A Modified Coacervation Method For Microencapsulation Biology Essay

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3.1 INTRODUCTION

Generally, the major problem encountered in microencapsulation of water soluble drugs as well as core (contains components with different solubilities) is the core partition effect. Several studies conducted by using different methods were highlighted the important roles in resolving the core partition effect (as explained in details in introduction, section1.?.?). Our previous work demonstrated the disability of the simple coacervation method to encapsulate the water soluble drugs (pseudoephedrine-HCl, diclofenac sodium and paracetamol) when drug-gelatin solution coacervated with aqueous alcohol (Aziz, 2006). It was shown that the drug particles was separated from the rubbery gelatin or deposited on the outer surface of gelatin. This limitation could be referred to the core partition effect. It can be explained that upon addition of a coacervating agent (ethanol) to an aqueous polymeric medium (gelatin) containing water-soluble core material, ethanol has the ability to remove water together with core from the dispersion medium due to the high mutual affinity, which leads to partitioning of core from gelatin (Figure 1). In addition, the degree of core partitioning is directly proportional to the degree of core solubility. Means, highly water soluble core materials could be completely partitioned and separated from the polymer. However, sparingly or slightly water-soluble core materials would be partially partitioned.

Dissolution

Coacervation

Lyophilization

Coacervating agent

Gelatin

Water

Water soluble core

Figure 3.1: Schematic diagram represented the partitioning effect in microencapsulation of water-soluble-core.

On the other hand, coacervation of a mixture containing water-soluble and insoluble cores, the core would partitioned in two (a dissolved part in the aqueous phase and a non-dissolved part in the gelatin phase), whereby gelatin entrapped the non-dissolved part while the dissolved part remained outside the microcapsule (Figures 2). As such, microencapsulation of a mixture of different compounds with different solubilities, the composition ratio entrapped into microcapsules could be negatively affected by the core partition problem.

Coacervation

Lyophilization

Dissolution/Dispersion

Coacervating agent

Gelatin

Water

Insoluble core

Soluble core

Figure 3.2: The partitioning effect in microencapsulation a mixture of water soluble and insoluble-cores.

One simple solution suggested in this part of study by using a material similar to gelatin property in the aqueous dissolution and different from gelatin property when expose to heating (gelatin dissolved and material solidified). This material can be act as barrier between core material and gelatin coat. Accordingly, chicken egg white (CEW) is the major source of ovalbumin (Datta et al., 2009, Awadé and Efstathiou, 1999, Chang and Chang, 2006). CEW have been often used as ingredients in food processing for their unique functional properties, such as gelling, foaming, heat setting and binding adhesion (Jerez et al., 2007). Ovalbumin a monomeric phosphoglycoprotein and the only protein containing free sulfhydril groups, four, which are buried in the protein core. Heat induced denaturation of ovalbumin results in the external exposure of these sulfhydril groups, accompanied by a decrease in the total sulfhydril content, due to the oxidation of SH groups to disulfide bonds (Van der Plancken et al., 2005).

Previous studies using egg albumin for microencapsulation of drugs by a capillary extrusion method (denaturation by dispersion of drug-egg-albumin solution into hot oil) or a w/o emulsion method (physical denaturation of the emulsified oil-drug-egg-albumin with different temperatures or by a chemical method using glutaraldehyde) resulted in rapid drug release (Brophy and Deasy, 1984, Ishizaka et al., 1981, Ishizaka and Koishi, 1983, Jun and Lai, 1983, Torrado et al., 1990). In the latter, the most important factor for control of drug release was the glutaraldehyde denaturation. However, a Schiff reaction between the glutaraldehyde and drug led to the formation of a brown product with unknown toxicological characteristics (Brophy and Deasy, 1984, Torrado et al., 1990). Such a phenomenon might explain the limitation of using ovalbumin as a coating in the microencapsulation process. In another study, the encapsulation of a hydrophilic compound (peptide) into microspheres was prepared by using a W1/O/W2 emulsion solvent evaporation method. Ovalbumin was used to stabilize the inner emulsion in order to prevent the destruction of the internal globules with the hope of avoiding leakage of the peptide to the outer aqueous phase (Blanco-Príeto et al., 1997). The peptide eluted from the surface of the microspheres through pores formed by ovalbumin aggregation that occurred during the washing process, and these pores constituted a pathway for the observed fast release (Blanco-Príeto et al., 1997).

In view of these aspects, this part of study aimed to modify the coacervation method by using ovalbumin as barrier to prevent the partitioning effect of the coacervating agent on the core materials and investigate the suitability of this model for microencapsulate of core(s) with different water solubility.

3.2 MATERIALS AND METHODS

3.2.1 Materials

Ovalbumin (grade 2) and gelatin (Type B, bloom 225) were purchased from Sigma, USA. Curcuminoid standard 98% was purchased from Acros Organic, USA. Pseudoephedrine-HCl, norpseudoephedrine-HCl, and norephedrine-HCl were purchased from Nutech, India. Paracetamol was purchased from Vangfou Pharma, China. Formaldehyde solution (37%) was purchased from Merck, Germany. Ethanol (95%) was purchased from Fisher Scientific, UK. Verapamil-HCl was purchased from Nicholas Piramal India Ltd, India. Propranolol-HCl was purchased from S.M. Pharmaceuticals, Malaysia.

3.2.2 Preparation of microcapsules using gelatin

Drug compounds, namely pseudoephedrine-HCl, norephedrine-HCl, verapamil-HCl, propranolol-HCl, norpseudoephedrine-HCl, paracetamol, and curcuminoid were used. The coacervation phase separation method was used. The coacervated layer determined was similar to that previously described (Aziz, 2006). The ratio of drug to gelatin was set at 1:1. For each gram of gelatin, 20 mL of water was used and coacervated with 20 mL of ethanol. Drug was dissolved or dispersed in the gelatin solution with a constant stirring using a magnetic hot plate stirrer (Heidolph, Germany). Ethanol was added to the drug dispersion using a syringe pump (Argus 600, Switzerland) at a feeding rate of 1 mL/min and a stirring speed of 500 rpm. The mixture was stirred continuously at 500 rpm for an additional hour at 10°C to ensure a complete deposition of gelatin onto the drug. Formaldehyde solution (37% v/v) was added to rigidize the gelatin coating. The volume of formaldehyde solution added was equivalent to the volume of coacervated layer obtained (1:1, v/v). The microcapsules collected were washed three times with ethanol, followed by cold water (5°C), re-dispersed in water, kept frozen at −70 °C for 24 hr, dried by lyophilization (Labconco, Missouri, US) and finally sieved through a 100-mesh sieve (150 μm).

3.2.3 Preparation of drug-ovalbumin microparticles

The drugs pseudoephedrine-HCl, norephedrine-HCl, verapamil-HCl, propranolol-HCl, norpseudoephedrine-HCl, paracetamol, and curcuminoid were used. The ovalbumin solution (20% w/v) was prepared in distilled water at room temperature using a magnetic stirrer (Heidolph, Germany). Known amount of drug (mg) was dissolved or dispersed in 6-40 mL of distilled water. The solutions or dispersions were dispersed into the ovalbumin solution, with core material to ovalbumin ratios of 1:3, 1:2, 2:3, 1:1, and 3:2 (w/w) as shown in Table 3.1. The mixtures were homogenized at 5,000 rpm for 1 min (Ultra-Turrax T18 Homogenizer) before transferred to a Teflon plate and dried in an air-oven (Carbolite, UK) at 30°C for 1 h. The resulting drug-ovalbumin particles were dismantled using the mortar and pestle and sieved through a sieve (150 µm).

3.2.4 Gelatin coating of drug-ovalbumin particles

5% w/v gelatin solution was prepared (Table 3.1). The drug-ovalbumin particles were dispersed in the gelatin solution with a constant stirring rate of 500 rpm for 10 min. Ethanol (20 mL) was added to the dispersion medium using a syringe pump (Argus 600, Switzerland) at a feeding rate of 1 mL/min under a constant stirring speed of 500 rpm. Formaldehyde solution was added to harden the gelatin coat. Formulations were collected and washed three times with ethanol, followed by cold water (5 °C). They were re-dispersed in water, kept frozen at -70 °C for 24 h, dried by lyophilization (Labconco, USA), and finally sieved through a sieve (150 µm). The experiments were carried out in triplicate for each sample.

3.2.5 Microencapsulation of combined cores

Composite cores containing pseudoephedrine-HCl (very soluble, 1g/0.5mL water) and curcuminoid (water insoluble) were mixed together at ratio of 1:1 (w/w). Furthermore, composite cores containing norpseudoephedrine-HCl (sparingly soluble, 1g/50mL water) and norephedrine-HCl (freely soluble, 1g/1.1mL water) were mixed together at ratio of 4:1 (w/w) to mimic the composition content of the extracted khat. The composite cores were microencapsulated as the procedures described in the above sections 3.2.3 and 3.2.4.

3.2.6 Morphology evaluation

The morphology of drug powder, drug-ovalbumin microparticles, and drug-ovalbumin-gelatin formulations were investigated using a light microscope (Leica DMLB, Cambridge, UK) connected to a camera (Leica DC 300, Cambridge, UK) and a compact workstation (Leica Compact, UK). The surface morphology of selected microcapsules was examined using a scanning electron microscope (Leica Cambridge S360, UK). The samples were first sputter coated with gold under an argon atmosphere (Emitech K750, Kent, UK).

3.2.7 Particle size determination

The particle size was determined with a Mastersizer S (Malvern Instruments, MAM5005, UK) fitted with a small sample dispersion unit (MS1) connected to a dispersion unit controller. A beam length of 2.4 mm and 300 RF lens (range 0.05-900 µm) was used. The core materials and the encapsulated formulations were sonicated in hexane or water for 1 min before loading into the small sample dispersion unit and stirred at a speed of 1,000 rpm until an obscuration value between 12-17% was obtained. Before running each sample, the system was aligned and a background measurement was taken using filtered hexane or water (0.45 µm, Nylon membrane) as dispersing solvent. The sample was measured thrice with 12,000 sweeps over 10 runs.

3.2.8 Drug analysis

Pseudoephedrine-HCl, verapamil-HCl, propranolol-HCl, and paracetamol were analyzed using UV-spectrophotometry (Hitachi, model U-2000, Tokyo, Japan) at detection wavelengths of 206, 278, 290 and 242 nm, respectively. The alkaloid (norpseudoephedrine-HCl and norephedrine-HCl) was analyzed as described in chapter 2, section 2.2.6. The curcuminoid content were analyzed using HPLC methods (Aziz, 2006).

3.2.9 Determination of yield, drug loading and entrapment efficiency

The percentage of microcapsule yield, drug loading and entrapment efficiency of the drug-loaded microcapsules were calculated using the following equations (Hu et al., 2003):

Yield (%) =

Weight of microcapsules

Ã- 100%

Eq. (3.1)

Weight of polymer and drug fed initially

Loading (%) =

Weight of drug in microcapsules

Ã- 100%

Eq. (3.2)

Weight of microcapsules

*EE (%) =

Weight of drug in microcapsules

Ã- 100%

Eq. (3.3)

Weight of drug fed initially

*EE = Entrapment efficiency

In curcuminoid microcapsules, the percentage of compositions of CUR1: CUR2: CUR3 in powder or in microcapsules (gelatin or ovalbumin/gelatin) were compared. Furthermore, the percentages of compositions of pseudoephedrine-HCl to curcuminoid entrapped into the microcapsules were determined. In addition, the percentages of norpseudoephedrine-HCl to norephedrine-HCl entrapped into the microcapsules were calculated.

3.2.10 In-vitro release profile

The drugs releases from powders or microcapsules, and the releases of curcuminoid (CUR1, CUR2 and CUR3), and alkaloid (norpseudoephedrine-HCl & norephedrine-HCl) from powders and from microcapsules were investigated using a modified Franz diffusion cell. 25 mg of powder or microcapsules containing an equivalent amount of powder was placed in dialysis membrane (pre-soaked in double-distilled water for 12 h, having pore size 2.4 nm) (Venkateswarlu and Manjunath, 2004). The alkaloid dissolution medium of 25 ml, consisting of ethanol and water (1:2, v/v, adjusted to pH 2.4 using 0.1 N HCl), was stirred at 50 rpm at water bath temperature of 37°C. Water was used as the dissolution medium of the water soluble drugs. The curcuminoid dissolution medium used was ethanol. At preset time intervals of 5 min, 10 min, 20 min, 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 hours after the commencement of the study, 0.2 mL of samples was removed, and replaced with the same volume of fresh dissolution medium. The alkaloid concentration in the sample was quantified using the HPLC method. The study was run in triplicate for each experiment.

3.2.11 Statistical analysis

The results were treated statistically using SPSS software (Version 13, USA). One-way analysis of variance was employed for the analysis of results. When there was a statistically significant difference, post-hoc Tukey Honestly Significant Difference (Tukey-HSD) test was applied. Paired Samples T-test was used to compare the means of two variables for a single group. A statistically significant difference was defined as P < 0.05.

3.3 RESULT AND DISCUSSION

3.3.1 Microencapsulation using gelatin

The results showed that among the drugs studied only curcuminoid was microencapsulated. The curcuminoid microcapsules appeared as discrete particles and were spherical in shape. In contrast, pseudoephedrine-HCl, norephedrine-HCl, verapamil-HCl, propranolol-HCl, norpseudoephedrine-HCl and paracetamol did not be encapsulated. When pseudoephedrine-HCl, norephedrine-HCl, verapamil-HCl, propranolol-HCl, norpseudoephedrine-HCl and paracetamol were added to the aqueous gelatin solution at 40 °C, clear solutions were obtained but two turbid layers were observed upon cooling. When examined microscopically after freeze-drying, the drug particles were vividly separated from the rubbery gelatin, which could be distinguished by the difference in color. As for pseudoephedrine-HCl and curcuminoid, agglomerated particles were obtained after freeze-drying. When examined under the microscope, drug could be seen on the surface.

The limitation of using gelatin alone for microencapsulating water soluble drugs in other studies may be explained by the inability of gelatin to capture drug due to the partition effect. Generally, a coacervation (aqueous phase separation) method is used to encapsulate water-insoluble materials and the most common microencapsulating agent used is gelatin (Chang and Robinson, 1990, Deasy, 1984). Such a process involves removal of associated water molecules from the dispersed colloid by coacervating agents with a greater affinity for water, such as various alcohols (ethanol is typically used). Dehydrated molecules of polymer tend to aggregate with surrounding molecules to form the coacervate (Deasy, 1984). Core material for encapsulation in the coacervate may be liquid or solid and must obviously be water-insoluble or very poorly water-soluble, otherwise it will simply dissolve in the aqueous medium (Deasy, 1984).

3.3.2 Microencapsulation using ovalbumin and gelatin

A phase diagram (Figure 3.3) shows the suitable area for preparing drug-ovalbumin dispersion medium. When drug solution blended with ovalbumin solution (20%), the water rang used for preparing drug solutions were 6-10 mL (very and freely soluble drugs), 10-20 mL (soluble drug), 20-40 mL (sparingly soluble drugs), and 40 mL (insoluble drug). The optimum microparticles produced; pseudoephedrine-HCl (very soluble), norephedrine-HCl (freely soluble), verapamil-HCl and propranolol-HCl (water soluble) were obtained at drug to ovalbumin ratio of 1:1. However, the norpseudoephedrine-HCl, and paracetamol (sparingly water soluble) were produced at drug to ovalbumin ratio of 2:3. In addition, curcuminoid microparticles were produced at ratio of 1:3.

Figure 3.3: Phase diagram showing the optimum area obtained for producing the drug-ovalbumin microparticles.

The results given in Table 3.1 show that formulations PE235, PE112, NE235, NE112, VP235, VP112, PP235, PP112, NPE235, NPE112, PC235, PC112, CU134, and CU123 were encapsulated, while formulations PE134, PE123, PE325, NE134, NE123, NE325, VP134, VP123, VP325, PP134, PP123, PP325, NPE134, NPE123, NPE325, PC134, PC123, PC325, CU235, CU112, and CU235 were not encapsulated. The morphological characterization of the encapsulated formulations showed that formulations PE112, NE112, VP112, PP112, NPE235, PC235, and CU134 appeared as discrete microcapsules. However, formulations PE235, NE235, VP235, PP235, NPE112, PC112, and CU123 appeared as agglomerated particles.

Table 3.1: Preparation of drug-microcapsules using ovalbumin and gelatin.

Drug

Water solubility

Drug solution

(mg/mL)

20% Ovalbumin solution

(mg/mL water)

Ratio

(D:O)

Code

5% Gelatin solution

(mg/mL water)

Ratio

(D:O:G)

Code

PE

Very soluble

1 g/0.5mL

250/10

750/3.75

1:3

PE13

1000/19

1:3:6

PE134

No-encapsulation

333/10

666/3.33

1:2

PE12

1000/19

1:2:3

PE123

No-encapsulation

400/10

600/3.00

2:3

PE23

1000/19

2:3:5

PE235

Agglomeration

500/10

500/2.50

1:1

PE11

1000/19

1:1:2

PE112

Microcapsules

600/10

400/2.00

3:2

PE32

1000/19

3:2:5

PE325

No-encapsulation

NE

Freely soluble

1 g/1.1mL

250/10

750/3.75

1:3

NE13

1000/19

1:3:6

NE134

No-encapsulation

333/10

666/3.33

1:2

NE12

1000/19

1:2:3

NE123

No-encapsulation

400/10

600/3.00

2:3

NE23

1000/19

2:3:5

NE235

Agglomeration

500/10

500/2.50

1:1

NE11

1000/19

1:1:2

NE112

Microcapsules

600/10

400/2.00

3:2

NE32

1000/19

3:2:5

NE325

No-encapsulation

VP

Soluble

1 g/15mL

250/20

750/3.75

1:3

VP13

1000/19

1:3:6

VP134

No-encapsulation

333/20

666/3.33

1:2

VP12

1000/19

1:2:3

VP123

No-encapsulation

400/20

600/3.00

2:3

VP23

1000/19

2:3:5

VP235

Agglomeration

500/20

500/2.50

1:1

VP11

1000/19

1:1:2

VP112

Microcapsules

600/20

400/2.00

3:2

VP32

1000/19

3:2:5

VP325

No-encapsulation

PP

Soluble

1 g/20mL

250/20

750/3.75

1:3

PP13

1000/19

1:3:6

PP134

No-encapsulation

333/20

666/3.33

1:2

PP12

1000/19

1:2:3

PP123

No-encapsulation

400/20

600/3.00

2:3

PP23

1000/19

2:3:5

PP235

Agglomeration

500/20

500/2.50

1:1

PP11

1000/19

1:1:2

PP112

Microcapsules

600/20

400/2.00

3:2

PP32

1000/19

3:2:5

PP325

No-encapsulation

NPE

Sparingly soluble

1 g/50mL

250/40

750/3.75

1:3

NPE 13

1000/19

1:3:6

NPE 134

No-encapsulation

333/40

666/3.33

1:2

NPE 12

1000/19

1:2:3

NPE 123

No-encapsulation

400/40

600/3.00

2:3

NPE 23

1000/19

2:3:5

NPE 235

Microcapsules

500/40

500/2.50

1:1

NPE 11

1000/19

1:1:2

NPE 112

Agglomeration

600/40

400/2.00

3:2

NPE 32

1000/19

3:2:5

NPE 325

No-encapsulation

PC

Sparingly soluble

1 g/70mL

250/40

750/3.75

1:3

PC13

1000/19

1:3:6

PC134

No-encapsulation

333/40

666/3.33

1:2

PC12

1000/19

1:2:3

PC123

No-encapsulation

400/40

600/3.00

2:3

PC23

1000/19

2:3:5

PC235

Microcapsules

500/40

500/2.50

1:1

PC11

1000/19

1:1:2

PC112

Agglomeration

600/40

400/2.00

3:2

PC32

1000/19

3:2:5

PC325

No-encapsulation

CU

Practically insoluble

250/40

750/3.75

1:3

CU13

1000/19

1:3:4

CU134

Microcapsules

333/40

666/3.33

1:2

CU12

1000/19

1:2:3

CU123

Agglomeration

400/40

600/3.00

2:3

CU23

1000/19

2:3:5

CU235

No-encapsulation

500/40

500/2.50

1:1

CU11

1000/19

1:1:2

CU112

No-encapsulation

600/40

400/2.00

3:2

CU32

1000/19

3:2:5

CU325

No-encapsulation

D:O (Drug : Ovalbumin), D:O:G (Drug : Ovalbumin : Gelatin), PE (Pseudoephedrine-HCl), NE (Norephedrine-HCl), VP (Verapamil-HCl), PP (Propranolol-HCl), NPE (Norpseudoephedrine-HCl), PC (Paracetamol), CU (Curcuminoid),

Microencapsulated core of pseudoephedrine/curcuminoid with gelatin alone, some crystals were deposited on the surface of the microcapsules. During the coacervation phase separation step, gelatin entrapped the insoluble portion (curcuminoid) and partitioned the very soluble (pseudoephedrine) part outside the microcapsule. As a result, the ratio of pseudoephedrine to curcuminoid entrapped into microcapsules was affected negatively. However, ovalbumin facilitated the entrapment of both soluble and insoluble parts into the gelatin coat, preventing the partitioning effect as well as drug deposition on the surface of microcapsules.

The present study hypothesized that the drug partition effect could be prevented by dissolving or dispersing a core into an ovalbumin solution prior to dispersion into the gelatin solution. At the coacervation phase separation step, ovalbumin could act as a drug coating barrier due to ovalbumin's denaturation property. When the coacervating agent was in contact with the ovalbumin surface, instant solidification of ovalbumin would occur, preventing the direct contact of the coacervating agent with drug thus protecting drug contents prior to gelatin coating. Hence, an ovalbumin protection feature could facilitate the successful formation of microcapsules through capturing and containing drug content irrespective of whether the drug is soluble or insoluble prior to gelatin coating (Figures 3.4 and 3.5).

Coacervation

Coacervating agent

Lyophilization

Dispersion

Gelatin

Water

Soluble core

Ovalbumin layer

Figure 3.4: Schematic diagram represented the effect of ovalbumin in microencapsulation of water-soluble-core.

Coacervation

Coacervating agent

Lyophilization

Dispersion

Insoluble core

Soluble core

Ovalbumin layer

Gelatin

Water

Figure 3.5: Schematic diagram represented the effect of ovalbumin in microencapsulation a mixture of soluble and insoluble-cores.

The results led to the assumption that the drug (core) was coated with two layers within microcapsules: an ovalbumin inner layer and a gelatin outer shell. The microcapsule affected by two shells differently, gelatin dissolved while ovalbumin solidified, thus increasing the consistency of the inner shell. This provided a protecting barrier to the core material to prevent core partition. Coating a drug with ovalbumin in hot dispersion medium, denature the ovalbumin but not the drug. As a result, the denatured ovalbumin could form a protective solidified layer around the drug.

3.3.3 Particle size

The results in Table 3.2 show that the particle size of water-soluble drugs (very, freely or soluble) was significantly decreased at drug: ovalbumin: gelatin ratios of 1:1:2 or 2:3:5 (PE112, NE112, VP112, PP112, PE235, NE235, VP235, and PP235). Increasing the amount of ovalbumin resulted in larger particle sizes. Formulations at a ratio of 1:1:2 (PE112, NE112, VP112, and PP112) exhibited significantly smaller particle size than formulations with a ratio of 2:3:5 (PE235, NE235, VP235, and PP235). However, the particle size of microcapsules prepared from sparingly (PC112, PC235, NPE112 and NPE235), or insoluble drug compounds (CU123 and CU134) increased significantly. An increase in the amount of ovalbumin also increased the particle size of curcuminoid formulations (CU123 < CU134). In the case of the norpseudoephedrine-HCl and paracetamol formulations, an increase in ovalbumin decreased the particle sizes (NPE112 > NPE235 and PC112 > PC235).

In short, the solubility of a drug in an ovalbumin solution affects the particle size of its formulation. Drugs dissolved into an ovalbumin solution showed smaller particle sizes than drugs that were dispersed. In addition, the particle size of microcapsules containing curcuminoid alone (CU134) or pseudoephedrine/curcuminoid (PECU134) formulated with ovalbumin/gelatin was relatively larger than curcuminoid formulated with gelatin only.

Table 3.2: Particle size of the formulations. Mean ± SD, N=3.

Formulations

Particle size D[4,3] µm

PE

NE

VP

PP

NPE

PC

CU or PECU

A- Core material (CM)

37.52±1.91

37.48±0.23

40.65±1.00

47.81±1.76

36.64±2.28

38.07±1.67

31.39±0.98

B- CM : ovalbumin : gelatin (1:1:2)

25.43±2.57

27.52±1.63

29.89±0.85

31.31±1.35

56.68±1.82

75.93±1.14

-

C- CM : ovalbumin : gelatin (2:3:5)

32.34±1.07

32.67±1.60

35.78±1.55

36.70±0.93

49.64±3.22

64.75±4.45

-

D- CM : ovalbumin : gelatin (1:2:3)

-

-

-

-

-

121.36±1.96

E- CM : ovalbumin : gelatin (1:3:4)

-

-

-

-

-

184.68±3.19

Statistical analysis

F

29.048

42.422

63.780

110.013

175.953

142.872

3565.634

Sig.

P<0.01

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

Tukey-HSD

A&B

P<0.01

P<0.001

P<0.001

P<0.001

P<0.001

P<0.001

-

A&C

P<0.05

P<0.05

P<0.01

P<0.001

P<0.001

P<0.001

-

B&C

P<0.05

P<0.01

P<0.01

P<0.01

P<0.01

P<0.01

-

A&D

-

-

-

-

-

-

P<0.001

A&E

-

-

-

-

-

-

P<0.001

D&E

-

-

-

-

-

-

P<0.001

3.3.4 Percent yield, drug loading, and entrapment efficiency

3.3.4 (a) Individual cores

As described earlier, the water soluble drugs were not successfully microencapsulated by using gelatin alone but were successfully microencapsulated by using ovalbumin/gelatin. The results (Table 3.3) of yield, drug loading, and entrapment efficiency of microcapsules of formulations PE112, NE112, VP112, PP112, NPE235, PC235, and CU134 were significantly higher than those of formulations PE235, NE235, VP235, PP235, NPE112, PC112 and CU123. The results showed that increasing ovalbumin decreased the yield, drug loading, and entrapment efficiency of freely-soluble or soluble drugs, but significantly enhanced the yield, drug loading, and entrapment efficiency of sparingly-soluble drugs. The microcapsules of formulations PE112, NE112, VP112, PP112, NPE235, and PC235 were successfully formulated with an optimum ovalbumin ratio, which exhibited relatively higher yield, drug loading, and entrapment efficiency. These microcapsules were selected for further study.

3.3.4 (b) Composite cores

Microencapsulated curcuminoid with gelatin alone, the yield, drug loading and entrapment efficiency were 82.6, 41.6 and 54.7% for CUR1, CUR2, and CUR3, respectively. However, microencapsulated curcuminoid (water-insoluble) using ovalbumin/gelatin, the results showed that increasing the ovalbumin ratio only increased the entrapment efficiency while the yield and drug loading were not affected significantly (Table 3.3).

Curcuminoid contain three compounds, curcumin (CUR1), mono-demethoxycurcumin (CUR2), and bis-demethoxycurcumin (CUR3) in a ratio of 75.9 ± 3.3 to 17.1 ± 1.4 to 6.4 ± 3.3, respectively (Ahmed and Gilani, 2009, Asai and Miyazawa, 2001, Chearwae et al., 2004, Yodkeeree et al., 2009). The present study attempted to investigate the effect of microencapsulation with gelatin alone or ovalbumin/gelatin on the composition ratios for the compounds entrapped in microcapsules. The results showed that the total content of curcuminoid entrapped in gelatin and/or ovalbumin/gelatin microcapsules was not significantly different while the ratios of CUR1 and CUR3 in gelatin microcapsules were significantly affected. However, the ratios of CUR1 and CUR3 were not significantly affected in ovalbumin/gelatin microcapsules (Table 3.4). The total content of curcuminoid in gelatin microcapsules was not affected due to the insolubility of curcuminoid in water where no partition occurred during the coacervation phase separation process. The small difference in the ratios of CUR1 and CUR3 could be due to the slight solubility of curcumin in ethanol (coacervating agent).

The results in Table 3.4 showed that the total composition content of pseudoephedrine/curcuminoid and the ratio of pseudoephedrine to curcuminoid were significantly affected in the gelatin microcapsules but not in ovalbumin/gelatin microcapsules. The variation in solubility of pseudoephedrine and curcuminoid contents was the main reason for the difference in the total content and composition ratios entrapped into gelatin microcapsules.

In addition, composite cores of norpseudoephedrine-HCl and norephedrine-HCl at ratio of about 5:1 (w/w) was microencapsulated at core to ovalbumin to gelatin ratio of 2:3:5. The resultant microcapsules (NPENE235) had yield, loading and entrapment efficiency of 91.14±2.60, 22.01±1.86 and 90.27±0.72% respectively. When yield, loading, and entrapment efficiency values were compared between KE235 and NPENE235, the results of Paired Samples T-test did not show any significant differences, p>0.05.

Table 3.3: Yield, drug loading, and entrapment efficiency of formulations. Mean ± SD, N=3.

Formulations

Yield

(%)

Loading

(%)

Entrapment efficiency

(%)

A- PE235

84.36±1.08

11.91±0.77

47.90±1.97

B- NE235

82.50±1.06

10.48±1.27

43.14±2.77

C- VP235

82.11±1.29

10.15±0.97

42.41±3.41

D- PP235

83.22±1.25

12.54±0.81

54.01±1.17

E- NPE235

92.01±0.05

19.35±1.23

88.51±2.81

F- PC235

90.10±1.70

19.83±0.87

89.32±3.67

G- CU134

91.31±2.46

12.43±0.78

90.73±3.64

a- PE112

94.42±1.20

23.96±1.63

90.56±7.23

b- NE112

91.34±1.27

24.50±1.26

89.57±5.81

c- VP112

89.85±1.09

23.84±0.84

85.71±4.02

d- PP112

89.77±1.13

23.00±1.53

82.65±6.48

e- NPE112

80.92±2.06

17.02±1.43

55.23±2.61

f- PC112

81.86±1.65

16.29±0.74

53.38±3.45

g- CU123

86.30±2.58

13.63±0.58

70.66±3.31

Paired Samples T-test

A & a

P<0.05

P<0.01

P<0.01

B & b

P<0.01

P<0.01

P<0.01

C & c

P<0.05

P<0.01

P<0.01

D & d

P<0.001

P<0.01

P<0.01

E & e

P<0.05

P<0.05

P<0.01

F & f

P<0.05

P<0.05

P<0.01

G & g

P>0.05

P>0.05

P<0.05

Table 3.4: Effect of microencapsulation on the composition percent. Mean ± SD, N=3.

Core

Composition

core

A- In Powder

B- In gel microcapsules

C- In oval/gel

microcapsules

Statistical analysis

Tukey-HSD

F

Sig.

A&B

A&C

B&C

CU

CUR 1

77.08±1.24

71.25±0.78

76.95±0.84

34.97

P<0.001

P<0.01

P>0.05

P<0.01

CUR 2

16.21±0.87

14.47±0.31

15.58±0.84

4.44

P>0.05

-

-

-

CUR 3

6.49±0.65

14.64±0.52

7.66±0.37

210.97

P<0.001

P<0.001

P>0.05

P<0.001

Total (%)

99.78±0.81

100.36±0.58

100.19±0.82

0.490

P>0.05

-

-

-

PECU

Pseudoephedrine

50.00±1.02

04.10±3.84

48.67±1.26

20.589

P<0.01

P<0.01

P>0.05

P<0.01

Curcuminoid

50.00±1.01

49.52±2.77

49.22±2.28

1.88

P>0.05

-

-

-

Total (%)

100.00±2.03

53.62±2.42

97.89±3.54

37.363

P<0.001

P<0.001

P>0.05

P<0.001

PNENE

Norpseudoephedrine-HCl

81.67±1.53

35.02±2.05

74.91±1.38

676.166

P<0.001

P<0.001

P<0.01

P<0.001

Norephedrine-HCl

17.00±1.00

2.26±1.13

15.36±0.98

181.362

P<0.001

P<0.001

P>0.05

P<0.001

Total (%)

98.33±2.08

37.27±3.07

90.27±0.72

694.762

P<0.001

P<0.001

P<0.01

P<0.001

3.3.5 In-vitro release profile

As depicted in Figure 3.6 and 3.7 the drug release of PE112, NE112, VP112, PP112, NPE235, PC235 and CU134 were exhibited sustained release rates common to powders. After eight hours, the drugs had released completely from all formulations except curcuminoid microcapsules (CU134), which was fully released only after 24 hours (due to the poor solubility of curcuminoid' in ethanol medium). When analyzed statistically using a Paired Samples T-test, results for the T50% (time for 50% of drug release) values of drug powders and microcapsules showed that PE, NE, VP, PP, NPE, PC and CU were significantly faster than the microcapsules PE112, NE112, VP112, PP112, NPE235, PC235 and CU134 (Table 3.5). A statistical analysis within the group (Tukey-HSD) of drug powders (PE, NE, VP, PP, PC and NPE) or drug microcapsules (PE112, NE112, VP112, PP112, NPE235, and PC235) revealed that the T50% of norpseudoephedrine-HCl (NPE) and paracetamol (PC) powders were higher than the other drug powders (PE, NE, VP, and PP). The T50% of norpseudoephedrine-HCl (NPE235) and paracetamol (PC235) microcapsules were also higher than the other drug microcapsules (PE112, NE112, VP112, and PP112) (Table 3.5).

In order to evaluate the consistency of release patterns, the release of CUR1, CUR2, and CUR3 from powder (CU) or microcapsules (CU134) were determined. The results showed (Figure 3.7) that the release profiles of CUR1, CUR2, and CUR3 from ovalbumin/gelatin-microcapsules were also similar to the release profiles from the powder in which the T50% values of CUR1, CUR2, and CUR3 in microcapsules were not significantly different compared with the curcuminoid powder (Table 3.5). The percentage of microencapsulated compositions of curcuminoid (CUR1 to CUR2 to CUR3) using ovalbumin/gelatin was not affected significantly upon release from the microcapsules.

Furthermore, the release of pseudoephedrine and/or curcuminoid from powder (PECU) or microcapsules (CU134) were determined to evaluate the consistency of release patterns of composite cores. The results showed (Figure 3.8 and Table 3.5) that the release profiles of pseudoephedrine and curcuminoid from ovalbumin/gelatin-microcapsules (PECU134) were consisted in which the T50% values of were 236.81±19.22 and 248.76±21.61 min respectively. However, in powder the pseudoephedrine (T50%=6.21) was released faster than the curcuminoid (T50%=89.35±2.16).

In addition, the in-vitro release of microcapsules of alkaloid (NPENE235) was exhibited sustained release rates common to powder of alkaloid (NPENE). After eight hours, the drugs had released completely from microcapsules. The releases of norpseudoephedrine-HCl and norephedrine-HCl, from powders or microcapsules were determined in order to evaluate the consistency of release patterns. The results showed that the release profile of norpseudoephedrine-HCl and norephedrine-HCl from the microcapsules were not similarly released from the powder. There was a significant difference in T50% (time for 50% of drug release) values between norpseudoephedrine-HCl and norephedrine-HCl in powder. However, in microcapsules (NPENE235) there is no significant different (Table 3.5, Figures 3.9, 3.10). In short, the percentage of microencapsulated alkaloid (norpseudoephedrine-HCl to norephedrine-HCl) was released in a consistent composition ratio. However, the release profiles of powders were not consistent.

Figure 3.6: Drugs releases from powder and microcapsules. Mean ± SD, N = 3.

Figure 3.7: Curcuminoid release from powder and microcapsules. Mean ± SD, N = 3.

Figure 3.8: Composite cores (pseudoephedrine/curcuminoid) release from powder and microcapsules. Mean ± SD, N = 3.

Table 3.5: T50% results of drug powders compared with drug-microcapsules. Mean ± SD, N=3.

T50% (min)

Paired Samples T-test

Powder

Microcapsules

Water soluble drugs

A- Pseudoephedrine-HCl

5.29±0.27

43.40±1.15

P<0.001

B- Norephedrine-HCl

5.20±0.21

43.22±1.12

P<0.001

C- Verapamil-HCl

5.39±0.36

44.01±2.32

P<0.01

D- Propranolol-HCl

5.36±0.31

44.76±2.01

P<0.01

E- Norpseudoephedrine-HCl

6.21±0.27

74.74±13.04

P<0.05

F- Paracetamol

6.46±0.22

79.32±14.82

P<0.05

Statistical analysis

F

10.376

16.317

-

Sig.

P<0.01

P<0.001

-

Tukey-HSD

E&F > (A,B,C&D)

P<0.01

P<0.01

-

Curcuminoid

A- CUR1

86.90±4.19

231.56±12.45

P<0.01

B- CUR2

89.18±1.51

246.45±13.22

P<0.01

C- CUR3

92.04±1.47

257.03±15.29

P<0.01

Statistical analysis

F

2.715

2.615

-

Sig.

P>0.05

P>0.05

-

Composite cores

Pseudoephedrine (very soluble)

5.85±0.50

236.81±19.22

P<0.001

Curcuminoid (insoluble)

89.35±2.16

248.76±21.61

P<0.001

Paired Samples T-test

P<0.001

P>0.05

-

Composite cores

Norpseudoephedrine-HCl

0.10±0.00

1.25±0.22

P<0.05

Norephedrine-HCl

0.09±0.00

1.32±0.25

P<0.05

Paired Sample t-test

P<0.05

P>0.05

-

Figure 3.9: In-vitro release of norpseudoephedrine-HCl and norephedrine-HCl from NPENE or NPENE235. Mean ± SD, N=3.

Figure 3.10: T50% values of norpseudoephedrine-HCl and norephedrine-HCl in NPENE or NPENE235. Mean ± SD, N=3.

3.4 CONCLUSION

The modified procedure of combining ovalbumin and gelatin resolved the disadvantages of treating each compound separately (ovalbumin or gelatin). During the microencapsulation processes, ovalbumin contained/captured drug into gelatin, which prevented the direct contact of drug-ovalbumin with the cross linking agent. As a result, the drug models were successfully microencapsulated by using ovalbumin/gelatin with higher percentage yields, drug loading, and entrapment efficiency. The capability of ovalbumin to capture pure drugs of different solubility (very water soluble, freely soluble, soluble, sparingly soluble, and practically insoluble) also rendered the microencapsulation of the drug compound (containing soluble and insoluble cores) or (containing very soluble and sparingly soluble cores) more consistent entrapment and release. Ovalbumin provided a protective barrier between the drug and the polymer-solvent phase; hence, it prevented the drug from partitioning out of the microcapsule. At the same time, the gelatin coat prolonged the release of pure drug from microcapsules as well as the drug compound with a consistent sustained release profile.

In conclusion, modifying the procedure for the coacervation phase separation method by including ovalbumin as barrier to prevent core partitioning effect was achieved. This procedure may be considered as a prospective technique for microencapsulation of core materials (containing more than one component) as well as composite cores with different solubility. As such this model can be considering the component of khat extract into microcapsules with a consistent entrapment and release.

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