Pharmaceutical Research

, Volume 26, Issue 5, pp 1217–1225 | Cite as

Solubility, Stability, Physicochemical Characteristics and In Vitro Ocular Tissue Permeability of Hesperidin: A Natural Bioflavonoid

  • Soumyajit MajumdarEmail author
  • Ramesh Srirangam
Research Paper



Hesperidin holds potential in treating age-related macular degeneration, cataract and diabetic retinopathy. The aim of this study, constituting the first step towards efficient ocular delivery of hesperidin, was to determine its physicochemical properties and in vitro ocular tissue permeability.


pH dependent aqueous solubility and stability were investigated following standard protocols. Permeability of hesperidin across excised rabbit cornea, sclera, and sclera plus retinal pigmented epithelium (RPE) was determined using a side-bi-side diffusion apparatus.


Hesperidin demonstrated poor, pH independent, aqueous solubility. Solubility improved dramatically in the presence of 2-hydroxypropyl-beta-cyclodextrin (HP-β-CD) and the results supported 1:1 complex formation. Solutions were stable in the pH and temperature (25, 40°C) conditions tested, except for samples stored at pH 9. Transcorneal permeability in the apical-basal and basal-apical directions was 1.11 ± 0.86 × 10−6 and 1.16 ± 0.05 × 10−6 cm/s, respectively. The scleral tissue was more permeable (10.2 ± 2.1 × 10−6 cm/s). However, permeability across sclera/choroid/RPE in the sclera to retina and retina to sclera direction was 0.82 ± 0.69 × 10−6, 1.52 ± 0.78 × 10−6 cm/s, respectively, demonstrating the barrier properties of the RPE.


Our results suggest that stable ophthalmic solutions of hesperidin can be prepared and that hesperidin can efficiently permeate across the corneal tissue. Further investigation into its penetration into the back-of-the eye ocular tissues is warranted.


hesperetin hesperidin ocular permeability solubility 



This project was supported by NIH grant numbers P20RR021929, from the National Center for Research Resources, and EY018426-01 from the National Eye Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Eye Institute, National Institutes of Health.


  1. 1.
    A. Ryskulova, K. Turczyn, D. M. Makuc, M. F. Cotch, R. J. Klein, and R. Janiszewski. Self-reported age-related eye diseases and visual impairment in the United States: results of the 2002 national health interview survey. Am. J. Public Health. 98:454–461 (2008) doi: 10.2105/AJPH.2006.098202.PubMedCrossRefGoogle Scholar
  2. 2.
    J. L. Wilkinson-Berka. Vasoactive factors and diabetic retinopathy: vascular endothelial growth factor, cycoloxygenase-2 and nitric oxide. Curr. Pharm. Des. 10:3331–3348 (2004) doi: 10.2174/1381612043383142.PubMedCrossRefGoogle Scholar
  3. 3.
    X. R. Chiou GC. Effects of some natural flavonoids on retinal function recovery after ischemic insult in the rat. J. Ocular. Pharmacol. Therap. 20:107–113 (2004) doi: 10.1089/108076804773710777.CrossRefGoogle Scholar
  4. 4.
    A. Garg, S. Garg, L. J. Zaneveld, and A. K. Singla. Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phytother. Res. 15:655–669 (2001) doi: 10.1002/ptr.1074.PubMedCrossRefGoogle Scholar
  5. 5.
    J. M. Beiler, and G. J. Martin. Inhibition of hyaluronidase action by derivatives of hesperidin. J. Biol. Chem. 174:31–34 (1948).PubMedGoogle Scholar
  6. 6.
    J. Zhang, R. A. Stanley, L. D. Melton, and M. A. Skinner. Inhibition of lipid oxidation by phenolic antioxidants in relation to their physicochemical properties. Pharmacologyonline. 1:180–189 (2007).Google Scholar
  7. 7.
    A. Hirata, Y. Murakami, M. Shoji, Y. Kadoma, and S. Fujisawa. Kinetics of radical-scavenging activity of hesperetin and hesperidin and their inhibitory activity on COX-2 expression. Anticancer Res. 25:3367–3374 (2005).PubMedGoogle Scholar
  8. 8.
    S. P. Ayalasomayajula, A. C. Amrite, and U. B. Kompella. Inhibition of cyclooxygenase-2, but not cyclooxygenase-1, reduces prostaglandin E2 secretion from diabetic rat retinas. Eur. J. Pharmacol. 498:275–278 (2004) doi: 10.1016/j.ejphar.2004.07.046.PubMedCrossRefGoogle Scholar
  9. 9.
    E. M. Galati, M. T. Monforte, S. Kirjavainen, A. M. Forestieri, A. Trovato, and M. M. Tripodo. Biological effects of hesperidin, a citrus flavonoid. (Note I): antiinflammatory and analgesic activity. Farmaco. 40:709–712 (1994).PubMedGoogle Scholar
  10. 10.
    M. T. Monforte, A. Trovato, S. Kirjavainen, A. M. Forestieri, E. M. Galati, and R. B. Lo Curto. Biological effects of hesperidin, a citrus flavonoid. (note II): hypolipidemic activity on experimental hypercholesterolemia in rat. Farmaco. 50:595–599 (1995).PubMedGoogle Scholar
  11. 11.
    E. M. Galati, A. Trovato, S. Kirjavainen, A. M. Forestieri, A. Rossitto, and M. T. Monforte. Biological effects of hesperidin, a citrus flavonoid. (Note III): antihypertensive and diuretic activity in rat. Farmaco. 51:219–221 (1996).PubMedGoogle Scholar
  12. 12.
    T. Tanaka, H. Makita, M. Ohnishi, H. Mori, K. Satoh, A. Hara, T. Sumida, K. Fukutani, T. Tanaka, and H. Ogawa. Chemoprevention of 4-nitroquinoline 1-oxide-induced oral carcinogenesis in rats by flavonoids diosmin and hesperidin, each alone and in combination. Cancer Res. 57:246–252 (1997).PubMedGoogle Scholar
  13. 13.
    T. Tanaka, H. Makita, K. Kawabata, H. Mori, M. Kakumoto, K. Satoh, A. Hara, T. Sumida, T. Tanaka, and H. Ogawa. Chemoprevention of azoxymethane-induced rat colon carcinogenesis by the naturally occurring flavonoids, diosmin and hesperidin. Carcinogenesis. 18:957–965 (1997) doi: 10.1093/carcin/18.5.957.PubMedCrossRefGoogle Scholar
  14. 14.
    B. Ameer, R. A. Weintraub, J. V. Johnson, R. A. Yost, and R. L. Rouseff. Flavanone absorption after naringin, hesperidin, and citrus administration. Clin. Pharmacol. Ther. 60:34–40 (1996) doi: 10.1016/S0009-9236(96)90164-2.PubMedCrossRefGoogle Scholar
  15. 15.
    A. Gil-Izquierdo, M. I. Gil, F. A. Tomas-Barberan, and F. Ferreres. Influence of industrial processing on orange juice flavanone solubility and transformation to chalcones under gastrointestinal conditions. J. Agric. Food Chem. 51:3024–3028 (2003) doi: 10.1021/jf020986r.PubMedCrossRefGoogle Scholar
  16. 16.
    T. H. Tsai, and M. C. Liu. Determination of extracellular hesperidin in blood and bile of anaesthetized rats by microdialysis with high-performance liquid chromatography: a pharmacokinetic application. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 806:161–166 (2004) doi: 10.1016/j.jchromb.2004.03.047.PubMedCrossRefGoogle Scholar
  17. 17.
    Y. Mitsunaga, H. Takanaga, H. Matsuo, M. Naito, T. Tsuruo, H. Ohtani, and Y. Sawada. Effect of bioflavonoids on vincristine transport across blood–brain barrier. Eur. J. Pharmacol. 395:193–201 (2000) doi: 10.1016/S0014-2999(00)00180-1.PubMedCrossRefGoogle Scholar
  18. 18.
    M. Ofer, S. Wolffram, A. Koggel, H. Spahn-Langguth, and P. Langguth. Modulation of drug transport by selected flavonoids: involvement of P-gp and OCT? Eur. J. Pharm. Sci. 25:263–271 (2005).PubMedGoogle Scholar
  19. 19.
    V. M. Breinholt, E. A. Offord, C. Brouwer, S. E. Nielsen, K. Brosen, and T. Friedberg. In vitro investigation of cytochrome P450-mediated metabolism of dietary flavonoids. Food Chem. Toxicol. 40:609–616 (2002) doi: 10.1016/S0278-6915(01)00125-9.PubMedCrossRefGoogle Scholar
  20. 20.
    S. Kobayashi, S. Tanabe, M. Sugiyama, and Y. Konishi. Transepithelial transport of hesperetin and hesperidin in intestinal Caco-2 cell monolayers. Biochim. Biophys. Acta. 1778:33–41 (2008) doi: 10.1016/j.bbamem.2007.08.020.PubMedCrossRefGoogle Scholar
  21. 21.
    H. Serra, T. Mendes, M. R. Bronze, and A. L. Simplicio. Prediction of intestinal absorption and metabolism of pharmacologically active flavones and flavanones. Bioorg. Med. Chem. 16:4009–4018 (2008).Google Scholar
  22. 22.
    C. Manach, C. Morand, A. Gil-Izquierdo, C. Bouteloup-Demange, and C. Remesy. Bioavailability in humans of the flavanones hesperidin and narirutin after the ingestion of two doses of orange juice. Eur. J. Clin. Nutr. 57:235–242 (2003) doi: 10.1038/sj.ejcn.1601547.PubMedCrossRefGoogle Scholar
  23. 23.
    A. K. Mitra, S. Macha, and P. M. Hughes. Ophthalmic Drug Delivery Systems. Marcel Dekker, New York, 2003.Google Scholar
  24. 24.
    M. M. Bradford. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254 (1976) doi: 10.1016/0003-2697(76)90527-3.PubMedCrossRefGoogle Scholar
  25. 25.
    N. M. Holekamp, M. A. Thomas, and A. Pearson. The safety profile of long-term, high-dose intraocular corticosteroid delivery. Am. J. Ophthalmol. 139:421–428 (2005) doi: 10.1016/j.ajo.2004.09.046.PubMedCrossRefGoogle Scholar
  26. 26.
    T. Jansen, B. Xhonneux, J. Mesens, and M. Borgers. Beta-cyclodextrins as vehicles in eye-drop formulations: an evaluation of their effects on rabbit corneal epithelium. Lens Eye Toxic. Res. 7:459–468 (1990).PubMedGoogle Scholar
  27. 27.
    P. Saarinen-Savolainen, T. Jarvinen, K. Araki-Sasaki, H. Watanabe, and A. Urtti. Evaluation of cytotoxicity of various ophthalmic drugs, eye drop excipients and cyclodextrins in an immortalized human corneal epithelial cell line. Pharm. Res. 15:1275–1280 (1998) doi: 10.1023/A:1011956327987.PubMedCrossRefGoogle Scholar
  28. 28.
    T. Loftssona, and T. Jarvinen. Cyclodextrins in ophthalmic drug delivery. Adv. Drug Deliv. Rev. 36:59–79 (1999) doi: 10.1016/S0169-409X(98)00055-6.PubMedCrossRefGoogle Scholar
  29. 29.
    S. Tommasini, M. L. Calabro, R. Stancanelli, P. Donato, C. Costa, S. Catania, V. Villari, P. Ficarra, and R. Ficarra. The inclusion complexes of hesperetin and its 7-rhamnoglucoside with (2-hydroxypropyl)-beta-cyclodextrin. J. Pharm. Biomed. Anal. 39:572–580 (2005) doi: 10.1016/j.jpba.2005.05.009.PubMedCrossRefGoogle Scholar
  30. 30.
    D. K. Wilcox. Extracellular release of acid hydrolases from cultured retinal pigmented epithelium. Invest. Ophthalmol. Vis. Sci. 28:76–82 (1987).PubMedGoogle Scholar
  31. 31.
    A. J. Adler. Selective presence of acid hydrolases in the interphotoreceptor matrix. Exp. Eye Res. 49:1067–1077 (1989) doi: 10.1016/S0014-4835(89)80027-2.PubMedCrossRefGoogle Scholar
  32. 32.
    E. Mannermaa, K. S. Vellonen, and A. Urtti. Drug transport in corneal epithelium and blood-retina barrier: emerging role of transporters in ocular pharmacokinetics. Adv. Drug Deliv. Rev. 58:1136–1163 (2006) doi: 10.1016/j.addr.2006.07.024.PubMedCrossRefGoogle Scholar
  33. 33.
    W. S. Marshall, and S. D. Klyce. Cellular and paracellular pathway resistances in the “tight” Cl- -secreting epithelium of rabbit cornea. J. Membr. Biol. 73:275–282 (1983) doi: 10.1007/BF01870542.PubMedCrossRefGoogle Scholar
  34. 34.
    M. R. Prausnitz, and J. S. Noonan. Permeability of cornea, sclera, and conjunctiva: a literature analysis for drug delivery to the eye. J. Pharm. Sci. 87:1479–1488 (1998) doi: 10.1021/js9802594.PubMedCrossRefGoogle Scholar
  35. 35.
    V. Kansara, Y. Hao, and A. K. Mitra. Dipeptide monoester ganciclovir prodrugs for transscleral drug delivery: targeting the oligopeptide transporter on rabbit retina. J. Ocul. Pharmacol. Ther. 23:321–334 (2007) doi: 10.1089/jop.2006.0150.PubMedCrossRefGoogle Scholar
  36. 36.
    S. Majumdar, K. Hippalgaonkar, and M. A. Repka. Effect of chitosan, benzalkonium chloride and ethylenediaminetetraacetic acid on permeation of acyclovir across isolated rabbit cornea. Int. J. Pharm. 348:175–178 (2008) doi: 10.1016/j.ijpharm.2007.08.017.PubMedCrossRefGoogle Scholar
  37. 37.
    S. Majumdar, Y. E. Nashed, K. Patel, R. Jain, M. Itahashi, D. M. Neumann, J. M. Hill, and A. K. Mitra. Dipeptide monoester ganciclovir prodrugs for treating HSV-1-induced corneal epithelial and stromal keratitis: in vitro and in vivo evaluations. J. Ocul. Pharmacol. Ther. 21:463–474 (2005) doi: 10.1089/jop.2005.21.463.PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  1. 1.Department of Pharmaceutics, School of PharmacyThe University of MississippiUniversityUSA
  2. 2.Research Institute of Pharmaceutical SciencesThe University of MississippiUniversityUSA

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