appl. Immunol. 1990 92 209 216 10.1159/0002351791703513 Akagi M. Katakuse Y. Fukuishi N. Kan T. Akagi R. Superoxide anion-induced histamine release from rat peritoneal mast cells Biol. Pharm. Bull. 1994 17 732 734 10.1248/bpb.17.7327522732 Shimoda K. Kobayashi T. Akagi M. Hamada H. Hamada H. Synthesis of oligosaccharides of genistein and quercetin as potential anti-inflammatory agents Chem. Lett. 2008 37 876 877 10.1246/cl.2008.876 Saija A. Scalese M. Lanza M. Marzullo D. Bonina F. Castelli F. Flavonoids as antioxidant agents: importance of their interaction with biomembranes Free Radic. Biol. Med. 1995 19 481 486 10.1016/0891-5849(94)00240-K7590397 Ficarra R. Tommasini S. Raneri D. Calabrò M.L. Di Bella M.R. Rustichelli C. Gamberini M.C. Ficarra P. Study of flavonoids/beta-cyclodextrins inclusion complexes by NMR, FT-IR, DSC, X-ray investigation J. Pharm. Biomed. Anal. 2002 29 1005 1014 12110385 10.1016/S0731-7085(02)00141-3 Tommasini S. Calabrò M.L. Stancanelli R. Donato P. Costa C. Catania S. Villari V. Ficarra P. Ficarra R. The inclusion complexes of hesperetin and its 7-rhamnoglucoside with (2-hydroxypropyl)-beta-cyclodextrin J. Pharm. Biomed. Anal. 2005 39 572 580 10.1016/j.jpba.2005.05.00915985355 Mishra P.R. Al Shaal L. Müller R.H. Keck C.M. Production and characterization of Hesperetin nanosuspensions for dermal delivery Int. J. Pharm. 2009 371 182 189 10.1016/j.ijpharm.2008.12.03019162147 Shimoda K. Kondo Y. Akagi M. Abe K. Hamada H. Hamada H. Synthesis of α-tocopheryl disaccharides as potential antiallergic agents Chem. Lett. 2007 36 570 571 10.1246/cl.2007.570