Changes in the anti-inflammatory activity of soy isoflavonoid genistein versus genistein incorporated in two types of cyclodextrin derivatives
© Danciu et al.; licensee Chemistry Central Ltd. 2012
Received: 24 April 2012
Accepted: 8 June 2012
Published: 20 June 2012
The isoflavonoid genistein represents the major active compound from soybean, the vegetal product from Glycine max (Fabaceae). The aim of this study is to prove that genistein was incorporated in two semisynthetic cyclodextrins, beta-cyclodextrin derivatives: hydroxypropyl-beta-cyclodextrin and randomly-methylated-beta-cyclodextrin as well as to compare the anti-inflammatory activity of genistein with that of genistein incorporated in these two types of semisynthetic cyclodextrins.
The animal studies were conducted on 8-week old C57BL/6 J female mice. Inflammation was induced in both ears of each mouse by topical application of 10 micrograms 12-O-tetradecanoylphorbol-3-acetate dissolved in 0.1 ml solvent (acetone : dimethylsulfoxide in a molar ratio 9:1). Thirty minutes later treatment was applied. The inflammatory reaction was correlated with increased values in ear thickness. Treatment with genistein and genistein incorporated in the two cyclodextrins led to decreased values for ear thickness. Better anti-inflammatory action was found for the complexes of genistein. Both haematoxylin-eosin analysis and CD45 marker expression are in agreement with these findings.
Results allow concluding that genistein is an active anti-inflammatory phytocompound and its complexation with hydrophilic beta-cyclodextrin derivatives leads to a stronger anti-inflammatory activity.
Flavonoids (flavus = yellow) are a class of secondary plant metabolites which function mainly as vegetal pigments, as antibiotic defence substances and as signal molecules for beneficial micro organisms in the rhizosphere . Researchers continue to find new functions for this class of compounds, such as components of a healthy human diet [1, 2].
Inflammation is a localized reaction of tissues in response to an aggressive action, characterized by redness, warmth, swelling, pain, and sometimes loss of mobility or function. Inflammation is considered a beneficial and necessary attempt of the organism to eliminate the aggressive agent and to start the healing process, which can consist in suppression of tumour initiation or progression. As a consequence, when the control mechanism of inflammation does not function properly and the inflammation persists, diseases including cancer may develop [7, 8]. Previous studies have demonstrated that genistein and other flavonoids exhibit an anti-inflammatory effect both for human and mouse skin, as well as inhibitory action against the activation of nuclear factor-kB and secretory phospholipase A2 [9–11].
Genistein shows a high solubility in organic solvents such as dimethylsulfoxide (DMSO), dimethylformamide, acetone and ethanol. Due to its chemical structure, it has, however, a very poor solubility in water, which drastically reduces its bioavailability . Inclusion complexes are nowadays largely used in the pharmaceutical field in order to optimize the solubility, chemical stability and bioavailability of guest molecules. Recently, much interest has focused on cyclodextrins (CD), because of their remarkable ability to form host–guest inclusion complexes with a wide variety of molecules, changing the physical-chemical properties of the guests [13, 14]. β-cyclodextrin derivatives are cyclic oligomers formed by seven units of glucose via α-(1,4)-linkages, having a toroidal shape with hydrophobic cavity and hydrophilic exterior. Due to this specific structure, they act as molecular hosts for a large variety of guest molecules, both polar and non-polar, through non-covalent interactions .
The aim of this study is to compare the anti-inflammatory activity of genistein with the one of genistein incorporated in two ramified beta-cyclodextrins: hydroxypropyl-;beta-cyclodextrin (HPBCD) and randomly–metylated-beta-cyclodextrin (RAMEB); cyclodextrin-genistein products were physical-chemically analysed by X-ray diffraction, thermal analysis and electronic microscopy in order to evaluate the formation of real inclusion complexes. The study intends to examine the correlation between the increased water solubility of genistein, due to complexation, and its anti-inflammatory activity on animal model.
Results and discussion
Genistein-β-CD complexes were previously prepared, as reported in the literature , mainly through the insertion of the guest A-ring into the cyclodextrin cavity. The complexes of genistein with the natural beta-cyclodextrin proved a higher solubility and bioavailability compared to the drug alone . Considering the rather low water solubility of natural β-CD, studies have been conducted on the complexation of genistein with derivatisedcyclodextrins . To the best of our knowledge, no studies have been performed on the biological properties (i.e. anti-inflammatory activity) of genistein complexes with semisynthetic hydrophilic derivatives of β-CD.
Complexes were analysed by X-ray diffraction, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) in order to establish the true inclusion nature of the final product.
DSC analysis was used in order to reveal the interaction between the drug and its host molecules, HPBCD and RAMEB. When guest molecules are trapped inside cyclodextrin cavities, their physical-chemical parameters (such as melting, boiling, sublimation points) change: they either disappear or shift to other temperatures .
Genistein consists of large, pure crystals with a smooth surface and a regular prismatic shape. The size of tetragonal particles is between 5–30 μm. The SEM pictures reveal the morphological changes of the crystals after complexation: the regular, smooth surface disappeared, and, because of the interaction between the drug and cyclodextrins, following the preparation procedure, aggregation can be seen. The cyclodextrins presumably cover the surface of genistein. HPBCD and RAMEB are amorphous materials and complexation agents, therefore amorphisation and/or inclusion complexation of genistein is presumable. The regular shape of the drug is also affected; the amorphous HPBCD and RAMEB can be seen on the particle surface.
For the control group, mice treated with indomethacin, Figure 7f, a loose stroma, oedematous, with low cellularity was detected. Indomethacin is a consecrated typical nonsteroidal anti-inflammatory compound [32, 33].
As conclusion, genistein can be reconsidered as an active anti-inflammatory phytocompound on C57BL/6 J animal model. Complexation of this active phytocompound with ramified β-CD derivatives was feasible and led to a stronger anti-inflammatory activity, proving to be a good method to improve the water solubility of genistein in order to obtain a better therapeutic response. These findings are of special interest since pharmaceutical formulations that produce increased drug concentration at the delivery site represent a better therapeutic option for patients.
Genistein was purchased from Extrasynthese (France, purity >95%), hydroxylpropyl-beta-cyclodextrin (HPBCD) and randomly-metylated-beta-cyclodextrin (RAMEB) from Cyclolab Hungary, 12-O-tetradecanoylphorbol-13-acetate (TPA) from Sigma Aldrich, Germany. All substances were used as received.
Preparation of complexes
simple powder mixing, using a mortar and a pestle;
kneading with a 50% (w/w) water : ethanol solution until the bulk of solvent evaporated and a paste-type product was formed; the mixture was then dried at room temperature for 24 hours and put in the oven, at 105°C, for several hours until reaching constant weight. The final product was pulverized and sieved.
All the binary products were prepared using 1:2 genistein : CD molar ratio (MwGen = 270,25, MwHPBCD = 1396, MwRameb = 1303). The molar ratio of 1:2 was chosen in order to achieve a better water solubility for genistein.
Differential scanning calorimetry (DSC)
The DSC measurements were made with a Mettler Toledo DSC 821e thermal analysis system with the STARe thermal analysis program V9.1 (Mettler Inc., Schwerzenbach, Switzerland). Approximately 2–5 mg of genistein or its product was examined in the temperature range between 25°C and 350°C. The heating rate was 5°C min-1. Argon was used as carrier gas, at a flow rate of 10 l h-1 during the DSC investigation.
Scanning electron microscopy (SEM) assay
The shape and surface characteristics of genistein and complex were visualized using a scanning electron microscope (Hitachi S4700, Hitachi Scientific Ltd., Japan). The samples were sputter coated with gold–palladium under an argon atmosphere using a gold sputter module in a high vacuum evaporator and the samples were examined using SEM set at 15 kV.
X-Ray-diffraction patterns were obtained on a Philips PW 1710 diffractometer, where the tube anode was Cu with Kα = 1.54242 Å. The pattern was collected with a tube voltage of 50 kV and 40 mA of tube current in step scan mode (step size 0.035, counting time 1 s per step).
Ethical statement: The work protocol followed all NIAH-National Institute of Animal Health rules: animals were maintained during the experiment in standard conditions: 12 h light–dark cycle, food and water ad libitum, temperature 24°C, humidity above 55%. The experiment was conducted according to the rules of the Ethical Committee of UMF “Victor Babes” Timisoara.
group A: blank group
group B: mice on which TPA in acetone/DMSO was applied on the ear
group C: mice on which TPA in acetone/DMSO and genistein (30 minutes later) were applied on the ear
group D: mice on which TPA in acetone/DMSO and genistein : HPBCD 1:2 (30 minutes later) were applied on the ear
group E: mice on which TPA in acetone/DMSO and genistein : RAMEB 1:2 (30 minutes later) were applied on the ear
group F: mice on which TPA in acetone/DMSO and indomethacin (30 minutes later) were applied on the ear as control.
Inflammation was induced in both ears of each mouse by the topical application of 10 μg TPA dissolved in 0.1 ml acetone : DMSO in a molar ratio 9:1 to both the inner and outer ear surfaces. Thirty minutes after the application of TPA, the inner and outer surface of each ear was treated with 2 mg of genistein, or the equivalent quantity of 2 mg genistein corresponding from the complex Gen : HPBCD and Gen : RAMEB. The same quantity of indomethacin, 2 mg, was used as control. The solvent was acetone : DMSO in a molar ratio 9:1 and the quantity administered was 0.1 ml.[25, 41] After 24 hours from the moment of application skin inflammation was induced, indicated by the increase of ear thickness on C57BL6/J mice. Ear thickness was measured with callipers, before treatment (value a) and 24 hours after TPA application (value b = TPA alone and value c = TPA + active substance). The experiment was repeated three times and results are expressed as mean ± standard deviation. The following values were also calculated:
Oedema X induced by TPA alone (b-a),
Oedema Y induced by TPA plus a sample (c-a),
Inhibitory rate (%) [(Oedema X-Oedema Y)/Oedema X] x 100.
After that, mice were killed by cervical dislocation and 6 mm2 diameter ear punch biopsies were collected and H&E analysis was carried out [24, 26]. The Prism software package (Graph Pad Prism 4.03 for Windows) was used for data presentation. The experiment was repeated three times and results are presented as mean ± SD. Paired Student’s t tests was applied to evaluate statistical significance (∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001).
For the histological analysis, tissue samples (skin) were fixed in 10% formalin solution, embedded in paraffin and cut at 4 microns. Finally, deparaffinized, the samples were stained with H&E (hematoxylin-eosin) and microscopically analysed. Immunohistochemistry was performed by using CD45 antibodies against inflammatory cells from the dermis. After dewaxing and rehydration, the sections were incubated with CD45 for 30 minutes, and then, the antigen-antibody reaction was detected by using an avidin biotin system from Novocastra. Visualization of the final product was done by using 3,3’diaminobenzidine as chromogen. Counterstain was performed with Lille’s modified haematoxylin.
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