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Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique
J. DRUG DEL. SCI. TECH., 15 (5) 363-369 2005
Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique M.R. Lauro1*, L. Maggi2, U. Conte2, F. De Simone1, R.P. Aquino1 Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo, Invariante 11/C, 84084, Fisciano, Salerno, Italy 2 Dipartimento di Chimica Farmaceutica, Università di Pavia, Via Taramelli, 12, 27100, Pavia, Italy *Correspondence: [email protected] 1
Quercetin and rutin gastro-resistant microparticles were prepared by spray-drying using cellulose acetate trimellitate (CAT) or cellulose acetate phthalate (CAP) as coating polymers. The influence of parameters such as the initial organic or aqueous feed solutions and polymer/drug ratio on the particle yield, behaviour and morphology was investigated. By spray-drying 2% buffer aqueous feed solutions in different polymer/drug ratio (1:1, 3:1 and 5:1) microparticles loaded with rutin were obtained. The microsystems were characterized by scanning electron microscopy (SEM), fluorescence microscopy (FM), and differential scanning calorimetry (DSC). In vitro dissolution studies, carried out using a pH change method, showed a typical biphasic drug release trend due to the pH dependent solubility of the enteric polymers. Key words: Rutin – Quercetin – Gastro-resistant microsystems – CAT – CAP – Spray-drying technique.
Flavonoids represent a large group of therapeutically active substances found in medicinal and edible plants, and present in more than 100 medicinal preparations marketed in Europe [1]. Particularly, the aglycon quercetin (Q) and its glycoside rutin (Rt) (Figure 1) are the naturally most widespread flavonoids contained in various herbal remedies showing various pharmacological effects. Some rutin derivatives are employed to treat cardiovascular chronic pathologies [2] such as venous insufficiency, capillary fragility and permeability, hæmorrhoids and perivascular œdema [3]. Quercetin has shown anti-ulcer activity in vivo [4] and antiproliferative and antimutagenic effects in vitro [5]. Several studies have reported that rutin and quercetin, present in fruits and vegetables, possess antioxidant properties and may play a dietary role in reducing the risk of chronic diseases such as cardiovascular pathologies and cancer [5]. In addition to the antioxidant activity, flavonoids possess an interesting anti-inflammatory profile after i.p. administration [6] and related to their capacity to interfere in vitro with a variety of processes involved in mediator release (such as the arachidonate metabolism by inhibition of both cyclooxygenase and 5-lipo-oxygenase pathways, the histamine release from mast cells and basophiles, Ca+2-regulated events and the respiratory burst of neutrophiles). Although the use of flavonoids as therapeutics in inflammatory conditions in vivo is dependent on the absorption, metabolism and excretion within the body after oral administration and on the properties of the resulting metabolites, our knowledge on the pharmacokinetics and bioavailability of Q and Rt in humans and animals is still limited. Some studies indicated that glycosides are hydrolyzed and partially degraded in the harsh pH and enzymatic conditions of the gastrointestinal tract. A first-pass effect of the orally administered drugs, convertion in conjugated or methylated metabolites with an intact flavonol structure, as well as breakdown in phenolic acids by the intestinal flora have been reported [7-9]. Nevertheless, relatively high levels of flavonoids can be absorbed through the small intestine mainly in the form of glucuronides of the parent aglycons or of the hydrolyzed glycosides. Therefore, a strategy to improve the bioavailability of these drugs may be to avoid the exposure of flavonoids
OH OH
HO
O
OR OH
R = -H
Quercetin
R = -rutinose
Rutin
O
Figure 1 - Structures of rutin (Rt) and quercetin (Q)
to the stressing conditions of the gastric tract and delivering them to the intestine. Thus, this study was aimed to optimize the performance of oral dosage forms of flavonoids developing gastroresistant microparticles prepared by spray-drying technique. Among the coating materials that looked adequate for this application, cellulose acetate phthalate (CAP) and cellulose acetate trimellitate (CAT) have been used. These gastroresistant polymers, which become soluble at slightly acidic pH values, are usually employed to coat oral dosage forms such as tablets for preventing their disintegration and drug release into the stomach [10, 11]. More recently, CAT and CAP have been used in the preparation of microparticles delivering therapeutical peptides and proteins to the intestine [12]. Spray-drying has previously been employed in the microencapsulation of non-steroidal anti-inflammatory drugs with enteric polymers [13]. This technique offers some advantages such as low amount of residual organic solvents in the final product and one-step process transforming a feed solution/suspension/emulsion into a dried particulate form. The influence of different polymer/drug ratios and different composition (aqueous or organic) of liquid feed on microparticles morphology and behavior was studied in comparison with those of non-gastroresistant HPMC (hydroxypropylmethylcellulose)/flavonoid microparticles and pure flavonoids. 363
J. DRUG DEL. SCI. TECH., 15 (5) 363-369 2005
Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique M.R. Lauro, L. Maggi, U. Conte, F. De Simone, R.P. Aquino
[14] higher than the aglycon (Q), due to the presence of the sugar moiety, CAT/Rt (Rt1, Rt2, Rt3) and CAP/RT (Rt4, Rt5, Rt6) microsystems were prepared only spray-drying buffer (intestinal fluid, IF, pH 7.5) 2% feed solutions in the polymer:drug ratios 1:1, 3:1 and 5:1, respectively. As control, non-gastroresistant HPMC:flavonoids systems (Q7 and Rt7) were obtained by spray-drying a 2% aqueous solution of HPMC and flavonoids in the ratio 3:1. The condition of the spray-drying process (Mini Spray Dryer Büchi 190 Flawil, CH) for CAT and CAP buffer solutions and HPMC aqueous solution were: nozzle 500 µm diameter; inlet air temperature 117-118°C; outlet air temperature 69-71°C; spray flow rate 5 ml/min; for CAT and CAP organic solutions were: nozzle 500 µm diameter; inlet air temperature 59-60°C; outlet air temperature 43-45°C; spray flow rate 5 ml/min. An organic and a buffer solution of both CAT and CAP (2%) were also sprayed to obtain drug empty microspheres used as comparison (Tables I and II). Each preparation was carried out in triplicate. The volume of feed solutions sprayed was 200 ml, and the total quantity of drug and polymer used was 6 g for the preparation of each batch. The solid microparticles were gathered from the apparatus and kept under vacuum for 48 hrs at room temperature. Only by using 2% CAT and CAP solutions, adequately coated microparticles were obtained.
I. MATERIALS AND METHODS 1. Materials
Rutin (Rt) and quercetin (Q) were supplied by Sigma-Aldrich Chemie Gmbh PO (Steinheim, Germany). Cellulose acetate trimellitate (CAT) and cellulose acetate phthalate (CAP), from Eastman Kodak (Kingsport, Tennessee, USA). Hydroxypropylmethylcellulose (HPMC; Methocel E3: 3 cPs, viscosity values stated by the supplier and measured at 20°C on 2% w/v aqueous solution, Ubbelohde apparatus) was supplied by Colorcon Limited (Orpington, UK). Acetone and ethanol 96%, were from Carlo Erba (Milan, Italy). All other chemical used were of reagent grade.
2. Microsystems preparation
The compositions of the different batches of spray-dried microparticles are reported in Tables I and II. Since quercetin (Q) (MW = 302) is very slightly soluble in water (7.7 mg/l) [14], organic and buffer feed solutions of CAT or CAP and drug were used. Organic solutions were prepared dissolving CAP or CAT at room temperature in 3:1 v/v mixture of acetone:ethanol, as previously reported [12] at 1 and 2% w/v concentration. Variable amount of Q (polymer:drug weight ratios 1:1, 3:1 and 5:1) was dissolved in the polymeric solutions by magnetic stirring to achieve the feed solutions. CAT/Q (Qs1, Qs2, Qs3) and CAP/Q (Qs4, Qs5, Qs6) microparticles were obtained by spray-drying these solutions. Buffer solutions were achieved dissolving CAT at 1 and 2% w/v concentration in intestinal fluid (IF, pH 7.5, according to USP 25 without enzymes). Then quercetin in different polymer:drug weight ratios (1:1, 3:1 and 5:1) was dispersed to give feed suspensions. CAT/Q microsystems (Q1, Q2, Q3) were obtained by spray-drying these feed solutions. Because rutin (Rt) (MW = 610) possess a water-solubility (45 mg/l)
3. Microparticles characterization
Particle size analysis of spray-dried drug-free blank and drugloaded microparticles were carried out with a laser light scattering granulometer (Beckman Counter LS 230, Particle Volume Module Plus, UK). The microparticles were suspended in water and the analysis was made in triplicate.
Table I - Composition, drug content, production yields and encapsulation efficiency of 2% CAP, CAT and HPMC spray-dried microspheres loaded with quercetin. Formulation
Polymer:drug ratio
Feed solutions
Theoretical drug content (%)
Actual drug content (%)
Production yields (%)
Encapsulation efficiency (%)
Q1 Q2 Q3 Q4 Q5 Q6 Q7 Qs1 Qs2 Qs3 Qs4 Qs5 Qs6
CAT:Q 1:1 CAT:Q 3:1 CAT:Q 5:1 CAP:Q 1:1 CAP:Q 3:1 CAP:Q 5:1 HPMC:Q 3:1 CAT:Q 1:1 CAT:Q 3:1 CAT:Q 5:1 CAP:Q 1:1 CAP:Q 3:1 CAP:Q 5:1
IF IF IF IF IF IF H 2O EtOH:Ac EtOH:Ac EtOH:Ac EtOH:Ac EtOH:Ac EtOH:Ac
50.0 25.0 16.6 50.0 25.0 16.6 25.0 50.0 25.0 16.6 50.0 25.0 16.6
37.2 18.0 10.7 38.0 16.0 10.2 23.3 48.0 24.0 16.0 40.0 22.0 14.0
75.0 56.0 56.8 72.0 60.0 56.0 45.0 50.0 52.0 54.0 54.0 60.0 54.0
74.4 72.0 64.4 76.0 64.0 61.4 93.2 96.0 96.0 96.4 80.0 88.0 84.3
IF: intestinal fluid, pH 7.5. EtOH:Ac: ethanol:acetone 3:1. CAT: cellulose acetate trimellitate. CAP: cellulose acetate phthalate. HPMC: hydroxypropylmethylcellulose. Q: quercetin. Table II - Composition, drug content, production yields and encapsulation efficiency of 2% CAP, CAT and HPMC spray-dried microspheres containing rutin. Formulation
Polymer:drug ratio
Feed solutions
Theoretical drug content (%)
Actual drug content (%)
Production yields (%)
Encapsulation efficiency (%)
Rt1 Rt2 Rt3 Rt4 Rt5 Rt6 Rt7
CAT:Rt 1:1 CAT:Rt 3:1 CAT:Rt 5:1 CAP:Rt 1:1 CAP:Rt 3:1 CAP:Rt 5:1 HPMC:Rt 3:1
IF IF IF IF IF IF H 2O
50.0 25.0 16.6 50.0 25.0 16.6 25.0
43.5 22.0 14.6 49.0 24.6 15.5 22.2
80.0 88.4 84.6 80.0 81.4 85.0 52.5
87.0 88.0 88.3 98.0 98.4 93.4 88.8
IF: intestinal fluid, pH 7.5. CAT: cellulose acetate trimellitate. CAP: cellulose acetate phthalate. HPMC: hydroxypropylmethylcellulose. Rt: rutin. 364
Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique M.R. Lauro, L. Maggi, U. Conte, F. De Simone, R.P. Aquino
Examination of the morphology of the microsystems was performed by scanning electron microscopy (SEM): the microparticles were coated with gold (degree of purity 99.9%) with an Edward S 150 A, sputter coated (BOC Edwards Italia, Milan, Italy) and then observed using scanning electron microscope (Cambridge Stereoscan 250; Cambridge Instruments Ltd, Cambridge, UK), operating at 15 kV. Analysis by fluorescence microscopy (FM) were carried out on samples of each batch of CAT and CAP/flavonoids and HPMC/flavonoids microparticles. Samples were viewed with a Zeiss Axiophot fluorescence microscope, with a 63 x 1.4 NA plan Apochromat oil immersion objective (Carl Zeiss Vision, München-Hallbergmoos, Germany) using standard DAPI (4ʼ, 6-diamidino-2-phenylindole) optics that absorb violet radiation (max 372 nm) and emit a blue fluorescence (max 456 nm).
J. DRUG DEL. SCI. TECH., 15 (5) 363-369 2005
than 1% solutions in coating the drugs. Rutin, which has higher water solubility than quercetin, was dispersed in a 2% buffer aqueous polymeric feed solutions. Quercetin, which doesnʼt possess sugar moiety in the structure and has lower molecular weight and very slightly water solubility, was dispersed in two different 2% feed solutions (organic and buffer aqueous). The obtained microparticles were characterized morphologically.
1. Microparticles analysis
Laser scattering particle size analysis showed that microparticles obtained spray-drying polymers:quercetin organic solutions (Qs1-Qs6) have a narrow size distribution with a mean diameter in the range 5-28 µm depending on the polymer:drug ratio. The microparticles obtained by spray-drying buffer feed solutions of both polymers showed much higher particle size and larger distribution, ranging from 70.7 to 180.0 µm for Q1-Q3 and from 81.7 to 185.7 µm for Rt1-Rt6. These results suggested the presence of microspheres aggregates as confirmed by SEM and FM analyses. The actual and theoretical drug contents of each batch, production yields and encapsulation efficiency are reported in Tables I and II. The UV analysis performed on microparticles showed a drug content close to the theoretical composition and good encapsulation efficiency of the different batches ranging from 80.0 to 96.4% for Qs1-Qs6, and from 87.0 to 98.4% for Rt1-Rt6, in comparison with that of HPMC/flavonoids microparticles (88.8% for Rt7 and 93.2% for Q7). The actual drug content of Q1-Q6 batches was lower as well as the encapsulation efficiency (61.4-76%). Because Q is very slightly soluble, a phase separation may occur and drug can be deposited in the spray-dried chamber. The production yields were about 50.0-60.0% for Qs1-Qs6, 56.075.0% for Q1-Q6 and from 80.0-88.4% for Rt1-Rt6 microparticles, depending on the microparticle compositions. These values were relatively good in view of the low quantity of materials used for the preparation of each batch, minding the usual loss of the smallest and lightest particles through the exhaust devices of the spray dryer apparatus.
3.1. Drug content, microencapsulation yields and encapsulation efficiency Samples (40 mg) of each batch of microparticles were dissolved in acetone:intestinal fluid (pH 7.5) 1:1 and the drug content was determined spectrophotometrically at λ 352 nm for Rt (1 mm cell) and at λ 366 nm for Q (1 cm cell) (Spectracomp 602, Advanced Products srl, Milan, Italy). Each analysis was made in triplicate and the results were expressed in average values. The production yields was expressed as the weight percentage of the final product compared with the total amount of polymer and drug sprayed. The encapsulation efficiency was calculated from the ratio of actual to theoretical drug content (Tables I and II). 3.2. Differential scanning calorimetry The thermal behavior of the microparticles was determined by differential scanning calorimetry (DSC) and it was compared with the thermal profiles of the blank microparticles and drugs as raw materials. DSC measurements were carried out using a Mettler Toledo DSC 821 module controlled by Mettler Star software. Samples were weighed (2-4 mg) with a microbalance (MTS Mettler Toledo, USA) and were scanned (10 K min-1) in open aluminum pans between 25-350°C under inert N2 dynamic atmosphere (200 ml/min).
2. Release studies
3.3. Infrared spectroscopy Sample of each batch of microparticles and the raw materials were tested as KBr discs in the 400-4000 cm-1 range using a Jasco FT-300IE (Jasco, Japan) Fourier transform IR (FTIR) spectrometer.
The release profiles of CAT and CAP gastroresistant microsystems are reported in Figures 2, 3 and 4 in comparison with non-gastro resistant microsystems containing HPMC of low viscosity (Q7, Rt7) and pure rutin (Rt) and quercetin (Q). Quercetin release profiles from Qs1-Qs6 microspheres obtained from organic solutions (Figure 2) showed that the amount of Q released/dissolved in 2 h, at pH 1.0, was about 15% from Qs4, but more than 20% from Qs5 and Qs6, and about 28-30% from Qs1-Qs3, in comparison with about 31% of pure Q dissolved in the same time. Thus, both polymers, CAT in the formulations Qs1-Qs3 and CAP in Qs4-Qs6, seemed not suitable to prepare Q gastroresistant microparticles by spray-drying organic feed solutions. In the second step, different microsystems were prepared by spraydrying buffer feed solutions of polymers and Q (Q1-Q6) in the ratios 1:1, 3:1 and 5:1. The dissolution profiles of all systems were almost superimposable (Figure 3). The amount of quercetin released/dissolved in 2 h, at pH 1.0, was less than 17%. Thus, these microparticles seem to be able to prevent Q exposure to pH acid-medium with slight differences. Quercetin was released in 15 min after pH change to 6.8 with a profile superimposable to that of the drug alone. Nevertheless, as reported in a previous study [14], only an incomplete release of Q (50-52%) from the formulations was obtained at pH 6.8, due to the drugʼs very slight solubility. As expected, non-gastroresistant HPMC:Q 3:1 (Q7) microsystem (Figure 3) showed dissolution profile similar to that of quercetin alone in both pH. Nevertheless the amount of drug dissolved is higher: within
3.4. Drug release tests In vitro dissolution/release tests of the drugs from the microparticles were carried out under sink conditions using a Sotax AT Smart Apparatus (Basel, CH) on line with a spectrophotometer (Spectracomp 602, Advanced Products srl, Milan, Italy) and USP 25 dissolution test apparatus No. 2: paddle, 100 rpm at 37°C. The pH change method (USP 25 drug release test, method A for Enteric Coated Articles) was used: 750 ml of HCl 0.1 N (pH 1) from 0 to 2 h, then the addition of 250 ml of 0.2 M tribasic sodium phosphate solution to give a final pH of 6.8 in a total volume of 1000 ml. All the dissolution/release tests were made in triplicate; only the mean values are reported in graph (standard deviations < 5%). Samples of spray-dried microparticles corresponding to about 5 mg of quercetin and to 20 mg of rutin were analyzed spectrophotometrically at λ 352 nm (Rt) and λ 366 nm (Q), respectively.
II. RESULTS AND DISCUSSION
Both drugs, rutin and quercetin (Figure 1), were spray-dried using CAP or CAT as coating polymers in different polymer:drug ratios (1:1, 3:1 and 5:1) in order to achieve gastroresistant microparticles, able to avoid the flavonoid exposure to harsh gastric conditions. Preliminary experiments revealed that 2% polymeric solutions were more efficient 365
J. DRUG DEL. SCI. TECH., 15 (5) 363-369 2005
Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique M.R. Lauro, L. Maggi, U. Conte, F. De Simone, R.P. Aquino
100
100
pH 6.8
pH 1.0
pH 6.8
pH 1.0
80
80
Qs5
% drug released
% drug released
Qs4
60
Qs6 Q Qs1 Qs2 Qs3
40
60
40
Rt4 Rt5 Rt6 Rt
20
20
Rt7 Rt1 Rt2 Rt3
0
0 0
30
60
90
120
150
0
180
30
60
90
120
150
180
time (min)
time (min)
Figure 2 - Dissolution profiles of CAT microparticles loaded with 50 (Qs1), 25 (Qs2), 16.6% (Qs3) of quercetin, or CAP microparticles loaded with 50 (Qs4), 25 (Qs5), 16.6% (Qs6) of quercetin, prepared by spray-drying organic feed solutions (ethanol 95%:acetone 3:1) in comparison with the dissolution profile of quercetin alone (Q).
Figure 4 - Dissolution profiles of CAT microparticles loaded with 50 (Rt1), 25 (Rt2), 16.6% (Rt3) of rutin, or CAP microparticles loaded with 50 (Rt4), 25 (Rt5), 16.6% (Rt6) of rutin, prepared by spray-drying buffer feed solutions (intestinal fluid, IF, pH 7.5), in comparison with the dissolution profiles of Rt7 (3:1 hydroxypropylmethylcellulose:rutin microparticles) and rutin alone (Rt).
100
% drug released
80
60
drug fraction, in the form of crystals, embedded at the surface of the microparticles and not effectively coated by the polymer (as shown by SEM and FM analysis, see below). Similar drug release profiles were obtained by dissolution studies of 1:1, 3:1 and 5:1 CAP:Rt microparticles (Rt4, Rt5, Rt6) (Figure 4). Also in this case the release rate of the drug is strongly improved in the neutral phase reaching 100% of the dose in 30 min. However, slight differences between the two polymers in reducing drug release at low pH values are present. CAP is considerably more effective than CAT in preventing the release of Rutin in the acidic medium; about 16-20% of drug is released from CAP Rt4-Rt6 microsystems at pH 1.0. For comparison, Figure 4 includes the release of Rt from non-gastroresistant HPMC/Rt particles (Rt7). The obtained results suggest that rutin gastroresistant formulations can be obtained by spray-drying an adequate solution of Rt and enteric polymers such as CAT or CAP. The different results obtained for particles loaded with quercetin (drug partially released in acidic medium and incomplete release in neutral phase) and rutin (drug retained to a major extent in acidic medium and complete release in neutral phase) can be justified on the basis of the different solubility and molecular weight of the drugs. Also these results are in agreement with those previously reported for other drugs; low MW molecules may easily leach from CAP particles, while larger ones are better retained by the polymeric structure [15].
pH 6.8
pH 1.0
Q4 Q5 Q6 Q HPMC:Q 3:1 Q1 Q2 Q3
40
20
0 0
30
60
90
120
150
180
time (min)
Figure 3 - Dissolution profiles of CAT microparticles loaded with 50 (Q1), 25 (Q2), 16.6% (Q3) of quercetin, or CAP microsystems loaded with 50 (Q4), 25 (Q5), 16.6% (Q6) of quercetin prepared by spray-drying buffer feed solutions (intestinal fluid, IF, pH 7.5), in comparison with the dissolution profiles of Q7 (3:1 hydroxypropylmethylcellulose:quercetin microparticles) and quercetin alone (Q).
30 min about 40% of Q was released from Q7 system compared with about 23% of Q alone. Furthermore, after pH change, an enhancement of Q dissolution rate was observed from Q7 system and it may be explained by an increase of drug-water interaction due to the presence of the hydrophilic polymeric substrate (lower viscosity HPMC), as observed in a previous paper [14]. Since Rt has a higher water-solubility than Q, microspheres were obtained by spray-drying aqueous buffer feed solutions. Figure 4 shows the release profile of rutin microparticles coated with CAT (Rt1-Rt3). There were no significant differences in the release rates or in the time of total release among particles prepared with 1:1, 3:1 and 5:1 CAT: Rt ratios. These formulations gave the classic release profiles of a gastro resistant dosage form: a very slow release (about 19.5-23%) of Rt in acid medium (pH 1.0) and the complete drug release (100% of the dose just in 30 min) after pH change to 6.8, compared with about 50% of rutin alone dissolving in the same time. The low amount of rutin released in the acid medium could be explained by the small
3. Particle morphology
The flavonoid powders and the microsystems were analyzed by FM (fluorescence microscopy) (Figures 5a, 5b and 6a, 6b) and SEM (scanning electron microscopy) (Figures 5c-5e and 6c). Figures 5c-5e shows photomicrographs of CAT microparticles loaded with 25% of rutin (Rt2), at different magnifications. The particles appeared to be grossly spherical even if in some cases sharp corners are visible. Some particles are aggregated, and some well formed and separated and some partially collapsed. The particle sizes are in the range 5-10 µm. Microspheres loaded with 50% (Rt1) and 16,6% of rutin (Rt3), and drug-free microparticles have similar size and morphology (data not reported). The photomicrographs at higher magnification (x 2500, x 5000) showed few drug crystals, either outside or adhering to the microsphere surface. SEM images of the particles loaded with 50% of quercetin (Q1) (Figure 6c) showed that these were formed by cluster of smaller micros366
Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique M.R. Lauro, L. Maggi, U. Conte, F. De Simone, R.P. Aquino
J. DRUG DEL. SCI. TECH., 15 (5) 363-369 2005
Figure 5 - Fluorescence microscopy images of rutin alone (control) (a) and Rt2 (25% rutin loaded CAT-microparticles) (b). Scanning electron micrographs (c, d, e) of Rt2 at different magnifications (x 1500, x 2500 and x 5000).
367
J. DRUG DEL. SCI. TECH., 15 (5) 363-369 2005
Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique M.R. Lauro, L. Maggi, U. Conte, F. De Simone, R.P. Aquino
4. Thermal and FT-IR analyses
Figures 7 and 8 show the thermograms of CAT blank microsystems, Q1 and Rt2 microparticles, respectively, and raw materials (Q and Rt). Quercetin peak at 116°C seems to be related to a decomposition process with water loss [16], and the melting peak was at 325°C (Figure 7a). In the thermograms of Q1 microparticles (Figure 7b) the detection of a new peak at 215°C gave an evidence of a polymer/drug interaction. Rutin showed the peak of the phase transition at 187°C (Figure 8a), probably related to the molecular rearrangement of the rutin polymorph in a plastic substance [16]. Peaks occurring over 215°C are due to the decomposition of rutin. In the thermograms of Rt2 microparticles (Figure 8b) the peak at 215°C was detected, suggesting, also in this case, a polymer/drug interaction. FTIR analysis confirmed this hypothesis showing in both the FTIR spectra of Q1 and Rt2 microsystems, compared with the spectra of raw materials (Q, Rt and CAT blank microparticles), a new band at 7a
7b
7c
50
00
100
55
150
10 10
200
15 15
250
20 20
300
25 25
°C
30 30
min min
Figure 7 - Differential scanning calorimetry thermograms of quercetin (a), CAT blank microparticles (c), and Q1 (50% quercetin loaded CATmicroparticles) (b).
Figure 6 - Fluorescence microscopy images of quercetin alone (control) (a) and Q1 (50% quercetin loaded CAT-microparticles) (b). Scanning electron micrographs of Q1 (50% quercetin loaded CAT-microparticles) at x 2500 magnification (c).
pheres (size about 10-20 µm), with aggregates of polymer particulates (size about 30-40 µm), and few needle shaped dry crystals adhering to the microspheres surface or inside them. Some of the microparticles were partially collapsed and derived from originally spherical particles distorted by loss of internal volume due to the temperature of spraydrying process, which involves rapid evaporation of the solvent. In fact the microspheres obtained by spray-drying feed organic solution (EtOH:acetone 3:1) were more collapsed and more irregular than microspheres obtained from buffer feed solutions (data not shown). The FM analysis led to similar conclusions (Figures 5a, 5b and 6a, 6b) showing microparticles with a distinct smooth membrane and a clear content, and few flavonoids crystals (red fluorescence for Rt, Figure 5a; orange fluorescence for Q, Figure 6a) not completely coated by the polymers but more evidently adhering to the microsphere surfaces (Figures 5b and 6b).
Figure 8 - Differential scanning calorimetry thermograms of rutin (a), CAT blank microparticles (c) and Rt2 (25% rutin loaded CAT-microparticles) (b). 368
Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique M.R. Lauro, L. Maggi, U. Conte, F. De Simone, R.P. Aquino
1693 cm-1 which reflected formation of ester linkages between hydroxyl groups of the drugs and carboxyl groups of the polymers. DSC curves and FTIR analysis of microsystems prepared with other polymer/drug ratios gave similar results and are not shown here.
4.
*
5.
Rutin and quercetin are very slightly soluble flavonoids with intermediate (Rt) or low molecular weight (Q), and unstable in the gastric environment in vivo. Previous studies showed slow dissolution rates and incomplete release for Quercetin in the gastric and intestinal fluids [14]. In the present study gastroresistant microparticles loaded with rutin and quercetin were prepared using spray-drying technique and CAT or CAP as coating enteric polymers. The series of experiments performed (variation of the polymers, of the initial feed solutions and of the polymer/flavonoid ratios) allowed to establish that 2% polymeric aqueous buffer solution (IF, pH 7.5) and 1:1 and 3:1 drug:polymer ratios were the optimal feed phase. The method used was effective in microencapsulating rutin, which has higher water solublility than quercetin, and the obtained microparticles showed adequate in vitro release patterns. Thus, CAT and CAP seemed to be efficient polymers to protect the glycoside, rutin (Rt), in the gastric medium and to release the maximum dose (100%) of the drug into the intestinal tract. On the contrary, the same microsystems loaded with very slightly soluble aglycon, quercetin (Q), were only partially able to protect the drug in the gastric medium, and Q release remained incomplete in the intestinal fluid, as previously described [14]. In conclusion, the present results suggest that spray drying technique may be used to obtain gastroresistant microparticles loaded with flavonol glycosides such as rutin. The obtained microsystems showed a quite good encapsulation efficiency, product yields and microparticles morphology and behaviour. This approach appears quite interesting for delivering flavonols, with chemical-physical properties similar to rutin, to the intestine avoiding gastric degradation of the molecule and improving their bioavailability.
15.
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and inhibition of arachidonic acid metabolism by flavonoids. - Agents Actions, 32 (3-4), 283-288, 1991. FERMÌN SANCHEZ DE MEDINA L. H., GALVEZ J., GOZALES M., ZARZUELO A. AND BARRETT K. E. - Effects of quercetin on epithelial chloride secretion. - Life Science, 61 (20), 20492055, 1997. KUO S. M. - Antiproliferative potency of structurally distinct dietary flavonoids human colon cancer cells. - Cancer Letters, 110, 41-48, 1996. GUARDIA T., ROTELLI A.E., JUAREZ A.O., PELZER L.E. - Anti-inflammatory properties of plant flavonoids, Effects of rutin, quercetin and hesperidin on adjuvant arthritis in rat. - Il Farmaco, 56, 683-687, 2001. ADER P., WESSMANN A., WOFFRAM S. - Bioavailability and metabolism by the flavonol quercetin in the pig. - Free Rad. Bio. Med., 28 (7), 1056-1067, 2000. MANACH C., MORAND C., DEMIGNÉ C., TEXIER O., REGERAT F., REMESY C. - Bioavailability of rutin and quercetin in rats. FEBS Letters, 409, 12-16, 1997. SPENCER J.P.E., CHOWRIMOOTOO G., CHOUDHURY R.,. DEBNAM E. S, SRAI S. K., RICE-EVANS C. - The small intestine can both absorb and glucoronidate luminal flavonoids. - FEBS Letters, 458, 224-230, 1999. GIUNCHEDI P., CONTE U. - Spray-drying as a preparation method of microparticulate drug delivery systems: an overview. - STP Pharm. Sci., 5 (4), 276-290, 1990. CONTI B., GIUNCHEDI P., CONTE U. - Cellulose microparticles in drug delivery. - STP Pharm. Sci., 7 (5), 331-342, 1997. GONCALVES DE BRITO AMORIN M. J. L., FERREIRA J. P. M. - Microparticles for delivering therapeutic peptides and proteins to the lumen of small intestine. - Eur. J. Pharm. Biopharm., 52, 39-44, 2001. GIUNCHEDI P., TORRE M. L., MAGGI L., CONTI B., CONTE U. - Cellulose acetate trimellitate ethylcellulose blends for nonsteroidal anti-inflammatory drug (NSAID) microspheres. - J. Microencapsulation, 13 (1), 89-98, 1996. LAURO M. R., TORRE M. L., MAGGI L., DE SIMONE F., CONTE U., AQUINO R.P. - Fast and slow-release tablets for oral administration of flavonoids: rutin and quercetin. - Drug Dev. Ind. Pharm., 28 (4), 371-379, 2002. SILVA J. P. S., FERREIRA J. P. M. - Effect of drug properties on the release from CAP microspheres prepared by solvent evaporation method. - J. Microencapsulation, 16, 95-103, 1999. MOREIRA DE COSTA E., BARBOSA FILHO J. M., GOMES DO NASCIMIENTO T., OLIVEIRA MACEDO R. - Thermal characterization of the quercetin and rutin flavonoids. - Thermochimica Acta, 392-393, 79-84, 2002.
MANUSCRIPT Received 3 January 2005, accepted for publication 12 May 2005.
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Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique M.R. Lauro1*, L. Maggi2, U. Conte2, F. De Simone1, R.P. Aquino1 Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo, Invariante 11/C, 84084, Fisciano, Salerno, Italy 2 Dipartimento di Chimica Farmaceutica, Università di Pavia, Via Taramelli, 12, 27100, Pavia, Italy *Correspondence: [email protected] 1
Quercetin and rutin gastro-resistant microparticles were prepared by spray-drying using cellulose acetate trimellitate (CAT) or cellulose acetate phthalate (CAP) as coating polymers. The influence of parameters such as the initial organic or aqueous feed solutions and polymer/drug ratio on the particle yield, behaviour and morphology was investigated. By spray-drying 2% buffer aqueous feed solutions in different polymer/drug ratio (1:1, 3:1 and 5:1) microparticles loaded with rutin were obtained. The microsystems were characterized by scanning electron microscopy (SEM), fluorescence microscopy (FM), and differential scanning calorimetry (DSC). In vitro dissolution studies, carried out using a pH change method, showed a typical biphasic drug release trend due to the pH dependent solubility of the enteric polymers. Key words: Rutin – Quercetin – Gastro-resistant microsystems – CAT – CAP – Spray-drying technique.
Flavonoids represent a large group of therapeutically active substances found in medicinal and edible plants, and present in more than 100 medicinal preparations marketed in Europe [1]. Particularly, the aglycon quercetin (Q) and its glycoside rutin (Rt) (Figure 1) are the naturally most widespread flavonoids contained in various herbal remedies showing various pharmacological effects. Some rutin derivatives are employed to treat cardiovascular chronic pathologies [2] such as venous insufficiency, capillary fragility and permeability, hæmorrhoids and perivascular œdema [3]. Quercetin has shown anti-ulcer activity in vivo [4] and antiproliferative and antimutagenic effects in vitro [5]. Several studies have reported that rutin and quercetin, present in fruits and vegetables, possess antioxidant properties and may play a dietary role in reducing the risk of chronic diseases such as cardiovascular pathologies and cancer [5]. In addition to the antioxidant activity, flavonoids possess an interesting anti-inflammatory profile after i.p. administration [6] and related to their capacity to interfere in vitro with a variety of processes involved in mediator release (such as the arachidonate metabolism by inhibition of both cyclooxygenase and 5-lipo-oxygenase pathways, the histamine release from mast cells and basophiles, Ca+2-regulated events and the respiratory burst of neutrophiles). Although the use of flavonoids as therapeutics in inflammatory conditions in vivo is dependent on the absorption, metabolism and excretion within the body after oral administration and on the properties of the resulting metabolites, our knowledge on the pharmacokinetics and bioavailability of Q and Rt in humans and animals is still limited. Some studies indicated that glycosides are hydrolyzed and partially degraded in the harsh pH and enzymatic conditions of the gastrointestinal tract. A first-pass effect of the orally administered drugs, convertion in conjugated or methylated metabolites with an intact flavonol structure, as well as breakdown in phenolic acids by the intestinal flora have been reported [7-9]. Nevertheless, relatively high levels of flavonoids can be absorbed through the small intestine mainly in the form of glucuronides of the parent aglycons or of the hydrolyzed glycosides. Therefore, a strategy to improve the bioavailability of these drugs may be to avoid the exposure of flavonoids
OH OH
HO
O
OR OH
R = -H
Quercetin
R = -rutinose
Rutin
O
Figure 1 - Structures of rutin (Rt) and quercetin (Q)
to the stressing conditions of the gastric tract and delivering them to the intestine. Thus, this study was aimed to optimize the performance of oral dosage forms of flavonoids developing gastroresistant microparticles prepared by spray-drying technique. Among the coating materials that looked adequate for this application, cellulose acetate phthalate (CAP) and cellulose acetate trimellitate (CAT) have been used. These gastroresistant polymers, which become soluble at slightly acidic pH values, are usually employed to coat oral dosage forms such as tablets for preventing their disintegration and drug release into the stomach [10, 11]. More recently, CAT and CAP have been used in the preparation of microparticles delivering therapeutical peptides and proteins to the intestine [12]. Spray-drying has previously been employed in the microencapsulation of non-steroidal anti-inflammatory drugs with enteric polymers [13]. This technique offers some advantages such as low amount of residual organic solvents in the final product and one-step process transforming a feed solution/suspension/emulsion into a dried particulate form. The influence of different polymer/drug ratios and different composition (aqueous or organic) of liquid feed on microparticles morphology and behavior was studied in comparison with those of non-gastroresistant HPMC (hydroxypropylmethylcellulose)/flavonoid microparticles and pure flavonoids. 363
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Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique M.R. Lauro, L. Maggi, U. Conte, F. De Simone, R.P. Aquino
[14] higher than the aglycon (Q), due to the presence of the sugar moiety, CAT/Rt (Rt1, Rt2, Rt3) and CAP/RT (Rt4, Rt5, Rt6) microsystems were prepared only spray-drying buffer (intestinal fluid, IF, pH 7.5) 2% feed solutions in the polymer:drug ratios 1:1, 3:1 and 5:1, respectively. As control, non-gastroresistant HPMC:flavonoids systems (Q7 and Rt7) were obtained by spray-drying a 2% aqueous solution of HPMC and flavonoids in the ratio 3:1. The condition of the spray-drying process (Mini Spray Dryer Büchi 190 Flawil, CH) for CAT and CAP buffer solutions and HPMC aqueous solution were: nozzle 500 µm diameter; inlet air temperature 117-118°C; outlet air temperature 69-71°C; spray flow rate 5 ml/min; for CAT and CAP organic solutions were: nozzle 500 µm diameter; inlet air temperature 59-60°C; outlet air temperature 43-45°C; spray flow rate 5 ml/min. An organic and a buffer solution of both CAT and CAP (2%) were also sprayed to obtain drug empty microspheres used as comparison (Tables I and II). Each preparation was carried out in triplicate. The volume of feed solutions sprayed was 200 ml, and the total quantity of drug and polymer used was 6 g for the preparation of each batch. The solid microparticles were gathered from the apparatus and kept under vacuum for 48 hrs at room temperature. Only by using 2% CAT and CAP solutions, adequately coated microparticles were obtained.
I. MATERIALS AND METHODS 1. Materials
Rutin (Rt) and quercetin (Q) were supplied by Sigma-Aldrich Chemie Gmbh PO (Steinheim, Germany). Cellulose acetate trimellitate (CAT) and cellulose acetate phthalate (CAP), from Eastman Kodak (Kingsport, Tennessee, USA). Hydroxypropylmethylcellulose (HPMC; Methocel E3: 3 cPs, viscosity values stated by the supplier and measured at 20°C on 2% w/v aqueous solution, Ubbelohde apparatus) was supplied by Colorcon Limited (Orpington, UK). Acetone and ethanol 96%, were from Carlo Erba (Milan, Italy). All other chemical used were of reagent grade.
2. Microsystems preparation
The compositions of the different batches of spray-dried microparticles are reported in Tables I and II. Since quercetin (Q) (MW = 302) is very slightly soluble in water (7.7 mg/l) [14], organic and buffer feed solutions of CAT or CAP and drug were used. Organic solutions were prepared dissolving CAP or CAT at room temperature in 3:1 v/v mixture of acetone:ethanol, as previously reported [12] at 1 and 2% w/v concentration. Variable amount of Q (polymer:drug weight ratios 1:1, 3:1 and 5:1) was dissolved in the polymeric solutions by magnetic stirring to achieve the feed solutions. CAT/Q (Qs1, Qs2, Qs3) and CAP/Q (Qs4, Qs5, Qs6) microparticles were obtained by spray-drying these solutions. Buffer solutions were achieved dissolving CAT at 1 and 2% w/v concentration in intestinal fluid (IF, pH 7.5, according to USP 25 without enzymes). Then quercetin in different polymer:drug weight ratios (1:1, 3:1 and 5:1) was dispersed to give feed suspensions. CAT/Q microsystems (Q1, Q2, Q3) were obtained by spray-drying these feed solutions. Because rutin (Rt) (MW = 610) possess a water-solubility (45 mg/l)
3. Microparticles characterization
Particle size analysis of spray-dried drug-free blank and drugloaded microparticles were carried out with a laser light scattering granulometer (Beckman Counter LS 230, Particle Volume Module Plus, UK). The microparticles were suspended in water and the analysis was made in triplicate.
Table I - Composition, drug content, production yields and encapsulation efficiency of 2% CAP, CAT and HPMC spray-dried microspheres loaded with quercetin. Formulation
Polymer:drug ratio
Feed solutions
Theoretical drug content (%)
Actual drug content (%)
Production yields (%)
Encapsulation efficiency (%)
Q1 Q2 Q3 Q4 Q5 Q6 Q7 Qs1 Qs2 Qs3 Qs4 Qs5 Qs6
CAT:Q 1:1 CAT:Q 3:1 CAT:Q 5:1 CAP:Q 1:1 CAP:Q 3:1 CAP:Q 5:1 HPMC:Q 3:1 CAT:Q 1:1 CAT:Q 3:1 CAT:Q 5:1 CAP:Q 1:1 CAP:Q 3:1 CAP:Q 5:1
IF IF IF IF IF IF H 2O EtOH:Ac EtOH:Ac EtOH:Ac EtOH:Ac EtOH:Ac EtOH:Ac
50.0 25.0 16.6 50.0 25.0 16.6 25.0 50.0 25.0 16.6 50.0 25.0 16.6
37.2 18.0 10.7 38.0 16.0 10.2 23.3 48.0 24.0 16.0 40.0 22.0 14.0
75.0 56.0 56.8 72.0 60.0 56.0 45.0 50.0 52.0 54.0 54.0 60.0 54.0
74.4 72.0 64.4 76.0 64.0 61.4 93.2 96.0 96.0 96.4 80.0 88.0 84.3
IF: intestinal fluid, pH 7.5. EtOH:Ac: ethanol:acetone 3:1. CAT: cellulose acetate trimellitate. CAP: cellulose acetate phthalate. HPMC: hydroxypropylmethylcellulose. Q: quercetin. Table II - Composition, drug content, production yields and encapsulation efficiency of 2% CAP, CAT and HPMC spray-dried microspheres containing rutin. Formulation
Polymer:drug ratio
Feed solutions
Theoretical drug content (%)
Actual drug content (%)
Production yields (%)
Encapsulation efficiency (%)
Rt1 Rt2 Rt3 Rt4 Rt5 Rt6 Rt7
CAT:Rt 1:1 CAT:Rt 3:1 CAT:Rt 5:1 CAP:Rt 1:1 CAP:Rt 3:1 CAP:Rt 5:1 HPMC:Rt 3:1
IF IF IF IF IF IF H 2O
50.0 25.0 16.6 50.0 25.0 16.6 25.0
43.5 22.0 14.6 49.0 24.6 15.5 22.2
80.0 88.4 84.6 80.0 81.4 85.0 52.5
87.0 88.0 88.3 98.0 98.4 93.4 88.8
IF: intestinal fluid, pH 7.5. CAT: cellulose acetate trimellitate. CAP: cellulose acetate phthalate. HPMC: hydroxypropylmethylcellulose. Rt: rutin. 364
Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique M.R. Lauro, L. Maggi, U. Conte, F. De Simone, R.P. Aquino
Examination of the morphology of the microsystems was performed by scanning electron microscopy (SEM): the microparticles were coated with gold (degree of purity 99.9%) with an Edward S 150 A, sputter coated (BOC Edwards Italia, Milan, Italy) and then observed using scanning electron microscope (Cambridge Stereoscan 250; Cambridge Instruments Ltd, Cambridge, UK), operating at 15 kV. Analysis by fluorescence microscopy (FM) were carried out on samples of each batch of CAT and CAP/flavonoids and HPMC/flavonoids microparticles. Samples were viewed with a Zeiss Axiophot fluorescence microscope, with a 63 x 1.4 NA plan Apochromat oil immersion objective (Carl Zeiss Vision, München-Hallbergmoos, Germany) using standard DAPI (4ʼ, 6-diamidino-2-phenylindole) optics that absorb violet radiation (max 372 nm) and emit a blue fluorescence (max 456 nm).
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than 1% solutions in coating the drugs. Rutin, which has higher water solubility than quercetin, was dispersed in a 2% buffer aqueous polymeric feed solutions. Quercetin, which doesnʼt possess sugar moiety in the structure and has lower molecular weight and very slightly water solubility, was dispersed in two different 2% feed solutions (organic and buffer aqueous). The obtained microparticles were characterized morphologically.
1. Microparticles analysis
Laser scattering particle size analysis showed that microparticles obtained spray-drying polymers:quercetin organic solutions (Qs1-Qs6) have a narrow size distribution with a mean diameter in the range 5-28 µm depending on the polymer:drug ratio. The microparticles obtained by spray-drying buffer feed solutions of both polymers showed much higher particle size and larger distribution, ranging from 70.7 to 180.0 µm for Q1-Q3 and from 81.7 to 185.7 µm for Rt1-Rt6. These results suggested the presence of microspheres aggregates as confirmed by SEM and FM analyses. The actual and theoretical drug contents of each batch, production yields and encapsulation efficiency are reported in Tables I and II. The UV analysis performed on microparticles showed a drug content close to the theoretical composition and good encapsulation efficiency of the different batches ranging from 80.0 to 96.4% for Qs1-Qs6, and from 87.0 to 98.4% for Rt1-Rt6, in comparison with that of HPMC/flavonoids microparticles (88.8% for Rt7 and 93.2% for Q7). The actual drug content of Q1-Q6 batches was lower as well as the encapsulation efficiency (61.4-76%). Because Q is very slightly soluble, a phase separation may occur and drug can be deposited in the spray-dried chamber. The production yields were about 50.0-60.0% for Qs1-Qs6, 56.075.0% for Q1-Q6 and from 80.0-88.4% for Rt1-Rt6 microparticles, depending on the microparticle compositions. These values were relatively good in view of the low quantity of materials used for the preparation of each batch, minding the usual loss of the smallest and lightest particles through the exhaust devices of the spray dryer apparatus.
3.1. Drug content, microencapsulation yields and encapsulation efficiency Samples (40 mg) of each batch of microparticles were dissolved in acetone:intestinal fluid (pH 7.5) 1:1 and the drug content was determined spectrophotometrically at λ 352 nm for Rt (1 mm cell) and at λ 366 nm for Q (1 cm cell) (Spectracomp 602, Advanced Products srl, Milan, Italy). Each analysis was made in triplicate and the results were expressed in average values. The production yields was expressed as the weight percentage of the final product compared with the total amount of polymer and drug sprayed. The encapsulation efficiency was calculated from the ratio of actual to theoretical drug content (Tables I and II). 3.2. Differential scanning calorimetry The thermal behavior of the microparticles was determined by differential scanning calorimetry (DSC) and it was compared with the thermal profiles of the blank microparticles and drugs as raw materials. DSC measurements were carried out using a Mettler Toledo DSC 821 module controlled by Mettler Star software. Samples were weighed (2-4 mg) with a microbalance (MTS Mettler Toledo, USA) and were scanned (10 K min-1) in open aluminum pans between 25-350°C under inert N2 dynamic atmosphere (200 ml/min).
2. Release studies
3.3. Infrared spectroscopy Sample of each batch of microparticles and the raw materials were tested as KBr discs in the 400-4000 cm-1 range using a Jasco FT-300IE (Jasco, Japan) Fourier transform IR (FTIR) spectrometer.
The release profiles of CAT and CAP gastroresistant microsystems are reported in Figures 2, 3 and 4 in comparison with non-gastro resistant microsystems containing HPMC of low viscosity (Q7, Rt7) and pure rutin (Rt) and quercetin (Q). Quercetin release profiles from Qs1-Qs6 microspheres obtained from organic solutions (Figure 2) showed that the amount of Q released/dissolved in 2 h, at pH 1.0, was about 15% from Qs4, but more than 20% from Qs5 and Qs6, and about 28-30% from Qs1-Qs3, in comparison with about 31% of pure Q dissolved in the same time. Thus, both polymers, CAT in the formulations Qs1-Qs3 and CAP in Qs4-Qs6, seemed not suitable to prepare Q gastroresistant microparticles by spray-drying organic feed solutions. In the second step, different microsystems were prepared by spraydrying buffer feed solutions of polymers and Q (Q1-Q6) in the ratios 1:1, 3:1 and 5:1. The dissolution profiles of all systems were almost superimposable (Figure 3). The amount of quercetin released/dissolved in 2 h, at pH 1.0, was less than 17%. Thus, these microparticles seem to be able to prevent Q exposure to pH acid-medium with slight differences. Quercetin was released in 15 min after pH change to 6.8 with a profile superimposable to that of the drug alone. Nevertheless, as reported in a previous study [14], only an incomplete release of Q (50-52%) from the formulations was obtained at pH 6.8, due to the drugʼs very slight solubility. As expected, non-gastroresistant HPMC:Q 3:1 (Q7) microsystem (Figure 3) showed dissolution profile similar to that of quercetin alone in both pH. Nevertheless the amount of drug dissolved is higher: within
3.4. Drug release tests In vitro dissolution/release tests of the drugs from the microparticles were carried out under sink conditions using a Sotax AT Smart Apparatus (Basel, CH) on line with a spectrophotometer (Spectracomp 602, Advanced Products srl, Milan, Italy) and USP 25 dissolution test apparatus No. 2: paddle, 100 rpm at 37°C. The pH change method (USP 25 drug release test, method A for Enteric Coated Articles) was used: 750 ml of HCl 0.1 N (pH 1) from 0 to 2 h, then the addition of 250 ml of 0.2 M tribasic sodium phosphate solution to give a final pH of 6.8 in a total volume of 1000 ml. All the dissolution/release tests were made in triplicate; only the mean values are reported in graph (standard deviations < 5%). Samples of spray-dried microparticles corresponding to about 5 mg of quercetin and to 20 mg of rutin were analyzed spectrophotometrically at λ 352 nm (Rt) and λ 366 nm (Q), respectively.
II. RESULTS AND DISCUSSION
Both drugs, rutin and quercetin (Figure 1), were spray-dried using CAP or CAT as coating polymers in different polymer:drug ratios (1:1, 3:1 and 5:1) in order to achieve gastroresistant microparticles, able to avoid the flavonoid exposure to harsh gastric conditions. Preliminary experiments revealed that 2% polymeric solutions were more efficient 365
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Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique M.R. Lauro, L. Maggi, U. Conte, F. De Simone, R.P. Aquino
100
100
pH 6.8
pH 1.0
pH 6.8
pH 1.0
80
80
Qs5
% drug released
% drug released
Qs4
60
Qs6 Q Qs1 Qs2 Qs3
40
60
40
Rt4 Rt5 Rt6 Rt
20
20
Rt7 Rt1 Rt2 Rt3
0
0 0
30
60
90
120
150
0
180
30
60
90
120
150
180
time (min)
time (min)
Figure 2 - Dissolution profiles of CAT microparticles loaded with 50 (Qs1), 25 (Qs2), 16.6% (Qs3) of quercetin, or CAP microparticles loaded with 50 (Qs4), 25 (Qs5), 16.6% (Qs6) of quercetin, prepared by spray-drying organic feed solutions (ethanol 95%:acetone 3:1) in comparison with the dissolution profile of quercetin alone (Q).
Figure 4 - Dissolution profiles of CAT microparticles loaded with 50 (Rt1), 25 (Rt2), 16.6% (Rt3) of rutin, or CAP microparticles loaded with 50 (Rt4), 25 (Rt5), 16.6% (Rt6) of rutin, prepared by spray-drying buffer feed solutions (intestinal fluid, IF, pH 7.5), in comparison with the dissolution profiles of Rt7 (3:1 hydroxypropylmethylcellulose:rutin microparticles) and rutin alone (Rt).
100
% drug released
80
60
drug fraction, in the form of crystals, embedded at the surface of the microparticles and not effectively coated by the polymer (as shown by SEM and FM analysis, see below). Similar drug release profiles were obtained by dissolution studies of 1:1, 3:1 and 5:1 CAP:Rt microparticles (Rt4, Rt5, Rt6) (Figure 4). Also in this case the release rate of the drug is strongly improved in the neutral phase reaching 100% of the dose in 30 min. However, slight differences between the two polymers in reducing drug release at low pH values are present. CAP is considerably more effective than CAT in preventing the release of Rutin in the acidic medium; about 16-20% of drug is released from CAP Rt4-Rt6 microsystems at pH 1.0. For comparison, Figure 4 includes the release of Rt from non-gastroresistant HPMC/Rt particles (Rt7). The obtained results suggest that rutin gastroresistant formulations can be obtained by spray-drying an adequate solution of Rt and enteric polymers such as CAT or CAP. The different results obtained for particles loaded with quercetin (drug partially released in acidic medium and incomplete release in neutral phase) and rutin (drug retained to a major extent in acidic medium and complete release in neutral phase) can be justified on the basis of the different solubility and molecular weight of the drugs. Also these results are in agreement with those previously reported for other drugs; low MW molecules may easily leach from CAP particles, while larger ones are better retained by the polymeric structure [15].
pH 6.8
pH 1.0
Q4 Q5 Q6 Q HPMC:Q 3:1 Q1 Q2 Q3
40
20
0 0
30
60
90
120
150
180
time (min)
Figure 3 - Dissolution profiles of CAT microparticles loaded with 50 (Q1), 25 (Q2), 16.6% (Q3) of quercetin, or CAP microsystems loaded with 50 (Q4), 25 (Q5), 16.6% (Q6) of quercetin prepared by spray-drying buffer feed solutions (intestinal fluid, IF, pH 7.5), in comparison with the dissolution profiles of Q7 (3:1 hydroxypropylmethylcellulose:quercetin microparticles) and quercetin alone (Q).
30 min about 40% of Q was released from Q7 system compared with about 23% of Q alone. Furthermore, after pH change, an enhancement of Q dissolution rate was observed from Q7 system and it may be explained by an increase of drug-water interaction due to the presence of the hydrophilic polymeric substrate (lower viscosity HPMC), as observed in a previous paper [14]. Since Rt has a higher water-solubility than Q, microspheres were obtained by spray-drying aqueous buffer feed solutions. Figure 4 shows the release profile of rutin microparticles coated with CAT (Rt1-Rt3). There were no significant differences in the release rates or in the time of total release among particles prepared with 1:1, 3:1 and 5:1 CAT: Rt ratios. These formulations gave the classic release profiles of a gastro resistant dosage form: a very slow release (about 19.5-23%) of Rt in acid medium (pH 1.0) and the complete drug release (100% of the dose just in 30 min) after pH change to 6.8, compared with about 50% of rutin alone dissolving in the same time. The low amount of rutin released in the acid medium could be explained by the small
3. Particle morphology
The flavonoid powders and the microsystems were analyzed by FM (fluorescence microscopy) (Figures 5a, 5b and 6a, 6b) and SEM (scanning electron microscopy) (Figures 5c-5e and 6c). Figures 5c-5e shows photomicrographs of CAT microparticles loaded with 25% of rutin (Rt2), at different magnifications. The particles appeared to be grossly spherical even if in some cases sharp corners are visible. Some particles are aggregated, and some well formed and separated and some partially collapsed. The particle sizes are in the range 5-10 µm. Microspheres loaded with 50% (Rt1) and 16,6% of rutin (Rt3), and drug-free microparticles have similar size and morphology (data not reported). The photomicrographs at higher magnification (x 2500, x 5000) showed few drug crystals, either outside or adhering to the microsphere surface. SEM images of the particles loaded with 50% of quercetin (Q1) (Figure 6c) showed that these were formed by cluster of smaller micros366
Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique M.R. Lauro, L. Maggi, U. Conte, F. De Simone, R.P. Aquino
J. DRUG DEL. SCI. TECH., 15 (5) 363-369 2005
Figure 5 - Fluorescence microscopy images of rutin alone (control) (a) and Rt2 (25% rutin loaded CAT-microparticles) (b). Scanning electron micrographs (c, d, e) of Rt2 at different magnifications (x 1500, x 2500 and x 5000).
367
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Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique M.R. Lauro, L. Maggi, U. Conte, F. De Simone, R.P. Aquino
4. Thermal and FT-IR analyses
Figures 7 and 8 show the thermograms of CAT blank microsystems, Q1 and Rt2 microparticles, respectively, and raw materials (Q and Rt). Quercetin peak at 116°C seems to be related to a decomposition process with water loss [16], and the melting peak was at 325°C (Figure 7a). In the thermograms of Q1 microparticles (Figure 7b) the detection of a new peak at 215°C gave an evidence of a polymer/drug interaction. Rutin showed the peak of the phase transition at 187°C (Figure 8a), probably related to the molecular rearrangement of the rutin polymorph in a plastic substance [16]. Peaks occurring over 215°C are due to the decomposition of rutin. In the thermograms of Rt2 microparticles (Figure 8b) the peak at 215°C was detected, suggesting, also in this case, a polymer/drug interaction. FTIR analysis confirmed this hypothesis showing in both the FTIR spectra of Q1 and Rt2 microsystems, compared with the spectra of raw materials (Q, Rt and CAT blank microparticles), a new band at 7a
7b
7c
50
00
100
55
150
10 10
200
15 15
250
20 20
300
25 25
°C
30 30
min min
Figure 7 - Differential scanning calorimetry thermograms of quercetin (a), CAT blank microparticles (c), and Q1 (50% quercetin loaded CATmicroparticles) (b).
Figure 6 - Fluorescence microscopy images of quercetin alone (control) (a) and Q1 (50% quercetin loaded CAT-microparticles) (b). Scanning electron micrographs of Q1 (50% quercetin loaded CAT-microparticles) at x 2500 magnification (c).
pheres (size about 10-20 µm), with aggregates of polymer particulates (size about 30-40 µm), and few needle shaped dry crystals adhering to the microspheres surface or inside them. Some of the microparticles were partially collapsed and derived from originally spherical particles distorted by loss of internal volume due to the temperature of spraydrying process, which involves rapid evaporation of the solvent. In fact the microspheres obtained by spray-drying feed organic solution (EtOH:acetone 3:1) were more collapsed and more irregular than microspheres obtained from buffer feed solutions (data not shown). The FM analysis led to similar conclusions (Figures 5a, 5b and 6a, 6b) showing microparticles with a distinct smooth membrane and a clear content, and few flavonoids crystals (red fluorescence for Rt, Figure 5a; orange fluorescence for Q, Figure 6a) not completely coated by the polymers but more evidently adhering to the microsphere surfaces (Figures 5b and 6b).
Figure 8 - Differential scanning calorimetry thermograms of rutin (a), CAT blank microparticles (c) and Rt2 (25% rutin loaded CAT-microparticles) (b). 368
Rutin and quercetin gastro-resistant microparticles obtained by spray-drying technique M.R. Lauro, L. Maggi, U. Conte, F. De Simone, R.P. Aquino
1693 cm-1 which reflected formation of ester linkages between hydroxyl groups of the drugs and carboxyl groups of the polymers. DSC curves and FTIR analysis of microsystems prepared with other polymer/drug ratios gave similar results and are not shown here.
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Rutin and quercetin are very slightly soluble flavonoids with intermediate (Rt) or low molecular weight (Q), and unstable in the gastric environment in vivo. Previous studies showed slow dissolution rates and incomplete release for Quercetin in the gastric and intestinal fluids [14]. In the present study gastroresistant microparticles loaded with rutin and quercetin were prepared using spray-drying technique and CAT or CAP as coating enteric polymers. The series of experiments performed (variation of the polymers, of the initial feed solutions and of the polymer/flavonoid ratios) allowed to establish that 2% polymeric aqueous buffer solution (IF, pH 7.5) and 1:1 and 3:1 drug:polymer ratios were the optimal feed phase. The method used was effective in microencapsulating rutin, which has higher water solublility than quercetin, and the obtained microparticles showed adequate in vitro release patterns. Thus, CAT and CAP seemed to be efficient polymers to protect the glycoside, rutin (Rt), in the gastric medium and to release the maximum dose (100%) of the drug into the intestinal tract. On the contrary, the same microsystems loaded with very slightly soluble aglycon, quercetin (Q), were only partially able to protect the drug in the gastric medium, and Q release remained incomplete in the intestinal fluid, as previously described [14]. In conclusion, the present results suggest that spray drying technique may be used to obtain gastroresistant microparticles loaded with flavonol glycosides such as rutin. The obtained microsystems showed a quite good encapsulation efficiency, product yields and microparticles morphology and behaviour. This approach appears quite interesting for delivering flavonols, with chemical-physical properties similar to rutin, to the intestine avoiding gastric degradation of the molecule and improving their bioavailability.
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MANUSCRIPT Received 3 January 2005, accepted for publication 12 May 2005.
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