ESPERIENZE DERMATOLOGICHE - DERMATOLOGICAL EXPERIENCES 2015;17:97-101

ARTICOLI ORIGINALI - ORIGINAL ARTICLES

Antiallergic and antioxidant properties of a lecithinbased delivery form of quercetin: an in vitro evaluation Proprietà antiallergiche e antiossidanti di una formulazione di quercitina basata su lecitina: una valutazione in vitro

IN C ER O V P A Y R M IG E H DI T C ® A

S. TOGNI 1, G. MARAMALDI 1, M. MENEGHIN 1, L. GIACOMELLI 2 R. CATTANEO 3, R. EGGENHOFFNER 2, S. BURASTERO 4 1Indena S.p.A, Milan, Italy;

2Department of Surgical Sciences and Integrated Diagnostics, School of Medicine, Genova University, Genoa, Italy 3Abich, Verbania, Italy

4San Raffaele Scientific Institute, Milan, Italy

Abstract - Riassunto

Aim. Quercetin is a very abundant flavonoid that has many potential benefits for human health, especially for its antioxidant, antiinflammatory and antiallergic properties. In this study we present two in vitro laboratory experiments performed to assess the antioxidative and antiallergic properties of quercetin. Methods. In the first experiment the effect of quercetin on UV-induced oxidative damage was tested on a stabilized human keratinocytes cell line, using a fluorogenic probe (dichloroflurescein diacetate) to measure the concentration of reactive oxygen species (ROS) inside the cells. Of note, we used a particular formulation of quercetin, characterized by a phospholipids based delivery system that increases the ability of quercetin to permeate the skin. In the second experiment we evaluated the ability of quercetin phospholipids to inhibit basophils degranulation on rat basophil cells expressing the human receptor FcεRI.The positive control was obtained by cross-linking the receptor FcεRI with a specific policlonal antibody; in addition the effect of quercetin was compared to that of hyaluronic acid (HA) which is capable to inhibit the effect of the polyclonal antibody. Results.The first experiment showed that quercetin phospholipids 1% was able to reduce ROS formation caused by UVA exposure in a dose-dependent manner and in response to irradiation levels ranging from 1 to 5 J. The second experiment revealed that quercetin was able to remarkably reduce basophils degranulation in a dose-dependent manner and at similar levels if compared to the positive control (HA). Conclusion. The results of our in vitro experiments provide further evidence on the ability of quercetin to protect the cells against various cellular insults, thanks to its antioxidant and antiallergic properties. Key words: quercetin - antioxidants - antiallergic agents - reactive oxygen species - basophil degranulation test. Obiettivo. Quercetina è un flavonoide particolarmente diffuso, con potenziali effetti benefici in particolare grazie alle sue proprietà antiossidanti, anti-infiammatorie e antiallergiche. In questo studio presentiamo due esperimenti in vitro condotti con l’obiettivo di valutare ulteriormente le proprietà antiossidanti e anti-allergiche di quercetina. Metodi. Nel primo esperimento gli effetti di quercetina sul danno ossidativo indotto da UV sono stati testati su una linea di cheratinociti umani stabilizzati, utilizzando una sonda fluorogenica (diclorofluerescina diacetato) per misurare la concentrazione intracellulare di radicali. È stata utilizzata una particolare formulazione di quercetina, caratterizzata da un sistema di delivery basato su fosfolipidi, che aumenta la penetrazione tessutale di quercetina. Nel secondo esperimento, abbiamo valutato la capacità di quercetina nell’inibire la degranulazione dei basofili in un modello murino esprimente il recettore umano FcεRI. È stato utilizzato un controllo positivo attraverso un cross-link del recettore FcεRI con un anticorpo specifico; inoltre, l’effetto di quercetina è stato confrontato con quello di acido ialuronico, in grado di inibire l’effetto dell’anticorpo. Risultati. Il primo esperimento ha mostrato che quercetina fosfolipidca 1% riduce la formazione di radicali liberi in modo dose-dipendente e in risposta a livelli di radiazione pari a 1-5 J. Nel secondo esperimento, quercetina ha nettamente ridotto la degranulazione dei basofili in modo dose-dipendente e in misura confrontabile al controllo positivo. Conclusioni. Questi risultati in vitro mostrano ulteriormente l’effetto protettivo di quercetina nei confronti di diversi tipi di danni cellulari, grazie alle sue proprietà antiossidanti e antiallergiche. Parole chiave: quercetina - antiossidanti - antiallergici - specie reattive dell’ossigeno - test di degranulazione dei basofili.

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reacts with intracellular ROS and is oxidized into dichlorofluorescin (DCF), a highly fluorescent molecule whose levels are directly proportional to the concentration of ROS inside the cells. The cells were initially exposed to the following samples/ controls and incubated overnight in standard conditions (37° C, 95% humidity, 5% CO2): —— negative control; —— positive control: 0.15 mg/mL ascorbic acid (vitamin C); —— Quercevita® 1% cream for external use (Quercetin phospholipids 1%): 2 µl/mL, 5 µl/mL, 10 µl/mL. Each condition was tested in triplicate. After the overnight treatment, the cells were exposed to increasing amounts of UVA rays, with a power of 2.85 mW/cm2 (irradiation doses: 1, 3 and 5 Joules respectively). A fourth set of cells was kept in the dark in identical conditions in order to evaluate the basal oxidative levels and the possible presence of non-specific fluorescence. After the irradiation, the cells were incubated for 20 minutes at 37° C in a 10 µM DCFDA solution and then the plates were read using a fluorimeter to measure the signal generated by ROS. Cells vitality was also measured using the MTT vitality assay.The test is based on the use of 3-[4,5-dimethylthiazol2-yl]-2,5- diphenyl tetrazolium bromide (MTT), which, is normally yellow in solution but forms purple crystals when its tetrazolium ring is cleaved by the mitochondrial dehydrogenases of living cells. The formazan produced can be quantified spectrophotometrically at 550 nm and is used to calculate the number of living cells, as follows: % of viable cells =(OD[550 nm] test product / mean OD[550 nm] negative control) x 100

Materials and methods

To evaluate the ability of quercetin phospholipids (powder) to inhibit basophils degranulation, we conducted an experiment on rat basophil cells (RBL-SX38) (a kind gift by M. H. Jouvin and J. P Kinet, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02115, USA), stably expressing human FcεRI alpha, beta and gamma chains.11 The cells were cultured in complete medium supplemented with 800 µg/mL Geneticin (Sigma), as previously described.12 The positive control was obtained by cross-linking the receptor FcεRI with a specific policlonal antibody against the α-chain; conversely, as negative control, no sample was added to the medium, to test the spontaneous cellular degranulation. The maximal degranulation of RBL-SX38 was obtained from a Triton X-100 lysate of the cell monolayer. The effect of quercetin phospholipid (1.25-10 µg/mL) was compared to that of hyaluronic acid monosodic (HA; 1.25-10 mg/mL). The mast cell stabilizer disodium cromoglycate (DSCG) in this experimental system was not capable to inhibit the effect of the polyclonal antibody against

IN C ER O V P A Y R M IG E H DI T C ® A

Quercetin is the most abundant flavonoid, which can be found in a variety of food, including vegetables and red wines.1, 2 As showed in a number of clinical and preclinical studies, this molecule has many potential beneficial effects on human health, including antihypertension, cardio and gastro-protective, analgesic and anticancer activities.3 One of the most interesting properties of quercetin is its antioxidant effect 3 attained through a strong scavenger activity against reactive oxygen species (ROS),4 the ability to chelate transition metals and a protective effect against lipid peroxidation.5 In particular, quercetin counteracts the cellular oxidative stress caused by UVA irradiation of the skin, which is responsible of DNA damage, photo-aging and solar elastosis.6 Quercetin presents also anti-inflammatory properties, as it inhibits the production of inflammatory producing enzymes, such as cyclo-oxygenase (COX) and lypoxigenase (LOX),7, 8 and various cytokines, including tumor necrosis factor (TNF) –α.9 In addition quercetin interacts with basophils and mast cells, which play a central role in the induction of allergic diseases. Their activation, triggered by the contact between specific antigens and human IgE receptors, leads in fact to a process called degranulation, characterized by a massive release of inflammatory mediators (histamine, leukotriens and prostaglandins).10 In this paper, we present two in vitro laboratory experiments conducted to evaluate the antioxidant and antiallergic properties of quercetin. In the former, we evaluated the ability of quercetin (formulated in a specific phytosome delivery system) to reduce ROS formation after UVA exposure of a cell line of human keratinocytes that mimic the cutaneous human tissue. In the latter, we tested the ability of quercetin to reduce rat basophils degranulation after their IgE-mediated activation.

The study was approved by the committee on research ethics at the institution in which the research was conducted and any informed consent from human subjects was obtained as required.

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S.Togni, G. Maramaldi, M. Meneghin, L. Giacomelli, R. Cattaneo, R. Eggenhoffner, S. Burastero

Antioxidant activity and UVA protection

In the first experiment the effect of quercetin on UVinduced oxidative damage was tested on a stabilized human keratinocytes cell line (HaCaT): this is a reliable model for the cutaneous human tissue since it is derived from adult human skin and maintains a complete epidermal differentiation capability. Of note, we used a particular formulation of quercetin (Quercevita®, 1% cream for external use, received by Indena S.p.A.), which is characterized by a phospholipids based delivery system used to increase the ability of quercetin to permeate the skin. The test performed was based on the use of dichloroflurescein diacetate (DCFHDA); this fluorogenic probe 98

Antiallergic effect and inhibition of basophils degranulation

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the α-chain, whereas HA acted as a very efficient inhibitor. We assume that this may depend on the different RBL cell derivative we used, as compared to other RBL cellular models (RBL-2H3) where DSCG was reported to be effective in inhibiting basophil degranulation.13 The extent of basophils degranulation was evaluated by measuring the release of β-hexosaminidase — a mediator secreted after basophils activation — whose levels are quantified spectrophotometrically at 405 nm, as previously described by Vangelista et al.14 The percentage of basophils degranulation inhibition was calculated as follows:

IN C ER O V P A Y R M IG E H DI T C ® A

% degranulation inhibition =100([OD sample+OD PC-OD NC]/ [OD PC-OD NC])*100,

with PC: positive control; NC: negative control; OD: optical density measured at 405 nm.

Results

10µl/ml with UVA 1 J. Treatment with Vitamin C resulted in a partial, not dose-dependent inhibition of ROS (Figure 1). Moreover, exposure to Quercetin phospholipids 1% did not relevantly reduce the viability of cell cultures. Cells viability ranged from 85.49% to 110.30% for different quercetin concentrations and different UVA exposures. Of note, administration of Vitamin C resulted in a slight increase in cells viability (range 103.39-108.33). The results of the second study are summarized in Tables I, II. As expected, non-stimulated cells reported limited degranulation and low OD values (mean OD 0.056), while in the positive control we observed a remarkable increase in β-hexosaminidase release (OD 0.422) and the maximum degranulation obtained with Triton X 1% resulted in the highest OD (0.697) (Table I). When HA or quercetin were added to the positive control, a remarkable reduction in basophils degranulation was observed. Indeed OD values were considerably decreased and were comparable to those registered in the negative control (0.050-0.063 for HA and 0.60-0.054 for quercetin) (Table I). Of note, the effect of both samples was dose-dependent and the inhibition of degranulation ranged from 52.2% (1.25 mg/mL) to 83.6% (10 mg/mL) for HA and from 67.5% (1.25 µg/mL) to 79.2% (10 µg/ml) for quercetin (Table II). Interestingly, the presence of HA or quercetin alone, with no IgE stimulation, did not result in a decrease in basophils degranulation (OD values ranged from 0.175 to 0.132 for HA and from 0.231 to 0.116 for quercetin). However a dose-dependent trend was observed since the higher the concentration of the sample, the lower the OD value measured.

The results of the first experiment showed that Quercetin phospholipids 1% was able to reduce ROS formation caused by UVA exposure at all tested concentrations (range 2 -10 µl/mL) and in response to different irradiation levels (UVA ranging from 1 to 5 J). Quercetin phospholipids 1% induced a dose-dependent inhibition of ROS; indeed the inhibition was limited at 2 µl/mL (from 10.87% to 16.08% at increasing UVA exposures) and considerably increased at 5 and 10 µl/mL, up to 39.87% for Quercetin phospholipids 1%

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Properties of quercetin - Proprietà della quercetina

Figure 1.—Percentage of ROS inhibition achieved with Quercetin phospholipids 1% or Vitamin C after UVA exposure.

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TABLE I.—Extent of basophils degranulation measured in different samples. % Samples

OD (405 nm)

No treatment (NC)

0.056

Anti-human IgE receptor antibody (PC)

0.422

Triton X 1% (maximum degranulation)

0.697

HA Quercetin + HA

PC

10 mg/mL

5 mg/mL

2.5 mg/mL

1.25 mg/mL

0.132

0.145

0.198

0.175

10 µg/mL

5 µg/mL

2,5 µg/mL

1.25 µg/mL

0.116

0.134

0.201

0.231

10 mg/mL

5 mg/mL

2.5 mg/mL

1.25 mg/mL

0.050

0.053

0.050

0.063

IN C ER O V P A Y R M IG E H DI T C ® A

PC

+ Quercetin

10 µg/mL

5 µg/mL

2.5 µg/mL

1.25 µg/mL

0.060

0.052

0.059

0.054

HA: hyaluronic acid; NC: negative control; OD: optical density; PC: positive control.

TABLE II.—Degranulation inhibition observed with hyaluronic acid and quercetin on basophils activated via IgE receptor. % degranulation inhibition

HA Quercetin HA: hyaluronic acid.

Discussion

10 mg/ mL

5 mg/mL

2.5 mg/ mL

1.25 mg/ mL

83.6

78.7

60.4

52.2

10 µg/ mL

5 µg/ mL

2.5 µg/ mL

1.25 µg/ mL

79.2

75.7

61.2

67.5

The results of our in vitro experiments provide further evidence on the ability of quercetin to protect the cells against various cellular insults, thanks to its antioxidant and antiallergic properties. Quercetin exerts a photoprotective role by reducing the release of ROS induced by UVA irradiation. The ability of quercetin to protect skin cells against UV-induced damages has been investigated in previous studies.15, 16 Erden Inal et al. observed that quercetin was able to limit the increase in malondialdheyde (MDA) — an end-product of lipid peroxidation — and the reduction in antioxidant enzymes (glutathione peroxidase, glutathione reductase, catalase and superoxide dismutase) caused by UVA irradiation of the skin in rats.15 In another in vivo study, Vicentini et al. reported that quercetin exerted a skin-protective action against UVB radiation, by preventing the irradiation-induced depletion of glutathione (GHS) — one of the most important endogenous antioxidant mechanism — and by reducing the secretion/activation of matrix metalloproteinases, responsible for skin collagen and elastin degradation that contributes to skin photoaging.16 Noteworthy, quercetin presents poor water solubility

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and low skin permeability,17 characteristics that can hamper its use as topical protective agent. Our results suggest that the use of a phytosome deliver system might be effective in overcoming these issues, favoring quercetin passage through the cells where it can exert its protective antioxidant action. This study also provides further evidence on the antiallergic properties of quercetin, that seems to be able to effectively inhibit basophils degranulation in a dose-dependent fashion, as already reported in previous studies.18, 19 Interestingly, the maximum inhibitory activity (80-100% inhibition) registered by Middelton et al. was achieved with quercetin 50µM (15 µg/mL), a value extremely similar to that registered in our study (10 µg/mL).18 In another in vitro experiment on flavonoids antiallergic action, Middleton et al. observed that the inhibitory activity of quercetin was higher when basophils were stimulated by IgE-dependent ligands (antigen, anti-IgE, and concanavalin A).19 In a similar way we observed that quercetin was able to reduce degranulation in case of IgE-dependent stimulus, but no interference was detected on spontaneous degranulation. Of note, quercetin has also proved to inhibit IgEmediated release of histamine, leukotrienes and pros-

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taglandin D (2) from mast cells, thus exerting an additional antiallergic activity.20 Last, it is important to note that quercetin exerts also an anti-inflammatory action, thanks to which it can reduce the negative effects produced by the inflammatory-mediators released from mast cells and basophils.

Conclusions Overall, our studies further support the important role of quercetin as a skin photoprotective and antiallergic agent. Further investigations and clinical trials will be important to confirm these activities in human subjects.

References 11. Wiegand TW,Williams PB, Dreskin SC, Jouvin MH, Kinet JP,Tasset D. High-affinity oligonucleotide ligands to human IgE inhibit binding to Fc epsilon receptor I. J Immunol 1996;157:221-30. 12. Vangelista L, Cesco-Gaspere M, Lorenzi R, Burrone O. A minimal receptor-Ig chimera of human FcepsilonRI alpha-chain efficiently binds secretory and membrane IgE. Protein Eng 2002;15:51-7. 13. Kuba-Miyara M, Agarie K, Sakima R, Imamura S, Tsuha K, Yasumoto T, et al. Inhibitory effects of an ellagic acid glucoside, okicamelliaside, on antigen-mediated degranulation in rat basophilic leukemia RBL-2H3 cells and passive cutaneous anaphylaxis reaction in mice. Int Immunopharmacol 2012;12:675-81. 14. Vangelista L, Soprana E, Cesco-Gaspere M, Mandiola P, Di Lullo G, Fucci RN, et al. Membrane IgE binds and activates Fc epsilon RI in an antigen-independent manner. J Immunol 2005;174:5602-11. 15. Erden Inal M, Kahraman A, Köken T. Beneficial effects of quercetin on oxidative stress induced by ultraviolet A. Clin Exp Dermatol 2001;26:536-9. 16. Vicentini FT, Simi TR, Del Ciampo JO, Wolga NO, Pitol DL, Iyomasa MM, et al. Quercetin in w/o microemulsion: in vitro and in vivo skin penetration and efficacy against UVB-induced skin damages evaluated in vivo. Eur J Pharm Biopharm 2008;69:948-57. 17. Bose S, Du Y, Takhistov P, Michniak-Kohn B. Formulation optimization and topical delivery of quercetin from solid lipid based nanosystems. Int J Pharm 2013;441:56-66. 18. Middleton E Jr, Drzewiecki G, Krishnarao D. Quercetin: an inhibitor of antigen-induced human basophil histamine release. J Immunol 1981;127:546-50. 19. Middleton E Jr, Drzewiecki G. Flavonoid inhibition of human basophil histamine release stimulated by various agents. Biochem Pharmacol 1984;33:3333-8. 20. Kim M, Lim SJ, Kang SW, Um BH, Nho CW. Aceriphyllum rossii extract and its active compounds, quercetin and kaempferol inhibit IgE-mediated mast cell activation and passive cutaneous anaphylaxis. J Agric Food Chem 2014;62:3750-8.

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 1. Bhatia N, Agarwal C, Agarwal R. Differential responses of skin cancer-chemopreventive agents silibinin, quercetin, and epigallocatechin 3-gallate on mitogenic signaling and cell cycle regulators in human epidermoid carcinoma A431 cells. Nutr Cancer 2012;39:292-9.  2. Pekal A, Biesaga M, Pyrzynska K. Interaction of quercetin with copper ions: complexation, oxidation and reactivity towards radicals. Biometals 2011;24:41-9.   3. Kelly GS. Quercetin monograph. Altern Med Rev 2011;16:172-94.   4. Cushnie TP, Lamb AJ. Antimicrobial activity of flavonoids. Int J Antimicrob Agents 2005;26:343-56.  5. Wagner C, Vargas AP, Roos DH, Morel AF, Farina M, Nogueira CW, et al. Comparative study of quercetin and its two glycoside derivatives quercetin and rutin against methylmercury (MgHg)-induced ROS production in rat brain slices. Arch Toxicol 2010;84:89-97.   6. Nechifor MT, Niculiţe CM, Urs AO, Regalia T, Mocanu M, Popescu A, et al. UVA Irradiation of Dysplastic Keratinocytes: Oxidative Damage versus Antioxidant Defense. Int J Mol Sci 2012;13:1671836.   7. Kim HP, Mani I, Ziboh VA. Effects of naturally-occurring flavonoids and bioflavonoids on epidermal cyclooxygenase from guinea pigs. Prostaglandins Leukot Essent Fatty Acids 1998;58:17-24.  8. Lee KM, Hwang MK, Lee DE, Lee KW, Lee HJ. Protective effect of quercetin against arsenite-induced COX-2 expression by targeting PI3K in rat liver epithelial cells. J Agric Food Chem 2010;58:5815-20.  9. Chuang CC, Martinez K, Xie G, Kennedy A, Bumrungpert A, Overman A, et al. Quercetin is equally or more effective than resveratrol in attenuating tumor necrosis factor-{alpha}-mediated inflammation and insulin resistance in primary human adipocytes. Am J Clin Nutr 2010;92:1511-21. 10. Heusser CH, Brinkmann V. Immune response and pathophysiology of the allergic reaction. Ther Umsch 1994;51:14-8.

Acknowledgments.—Editorial assistant for this manuscript has been provided by Ambra Corti. Funding.—This assistance was supported by internal funds. Conflicts of interest.—ST, GM and MM are employees of Indena S.p.A.; LG is a consultant for Indena S.p.A.. Received on October 18, 2015. - Accepted for publication on November 9, 2015. Corresponding author: G. Maramaldi, Indena S.p.A.,Viale Ortles, 12, Milano, Italy. E-mail: [email protected]

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Properties of quercetin - Proprietà della quercetina

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