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EXPERIMENTAL INVESTIGATIONS IN ENCAPSULATION OF PHARMACEUTICALLY ACTIVE INGREDIENTS

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A Synopsis of the Ph.D. Thesis

Submitted to

Gujarat Technological University, Ahmedabad

By

Yashawant Pralhad Bhalerao Enrollment No.: 129990905005 Branch: Chemical Engineering

Under the Supervision of

Dr.Shrikant J. Wagh Principal

Gujarat Power Engineering & Research Institute, Mehsana, Gujarat, India

Under Doctoral Progress Committee

Dr. S. A. Puranik Professor, Atmiya Institute of Technology,

Rajkot.

Dr. Sachin Parikh HOD, Chemical Engineering Department,

LDCOE, Ahmedabad.

GUJARAT TECHNOLOGICAL UNIVERSITY

JULY, 2019

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1. Abstract Microencapsulation comes as an important protection, storage technique, and controlled release

tool for several Pharmaceutically Active Ingredients (PAI), food, cosmetic and other medical

products. Purposes for encapsulation of PAI like thymol may be numerous, such as controlled

release, targeted controlled release, protection/preservation, economic utilization, formulations,

and modification/hiding undesirable property such as taste, odour and touch. Controlled release

helps to maintain an adequate drug concentration in the blood or in target tissues at a desired

value as long as possible and, for this, they are able to control drug release rate, hence control

release of PAI (thymol) is important. Controlled release formulation can be obtained by various

methods such as spray drying, solvent diffusion, nanoprecipitation etc.

The main objective of this study is to encapsulate thymol and determine its controlled or

sustained release by solvent diffusion and nanoprecipitation methods. In this work, the effect of

concentration of thymol (the core material), sodium alginate and ethyl cellulose (the shell

material), stirring speed for synthesis on sustained release and encapsulation efficiency of thymol

loaded beads and microparticles has been studied as discuss in table 1. For method optimization

the statistical design approach using 3 level 2 factors design and Plackett–Burman factorial

Design (PBD) was applied using the Design-Expert® (DoE) software(Version- 9.0.3.1, Stat-Ease

Inc., Minneapolis, MN). Obtained formulation of thymol loaded ethyl cellulose microparticles

shows maximum 98% drug release in 10 h. on the other hand, Formulation of thymol loaded

calcium chloride– sodium alginate beads showed that maximum 95.18±0.43 % drug release in 12

h. No chemical interaction between drug and polymer was found in FTIR study and

beads/particles obtained were spherical and distinct in nature.

Table 1. Overview of experimental methodology.

Core material

Shell material

Method Parameter studied Evaluation

Thymol Ethyl cellulose

Emulsification Solvent Diffusion

Process time, stirring speed for synthesis (RPM), Polymer concentration on encapsulation efficiency and control release

Optimization of the important process variables on Encapsulation Efficiency (EE) and Control Release (CR) by Emulsification solvent diffusion is achieved

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Thymol Ethyl cellulose

Nano precipitation

Process time, stirring speed for synthesis (RPM), Polymer concentration on encapsulation efficiency and control release

Optimization of the important process variables on EE and CR by Nano-precipitation techniques has been achieved

Thymol Sodium Alginate

Emulsification Solvent Diffusion

effect of concentration of thymol, sodium alginate, stirring speed on synthesis, and sustained release properties of thymol loaded sodium alginate beads using emulsion microencapsulation technique

Sustained release of thymol from Sodium Alginate Beads synthesized by Emulsification solvent diffusion Microencapsulation has been studied

2. Brief description on the state of the art of the research topic: The synopsis of the research work on “EXPERIMENTAL INVESTIGATIONS IN

ENCAPSULATION OF PHARMACEUTICALLY ACTIVE INGREDIENTS” gives a brief

description on i) Introduction ii) Literature review iii) Experiment and Analysis iv) Results and

Discussion v) Overall Conclusion and vi) Bibliography.

The introduction and literature review discuss merits, demerits, challenges, and factors affecting

microencapsulation, scope, aims &objectives of PAI microencapsulation, organization of thesis,

information about Emulsion Technique, Microencapsulation, Solvent diffusion,

Nanoprecipitation, Beads, Biodegradable polymers, and Design Expert.

The experimental methodology is to prepare thymol loaded ethyl cellulose micro particles using

solvent diffusion and nanoprecipitation techniques and methods of analysis is to study the effect

of concentration of thymol, sodium alginate, stirring speed on synthesis, and sustained release

properties of thymol loaded sodium alginate beads using emulsion microencapsulation

technique.

Main results are described in experiment and analysis and conclusions are described in results

are discussion and conclusion.

The following is the background of the overall research work undertaken:

Biodegradability of shell material along with its health compatibility and the half-life of the core

material and knowledge of microstructure for Control Release/Sustained Release (CR/SR) are

some of the main issues that must be addressed while studying encapsulation of PAI. The

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encapsulation of essential ingredients in core–shell or matrix particles has been investigated by

many researchers for various reasons, e.g. protection from oxidative decomposition and

evaporation, odour masking or merely to act as support to ensure controlled release. In order to

adapt to different types of active agents and shell materials different microencapsulation methods

have been developed generating particles with a variable shell thickness, range of sizes and

permeability, providing a tool to modify the release rate of the active principle.

Preparative conditions such as concentration ratio, temperature, stirring speed, and nature of

solvent used have deterministic effect on the polymer shell formed around the core material and

hence the release rate. Thus the final objective of encapsulation is controlled release of PAI. But

it (encapsulation) can be engineered according to the need. Microencapsulation can promote

pharmaceutical base products by introducing innovation, added functional properties and thus

added value.

Chemically thymol is 2-isopropyl-5-methylphenol phyto-constituent and classified as

monoterpene found in certain plants [1]. Thymol improves the digestion by relaxing smooth

muscles, prevents menstrual cramps, attenuates respiratory problems and also have wide used in

food industry [2, 3]. Thymol also shows potential antimicrobial properties, that thymol give the

impression to bind to the ergosterol in the membrane, which increases ion permeability and

ultimately results in cell death [4]. Thymol also shows antiseptic, anti-inflammatory, antioxidant

and healing properties and a broad spectrum of biological activity [5-7]. Thymol is able to inhibit

both Gram-positive 1 and Gram-negative bacteria 2 [8]. Antifungal agents like miconazole,

fluconazole, and ketoconazole may be prescribed instead of thymol [9–11]. However,

unselective use and the less number of available antifungal agents have encouraged the

development of resistant strains, especially in immune compromised individuals like thymol [12,

13].

Thymol used as an individual component shows greater predictability effects, allowing the

minimization of adverse effects of thymol. Rapid absorption limits the luminal availability for

antimicrobial activity. Hence, it is assumed that controlled release product could be improved

their effects on the microflora [14].Controlled release behavior of any drug displays control in

rate time and site of drug release in the body. The desired rate of drug release is obtained and can

1Gram-positive bacteria are bacteria that give a positive result in the Gram stain test. 2Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the gram-staining method.

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enhance the therapeutic effect of drug [15]. Numbers of biodegradable/biocompatible polymers

is used as controlled release carriers and play an important role in such system. Natural, semi

synthetic and synthetic polymers are nowadays widely used as controlled or sustained release

agent. Ethyl cellulose (EC) a hydrophobic polymer is commonly used as a drug carrier in

controlled drug delivery system [16] due to its low toxicity, film forming ability [17, 18].

Controlled release formulation can be obtained by various methods such as spray drying,

lyophilization, solvent diffusion, nanoprecipitation etc. [19]. Solvent diffusion is one of the

techniques widely used in the preparation of microparticles; shows better results than other

microencapsulation techniques such as interfacial polymerization and coacervation widely used

in pharmaceutical industries for various purposes such as protecting drugs from degradation,

protecting body from the toxic effects of the drugs, controlled drug delivery, masking the taste

and odour of drugs and its formulation process is easy and convenient. There are different single

and double solvent diffusion techniques like O/W, W/O, O/W/O, W/O/W. In O/W single

emulsion solvent diffusion method, the polymer is dissolved in a suitable water miscible organic

solvent, and drug solution is emulsified in this polymeric solution. In O/W solvent emulsion

method the oil (Organic solvent) is media while water is serves as continuous aqueous medium

[20, 21].

Controlled release of thymol:

Martin et al. have studied the release behaviour of thymol and p-cymene used as core materials

through Poly Lactic Alcohol (PLA) microcapsules. The microcapsules were obtained by a

coacervation process [22]. Milovanovic et al. have investigated that Cellulose Acetate (CA) is a

shell material for the controlled release of thymol [23].

Antilisterial activity of thymol:

Xiao et al. in their work produced spray-dried capsules from zein solutions (Zein is a class of

alcohol-soluble storage protein). They demonstrate that its non-ionic surfactant can effectively

improve antimicrobial functions in food systems. Spray drying is a practical technology for the

production of an antimicrobial capsule, which is possessed by a manipulated incorporating

surfactant such as Tween 20 [24]. Xue et al. have studied the ability of whey protein isolate

(WPI) and maltodextrin (R) to conjugate the thymol nano emulsifier with propylene glycol (PG)

to improve the antifungal properties of milk [25].

Antioxidant activity of thymol:

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Liolios et al. isolated carvacrol, thymol, p-cymene, and c-terpinene by hydro-distillation

technique and successfully encapsulated in phosphatidyl choline-based liposomes and the

possible improvement of their antioxidant and antimicrobial activities was tested against selected

microbial [26]. Davoodi et al. reveals the antioxidant capacity and physical properties of potato

starch dispersions enriched with polysorbate-thymol micelles. The results showed that potato

starch has essential antibacterial action only in the presence of polysorbatetimol but below

polysorbate thymol alone [27].

Antimicrobial activity of thymol:

Chang et al. prepared thyme oil-in-water nanoemulsions (pH 3.5) as potential antimicrobial

delivery systems. The nanoemulsions were highly unstable to droplet growth and phase

separation, which was attributed to Ostwald ripening due to the relatively high water solubility of

thyme oil. Nano stable thyme oil emulsions were tested for antimicrobial performance, as

opposed to acid-resistant spoilage yeast, Zygosaccharomyces bailii (ZB) [28]. Li et al. revealed

new antimicrobial films based on colloidal nanoparticles zein coated with sodium caseinate (SC)

emulsifier/stabilizer [29]. Li et al. prepared various sub-micron-thymol emulsions with a high

HLB (hydrophilic-lipophilic balance) surfactant by spontaneous emulsification. The emulsions

were then screened for various provocative pathogens to evaluate antimicrobial efficacy [30].

Antispasmodic activity of thymol:

Engelbertz et al. fractionated Thyme fluid extract by Fast Centrifugal Partition Chromatography

(FCPC), Low Pressure Liquid Chromatography (LPLC), and High Pressure Liquid

Chromatography (HPLC) and compounds isolated were identified by spectroscopic methods.

Thyme extracts have antispasmodic activity, which is at least due to synergistic effects of

phenolic volatile oil compounds and the flavone luteolin [31].

Anti-inflammatory activity of thymol:

Riella et al. assess the anti-inflammatory and cicatrizing activities of thymol in rodents, the

peritonitis models of inflammation and analysis, followed by the evaluation of myeloperoxidase

activity (MPO), total cell counts and histological analysis were used [32].

3. Definition of the Problem The active core material in present research work is thymol and study of its controlled and

sustained release is the main objective. It is obvious that the diffusive flux of the core API will

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be governed by the shell material membrane. Therefore, it is required to study the influence of

the parameter such as the effect of concentration of thymol (the core material), sodium alginate

and ethyl cellulose (the shell material), stirring speed for synthesis on sustained release and

encapsulation efficiency of thymol loaded beads and microparticles. We have selected two shell

materials viz. ethyl cellulose and sodium alginate. Both these materials are biodegradable, health

compatible and easily available. Thymol is an important PAI which has enormous applications

described earlier but its doses need to be properly designed in order to avoid side effects and

undesired reactions at the targeted sites due to high concentration. Control release is the answer

to this problem and encapsulation of the PAI is the technique used for this. To the best of our

knowledge, these systems have not been studied before with these objectives.

4. Objectives and Scope of the work i. To investigate suitable encapsulation techniques (Emulsion solvent diffusion

(emulsification diffusion), Nano-precipitation etc.) for Thymol.

ii. To optimize the important process variables (Process time, stirring speed for synthesis

(RPM), Polymer concentration) on encapsulation efficiency and control release by

Emulsion solvent diffusion and Nano-precipitation techniques: A Comparative study.

iii. To study the sustained release of thymol from Sodium Alginate Beads synthesized by

Emulsion solvent diffusion Microencapsulation.

5. Original contribution by the thesis Microencapsulation of thymol in liposomes, solid microparticles and microemulsions, and

polymeric micro/nanoparticles represents a promising strategy for overcoming thymol’s

limitations, lowering their dose and increasing long-term safety of thymol. Low dosages of

thymol are also found to be effective to lower its long term effects on various parts of body.

Standardization is necessary in terms of purity of product and stability. Microencapsulation

formulation can provide an effective alternative for thymol administration in relatively high or

low dosage depending upon application.

Thymol has potential for maintaining and promoting health, also preventing and potentially

treating some diseases such as eczema, cough, cold, dermatitis, antifungal, antibacterial infection

However, the generally low water solubility and stability as well as the high volatility and side

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effects associated with its application have limited its use in medicine. Encapsulation is an

approach that has potential applications in medicinal and health research.

6. Methodology of Research, Results / Comparisons 6.1. Methodology 6.1.1. Experimental System: Thymol loaded Ethyl Cellulose (ETHOCEL), Sodium Alginate micro particles were prepared by

1. Nanoprecipitation Techniques (precipitation in principle)

2. Emulsification Solvent Diffusion Method

3. Emulsified beads

6.2. Experimental Procedure: 6.2.1. Techniques of microencapsulation: Emulsion–diffusion method

Preparation of micro-capsules by the emulsion–diffusion method allows both lipophilic

and hydrophilic active substance encapsulation.

The experimental procedure performed to achieve this requires three phases: organic,

aqueous and dilution.

When the objective is the micro-encapsulation of a lipophilic active substance, the organic

phase contains the polymer, the active substance, oil and an organic solvent partially

miscible with water, which should be water-statured.

This organic medium acts as solvent for the different components of the organic phase. If it

is required, the organic phase can also include an active substance solvent or oil solvent.

The aqueous phase comprises the aqueous dispersion of a stabilizing agent that is prepared

using solvent-saturated water while the dilution phase is usually water.

a. Drug (PAI): thymol (pure thymol crystals , purchased from Loba Chemie, Mumbai )

b. Polymer: Ethyl cellulose “ETHOCEL” (as a Gift sample from ICT, Mumbai)

c. Stabilizer/surfactant: Tween 80 (purchased from Loba Chemie, Mumbai )

d. Organic solvent: Ethanol

6.2.2. Techniques of microencapsulation: nanoprecipitation

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Thymol loaded ethyl cellulose nanoparticles were prepared by means of the

nanoprecipitation method using the same concentrations of drug and other excipients as

per above method.

In this technique, the drug and polymer was dissolved in dichloromethane to form

diffusion phase (Solution A). This phase was then added into aqueous dispersing medium

(Tween 80 + distilled water) by using syringe under stirring speed 700 rpm. The

dispersing phase was constituted of a liquid in which the polymer is insoluble – the non-

solvent containing a surfactant (Tween 80).

In this technique the ethyl cellulose deposit on the interface between the aqueous and the

organic solvent, caused by fast diffusion of the organic solvent, leads to the formation of

a colloidal suspension. The freshly formed nanoparticles by this technique in the form of

colloidal particles are separated by Whatman filter paper and dried for further

characterization

6.3. Encapsulation efficiency % (EE) and % drug release (DR): The amount of thymol in thymol loaded ethyl cellulose nanoparticles was determined using a

UV-Vis spectrophotometer (Hitachi U2900). Percent encapsulation efficiency was calculated

using the equation (1):

% EE = (Wt – Ws) x 100/Wt (1)

Where, Wt is the total weight of the drug used in formulation and Ws is weight of the drug in

supernatant.

Drug release study was performed using Dissolution Testing Type II Apparatus at 37 ± 0.5°C

and at 100 rpm using 900 ml phosphate buffer having pH 6.8. Nanoparticles equivalent to 40 mg

of thymol were used for the dissolution study. At predetermined interval 5 ml of aliquot was

withdrawn and analyse spectrophotometrically and an equal amount of fresh buffer was added to

maintain the sink condition.

6.4. Results and Discussions The main objective of this study is to prepare thymol loaded ethyl cellulose micro particles using

solvent diffusion and nanoprecipitation techniques and study the effect of concentration of

thymol, stirring speed on synthesis, and sustained release properties of thymol loaded sodium

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alginate beads using emulsion microencapsulation technique. For optimization method, the

statistical design approach was employed and 3 level 2 factors design was applied using design

expert (version 9.0.3.1) software and design approach of Plackett–Burman factorial Design

(PBD) of experiment was performed using Design-Expert® (DoE) software (Version- 9.0.3.1,

Stat-Ease Inc., Minneapolis, MN).

6.4.1. Formulation and evaluation of thymol loaded ethyl cellulose microparticles using solvent diffusion and nanoprecipitation methods

The solvent diffusion technique and nanoprecipitation technique are used for micro particles

formulation to investigate suitable encapsulation technique for thymol. The Obtained

formulation shows maximum 98% drug release in 10 h. Thymol loaded ethyl cellulose

microparticles were successfully prepared by solvent diffusion as well as nanoprecipitation

method without any incompatibility. This study observed that nanoprecipitation method gives

quite better results than the solvent diffusion method and seems to be promising for sustained

delivery of thymol.

Encapsulation efficiency (EE) of thymol loaded ethyl cellulose micro particles shows direct

relationship with the polymer concentration i.e. encapsulation efficiency is higher at higher

polymer concentration and minimum with lower polymer concentration. The EE obtained in the

range of 69.11 % to 81.3 % for the solvent diffusion method and 63.12 % to 75.47 % for the

nanoprecipitation technique. Design expert software was employed; using 3 level 2 factor design

total 13 runs was carried out for each method to study the effect of independent variables on

dependent variables. Analysis of variance (ANOVA) and all statistical analysis were also

performed using the Design Expert (DoE) software (Version- 9.0.3.1, Stat-Ease Inc.,

Minneapolis, MN) shows linear model.

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Figure 1: 2D counter plot and 3D surface plot (A and B) showing the effect of independent

variables on encapsulation efficiency and drug loading prepared by solvent diffusion and

nanoprecipitation method respectively.

6.4.1.1.Encapsulation efficiency and In-vitro drug release studies:

Figure 2: Display of the release behavior of micro/nanoparticles by solvent diffusion

method (A) and nanoprecipitation method (B).

The EE obtained in the range of 69.11 % to 81.3 % for the solvent diffusion method and 63.12 %

to 75.47 % for the nanoprecipitation technique. Encapsulation efficiency is directly proportional

to the polymer concentration. Drug release profile for all the runs was carried out using in-vitro

release study at pH 6.8. Cumulative percent drug release for solvent diffusion runs are in the

range of 83.43% to 94.38% while in nanoprecipitation technique 84.91% to 98.71 % in 10 h

shows control release (Figure 1). The 2D counter plot and 3D response surface plot in Figure 2A

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shows the effect of polymer concentration and surfactant concentration on EE and DR for the

solvent diffusion technique. The EE efficiency increases with the increase in polymer

concentration and drug release also shows the same behavior. Similarly, in nanoprecipitation

technique 2D counter plot and 3D response surface plot (Figure 2B) shows the direct relationship

with the polymer and surfactant concentration. When we compare the EE and DR data in both

the methods the data obtained by solvent diffusion method is quite better than nanoprecipitation

method in especially in terms of release behavior.

In this study thymol was successfully entrapped in the biodegradable polymer ethyl cellulose by

solvent diffusion and nanoprecipitation method. Drug release from prepared polymer matrix was

observed slowly in in-vitro release profile up to 10 h. Both methods are suitable for the

nanoparticle preparation. No chemical interaction was found in FTIR study and particles

obtained are spherical and distinct in nature. Comparison of two methods showed that

nanoprecipitation method gives better encapsulation efficiency results while particles prepared

by solvent diffusion method gives the more controlled action in in-vitro release.

Formulation shows maximum 98 % drug release in 10 h. Thymol loaded ethyl cellulose

microparticles were successfully prepared by solvent diffusion as well as nanoprecipitation

method without any incompatibility. This study observed that nanoprecipitation method gives

quite better results than the solvent diffusion method and seems to be promising for sustained

delivery of thymol.

6.4.2. Sustained release of thymol from Sodium Alginate Beads synthesized by Emulsion solvent diffusion Microencapsulation

The concentration of polymer (X1) and concentration of surfactant (X2) are considered as

independent variables while percent encapsulation efficiency (Y1) and percent drug release (Y2)

are as dependent variables for both methods Preparation of thymol loaded calcium chloride –

sodium alginate beads was carried out using the emulsion microencapsulation technique.

The PBD is an efficient approach to evaluate the results which are shown in Table 1.

Table 1: The Plackett-Burman Experimental Design matrix (in coded level) and

experimental results

Runs Variables Response A B C D E F G H J K L EE (%) DR (%) 1 -1 1 1 -1 -1 1 1 -1 1 -1 -1 65.31± 0.56 63.44±0.54

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2 -1 1 1 -1 1 1 -1 1 -1 -1 -1 96.81± 1.59 87.03±0.65 3 -1 1 -1 -1 1 1 1 1 -1 1 1 91.86± 2.74 92.9±0.74 4 -1 -1 -1 -1 -1 -1 1 -1 -1 -1 -1 95.04± 2.12 30.06±0.73 5 1 1 -1 1 -1 -1 -1 1 1 1 -1 96.20± 1.25 95.07±0.72 6 -1 -1 -1 1 -1 -1 -1 1 -1 1 -1 87.79± 2.55 79.96±0.92 7 1 -1 1 -1 1 1 1 -1 1 1 -1 94.66± 0.57 78.98±0.12 8 1 -1 -1 -1 1 -1 1 1 -1 -1 1 80.27± 2.43 95.18±0.43 9 1 1 -1 1 1 -1 1 -1 1 -1 1 93.24± 1.86 85.72±0.70 10 1 -1 -1 1 1 1 -1 1 1 -1 1 31.18± 0.94 94.63±0.54 11 -1 1 1 -1 1 -1 -1 -1 1 1 1 92.34± 0.83 91.26±0.43 12 1 -1 1 1 -1 1 -1 -1 -1 1 1 84.31± 2.24 92.03±0.87

The release of thymol was evaluated using phosphate buffer (pH 6.8) as the release medium.

Sustained drug release was observed in the range of 30.06±0.73 to 95.18±0.43 % in 12 h study of

all the experimental runs. The EE of different experimental runs of the beads is reported in Table

2. The 3D response surface plots are useful in understanding about the main and interaction

effects of the independent variables. To visualize the effect of independent variables on each

response, 3D response surface plots (Figure 3) were constructed. The % EE of the beads was

calculated to be in the range 31.18 % to 96.81 %.

(A) (B)

Figure 3: 3D surface plots showing independent and interaction effect of thymol and

sodium alginate on (A) encapsulation efficiency; (B) drug release, prepared by

microencapsulation.

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To examine the interaction between the drug and polymer FTIR spectra of pure thymol, ethyl

cellulose and formulation were obtained using Fourier transform infrared spectrophotometer.

No chemical interaction between thymol and sodium alginate was found in FTIR study and

beads obtained were spherical and distinct in nature. Formulation showed that

microencapsulation method gives sustained action in in-vitro release. Formulation shows

maximum 95.18±0.43 % drug release in 12 h. Thymol loaded sodium alginate beads can be

successfully prepared by emulsion microencapsulation method without any incompatibility. This

study observed that the emulsion method gives promising results for sustained delivery of

thymol.

XRD spectra of pure thymol produced peaks at different 2Ɵ angles and found to be crystalline in

nature while, in case of ethyl cellulose, sodium alginate and the formulations, less intense peaks

are observed than that of pure thymol. This result tells that majority of the drug was entrapped

within the polymer and dispersed homogeneously at molecular level. The XRD patterns of

optimized formulation prepared by both methods showed 43.7% crystalline and 56.3 %

amorphous regions. Thus, indicating absence of distinct diffraction peaks of thymol. This result

tells that majority of the drug was entrapped within the polymer and is dispersed homogeneously

at molecular level

Decrease in particle size shows increase in dissolution rate and ultimately the absorption.

The PDI of optimized batch for solvent diffusion method is 0.122(nm) while the PDI of batch for

nanoprecipitation method is 0.223(nm). The results indicate that the particles obtained by the

solvent diffusion method shows narrow particle size distribution than the particles obtained by

nanoprecipitation method (Figure 4).

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Figure 4: Showing the particle size distribution of optimized batches by solvent diffusion

and nanoprecipitation method.

The surface morphology of thymol loaded ethyl cellulose micro particles was examined by FE-

SEM. The obtained micro particles are smooth, discrete, and spherical with sharp edges without

any cracks or erosion at the surface. The particles prepared by solvent diffusion method are in

the range of 6 to 19 μm and particles prepared by nanoprecipitation method have size range 27 to

38 μm. The surface morphology of thymol loaded 2% sodium alginate beads was examined by

FE-SEM as depicted in Figure 5 (A & A1). Thymol loaded 4% sodium alginate beads were

examined by FE-SEM (Figure 5 (B & B1)). The obtained beads are smooth, discrete, and

spherical with sharp edges without any cracks or erosion at the surface before drying (Figure 5

(A & B)), but after drying, an irregular and rough surface was observed (Figure 5 (A1 & B1)).

The beads prepared by microencapsulation method are in the range of 6 to 38 μm

Figure 5: Microscopic analysis of beads obtained by microencapsulation : A, A1 before and

after drying at 2% sodium alginate beads respectively; and B, B1 before and after drying

at 4% sodium alginate beads respectively.

A1 A

B1 B

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7. Achievements with respect to objectives i. Microencapsulation of thymol is successfully carried out with three methods/systems. For

optimization method, the statistical design approach of Plackett–Burman and factorial

design of experiment was performed using Design-Expert® (DoE) software.

ii. Optimization of the important process variables on encapsulation efficiency and control

release by Emulsion solvent diffusion is achieved.

iii. Optimization of the important process variables on encapsulation efficiency and control

release by Nano-precipitation techniques has been achieved.

iv. Sustained release of thymol from Sodium Alginate Beads synthesized by Emulsion

solvent diffusion Microencapsulation has been studied.

v. Comparison of Emulsion solvent diffusion Microencapsulation and Nano-precipitation

techniques on microencapsulation of thymol has been done.

8. Conclusion In this study thymol was successfully entrapped in the biodegradable polymer ethyl cellulose by

solvent diffusion and nanoprecipitation methods. Drug release from prepared polymer matrix

was observed slowly in in-vitro release profile up to 10 h. Both methods are suitable for the

microparticle preparation. No chemical interaction was found in FTIR study and particles

obtained are spherical and distinct in nature. When we compare both the methods with respect to

the encapsulation efficiency and drug release, both methods have their own better results i.e.

nanoprecipitation method gives better encapsulation efficiency while particles prepared by

solvent diffusion method gives more controlled action in in-vitro release.

Thymol was successfully entrapped in the calcium-alginate by microencapsulation method. The

% EE of the beads ranged from 31.18 % to 96.81 %. Drug release from prepared polymer matrix

was observed slowly in in-vitro release profile up to 12 h. Formulation showed that

microencapsulation method gives more sustained action in in-vitro release. Formulation shows

maximum 95.18±0.43 % drug release in 12 h. No chemical interaction was found in FTIR study

and beads obtained were spherical and distinct in nature.

Thymol loaded calcium-alginate beads were successfully prepared by microencapsulation

method without any incompatibility. This study observed that microencapsulation method gives

satisfactory results and seems to be promising for sustained delivery of thymol. As the thymol

concentration increases, the EE also increases. On the other hand with increasing concentration

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of sodium alginate the EE slightly decreases. Whereas an increasing pattern of drug release was

observed with increase in the concentration of thymol and sodium alginate.

9. Copies of papers published and a list of all publications arising from the thesis i. Yashawant. P. Bhalerao, Shrikant J. Wagh. Formulation and evaluation of thymol loaded

ethyl cellulose microparticles using solvent diffusion and nanoprecipitation methods: A

comparative factorial design approach. International Journal of Pharmaceutical Research

July- Sept 2018. Vol 10, Issue 3.

ii. Yashawant. P. Bhalerao, Shrikant J. Wagh. In vitro sustained release study of Thymol from Sodium Alginate Beads synthesized by Emulsion Microencapsulation. International

Journal of Pharmaceutical Research, Apr - June 2019. Vol 11, Issue 2.

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