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INTRODUCTION

Isoflavonoid genistein (4’,5,7-trihydroxyisoflavone), the aglicon of heteroside genistin, represent the major active compound from soybean, the vegetal product from Glycine max Fam. Fabaceae. Previous studies have demonstrated that genistein possess many biological functions such as prevention of coronary heart disease, as well as osteoporosis, antioxidant,

ORIGINAL ARTICLES

ABSTRACT

REZUMAT

INCORPORATION OF ISOFLAVONOID GENISTEIN IN BETA RAMIFIED CYCLODEXTRINS - AN OPTION FOR IMPROVING WATER SOLUBILITY

Corina Tiulea Danciu1, Cristina Dehelean2, Codruta M. Soica3, Camelia Peev1, Andrei Motoc4

Isoflavonoid genistein (4’,5,7-trihydroxyisoflavone), the aglicon of heteroside genistin, represent the major active compound from soybean. It is solubile in organic solvents such as DMSO , dimethyl formamide, acetone, ethanol. Due to its chemical structure it shows, however, poor solubility in water, that of course drastically reduces its bioavailability. The aim of this study is to demonstrate that genistein can be incorporated in different tyes of ramified cyclodextrins, compounds that increase water solubility: hydroxyl-propyl-beta-cyclodextrin (HPBCD) , randomly –metylated- beta-cyclodextrin (RAMEB) and 6-O –Maltosil- beta-cyclodextrin (G 2BCD) . The scanning electron microscopy images show a difference between the structure of the pure substance, genistein and the structure of genistein after the kneading with the three cyclodextrins. Another analyze that was made in order to prove that complexation took place was the differencial scanning calorimetry. Genistein has an endothermic peak which reflect its melting point around 300 oC. HPBCD , RAMEB and G 2BCD are amorphous materials, in these cases complexation phenomena is presumable, because the melting point disappeared. Presented data suggest that incorporation of genistein in hydroxy-propyl- beta-cyclodextrin , randomly –metylated- beta-cyclodextrin and 6-O–maltosil-

Genistein Isoflavonoid (4 ‘,5,7-trihydroxyisoflavone), aglicon de heteroside genistin, reprezinta compusul activ principal din soia. El este solubil în solvenţi organici, cum ar fi DMSO, dimetil formamidă, acetonă, etanol. Cu toate acestea, datorita structurii sale chimice, prezinta solubilitate slaba în apă, ceea ce desigur că ii reduce in mod drastic biodisponibilitatea. Scopul acestui studiu este de a demonstra că genisteina poate fi încorporata în diferite tipuri de ciclodextrine ramificate, compusi care cresc solubilitatea în apă: hidroxil-propil-beta-ciclodextrina (HPBCD), aleator-metilata-beta-ciclodextrină (RAMEB) şi a 6-O-Maltosil-beta-ciclodextrină (G 2BCD). Imaginile de microscopie electronica de baleiaj arată o diferenţă între structura substanţei pure, genistein si structura genistein după frământare cu cele trei ciclodextrine. O alta analiza care a fost făcută în scopul de a dovedi că procesul a avut loc a fost calorimetria de scanare differentiala. Genisteina are un vârf endoterm care reflectă punctul de topire în jurul valorii de 300 oC. HPBCD, RAMEB şi G 2BCD sunt materiale amorfe; în aceste cazuri, sunt presupuse a exista fenomene de complexare, deoarece punctul de topire a dispărut. Datele prezentate sugerează că încorporarea de genisteina în hidroxi-propil-beta-ciclodextrina, aleator-metilata-beta-ciclodextrina şi 6-O-maltosil-beta-ciclodextrină a avut loc din cauza schimbărilor aparute in proprietăţile fizico-chimice ale compuşilor.Cuvinte cheie: genisteină, hidroxi-propil-beta-ciclodextrina, aleator-metilata-beta-ciclodextrina, 6-O-maltosil-beta-ciclodextrină, SEM, DSC

Received for publication: Oct. 11, 2011. Revised: Nov. 14, 2011.

1 Department of Pharmacognosy, 2 Department of Toxicology, Faculty of Pharmacy, 3 Department of Pharmaceutical Chemistry, 4 Department of Anatomy, Faculty of Medicine, Victor Babes University of Medicine and Pharmacy, Timisoara

Correspondence to:Corina Tiulea, Victor Babes University of Medicine and Pharmacy, 2A E. Murgu Sq., 300041 Timisoara, Romania, Tel. +40-744-648855.Email: corina_tiulea@yahoo.com

anticancerous and anti-inflamatory activity.1-4

Genistein is solubile in organic solvents such as DMSO , dimethyl formamide, acetone, ethanol. Due to its chemical structure it shows, however, poor solubility in water, that of course drastically reduces its bioavailability. Inclusion complexes are now widely used in pharmaceutical industry, for improving the solubility, stability and bioavailability of the guest molecules. Recently, much interest has been focused on cyclodextrins, because of their remarkable ability to form, via noncovalent interactions, “host–guest” inclusion complexes with a wide variety of molecules, altering the physico-chemical characteristics of the guest.5,6 Beta-cyclodextrines are cyclic oligomers formed of seven units of glucose via α-(1,4)-linkages, having a toroidal shape with a non-polar inside and two hydrophilic rings. Due to this specific structure they act as molecular hosts for a large variety of guest molecules, polar and non-polar ones, through non-covalent interactions.7

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AIM AND OBJECTIVES

The aim of this study is to demonstrate that genistein can be incorporated in different tyes of ramified cyclodextrins, compounds that increase water solubility: hydroxyl-propyl-beta-cyclodextrin (HPBCD), randomly-metylated-beta-cyclodextrin (RAMEB) and 6-O-Maltosil-beta-cyclodextrin (G2BCD).

MATERIAL AND METHODS

ReagentsGenistein was acquired from Extrasynthese (France),

hydroxyl-propyl-beta-cyclodextrin (HPBCD), randomly-metylated-beta-cyclodextrin (RAMEB) and 6-O-Maltosil-beta-cyclodextrin (G2BCD) from Cyclolab Hungary.

Scanning electron microscopy (SEM) assayGenistein was prepared in three complex one

with hydroxy-propyl-beta-cyclodextrin one with randomly-metylated-beta-cyclodextrin and one with and 6-O-maltosil-beta-cyclodextrin in a molar ratio 1:2 by kneading method. 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.

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 MEL or its product was examined in the temperature range between 25oC and 300oC. The heating rate was 5 oC min-1. Argon was used as carrier gas, at a flow rate of 10 l h-1 during the DSC investigation.

RESULTS

Genistein was incorporated in the three ramyfied beta-cyclodextrins in a molar ratio of 1:2 by kneading method. The scanning electron microscopy images show a difference between the structure of the pure substance, genistein and the structure of genistein after the kneading with the three cyclodextrins, HPBCD , RAMEB and G 2BCD. This analyze confirms the fact that the complexation took place and physical interactions were performed. These preliminary observations are important to establish the solvent and formulation type for active compound application. Results are presented in Figures 1-4.

Figure 1. SEM picture of pure genistein.

Figure 2. SEM picture of genistein incorporated in HPBCD.

Figure 3. SEM picture of genistein incorporated in RAMEB.

Figure 4. SEM picture of genistein incorporated in G2BCD.

Figure 1

Figure 2

Figure 3

Figure 4

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Corina Tiulea et al 223

Figure 1 presents the aspect of genistein: the large, pure crystals have a smooth surface with a regular prismatic form. The size of tetragonal particles is between 5-30 μm. The SEM pictures demonstrate the changes in habit of the crystals after complexation: the regular, smooth surface disappeared. (Figs. 2-4) Because of the interaction between the drug and excipient and the preparation procedure, aggregation can be seen. Presumably the excipient covered the surface of genistein. HPBCD, RAMEB and G 2BCD are amorphous materials and complexing agents, therefore amorphization or inclusion complexation of genistein is presumable. The regular shape of the drug is also splitted. It can be seen the amorphous HPBCD, RAMEB and G 2BCD with irregular shape on the particle surface. (Figs. 2-4) These results suggest that complexation took place.

Another analyze that was made in order to prove that complexation took place was the differencial scanning calorimetry. Results can be seen in Figure 5. Genistein has an endothermic peak which reflect its melting point around 300 oC. HPBCD , RAMEB and G 2BCD are amorphous materials, in these cases complexation phenomena is presumable, because the melting point disappeared. These data regarding DSC analysis are preliminary studies. Further studies expanding the range of temperature at 350 degrees for better observing the endothermic pick will be made.

Figure 5. DSC results for genistein and genistein incorporated in the

three types of cyclodextrins.

DISCUSSION

Recently, one of the most used methods to improve watter solubility, stability, safety and bioavailibility of drug molecules is complexation with cyclodextrins.8,9 Genistein has a poor water solubility, and in order to improove the drug delivery parameters many scientist have resorted this solution.10-12

β-cyclodextrins seem to be the best natural cyclodextrin for complexation because of their cavity size, efficient drug complexation and availability in pure form.13,14 Although inclusion complex formation

of beta-cyclodextrins with drugs have been studied extensively the lower solubility of these complexes to some extent limits their use.15 Therefore in this study we have focused on the preparation of derivates of beta-cyclodextrin with a higher water solubility. Genistein was incorporated in the three ramyfied beta-cyclodextrins, HPBCD, RAMEB and G 2BCD in a molar ratio of 1:2 by kneading method. Scanning electron microscope analyse show physico-chemical changes in the structure of the complexes compared to pure genistein, a proof that complexation took place. Crupi et al also concluded that these type of cyclodextrins are suitable for a myriad of lipophilic substances and Bergonzi et al studied the complex formed by these cyclodextins with other flavonoids. Borghetti et al also studied the formation of the complex between ramyfied beta-cyclodextrins and the second most plentiful isoflavonoid from soybean, daidzein and concluded that the complexation increased water solubility.16-18 Another analyze that was made in order to prove that complexation took place was the differencial scanning calorimetry. This analyse also suport the fact that incorporation took place because the melting point of genistein which it was detected at 300oC disappeared, but in order to explain better the process furtherer studies expanding the range of temperature at 350 degrees for better observation of the endothermic pick will be made. Shuang et al studied the formation of the complex between genistain and beta-cyclodextrin using DSC method and concluded that incorporation took place.19

SEM and DSC analyses confirm that incorporation of genistein in ramyfied beta cyclodextrins took place.This result is of special interest for practical purposes in the pharmaceutical field, since formulations that produce higher drug concentrations in solution may provide improved therapeutic options for patients.

CONCLUSION

Presented data suggest that incorporation of genistein in hydroxy-propyl-beta-cyclodextrin, randomly- metylated-beta-cyclodextrin and 6-O-maltosil-beta-cyclodextrin took place because of the changes in the physico-chemical proprieties of the compounds.

ACKNOWLEDGEMENTS

During the research described in this paper, the first author, Corina Tiulea, benefitted by a grant from the PhD programme POSDRU/88/1.5/S/63117.

Figure 5

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REFERENCES1. Dalais FS, Ebeling PR, Kotsopoulos D, et al. The effects of soy protein

containing isoflavones on lipids and indices of bone resorption in postmenopausal women. Clin. Endocrinol. 2003; 58: 704–70.

2. Wei H, Saladi R, Lu Y, et al. Isoflavone Genistein: Photoprotection and Clinical Implications in Dermatology, J. Nutr. 2003; 133:3811S-3819S.

3. Kao TH, Huang RF, Chen BH. Antiproliferation of hepatoma cell and progression of cell cycle as affected by isoflavone extracts from soybean cake. Int. J. Mol. Sci. 2007; 8: 1095–1110.

4. Bhatia AL , Gaur A, Sharma A. Radiation protection by an isoflavone, genistein: a study on the survivability of mice, Nuclear Technology & Radiation Protection 2007; 22: 34-39.

5. Crupi V, Ficarra R , Guardo M, et al. UV-vis and FTIR-ATR spectroscopic techniques to study the inclusion complexes of genistein with beta-cyclodextrins, J Pharm Biomed Anal. 2007; 9, 44(1):110-7.

6. Cannavà C, Crupi V, Ficarra P, et al. Physico-chemical characterization of an amphiphilic cyclodextrin/genistein complex, J Pharm Biomed Anal 2010; 6, 51(5):1064-8.

7. Menuel S, Joly JP, Courcot B, et al. Synthesis and inclusion ability of a bis-β-cyclodextrin pseudo-cryptand towards Busulfan anticancer agent. Tetrahedron, 63, 2007; 7:1706-1714.

8. Rasheed A, Kumar A, Sravanthi VV. Cyclodextrins as Drug Carrier Molecule: A Review, Scientia Pharmaceutica, 2008; 76: 567–598.

9. Patil JS, Kadam DV, Marapur SC. Inclusion complex system; a novel technique to improve the solubility and bioavailability of poorly soluble drugs: a review, International Journal of Pharmaceutical Sciences Review and Research ,2010, 2 (2):29-34.

10. Daruházi AE, Szente L, Balogh B, et al. Utility of cyclodextrins in the formulation of genistein part 1. Preparation and physicochemical

properties of genistein complexes with native cyclodextrins, J Pharm Biomed Anal., 2008, 8(3):636-40.

11. Stancanelli R, Guardo M, Cannavà C et al. Amphiphilic cyclodextrins as nanocarriers of genistein: A spectroscopic investigation pointing out the structural properties of the host/drug complex system, J Pharm Sci., 2010 , 99(7):3141-9.

12. Xavier CR, Silva PC, Schwingel LC et al. Improvement of genistein content in solid genistein/β-cyclodextrin complexes, Quím. Nova , 2011, 34 (9) :1534-1538.

13. Fernandes SA, Cabeca LF, Marsaioli AJ, Eneida P. Investigation of tetracaine complexation with beta-cyclodextrins and p-sulphonic acid calixarenes by nOe and PGSE NMR. J Incl Phenom Macrocycl Chem , 2007, 57:395–401.

14. Crini G. Studies on adsorption of dyes on beta-cyclodextrin polymer. Bioresource Technology, 2003, 90 (2):193-198.

15. Haiyun D, Jianbin C, Guomei Z et al. Preparation and special investigation on inclusion complex of beta-cyclodextrins with rutin Spectrochim Acta A Mol Biomol Spectrosc ,2003, 59(14):3421-9.

16. Crupi V, Majolino D, Paciaroni A, et al., The effect of hydrogen bond on the vibrational dynamics of genistein free and complexed with β-cyclodextrins, Journal of Raman Spectroscopy, 2010, 41(7) :764-770.

17. Borghetti GS, Pinto AP, Lula IS, et al. Daidzein/cyclodextrin/hydrophilic polymer ternary systems, Drug development and industrial pharmacy, 2011, Epub ahead of print

18. Bergonzi MC, Bilia AR, Bari LD et al. Studies on the interactions between some flavonols and cyclodextrins. Bioorg.Med.Chem.Lett 2007, 17(21): 5744-8.

19. Shuang C, Ling-Yun DU, Mei-Ju N et al. Study on inclusion interaction of β-cyclodextrin and genistein. , Food Science, 2006, 27 (2): 94–99.