Letters in Drug Design & Discovery, 2012, 9, 213-217
213
Fat-Soluble Vitamins Release Based on Clinoptilolite Zeolite as an Oral Drug Delivery System Mehdi Rahimi*,a , Hamid Mobedib, Aliasghar Behnamghaderc, Alireza Nateghi Baygib, Houri Mivehchib and Esmaeil Biazard a
Department of Biomedical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran
b
Department of Novel Drug Delivery Systems, Iran Polymer & Petrochemical Institute, Tehran, Iran
c
Material and Energy Research Center, Tehran, Iran
d
Department of Engineering , Tonekabon Branch, Islamic Azad University, Tonekabon, Iran Received September 22, 2011: Revised October 17, 2011: Accepted November 03, 2011
Abstract: The purpose of this research was to study the effect of zeolite powder, with particles size range between 53 and 1180 μm, on the stability and release of vitamins A, D3 and E. Zeolite type, chemical and morphological features were studied using SEM and XRF. High performance liquid chromatography was used to investigate the extracted vitamins from zeolite powder. The powder emanating from natural Iranian zeolite was first plunged into each saturated solution of vitamins A, D3 and E. The powders were then taken in laboratory condition for 2 h, 1, 2, 3 and 4 weeks at room temperature. The amount of the extracted vitamin constantly decreased with time in the control sample (without zeolite powder), whereas it remained steady in the zeolite containing sample. The amount of stable vitamin in ambient environment after 4 weeks was higher than the control sample due to the zeolite with particles size range between 710 and 850 μm. For simulated gastrointestinal pH, the amount of release from the sample with zeolite was also more than the control sample. Based on the results obtained from HPLC experiments, zeolite powder enhanced the stability of the system in acidic pH and increased the amount of vitamins released in gastrointestinal condition.
Keywords: Natural clinoptilolite, Fat-soluble vitamins, Stability, Drug delivery, HPLC. 1. INTRODUCTION Zeolites are hydrated aluminosilicates of alkali and alkaline earth elements with unique crystalline structures consisting of a three-dimensional framework of SiO2 and tetrahedral Al2O3 [1,2]. This structure causes zeolite to have negatively charged surface. Negatively charged surface of zeolites can be used for adsorption of alkali earth metals [35]. Clinoptilolite is a natural mineral zeolite from the tectosilicates group. Its typical unit cell formula is Na [(Al2O3)6(SiO2)30].24H2O [3]. The most important property of clinoptilolite is its high cation exchange capability which makes it a potential candidate in acidic solution for the removal of H + [3]. Indeed, the cation exchange capacity (CEC) of clinoptilolite mostly results from the unbalanced charges [3,6]. The most important contribution to its CEC comes from the isomorphic substitutions of Al+ for Si4+ leading to some ion exchangeable cations such as Na+, K+, Ca2+ and Mg2+ entering into the clinoptilolite channels to compensate the positive charge deficiencies of the clinoptilolite lattice [3]. In addition, the H+ ions may be considered as exchangeable cations present in Si–OH and Al–OH as in clay minerals [3]. Therefore, the variation of pH in a liquid system is envisaged to affect the ion exchange behavior, which is controlled by electrostatic forces. Other
*Address correspondence to this author at the Department of Biomedical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran; Tel: +98 912 1079083; Fax: +98 21 26211894; E-mail:
[email protected] 1875-628X/12 $58.00+.00
factors such as the particle size of the ion exchanger, solvent type, heat of solution, ion concentration, and cation type modify the ion exchange behavior of clinoptilolite and other ion exchangers [6,7]. Natural zeolites have been widely used in the industrial, agricultural and waste treatment applications [8,9]. Medical and pharmaceutical applications of natural zeolite type clinoptilolite have expanded considerably over the last decade, due to the good performance of this material in ion exchange, adsorptive and biocatalytic processes, along with its high chemical stability [9]. Several biomedical applications of zeolites and other mesoporous and microporous solids have been reported: for example, the use of clinoptilolite as a potential adjuvant in anticancer therapy[8-10]. The numerous research papers over many years demonstrate that clinoptilolite is safe and nontoxic [11]. One of the many exciting potential pharmacological applications of zeolites is the possible encapsulation and/or adsorption of different ions and molecules in their open framework, and their subsequent controlled release [12-14]. Vitamins A, D3 and E are the most abundant lipid-soluble antioxidants found in the plasma and cells of higher mammals. Synthetic retinyl acetate (C22H32O2, MW 328.5) and retinyl palmitate (C36H60O2, MW 524) are used commercially to supplement the vitamin A content of foodstuffs. Tocopherols are methyl-substituted derivatives of tocol, which comprises a chroman-6-ol ring attached at C-2 to a saturated isoprenoid side chain [15]. In nature, there are four tocopherols and four corresponding tocotrienols designated as: alpha (), beta (), gamma () and delta () according to the number and position of the methyl substituents in the chromanol ring. The most biologically © 2012 Bentham Science Publishers
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active vitamin is the naturally occurring -tocopherol (C29H50O2, MW 430.7). Vitamin D2 (C28H44O, MW 396.6) and vitamin D3 (C27H44O, MW 384.2) differ structurally only in the C-17 side chain, which in vitamin D2 has a double bond and an additional methyl group [15-17]. Absorption of the fat-soluble vitamins takes place mainly in the proximal jejunum. Vitamins are sensitive to acidic pH of stomach [15] and therefore, to increase the bioavailability of vitamins there is a need for a carrier to transport vitamins from stomach to the intestine. Clinoptilolite adsorbs vitamins safely as it has a buffering property. The aim of this study was to investigate the effect of zeolite powder on the stability and release of fat-soluble vitamins under gastrointestinal tract condition. 2. MATERIALS AND METHODS
different vessels with degrees of acidity of 1.7, 6.8, and 11.5, respectively and were all evaluated every 30 min with pH meter. 2.4. Quantitative Vitamins Analysis The amount of vitamin was investigated by HPLC. Detector was set at a wavelength of 284 nm. The mobile phase was prepared by mixing water with methanol (2:98, v/v). The mobile phase was filtered through 0.45 m nylon membrane and degassed before use. Elution was performed at a flow rate of 1.5 mL/min. The mean linear regression equation (y = mx + c) of calibration curve for the vitamin was y = 351.55x - 81.954, where y was the peak area ratio of the vitamin and x was the concentration of the vitamin. The correlation coefficient (R2) for the vitamin was above 0.9947 over the concentration range used.
2.1. Materials Clinoptilolite (Anzymite®) was supplied by the Afrand Tooska Co. (Tehran, Iran) from Semnan Zeolite Mines located in the central region of Iran. The natural zeolite was grinded and sieved. The particle size diameter of the selected zeolite to carry out the experiments was within the range of 53-1180 microns. The powder was dried at 105 ºC for 2 h. Vitamin A (palmitate), vitamin D3 and vitamin E (tocopherol) were provided by DSM Nutritional Products (Grenzach, Germany). Buffer solutions were produced fresh in the laboratory. 2.2. Methods For chromatographic analysis, Agilent® HPLC (1200 series, Palo Alto, USA) system equipped with a quaternary pump (Model G1311A, Quat pump), a column oven (Model G1316A, Tcc), an automated sample injector (Model 7725/7725i) holding 100 L loop, UV-visible detector (Model G1314B, VWD) and a data system (Chemstation version B.04.01) were used. The separation of the compounds was made on a C-8 symmetry column (150mm 4.6 mm, 5m, Thermo Separation Co.) at temperature 25 ºC. The amount of release and pH were tested in a EU802 dissolution tester (Hangzhou Tailin Bioengineering Equipments Co., China) and pH meter (C533X model, Consort, Belgium), respectively. Zeolite chemical composition was studied by XRF (D4, Siemens, Bruker Co, Germany). A scanning electron microscope (Philips, XL30, Holland) was used to investigate the size and morphological characteristics of the particles. 2.3. Buffer Action of Natural Clinoptilolite The studies on natural clinoptilolite buffer characteristics were conducted by a dissolution apparatus as follows: a constant amount of clinoptilolite (5 g) was added into each 3 Table 1.
2.5. The Optimum Size Range of Zeolite Particles The protective action of clinoptilolite on vitamin E stability was studied against its environmental conditions. A stock solution containing 10 g of vitamin E was prepared by shaking with 1 mL of ethanol. At first the solutions were covered by aluminum foil and stored at 3°C for the safe protection of environment conditions. For stability testing 6 different particles size ranges of clinoptilolite powder were obtained by sieving. The ranges of sizes were 53-106, 150180, 250-300, 425-500, 710-850, 1000-1180 m. Six samples were made by introducing 1 g of each range in a test tube followed by the addition of 1 ml of stock solution. The remaining vitamin E in samples was extracted by methanol after 4 weeks and its quantity was determined. All samples were maintained in environmental condition before testing. Extraction was performed in a new test tube. The particles of clinoptilolite were separated by a Minisart® filter (0.2 m) and then the solution was kept at 3°C. The samples were injected into HPLC instrument. The control sample had no clinoptilolite. 2.6. Release Study To examine the amount of vitamin released in in-vitro condition, first 5 mL stock solution of 10 microgram/ml ethanol was added to 5 g of clinoptilolite with particle size range between 710 and 850 μm and the petri dish was kept under the hood for 2 hours to evaporate ethanol. A known quantity of the clinoptilolite (5 g) was introduced into two separate vessels in which one contained 100 mL of pH 1.7 and the pH of the other vessel increased to 6.8 by 2 N NaOH at the 90th min, simulating the intestine condition. At the same time there were two vessels prepared which contained 5 ml of stock solution without zeolite under the same condition as control samples. The parallel evaluations were carried out every 30 min for total 330 min, at 37°C. The
Chemical Composition of Iranian Natural Clinoptilolite
Component
SiO2
Al2O3
CaO
K 2O
Na2O
Fe2O3
MgO
TiO
MnO
P2O5
LOI (loss on ignition)
Wt (%)
66.50
11.80
3.10
2.10
2.00
1.30
0.80
0.30
0.04
0.01
12.00
Fat-Soluble Vitamins Release Based on Clinoptilolite Zeolite
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 2
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Fig. (1). SEM image of clinoptilolite powder (5000).
zeolite suspension was agitated (at 50 rpm) in dissolution chamber. In these experiments the control sample was without any clinoptilolite. The samples were maintained at 3°C and tested by HPLC analysis.
clinoptilolite suspensions exhibit a buffer pH around pH 8 ± 1, which has also been reported via B. Ersoy et al. [3, 18].
3. RESULTS AND DISCUSSION 3.1. Characterization of Clinoptilolite XRF analysis revealed the powder structure as a clinoptilolite type of zeolite. The main composition of clinoptilolite consisted of SiO2 and Al2O3. The chemical composition of Iranian natural clinoptilolite is shown in Table 1. Fig. (1) shows SEM image of clinoptilolite powder as porous particles. These pores are suitable location sites for molecules such as vitamins. Chemical analysis by EDAX shows that the ratio of Si to Al is 5.2 as also confirmed by XRF.
Fig. (2). The variations in pH in the presence of clinoptilolite solutions at pH 1.7 (a), 6.8 (b), 11.5(c) and the simulated pH (d).
3.2. Clinoptilolite Buffer Actions Results of simulated gastrointestinal pH of aqueous solutions and clinoptilolite buffer action are shown in Fig. (2). Clinoptilolite has acted as a buffering agent to increase the pH 1.7 to 5.2 and 6.8 to 8.2 and decreasing the pH 11.5 to 9.5. For simulation of gastrointestinal condition the pH was adjusted on 6.8 at 90th min. The variation of gastrointestinal pH can affect pH-sensitive drugs. Clinoptilolite can be considered as a permanently charged mineral because of the isomorphic substitution [2,18]. Thus,
3.3. Particle Size Selection The amounts of extracted vitamin E after 4 weeks for different particles size range are shown in Fig. (3). This experiment was carried out to find the best particles size of 710 to 850 m. Fig. (4) shows the extracted quantity of vitamin E in each period of time (2 h, 1, 2, 3 and 4 weeks) for particle size range between 710 to 850 m and control sample. Zeolite powder has enhanced the stability of vitamin. The extracted vitamin after 3 and 4 weeks in
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Fig. (3). The amount of extracted vitamin E versus particle size after 4 weeks.
zeolite-contained samples was found to be 5% and 17% higher than the controlled sample (without zeolite). There was a steady decrease in the extracted vitamin from the controlled sample compared to zeolite-contained sample. This enhanced stability can be attributed to the porosity and buffer action of zeolite powder (Fig. 1).
addition of 2 N NaOH (Fig. 5). The changes in control sample probably are related to degradation of more vitamin molecules due to their sensitivity to acidic pH. At first, in the zeolite containing sample only the vitamins on the surface had been in contact with acid. The pH value of the solution increases because of the buffered properties of zeolite. The decreasing acidic properties with time are due to the stability of released vitamins in aqueous medium pH. After 90 min, with addition of NaOH the pH increases and the amount of release increases up to 270 min, beyond which the amount of release remains constant. Finally, the amounts of release from the samples with zeolite were about 24, 23 and 20% higher than the control sample for vitamins A, D3 and E respectively (Table 2).
Fig. (4). Amount of remaining vitamin E versus time under laboratory condition in the zeolite-contained samples (retention time: 672 hours).
3.4. Vitamin Release Study In the simulated gastrointestinal pH test the amounts of vitamins A, D3 and E which remained in the process of the control sample (up to 30 min) were 34, 41 and 59%, and then the rate of degradation was decreased with time. In contrast, in the first 30 min in the sample with zeolite there was only about 8% vitamin left. The amount of vitamin in environment due to the presence of zeolite was more than the control sample over a long period of time for 330 min. At first (between 0 to 90 min), the rate of release of vitamin in samples with zeolite was steady and then the rate increased between 90 to 270 min when pH was adjusted to 6.8 by
Fig. (5). The changes in vitamin quantity versus time under simulated gastrointestinal pH with and without zeolite (retention time: 330 minutes).
Zeolite is a powerful support in protecting vitamins with their related physical and chemical properties. The porosities increase the active surface for protection of vitamins in appropriate pH (Fig. 1).
Fat-Soluble Vitamins Release Based on Clinoptilolite Zeolite
Table 2.
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 2
The Amount of Vitamins Released at Simulated Gastrointestinal pH with and Without Zeolite Vitamin A (%)
Time (min)
Vitamin D3 (%)
control
Sample
Control
sample
Control
0
8
100
10
100
9
100
30
12
34
12
41
15
59
60
15
33
17
41
23
59
90
18
33
20
40
29
58
120
28
33
30
40
44
57
150
34
32
36
39
54
57
180
40
32
41
39
60
57
210
47
32
49
39
66
57
240
52
32
52
38
71
56
270
53
32
58
37
73
56
300
54
31
58
37
75
56
330
54
30
59
36
75
55
SEM images have shown that the clinoptilolite particles are porous. These porosities are suitable sites for holding vitamin molecules. Also clinoptilolite has a buffer property and it can protect vitamins against acidic pH. After 4 weeks, the amount of remaining vitamin in zeolite samples is higher than control samples, and besides clinoptilolite can increase the shelf life of fat-soluble vitamins. The clinoptilolite that was used in this study performed well in high acidic pH of 1.7 and medium acidic pH of 6.8. The amount of vitamins released in samples with zeolite at first was about 8% and finally the amounts increased to 24, 23 and 20%, higher than the control samples and therefore fat-soluble vitamins have higher bioavailability in the presence of zeolite. ACKNOWLEDGMENTS The authors gratefully acknowledge the Helia Trade Persian Co. for financial support. Also special thanks to Iran Polymer and Petrochemical Institute, Materials and Energy Research Center for testing instruments and finally Dr. J. Eftekharnezhad for his helpful comments.
[4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
CONFLICT OF INTEREST The authors have reported no conflict of interest.
[15] [16]
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Vitamin E (%)
sample
4. CONCLUSION
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