Evaluation of Some Biopolymers for Various Pharmaceutical Applications

EVALUATION OF SOME BIOPOLYMERS FOR VARIOUS PHARMACEUTICAL APPLICATIONS

BY SHAZMA

MASSEY

ROLL NO.

105-Ph.D-Chem-2009 SESSION: 2009-2014

DEPARTMENT OF CHEMISTRY GC UNIVERSITY, LAHORE EVALUATION OF SOME BIOPOLYMERS FOR VARIOUS PHARMACEUTICAL APPLICATIONS

A thesis submitted to the GC University Lahore in partial fulfillment of the requirements for the award of the

degree of

DOCTOR OF PHILOSOPHY IN CHEMISTRY

BY SHAZMA MASSEY

ROLL NO. 105-Ph.D-Chem-2009

SESSION: 2009-2014

DEPARTMENT OF CHEMISTRY GC UNIVERSITY, LAHORE

IN THE NAME OF THE MOST MERCIFUL AND GRACIOUS GOD “WHO EVER BELIEVES IN HIM WILL NOT BE

DISAPPOINTED”

Romans 10: 11 DEDICATED TO MY DEAREST AND LOVING

PARENTS

PROF. ISAAC MASSEY (Late)

AND

MRS SHAKUNTALA MASSEY (Late)

RESEARCH COMPLETION CERTIFICATE

This is to certify that the research work contained in the thesis titled “Evaluation of some biopolymers for various pharmaceutical applications” has been carried out and completed by Ms.Shazma Massey, Roll No. 105-PhD -Chem-2009, Reg. No. 46 -PhD-Chem-2009 under my supervision during her PhD (Chemistry) studies in the laboratories of the

Department of Chemistry. The quantum and the quality of the work contained in this thesis is adequate for the award of degree of Doctor of Philosophy.

Dated: June27, 2014

______Prof. Dr. Mohammad Saeed Iqbal Dr. Irfana Mariam Supervisor Co-Supervisor

Submitted through

______Prof. Dr. Adnan Ahmad Controller of Examination

Chairman GC University, Lahore Department of Chemistry, GC University, Lahore.

DECLARATION

I, Ms. Shazma Massey, Reg. No. 046-PhD-Chem-2009 student of PhD in the subject of

Chemistry, session 2009-2014, hereby declare that the matter printed in the thesis titled

“Evaluation of some biopolymers for various pharmaceutical applications” is my own work

and has not been printed, published and submitted as thesis or publication in any form in

any university, research institute etc. in Pakistan or abroad.

Dated: June27, 2014

______Shazma Massey

CONTENTS

ACKNOWLEDGEMENT I - II

ABSTRACT III - IV

LIST OF ABBREVIATIONS V - VII

LIST OF FIGURES VIII - XIV

LIST OF TABLES XV – XVI

1. Introduction 1-30

1.1. General 1

1.2. Polymers in pharmaceuticals 2

1.2.1. Binders 2

1.2.2. Thickners 2

1.2.3. Suspending agents 3

1.2.4. Film coating agents 3

1.2.5. Drug delivery 3

1.3. Polymers from plant materials 4

1.3.1. Materials in use 4

1.3.2. Materials used in the present work 5

1.4. Some important properties of carbohydrate polymers 18

1.4.1. Structure 18

1.4.2. Surface morphology 19

1.4.3. Rheology 19

1.4.4. Thermal behavior 20

1.4.5. Monosaccharide analysis and protein analysis 20

1.4.6. NMR analysis 21

1.4.7. Swelling behavior and water retention 22

1.4.8. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) 23

1.4.9. Gel Permeation Chromatography (GPC) 23

1.4.10. Mechanical strength 24

1.4.11. Drug release models and mechanism 24

1.4.12. Empirical/Semi-Empirical models 27

1.4.12.1. Power law 27

1.4.12.2. Zero and First order models 28

1.4.12.3. Mechanistic realistic theories 29

2. Materials and methods 31-46

2.1. Materials 31

2.2. Methods 31

2.2.1. Isolation of biopolymers 31

2.2.2. Characterization 32

2.2.2.1. Elemental analysis 32

2.2.2.2. Moisture content 33

2.2.2.3. FT-IR spectroscopy 33

2.2.2.4. Thermal analysis 33

2.2.2.5. Scanning electron microscopy 35

2.2.2.6. Atomic force microscopy 35

2.2.2.7. Monosaccharide analysis by HPLC 35

2.2.2.8. Protein analysis 36

2.2.2.9. NMR study 37

2.2.2.10. Rheology 38

2.2.2.11. Determination of molar mass 39

2.2.2.12. ToF-SIMS 41

2.2.2.13. Mechanical strength 42

2.2.2.14. Swelling index 42

2.2.2.15. Water retention 42

2.2.3. Evaluation of biopolymers as drug carriers 43

2.2.4. Evaluation as binders in tablets 45

2.2.5. Evaluation as suspending agents 45

2.2.6. Evaluation as thickening agents 46

2.2.7. Evaluation as film coating materials 46

3. Results and discussion 47- 151

3.1. Isolation of biopolymers 47

3.2. Characterization 48

3.2.1. Elemental analysis 48

3.2.2. Moisture content 48

3.2.3. FT-IR spectroscopy 48

3.2.4. Thermal analysis 51

3.2.5. Electron microscopy 61

3.2.6. Atomic force microscopy 61

3.2.7. Monosaccharide analysis by HPLC 66

3.2.8. Protein analysis 66

3.2.9. NMR study 71

3.2.10. Rheology 76

3.2.11. Determination of molar mass 76

3.2.12. Mechanical strength 81

3.2.13. Swelling index 84

3.2.14. Water retention 84

3.3. Evaluation of biopolymers as drug carriers 84

3.3.1. Electron microscopy 86

3.3.2. ToF-SIMS 86

3.3.3. Dissolution study 91

3.3.3.1. Release profile of diclofenac sodium loaded polymer films in phosphate buffer 97

3.3.3.2. Release profile of diclofenac sodium loaded polymer films in 0.1 N HCl 98

3.3.3.3. Release profile of caffeine loaded polymer films in distilled water 98

3.3.3.4. Release profile of diclofenac sodium loaded polymer tablets in phosphate 120

3.3.3.5. Release profile of diclofenac sodium loaded polymer tablets in 0.1 N HCl 120

3.3.3.6. Release profile of caffeine loaded polymer tablets in distilled water 121

3.3.4. Targeted delivery 146

3.3.5. Disintegration study 146

3.4. Evaluation as binders in tablets 146

3.5. Evaluation as suspending agents 146

3.6. Evaluation as thickening agents 149

3.7. Evaluation as film coating materials 149

3.8. Concluding remarks 152

3.9. Research publication by the author from this work 153

1. Introduction

1.1. General

Polymers are extensively used in formulation of various dosage forms of pharmaceuticals.

They play their roles as binders, viscosity enhancers, suspending agents, retarded release materials, targeted delivery devices and scaffolds in tissue engineering. The polymers employed for these applications are mostly synthetic or semi-synthetic materials [1, 2].

Early research was focused on synthetic non-biodegradable materials such as polyethylene glycol (PEG) copolymers, which are used in cardiovascular devices. Similarly polyvinyl alcohol (PVA) gels are used for contact lenses, lining for artificial hearts and in drug delivery devices. The synthetic devices need to be implanted and then removed by surgery.

Thus for biomedical applications it is desirable that the materials should preferably be biocompatible and biodegradable. The synthetic polymers are made up of highly toxic monomers and as such lack biocompatibility. Natural sources of very useful polymers, also known as biopolymers, are abundantly available, which can be developed as important pharmaceutical ingredients.

Biopolymers have been isolated from animal or plant sources. Gelatin, collagen and chitosan are among the extensively used biopolymers from animal sources. Gelatin is widely used for fabrication of capsule shells. Plants produce large quantities of polysaccharides; the most important are starches, celluloses and hemicelluloses. In pharmaceutical applications the use of natural hydrogels such as guar gum, pectin, cellulose ether, chitosan, carrageenan, hyaluronic acid and alginic acid is quite common.

Polylactide (PL), polyglycolide (PG) and their copolymer polylactide-co-glycolide

(PLGA), being biodegradable, have long been used for designing controlled drug delivery devices. These degrade into glycolic and lactic acids in the body and are easily handled via normal body metabolism.

1.2. Polymers in pharmaceuticals

The polymers being used as inactive ingredients (adjuvants) in pharmaceutical formulations are described as follows.

1.2.1. Binders

The most commonly used polymers as binders for tablets are synthetic and include polyvinyl pyrolidone (PVP), hydroxypropylmethyl cellulose (HPMC), hydroxypropyl cellulose

(HPC), hydroxyethyl cellulose (HEC) and carboxy methyl cellulose (CMC) [3, 4]. They produce harder granules with greater stability, higher binding, low friability and good flowability [5,6]. Among the natural polymers guar gum, pectin, high methoxy pectin [7-

10] have found their way in this application. These are biocompatible, low cost, environmentally friendly and easily available materials. Some of the natural materials including Lallemantia royleana (LR) and Astragalus tragacantha (AT) are subject of several studies to evaluate their potential in this respect [11].

1.2.2. Thickeners

Different grades of synthetic polymers described as binders are also used thickening or viscosity enhancing agents in formulation of oral liquids and ophthalmic solutions.

Among the natural materials gum Arabic, guar gum, xanthan gum and gum tragacanth are in common use in formulation of oral liquids. Glucomannan, a food additive, is used as an emulsifier and thickener with the E number E425(ii) [12] in candies and cosmetics.

1.2.3. Suspending agents

PVP and PVA are synthetic suspending agents mostly used in formulation of oral liquids and ophthalmic solutions [13, 14]. But now these polymers are being replaced by natural polymers such as guar gum and Acacia nilotica (AN) which are used as stabilizers, emulsifier, thickening, and suspending agent in liquid formulations [15]. AN has been listed as edible material with E number as E 414.

1.2.4. Film coating agents

Film coating of pharmaceutical tablets is generally being carried out by use of synthetic polymers including PVP and HPMC. Now film coating materials are undergoing a transition from synthetic or semi-synthetic to natural products. Hypromallose-pectin and ethyl cellulose aqueous dispersion also as mixtures with chitosan are becoming popular for film coating the tablets [16].

1.2.5. Drug delivery

All the sustained release pharmaceutical formulations invariably involve the use of polymers. The polymers currently in use are mostly synthetic or semi-synthetic materials.

The most common polymers are PVA, HPMC, polymethyl methacrylate (PMMA) and polylactide-co-glycolide (PLG) [17, 18]. For bioadhesive applications, high molecular weight acrylic acid polymer crosslinked with divinyl glycol are extensively used in various

drug delivery systems. Buccal, intestinal, nasal, vaginal and rectal bioadhesive products can all be formulated with such polymers [19]. These polymers, in addition to having very high cost, have biocompatibility issues [20]. Owing to these concerns, people started exploring natural materials for such applications. In this regard proteins and carbohydrate polymers such as pectin, guar gum, MP, hyaluronic acid and alginic acid are being suggested as biocompatible and biodegradable drug carriers. Hydrophilic swellable natural polymers are promising materials for use in controlled drug delivery systems [21]. These polymers would absorb water when in contact with body fluids, swell, and release the encapsulated drug in a programmed manner.

Pectin have been used in controlled-release matrix tablet formulations and colonic drug delivery applications [22]. Guar gum has been shown to retard drug release in colonspecific drug delivery systems [23-25]. Formulations containing MP have produced release profile similar to a commercial sustained-release formulation of diclofenac [26, 27]. Hyaluronic acid has been used in the preparation of gels for ophthalmic drug delivery [28]. Starch is also used for sustained release due to its gel-forming ability, biodegradability, and biocompatibility [29].

During the last three decades or so, stent design has witnessed a fairly rapid evolution from bare metal stents of increasing complexity, through shape memory alloy stents, polymer coated, drug eluting stents to biodegradable stents made by use of polymers [30].

1.3. Polymers from plant materials

1.3.1. Materials in use

As discussed above only a few of several natural polymeric materials could find their use in pharmaceutical formulations. The reason for this is that the natural materials are still

passing through various evaluation processes. Most of the evaluations could not reach the level of approval due to lack of standardization and authenticity. The object of the present work was to employ authentic methodology to study various properties of selected plant materials, which could substantiate the claims that natural materials are better substitutes for the synthetic or semi-synthetic polymers used for pharmaceutical applications.

1.3.2. Materials used in the present work

The materials selected for the present work were swellable when in contact with water. The criteria for their selection included biocompatibility, biodegradability, non-toxicity and abundant availability [31]. The plant materials thus selected were: Ocimum basillium (OB) seeds, Mimosa pudica (MP) seeds, Lallemantia royleana (LR) seeds, Acacia nilotica (AN) gum, Acacia modesta (AM) gum, Salvia plebian (SP) seeds, , Plantago ovata (PO) seeds and seed husk, Astragalus tragacantha (AT). A brief description of these materials is presented as follows.

OB plant

OB, commonly known sweet basil and locally known as „tukhm-e-raihan‟ and „niazbo‟

(Pakistan and India), is a soft green plant having approximately 2 ft height with alternate leaves and white or pink flowers (Fig. 1). It grows in dry-hot weather (like in Asia and

Middle East) and can be grown in door with exposure to sunlight in colder parts of the world. Its seeds are small, oval in shape and jet black in color. Its botanical classification

a b

Fig. 1. Pictures of a) OB plant, b) dry seeds and c) seeds soaked in water

is: kingdom: Plantae; division: Magnoliophyta; class: Magnoliopsida; order: Lamiales; family: Labiatae; genus: Ocimum; species: bacilicum [32]. There are about 150 varieties, including sweet basil, holy basil, lemon basil, of basil found throughout the world.

Almost all parts of the plant, including seeds, flowers, leaves and roots, are used for health purposes and as such they do not exhibit toxic effects when consumed in normal dose. Since ancient time OB leaves are used as flavoring agent in cooking and dried leaves for treatment of acne, insect bites and snakebites [33-36]. The plant extracts can be used as perfumes or room freshener. There exist several herbal remedies for treatment of brain, heart, lungs, bladder and kidney related diseases [37], and as antiviral [38], antiinflammatory [32], antiseptic [32, 39, 40], antifungal [33, 40-42], antispasmodic [33, 39], antivenom [39], antioxidant [33, 43], antimicrobial [44, 45], antiulcer [32] agents.

MP plant

MP, commonly known as touch-me-not in English, „chui mui‟ and „lajwantee‟ in Pakistan and India [46, 47]. It is a plant which closes it leaves when touched and reopens them within few minutes. The plant grows in sunny weather a height of about 50 cm with a spread of 30 cm (Fig. 2). It can grow in a variety of soils. Seeds of MP are locally called as „tukhm-e- lajventee‟. The seeds are reddish brown, spherical or flat; they produce mucilage when soaked in water. The mucilage has been characterized to be mainly composed of D-xylose and D-glucuronic acid. Its botanical classification is: kingdom: Plantae; division:

Angiospermae; class : Eudocots ; order : Fabales ; family: Fabaceae; genus: Mimosa; species: Pudica.

All parts of this plant are being used as aphrodisiac and for treatment of various ailments,

a b

Fig. 2. Pictures of a) MP plant, b) dry seeds and c) seeds soaked in water

such as asthma [48], depression [49] , pain [48], infections, toxic effects of venom [50,51], early aging [52-54] , diabetes [55,56]. It also plays a role in regeneration of nerves [57] and wound healing [58].

LR plant

LR, commonly known as holy basil also called balango in Pakistan and India, grows in

Asia, Europe and Middle East. The height of this plant is approximately 2 ft. Its seeds are locally known as „tukhm-e-balango‟ or „tukmalanga‟, and black psyllium seeds in English.

They are oval, jet black with a white spot at one end but bigger in size than tukhm-eraihan

(Fig. 3). The seeds are widely used in ayurvedic medicine [59]. Its botanical classification is: kingdom : Plantae; division: Angiospermae; class: Eudicots; order : Lamiales; family:

Lamiaceae; genus: Lallemantia; species: Royleana [60].The Labiatae family (Lamiaceae) is one of the largest family of flowering plants, with almost 4000 species and about 220 genera existing worldwide.

Balangu seed gum (BSG) is a low cost source of hydrocolloid with high molecular weight

(3.65×106 g/mole) and intrinsic viscosity (7236.18 g/ml) [61].The seeds of this plant have cool effect on body and mind and their extract cures many diseases including inflammation

[62], heart problems [63, 64] , women specific diseases [65]; it lowers blood pressure, removes stress and acts as sedative [66]. The paste of this plant helps cure abscesses produced by pus.

AN plant

AN, commonly known gum Arabica (Fig. 4) and gum keekar or babul in Pakistan and

a b

Fig. 3. Pictures of a) LR plant, b) dry seeds and c) seeds soaked in water

Fig. 4. Pictures of a) AN plant, b) gum

India. Its botanical classification is: kingdom: Plantae; division: Angiosperms; class:

Magnoliopsida; order: Fabales; family: Fabaceae; genus: Acacia; species: Nilotica. The gum is used in treating hypotension caused by surgical shock or hemorrhage. The gum has been successfully used in plastic surgery for grafting of destroyed peripheral nerves [67].

Use of AN pods reduces blood sugar level, plasma cholesterol, triglyceride and lowdensity lipids but increases plasma high-density lipids [68]. Almost all mature and immature parts of AN plant have shown to be active against a number of diseases such as cancer, asthma, diabetes, hepatitis C, high blood pressure, bacteria, AIDS, fungal and bacterial infections.

The gum acts as antipyretic, emollient, astringent, anti-asthmatic and liver tonic [69,70].

Gum arabic is a branched-chain complex polysaccharide, may be neutral or slightly acidic, found as mixed calcium, magnesium and potassium salt of polysaccharidic acid. Main components of this acid are D-galactose (Gal), L-arabinose (Ara), L-rhamnose (Rha), and D- glucuronic acid (GlcA) with the structure as:

where A = L-arabinose, R = L-rhamnose, G = D-galactose, U = D-glucuronic acid. The backbone consists of 1,3 -linked beta- D-galactopyranosyl units. The side chains are composed of two to five 1,3-linked beta- D-galactopyranosyl units, joined to the main chain through 1,6-linkages. The gum is known to act as an anti-oxidant and protects hepatic-, renal- and cardiac toxicities in rats. It enhances dental remineralization and has antimicrobial activity. It showed adverse effects on electrolyte balance and vitamin D in

mice [71]. The gum is collected after 20-30 days after an incision has been made on the tree. The gum is also known to contain some peroxidases. For this reason, it should not be mixed with easily oxidizable compounds.

AM plant

AM , commonly known as blackwood and locally as gum phulai or Amritsar gum in

Pakistan, Afghanistan and India (Fig. 5). It belongs to a large genus acacia having about

1500 species. It grows mostly in hot weather. Its botanical classification is: kingdom:

Plantae; division: Angiosperms; class: Magnoliopsida; order: Fabales; family: Fabaceae; genus: Acacia; species: Modesta.

AM gum has been used for effectively treating lumbago, skeleto-muscular problems and chronic stomach disorders [72]. Ash of the bark of AM finds use in treating paralysis and asthma. Chest pains and dysentery can also be treated by powder of dry bark with a little quantity of salt and sugar [73,74]. AM twigs are used as tooth brush (miswak) for cleaning teeth and is good for bleeding gums. The extracts of AM leaves was found to be effective against most bacterial and fungal infections. The alcoholic extracts of AM leaves are known to reduce blood glucose (Glc) level in rats significantly [75].

SP plant

SP, commonly known as sage and locally known as „kamarkas‟ and „samundersok‟, grows on the sides of streams and rivers as a small herb (Fig. 6). Its botanical classification is:

a b

Fig. 5. Pictures of a) AM plant, b) gum

a b

Fig. 6. Pictures of a) SP plant, b) seeds soaked in water

kingdom: Plantae; division: Magnoliophyta; class: Magnoliopsida ; order: Lamiales; family:

Lamiaceae; genus : Salvia ; species : Plebia. Its seeds and leaves have a lot of medicinal value. The popular uses include their use in: sore throat and headach [65].

When seeds are soaked in water they develop mucilage which is carbohydrate biopolymer

[76].

PO plant

PO, commonly known as psyllium and locally known as ispaghula (Fig.7) [77], is cultivated all over the world due to its importance as a food. Its seeds are oval in shape and brown in color with one side smooth and the other side depressed. The seed husk is soft and needle like fiber (Fig.7). The husk and seeds swell in water and produce a mucilage characterized to be polysaccharides. The botanical classification is: kingdom: Plantae; division:

Magnoliophyta; class: Magnoliopsida; order: Lamiales; Family: plantaginacea; genus:

Plantago; species: Ovata.

Both the husk and seeds possess medicinal value and are used as health foods. They are used in diarrhea and constipation, for control of body weight, blood pressure and cholesterol level [78-81]. The mucilage is also used in frozen dairy desserts as thickener or stabilizer.

AT plant

AT, commonly known as goat's-thorn, is cultivated in middle east and Iran. It is among

3,000 species of astragalus herbs and shrubs, belonging to the legume family (Fig. 8). The genus is native to temperate regions of the Northern Hemisphere. Botanically it is

a b

c d

Fig. 7. Pictures of a) PO plant, b) dry seeds, c) seeds soaked in water and d) seed husk soaked in water

Fig. 8. Pictures of a) AT plant, b) gum

classified as kingdom: Plantae, division: Spermatophyta; class: Dicotyledones; order :

Fabales; Family: Fabaceae; genus: Astragalus; species: Tragacantha. Its gum is locally called „gond katira‟.

Gum tragacanth looks like twisted ribbons or flakes of polysaccharides (Fig. 8) having no specific color and taste. The gum absorbs water to form gel, which can be converted into paste. It is very commonly is used in veg-tanning the leather, as stiffener in textile industry and binder in making artist's pastels. Its paste is used in floral sugar craft to make flower decorations for cakes. It is also used to treat cough and diarrhea in herbal medicines.

Tragacanth mucilage has fast wound healing capacity because hydrolysis of tragacanth into

Arabinose and glucoronic acid coagulate the surface proteins for fast recovery and prevention of infections [82, 83].

1.4. Some important properties of Polysaccharides

In order to qualify their use as pharmaceutical ingredients the polysaccharides must pass some specific tests for their intended use. In the following paragraphs some of the most important properties of the polymers and the methods of testing thereof have been reviewed briefly.

1.4.1. Structure

It is one of the essential requirements for the prospective pharmaceutical ingredients that their precise chemical composition and structure must be known. The first step towards structural determination is the elemental analysis. This analysis can be carried out by use of automatic CHNSO analyzers very precisely. Carbohydrate polymers, natural polysaccharides, are reported to have C and H around 45% and 6% respectively; these

values largely depend upon the moisture content and some other components such as uronic acids in the materials [84-86]. The moisture content can be determined as loss on drying or more precisely by Karl-Fischer titration.

Infrared spectroscopy is generally helpful in identifying the nature of the polysaccharides.

In case of hemicelluloses, a strong broad bond at 3414 cm-1 (due to hydrogen-bonded hydroxyl groups) and a band at 2919 cm-1 (due to symmetric C–H vibration) are generally observed along with other characteristic bands at 1419, 1384, 1244, 1040, and 897 cm-1

[87]. The spectra are generally dominated with stretching and bending vibrations of C-H,

C-O, C-C, C-OH, and C-O-C groups. The band at 1039 cm-1 is mainly due to a glycosidic linkage (C–O–C). The band at 1600 cm-1 is principally associated with absorbed water.

The peak at 630 cm-1 and 500 cm-1 are due to polymer backbone.

1.4.2. Surface morphology

Surface morphology of polymeric materials plays an important role in controlling drug loading, distribution and release. Scanning electron (SEM) and atomic force microscopic

(AFM) techniques are powerful tools to study the surface morphology of materials. The

SEM and AFM images can be used to identify the types of voids, layers, surface roughness and nanostructure in the polymers.

1.4.3. Rheology

Rheology involves the study of the effect of shear stress on viscosity of the dissolved polysaccharides, which provides important information about the viscosity and elasticity of a polymer. Elasticity is a phenomenon where a polymer is stretched on application of a stress and readjusts to its original structure as soon as the stress is removed. It also provides

information about the flow (Newtonian or non-Newtonian) of viscous solutions formed by polymers.

1.4.4. Thermal behavior

Thermal stability of polymeric materials is extremely important for determining their potential in various applications [88,89]. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) are significant and most widely used techniques to study the stability, degradation, moisture content, glass transition temperature and other properties of polymeric materials [90]. Thermal stability of hemicelluloses generally decreases with decrease in molar mass [91]. Loss of moisture is associated with an endothermic peak at 85-110°C in the DSC scans. The stability of polymers is characteristically judged by determining its integral procedural decomposition temperature

(IPDT) and comprehensive index of thermal stability (ITS). The life-time of polymers can also be predicted by two standard ASTM methods based on thermal analysis. However, according to thermal community it is emphasized that better estimate of life-times can be obtained by isoconversional methods [92]. Pyrolytic GC-MS analysis of the volatiles formed by degradation of polysaccharides can give an insight into the mechanism of the degradation pattern of the polymer [93-95].

1.4.5. Monosaccharide analysis and protein analysis

Monosaccharide analysis is done by hydrolyzing the polysaccharides generally by

Seamon‟s method [96]. The hydrolysis may be severe or mild. In severe hydrolysis 12M and in mild 1M sulphuric acid is used. After hydrolysis the monosaccharides are determined by HPLC. The monosaccharide composition of the polymer give us an idea

about the major and minor sugar content. Protein analysis is carried out to get a confirmation that the biopolymers are pure carbohydrate polymers or contain some proteins. Protein analysis is conviently performed by the use of bicinchoninic acid (BCA) kit [97].

1.4.6. NMR analysis

NMR spectroscopy is a very powerful technique and can be used to find detailed structural

information of the sample. In case of polysaccharides, NMR peak broadening can cause

problems due to the long relaxation times involved. However, after degradation, fine

structure information regarding relationship between proton-proton and proton-carbon by

different advance NMR techniques such as two-dimensional correlation spectroscopy (2D-

COSY), total correlation spectroscopy (TOCSY), heteronuclear singlequantum correlation

(HSQC), heteronuclear multiple-quantum correlation (HMQC), and heteronuclear

multiple bond correlation (HMBC) can be obtained. Distortionless Enhancement of

Polarization Transfer using a 135 degree decoupler pulse (135-DEPT) can differentiate

between carbons having even number of protons and carbons having odd number protons.

One-dimensional 1H and 13C NMR spectra have been used in combination with two

dimensional COSY, HSQC-DEPT techniques for investigation of anomeric protons and

carbons of AN and AM [98-101]. Information on the nature, relative content of

monosaccharide residues, configuration, and the type and amount of specific linkages in

AN and AM have been determined using 13CNMR [102-104]. Signals from non- and

monosubstituted xylose residues in 13C NMR spectrum has been assigned using 13C-

HSQC-DEPT and COSY techniques [105]. Structure of AX from ispaghula seed husk has

been discussed in detail using HMQC and HMQC-TOCSY NMR

techniques after partial hydrolysis [106]. AN and AM were also analyzed by solid state

13C CP-MAS NMR technique [107].

1.4.7. Swelling behavior and water retention

For the polymers to be used in drug delivery it is important to know swelling behavior and water retention of polymers used for formulation of drug delivery devices [108]. The polymers absorb water, swell, and release the encapsulated drug in a sustained manner.

Swelling index is determined by the formula

Swelling Index = [(Weight of wet sample –Weight of dry sample) / Weight of dry sample)]

×100

The water retention was calculated by the formula:

Ww - Wd 100

Water Retention(%) Wd where, Ww = weight of sample

in wet state, Wd = weight of sample after drying at 105 C.

1.4.8. Time-of-flight secondary ion mass spectrometry (ToF-SIMS)

ToF-SIMS is a powerful technique that can map distribution of a chemical compound dispersed in a polymer matrix with high spatial resolution. It has emerged as a rapid technique to study surface chemistry of materials at a spatial resolution around 1 m, and has been used extensively to characterize a range of materials including polymers and biological samples [109, 110]. Only a few studies involving imaging of drug delivery

systems by ToF-SIMS [111] have been reported. The technique involves rastering of a cluster ion beam onto the surface of the sample, which results in generation of secondary ions through a cascade of collisions. The secondary ions are then accelerated into time- offlight tube. The advantages of ToF-SIMS include high mass resolution (>7,000), a large mass range (element to a complex molecular mass), excellent spatial resolution and an ability to simultaneously detect fragment ions over a large mass range [112]. The technique allows several samples to be loaded on to the cryo-stage and analyzed consecutively. The technique produces images.

1.4.9. Gel Permeation Chromatography (GPC)

The molar mass distribution and polydispersitivity index (PDI) of a polymer are important characteristics that indicate the bulkiness and hetero-/homogenic nature of the polymer. The mass averages generally determined are Mn, Mw and Mz, which are numberaveraged, weight-averaged and z-averaged molar masses respectively. The PDI is defined as the ratio

Mw/Mn. For an ideal monodisperse polymer, the molar mass averages are equal i.e.

Mn=Mw=Mz and therefore PDI value is 1. However, for a polydispersed system the relationship is Mn

PDI value >1 [113]. 1.4.10. Mechanical strength

Mechanical strength also known as tensile strength greatly affect the film formation ability and is measured by universal testing machine. The knowledge of strength of materials is important and useful at the time of fabrication of devices from the polymers.

s = F/A Where; s = the breaking strength (stress)

F = the force applied that caused the failure in N.

A = the least cross- sectional area of the material in m2 (original width ˣ original thickness)

1.4.11. Drug release models and mechanism

In order to study the release profiles of polymers various models have been proposed, which describe the release mechanisms. The work on modeling started with publication of the so called „Higuchi equation‟ by Prof. T. Higuchi [114, 115], which described the release of drug molecules from polymer films.

1/2 M = kH t (1) where M is the amount of drug released in

time t, kH is the Higuchi release constant. Since this work, a number of empirical/semi-

empirical and mechanistic releasic models of drug release process have been suggested.

The later type models are more accurate and being based on real phenomenon, can give

insight into the phenomenon of drug release as compare to the former which lack these

capabilities. The continuous increased importance of hydrogels in CDDS, a number of

mathematical models has been suggested for such systems [116-127].

The drug release from a hydrophilic matrix is generally described by two contending

mechanisms: Diffusion-controlled release and relaxation-controlled release. The swelling

of hydrogel on contact with biological fluid changes its glassy state to the rubbery state.

The absorption of water into the hydrogel bring about its expansion due to lowering of the

glass transition temperature (Tg) being controlled by the concentration of the swelling

agent. The strength of the swollen gel is important for the matrix performance and it depends upon the viscosity, concentration and structure of the rubbery polymer.

Colombo et al [124] described the swelling of heterogeneous swellable matrices by three front positions, where „front‟ is the region in the matrix where the physical changes are occurring sharply. The three fronts are, as shown in Fig. 9.

• the „swelling front‟; between the rubbery and the glassy region.

• the „erosion front‟; between the solvent and the matrix. The time variation of the

thickness of the gel-layer is controlled by the positions of the erosion and the

swelling fronts.

• the „diffusion front‟; between the erosion and the swelling fronts. It forms a

boundary that separates solid from dissolved drug.

The position of the diffusion front in the gel depends upon the solubility and loading of the drug. The movement of diffusion front is a function of the dissolution rate of the drug in the gel.

Fig. 9. Schematic representation of a swelling controlled drug delivery system showing three fronts of movement is shown below.

The rate of drug release is dependent upon the interactions between polymer, water and drug. The thickness of the gel layer and drug gradient in the gel determines the release kinetics. The increase in thickness of gel layer is fast in the beginning owing to rapid penetration of solvent that causes chain disentanglement, slows down as the process goes on due to increase in distance for diffusing solvent. It therefore, follows that the gel-layer formation and its permeability to the drug molecules is the key factor that controls the drug release and these are governed by solvent penetration, drug diffusion and dissolution, polymer swelling and matrix erosion. There are several other factors which can influence the drug release phenomenon, some of which have been considered in other mechanistic realistic theories a brief description of which follows.

In addition to the Higuchi equation discussed above, below is given a brief account of models that appear frequently in research articles for analyzing the drug release from hydrogels.

1.4.12. Empirical/Semi-Empirical models

These models can describe the drug release from a given polymeric device. The models generally used are described below.

1.4.12.1. Power law

An empirical relationship called Power Law is frequently used to describe the Fickian,

non-Fickian, case-II transport mechanisms of drug release from a polymer matrix

[128131].

t M lnkp nlnt (2) ln M

Where Mt/M∞ is the fraction of drug released in time t, kp is the Power Law constant

characteristic of the drug-matrix system and n is the diffusion exponent. The value of n

identifies different mechanism for drug release. For different geometries the limits of n are

different and are summarized in Table 1.

Case I mechanism occurs when the diffusion rate is far less than the relaxation rate and

case-II mechanism is seen otherwise. If both rates are comparable then anomalous

transport is the dominant mechanism, and for value of n greater than certain limits the drug

release become constant for a longer period (time-independent) and termed as super case-

II transport [132]. It is generally believed to be controlled by polymer erosion process

which cause and exponential increase in the release of drug towards the later stages.

Table 1: The limits of release exponent n for different geometries

Release exponent, n Mechanism of drug release Thin Film Cylinder Sphere Diffusion-controlled 0.5 0.45 0.43 (Fickian, Case-I, transport) Diffusion-/swelling-controlled 0.5

Swelling-controlled (Case-II 1.0 0.89 0.85 tansport) Time-independent release (Super n>1.0 n>0.89 n>0.85 case-II tansport)

1.4.12.2. Zero and First order models

Zero order (Eq. 3) [133] and first order (Eq. 4) [134] kinetic equations are also widely used to describe drug release from matrices. However, these models do not provide an explanation of the physical/chemical phenomenon involved in drug release rather they are employed to simply fit the release profile.

M=k0t (3)

where k0 is the zero order release constant, M is the amount of drug released in time t.

lnM = –k1 t + lnM0 (4) where k1 is the first order release constant, M is the remaining

amount of drug in the tablet after time t and M0 is the initial amount of drug in the tablet.

1/3 1/3 Hixon-Crowell cube root law (Mo – M =kHCt ) (5) where M is the amount

of drug released in time t, kHC is the Hixson–Crowell release constant and Mo is the initial amount of the drug in the tablet [135].

1.4.12.3. Mechanistic realistic theories

Mechanistic realistic models tried to develop a real story for the drug release phenomenon from a given device. These take into account a number of factors that may influence the drug release. A good detail of the possible physical factors that may be related to this phenomenon has been provided by Siepmann and Siepmann [136]. The model developed by Korsmeyer et al. describes the diffusion of water (penetrant) and a solute for a swellable

polymer slab [137,138]. The developed model was successfully applied to a hydrophilic polymer with a water-soluble drug. This interesting model suggested that the water

(penetrant) is sorbed and the drug is desorbed and released. Any form of diffusion coefficient can be used in the model.

Ju and co-workers [139-141] developed a comprehensive mathematical model to describe the swelling/dissolution behaviors and drug release from HPMC matrices. The major thrust of this model is to employ an important physical property of the polymer, the polymer disentanglement concentration, r, the polymer concentration below which polymer chains detach of the gelled matrix. Furthermore, matrix dissolution is considered similar to the dissolution of an object immersed in a fluid. As a result, a diffusion layer separating the matrix from the bulk solution is incorporated into the transport regime. In addition, an anisotropic expansion model is introduced to account for the anisotropic expansion of the matrix, the surface area in the radial direction dominating over the surface area in the axial direction. They predicted that the overall tablet size and characteristic swelling time correlate with r qualitatively. Two scaling laws were established for the fractional polymer

[M (t) /M (infinity)] and drug Seipmann et al. [126127] developed a comprehensive mathematical model describing drug release from HPMC-based matrix tablets, taking into account the diffusion of water and drug, nonconstant diffusivities, moving boundary conditions, the swelling of the system, polymer and drug dissolution, and radial and axial mass transfer in cylindrical geometries (Fig. 10). The model was successfully fitted to drug release kinetics of the ophylline from HPMC matrices.

Fig. 10. Mathematical modeling of drug release from HPMC-based matrix tablets; (a)

scheme of cylindrical tablet for mathematical analysis, (b) swelling matrix tablet.

2. Materials and methods

2.1. Materials

The materials and chemicals used in this study were: seeds of OB, MP, LR, PO and SP; gums AN, AM and AT; PO Husk (purchased from local market); L-(+)-arabinose, D-

(+)galactose, D-glucose, D-(+)-xylose, L-rhamnose monohydrate, sodium azide

(SigmaAldrich, USA); BCA protein assay reagent A (cat # 23228) and B (cat # 23224)

and albumin standard (cat # 23209) (Thermo Scientific, Pierce, USA); lactose (Sheffield

BioScience, UK); citric acid and sodium citrate (Riedel-de Haën Chemicals, Germany); paracetamol (NovaMed, Pakistan); titanium dioxide (Colorcon, UK); talc (Specialty

Minerals Inc., USA); Opadry II yellow (Colorcon, UK) ; dextrose (Bio-Rad, USA); disodium hydrogen phosphate and hydrochloric acid (E. Merck, Germany). All the chemicals were used without further purification. Distilled water was used throughout this study.

2.2. Methods

2.2.1. Isolation of polysaccharides

Mucilage of PO, OB, MP and LR seeds

The seeds were dedusted by sifting and 50 g of them were soaked in distilled water

(seedwater ratio 1:50 w/v) separately for about 24 h. The swollen material was blended by use of a kitchen blender for 2-3 min intermittently, taking care that the seeds are not broken.

The mucilage was separated by filtration through muslin cloth under vacuum. The water in the mucilage was removed by evaporation in a rotary evaporator at about 30 C and the semi-dry material was spread on polyethylene sheet and allowed to air-dry at room temperature ( 25°C) for one week to obtain a film having thickness 0.07-0.15 mm.

Mucilage of SP seeds

The SP mucilage was prepared from the seeds according to the procedure as above by excluding the blending step because of the softness of the SP seeds.

Mucilage of PO Husk

The PO husk (20 g) was soaked in water (1000 mL) for 24 h. This was followed by blending with the kitchen blender. The excessive water was separated by filtration under vacuum (1.5

× 10-2 mbar; Edwards rotary pump E2M28) through muslin cloth (maximum pore size 1 mm) followed by further removal of water by use of the rotary evaporator at about 30 C.

The wet mucilage was spread on the polyethylene sheet and air-dried to obtain a thin film.

Purification of gums AM, AN and AT

The gums AM and AN (20 g each) as obtained from the market were freed from extraneous matter by dissolving them in water (150 mL) separately. The solutions were filtered through muslin cloth (maximum pore size 1 mm) under vacuum (1.5 × 10-2 mbar; Edwards rotary pump E2M28). The volume of the filtrate was reduced to approximately 30 mL by evaporation in a rotary evaporator at about 30°C. The thick paste was spread on polyethylene sheets and air-dried at room temperature (~ 25°C) for five days to obtain a film having thickness 0.22-0.25 mm. The yields were approximately 98%.

2.2.2. Characterization

2.2.2.1. Elemental analysis

Elemental analysis of the materials were performed on CHNS analyzer Vario MICRO

V1.4.2 (Elementar Analysensysteme, GmbH, Germany).

2.2.2.2. Moisture content

Moisture content was determined by Karl-Fischer titration using 701KF Titrino (Metrohm,

Switzerland) after drying the materials at 25 ᵒC.

2.2.2.3. FT-IR spectroscopy

The FT-IR spectra were recorded on FT-IR 640 (Varian, USA) by use of KBr disc and film in the range 4000 – 400cm-1.

2.2.2.4. Thermal analysis

Thermogravimetric analysis was performed in the range ambient to 600 C on SDT

(Q600) thermal analyzer (TA Instruments, USA) in the TGA, DTA and DSC modes under

nitrogen at different heating rates 5, 10, 15 and 20 C min-1. The DSC scans were also

obtained in the range -40 – 300 C at 10 C min-1 heating rate. In order to determine

activation energy the data were analyzed by isoconversional Flynn–Wall–Ozawa (FWO)

method (Eq.6)

(6) where , the heating rate; A the pre-exponential factor; R the general gas constant and T is the temperature (K) at the conversion . The FWO method was the first isoconversional linear integral method developed by Flynn, Wall [142] and Ozawa [143].

This method is based on the assumption that for a fixed extent of conversion, the reaction rate depends upon temperature only. Thus it eliminates the dependence of reaction kinetics on any model, that may be represented by an integral form g( ). Therefore, this may be termed as a model-free approach. This method uses data obtained at different heating rates, thus at fixed , the plot of log vs 1/T will be a straight line, the slope of which permits the

calculation of Ea. The is defined as (w0 – wt) / (w0 – wf), where wt is the weight of the

sample at any temperature T, w0 the initial weight and wf is the final weight at the temperature where the mass loss is approximately negligible. Thermal stabilities of the polysaccharides were determined by integral procedural decomposition temperature (IPDT) and comprehensive index of intrinsic thermal stability (ITS) by Doyle [144]. This method is considered to be most appropriate and reliable to determine these parameters because it takes into account the whole TGA curve by measuring area under it. The ITS and IPDT values were determined from TGA of all four heating rates and mean values are reported for each polysaccharide. The life-times of polysaccharides were also predicted by model- free approach based on Eq. 7. The model-free approach eliminates the limitations of other methods such as ASTM E1641 and E698 methods which assume that the Ea remains constant throughout the degradation step. Therefore, in cases where Ea is not constant in a step the model-free approach is more appropriate [145].

(7) This relationship exploits the variation of activation energy with . The integral in the numerator has no analytical solution, however, it can be evaluated by different approximations. In this study the Senum–Yang fourth degree approximation was used.

The data were analyzed by the use of Universal Analysis 2000 software, version 4.2E (TA

Instruments, USA), and MS Excel 2010. Hierarchical cluster analysis (HCA) was performed to classify the materials with similar thermal properties by use of Statistica 8 and dendrogram were drawn using weighted pair-group average and Euclidean distance.

2.2.2.5. Scanning electron microscopy

Surface morphology was studied by recording images on scanning electron microscope

(SEM) Hitachi S-3400N or Joel JSM-6060 LV after sputter coating with gold with Leica

EM SCD005. The micrographs were recorded at different magnifications.

2.2.2.6. Atomic force microscopy

Surface roughness was studied by atomic force microscopy (AFM). The images were optained from the samples films in a non-contact mode on the scanning probe microscope

CP-11 (Veeco, USA) at room temperature.

2.2.2.7. Monosaccharide analysis by HPLC

Monosaccharide analysis was performed after hydrolysis [96] by using Dionex ICS 3000

HPLC system consisting of: CarboPacPA20 column (0.4 × 150 mm) and electro chemical detector according to a reported method (CarboPack PA20: a new monosaccharide separator column with electrochemical detection with disposable gold electrodes) [146].

The samples were subjected to both mild and severe hydrolysis treatment. The mild procedure is a single-step method. It is used to reduce the formation of by-products. For more stable polysaccharides severe hydrolysis treatment, the two-steps method is required.Sugar composition determined gave slight variation depending on the one-step or two-step hydrolysis methods used. In severe treatment the sample (30 mg) was heated with of 12M sulphuric acid (1 mL) at 37 C for 1 h followed by addition of water (11 mL), heating at 100 C for 2 h and quick cooling. In mild treatment the sample (30 mg) was heated with 1M sulphuric acid (12 mL) at 100 C for 2 h and then cooled quickly. The samples from both the treatments were diluted 100 time with 10mM NaOH. To these solutions mannitol (50 µL) was added as an internal standard and measurements were performed in triplicate.

Calibration curves were constructed for Ara, Xyl, Gal, Rha and Glc. The concentrations used of these sugars were: 400, 200, 100, 50, 25, 12.5, 6.25, 3.125 µM.

2.2.2.8. Protein analysis

The protein content was determined by use of bicinchoninic acid (BCA) kit [97]. Briefly,

The sample (0.11g) was dissolved in distilled water (1 mL) by heating to 37 C in water bath for 24 h and shaking. Only the materials from SP, AM, AN, MP and PO produced clear solutions, therefore, the other materials could not be analyzed for the protein content.

The solutions were centrifuged for 3 minutes for further clarity. The albumin standard dilutions (1000, 750, 500, 250, 125, 25 and 0 g mL-1) were prepared similarly. The sample and the standard preparations were carried out in duplicate.

To each of the standard and sample solutions (0.1 mL) 2.0 mL of the coloring reagent composed of reagent A (50) and reagent B (1) were added and mixed well. These solution mixtures were covered and incubated at 37 C for 30 min. After cooling to room temperature, absorbance was measured, within 10 min, at 562 nm by using water as reference. The calibration curve was constructed by use of MS Excel spread sheet.

2.2.2.9. NMR study

NMR methods: sample preparation

AN and AM gums, being soluble in water, were subjected to NMR analysis. The sample

(2.00 g) were dissolved in D2O (20 mL), freeze-dried, redissolved in D2O (20 mL), freeze-

dried, and finally dissolved again in D2O (20 mL). For the solid state NMR the non-

deuterated and deuterated samples were used.

NMR methods: solid state experiments

13C CPMAS NMR spectra were recorded on a Bruker (Karlsruhe Germany) AVANCE 600

NMR Spectrometer with narrow bore magnet and 4mm triple resonance probe. The parameters used in CPMAS experiments were as follows. The Proton 90º pulse length was

5 µs. Field strength of the proton and spin locking fields was 50 KHz. Samples were packed into 4 mm rotors and spun at 10 KHz. ppm scales were referenced to the high field line of adamantane (29.5 ppm) run as an external standard under identical conditions to the samples. Proton decoupling was provided by a WAHUHAHA sequence and the proton power levels during the contact time and decoupling stage could be varied independently to provide optimum signal to noise levels.

NMR methods: high resolution experiments

All single and multi-dimensional NMR experiments were carried out on a Bruker 800 MHz

Avance III Spectrometer equipped with a QCI cyroprobe. For each sample the 90 pulse and transmitter frequency were calibrated. The number of scans collected in each dimension for each experiment was determined by the carbohydrate concentration. Data acquisition and processing were carried out using Topspin 3.1.b.53 software. The 1-D experiments were apodised using an exponential window function with 2 Hz line broadening. For multi-dimensional datasets a shifted squared sine bell was used with the offset being optimised to achieve the best balance between resolution and signal to noise.

All data were zero-filled by at least a factor of 2. For heteronuclear dimensions linear prediction was employed.

1-Dimensional Experiments

The 1-D proton spectra were recorded using excitation sculpting water suppression, with

a spectral width of 14 ppm. The proton transmitter frequency was set to 4.702 ppm and

typically 64 scans were acquired.

2-Dimensional Experiments

The 2-D carbon protonheteronuclear single quantum coherence (HSQC) spectra were

acquired over a spectral width of 14 ppm in the 1H dimension and 200 ppm in the 13C

dimension. The transmitter frequency for carbon was centred at 100 ppm. Between 16

and 64 scans were acquired, with 128 complex points in F1. Quadrature detection in the

carbon channel was achieved using States-TPPI.

3-Dimensional Experiments

The 3-D data were acquired for the assignment of spin systems of individual sugar subunits

of the gums. The carbon and proton dimensions were optimized as for the 2-D HSQC

experiments with the carbon transmitter frequency being set at 100 ppm. A third proton

dimension enabled a TOCSY experiment to be correlated with each HSQC cross peak. 16-

64 scans and 98 - 128 points were acquired in the first proton dimension, whilst for the

second proton dimension scans and 128 points were acquired. The 3-D processing was

handled as per HSQC.

2.2.2.10. Rheology

Solution of gums and gels were extracted from the seeds and husk and weight of dry polymer was calculated from dry and wet weight. pH of all the solutions were measured at 25 C.

Rheology was studied on Anton Par Physica MCR301. Double gap (Dg) cylinder was used to measure the effect of shear rate on viscosity. Shear rate was varied from 10-2 to 10+3 and vice versa.

2.2.2.11. Determination of molar mass

Molar mass of the polymers, under investigation, were determined by size exclusion chromatography coupled with multi-angled light scattering (SEC-MALS) and an online viscometer (Torqometer, Beckman optima XL-A, USA), analytical ultracentrifuge

(Proteome LabTM XL with scanning absorption optics, USA) and from rheology data.

Intrinsic viscosity data can be used to determine shape and molar mass of polymers [147].

The concentrations of the polymer solutions were determined by use of Atago DD-5

Differential Refractometer (Jencons Scientific, UK). The intrinsic viscosity was measured in distilled water using Anton Par Physica MCR 301and double gap cylinder. The pH of the solutions were 4.41 (AN) and 4.29 (AM) at 24 C. Molar masses were determined in phosphate buffer (pH 6.8) by light scattering using 1% solution of each gum and in water for rheological data using 25% stock solution using Mark-Houwick equation (8)

[ɳ] = KMa (8)

Size Exclusion Chromatography coupled to multi-angled light scattering and an online viscometer (SEC-MALS).

Analytical fractionation was carried out using a series of SEC columns TSK G6000PW,

TSK G5000PW and TSK G4000PW protected by a similarly packed guard column (Tosoh

Bioscience, Tokyo, Japan) with on-line MALLS (Dawn DSP, Wyatt Technology, Santa

Barbara, USA) and refractive index (Optilab rEX, Wyatt Technology, Santa Barbara,

USA) detectors. The eluent ( pH 6.8, phosphate buffer) was pumped at 0.8 ml min-1

(PU1580, Jasco Corporation, Great Dunmow, UK) and the injected volume was100 l ( ~

1.0 ˣ 10-3g ml-1) for each sample. Absolute weight- average molar masses (Mw) were calculated using the ASTRA (Version 5.1.9.1) software (Wyatt Technology, Santa

Barbara, USA), using the refractive index increment, dn/dc = 0.163 ml g-1 [148].

. Gel permeation chromatography (GPC)

Gel permeation chromatorgraphy of some of the suitable gels were performed by Agilent

1200 series (Agilent, Germany) equipped with Quat pump (G1311A) and refractive index detector (G1362A) using water as eluent (flow rate 1.0 cm3 min-1 at 70 C) and injection volume of 10 L. The parameters calculated were molar mass averages (Mw, Mn, Mz), molar masses at peak top (Mp) and volumes at peak top (Vp) and polydispersity index

(PDI). The data were analyzed by use of Chem Station GPC Data Analysis software Rev.

A.02.02 (Agilent, Germany).

Ultracentrifugation

Sedimentation velocity experiments were performed using a Beckman Instruments (Palo

Alto, USA) Optima XLI Analytical Ultracentrifuge. The polymer solutions (380 L) of various concentrations (0.25–1.0 mg/ml), pH 6.8, phosphate buffer (400 L) were injected into the solution and reference channels, respectively, of a double sector 12 mm optical path length cell. Samples were centrifuged at 45000 rpm at a temperature of 20.0 C

[149]. The data were analysed by using the „„least squares, ls-g(s) model” incorporated into the SEDFIT (Version 9.4b) program [150, 151].

2.2.2.12. ToF-SIMS

Dispersion of drug particles into the polymer matrix was studied by ToF-SIMS. The samples were prepared as follows. A piece (1 × 1 cm) of the dried films of MP, LR, OB, PO husk and seed gel were immersed in 20 mL each of 1% caffeine and 2% diclofenac sodium solutions separately for 2 h, and the films of SP gel, AN and AM gum were immersed in the drug solutions for 10 min as the longer immersion would result in erosion of these films.

The drug-loaded polymer films were removed from the solutions, air-dried on the polythene sheets at room temperature for about 24 h and subjected to ToF-SIMS analysis.

Measurements were carried out by use of a ToF-SIMS Ion-TOF IV (ION-TOF GmbH,

Münster, Germany) system equipped with a Bi3+ cluster source and a single-stage reflectron analyzer. The system was evacuated to 1 × 10-6 millibar. Spectra were acquired in positive and negative modes by rastering a primary ion energy of 25 kV along with a pulsed target current of approximately 1 pA and post-acceleration energy of 10 kV across the sample surface (area 225 × 225 µm at a resolution of 225 × 225 pixels). The primary ion dose density was maintained at <1012 ions cm-2 to ensure static conditions. Data were processed by use of imaging software (SurfaceLab 6 Image; ION-TOF GmbH).

2.2.2.13. Mechanical strength

Mechanical strength of the polymer films (1cm × 6cm ) of SP, AN, MP, OB, LR, AM, PO seed and husk, as prepared in section 2.2.1., were measured by Universal Testing machine

AGS-J (Shimadzu, Japan) using 1 kN force at 25 2 C.

2.2.2.14. Swelling index

The polymer (0.10 g) was soaked in distilled water (10 mL) and wet weights were recorded after drying externally by use of a blotting paper, after every five min for the first hour and every hour till a constant weight was obtained. Swelling Index was calculated by the formula.

Swelling Index = [(Weight of wet sample –Weight of dry sample) / Weight of dry sample)]

×100

2.2.2.15. Water retention

Water retention by the polymers was determined by centrifugation method [160-162].

Accurately weighed sample (0.01 – 0.3 g) was soaked in water (about 10 mL) at 30°C) in a petri plate for 2 h (30 min for gums). The swollen material was centrifuged at 4000 rpm for 15 min to remove excessive water. The wet samples were dried at 105 2°C in an hotair oven to a constant weight. Water retention was calculated by the formula.

Ww - Wd 100

Water Retention(%) Wd

where, Ww = weight of sample in wet state, Wd = weight of sample after drying at 105 C.

2.2.3. Evaluation of the isolated polysaccharides as drug carriers

Preparation of tablets

Tablets (620 mg total weight) were prepared by thoroughly mixing the drug (100 mg) with the biopolymer (100 mg), lactose (400 mg) and magnesium stearate (20 mg); grinding by use of laboratory grinder, sieving through 0.8 mm mesh and subjecting to direct compression at about 116 N in a 5-mm die.

Preparation of dissolution media

The dissolution was studied in distilled water (for caffeine), 0.1 N HCl (for diclofenac sodium) and pH 6.8 phosphate buffer (for diclofenac sodium) as directed by US

Pharmacopeia. For the preparation of the buffer disodium hydrogen phosphate (71.5000 g) was dissolved in water (1000 mL). Out of this 77.3 mL was mixed with 22.7 mL of a 2.1

% citric acid solution.

Drug release study

The release study was carried out in the appropriate dissolution medium (900 mL) using

USP Paddle Dissolution apparatus II at 37±0.1°C and 50 rpm for diclofenac sodium and at

100 rpm for caffeine. Samples (2 mL) were withdrawn at 15 min, 30 min, 45 min, 60 min,

120 min, 180 min intervals, filtered, suitably diluted and assayed spectrophotometrically at

273 nm ( =9124.045) for caffeine, 275 nm ( =10181.45) for diclofenac sodium in the buffer solution and 276 nm ( =380.084) for diclofenac sodium in 0.1N HCl. The values were determined experimentally. The measurements were made on UV-Vis spectrophotometer (Schimadzu, Japan). After each withdrawl an equal volume of the dissolution medium was replaced immediately. The cumulative release (percent of the drug amount in the tablet) was plotted against time. The data was fitted into zero order, first order

[133, 134], Higuchi [152, 153] and Hixson–Crowell cube root law models in order to determine the release pattern. Drug release data obtained via dissolution was analyzed using following release models to investigate the true kinetics of drug release. The release mechanism (Fickian, non-Fickian, case-II transport) was determined by applying a generally used equation, the so-called Power Law [154-157] as follow.

t M lnkp nlnt ln M where Mt/M∞ is the fraction of drug released in time t, kp is the Power Law constant characteristic of the drug matrix system and n is the diffusion exponent. The value of n identifies different mechanisms for drug release. For best model selection, a modified

Akaike Information Criterion called Model Selection Criterion (MSC), Eq. (9) [158], was used.

n _ 2 wi(Yobsi

i 1 Yobs ) 2p (9) MSC ln n n

wi(Yobsi Ycali )2 i 1

th where Yobsi and Ycali are the observed and calculated value of i data point respectively, the mean of observed data points, wi the optional weight factor, n the number of data points and p the number of parameters. MSC is independent of the scaling of data points and the model with largest MSC value is considered to be the most appropriate. All the calculations were performed by use of MS Excel® 2003.

2.2.4. Evaluation as binders in tablets

The polymers under investigation were evaluated for their intended use as tablet binders.

For this purpose, 20 tablets composed of acetaminophen (10 mg), the polymer (10 mg), lactose (80 mg) and magnesium sterate (1 mg) were mixed and ground well. The homogeneous mixture was passed through 0.8 mm sieve and pressed into bi-planar tablets

(9 mm diameter) under 13.9 N mm-2 force. Hardness (crushing strength) of the tablets was determined by use of a digital hardness tester (Curio, Pakistan). The hardness was compared with those of similarly prepared tablets by use of microcrystalline cellulose as binder.

Disintegration time was determined by use of six tablets and water as medium under standard conditions; the disintegration apparatus used was tester VD-2 Vision Scientific

(China).

2.2.5. Evaluation as suspending agents

Acetaminophen (50 mg mL-1) suspensions were prepared as: dextrose (7.5 g) was dissolved in water (15 mL) with stirring to obtain a clear solution; to this citric acid

(0.125g), sodium citrate (0.125 g), the appropriate mucilage (0.125 g) from OB, MP, LR,

SP, PO seeds and husk or the gum (1.25 g) of AN, AM and AT, and acetaminophen (1.25 g) were added under stirring in that order. The suspensions thus prepared were transferred to

50-mL graduated cylinders and the volume was made up to 25 mL with water and mixed well. The cylinders were placed undisturbed in the dark at room temperature (25 2 C) at a safe place. Sedimentation was recorded was recorded daily after 24 h for 90 days.

2.2.6. Evaluation as thickening agents

Thickening power of the polymers was determined in terms of viscosity at a concentration

(1%) and room temperature (25 C). The results were compared with those of carboxymethylcellulose.

2.2.7. Evaluation as film coating materials

The mucilage (1.5 g) of OB, MP, LR, SP , PO seeds and husk ) or the gum, AN (24 g) and

AM (30 g), was suspended in distilled water (300 mL) and heated to about 60 C on hotplate with constant stirring for 1-2 h to allow the polymers to swell. To this Opadry yellow (1 g), titanium oxide (5 g) and talc (9 g) were added and the mixture was heated again to 60 C with constant stirring to get a homogenous mixture, which was used to coat the lactose tablets (round and oval) with the help of THAI COATER® (China). The coating was subjected to film rupture test also known as drop test.

3. Results and discussion

3.1. Isolation of polysaccharides

In the present study nine biopolymers, characterized to be polysaccharides, were isolated from the plant seeds or husk, purified and characterized by use of different analytical techniques such as FTIR, elemental analysis, thermal analysis, AFM, SEM, ToF-SIMS, protein and sugar analysis. The materials were also evaluated for their potential applications in pharmaceuticals as binders, suspending agent, coating agents for tablets, thickeners and as drug releasing device.

The polymers, from colorless to light brown or dark brown, were isolated in good yield (10-

98 % on dry substance basis) as listed in Table 2. The isolations could be quickened by use of organic solvents, such as methanol, acetone or acetic acid to coagulate the polymers dispersed in water, but in that case residual solvents were found to be present in the product.

The presence of residual solvents in pharmaceutical adjuvants is restricted because of their toxicity. Therefore, all the isolations were achieved without the use of any solvent. It was noted that the drying should be carried out at temperatures lower than 40 C as the color of the product darkens at higher temperatures. Taking into account the yields obtained the cost of the polymers was calculated based on the current local market rates of the plant materials.

It appears that the isolated materials can produced at very low costs as compared with the commercially available synthetic polymers like hydroxypropylmethylcellulose (HPMC) etc.

3.2 Characterization

3.2.1 Elemental analysis

Average percentages of carbon and hydrogen in the isolated polymers was found to be

28.75% (Table 3) ratio of % C to % H reported in the natural polysaccharide is 7.38, these lower values than those of natural polysaccharides may be attributed to adsorbed moisture or the presence of uronic acids in the materials [159-161]. Average percentage of sulphur and nitrogen is 0.14 and 0.61 which is less than 1%. Thus the absence of nitrogen proves that these are polysaccharides.

3.2.2. Moisture content

Moisture content as determined by Karl-Fischer method ranged from 0.40% to 14.81%, which was used for the purpose of calculations on dry substance basis and explaining the mechanical properties.

3.2.3. FT-IR spectroscopy

The absorption bands observed in the FT-IR spectra of the polymers were assigned by comparison with literature values [162-165]. The assignments are given in Table 4. The characteristic bands due to (OH) at 3359 – 3463 cm-1, (C–C) in arabinosyl side chain at

1000 – 1059 cm-1, -glycosidic C-H bending at 849 – 910 cm-1, and the out-of-phase bending of hydrogen bonded hydroxyl groups in the polymer backbone at 600 – 668 cm-1 were observed in all the polymers along with some other bands. The absence of

characteristic bands of proteins and ferulic acid indicates that the polymers under investigation are free from these materials.

Table 2. Physical appearance and yields of isolated polymers Material Yield (%) Color Cost (1kg) Remarks SP 10 Light Brown $6.00 The colour AN 98 Light Brown $6.00 appears during drying process MP 12 Light Brown $9.00 due to air PO husk 25 White $7.50 oxidation at trace level. AM 98 Light Brown $9.00 LR 12 Light Brown $4.50 OB 12 Light Brown $6.00 PO seeds 12 Light Brown $3.00

AT 98 White $15.0

Table 3. Elemental (% on dry substance basis) and moisture analysis data Sample N* C H S C/N C/H Moisture

SP 0.89 19.45 2.89 0.24 21.85 6.73 7.15

AN 0.34 32.21 4.94 0.00 94.74 6.52 4.77

MP 0.78 29.72 4.62 0.22 38.12 6.43 0.40

PO Husk 1.10 28.99 4.28 0.53 26.35 6.77 8.24

AM 0.20 32.47 4.98 0.00 162.35 6.52 5.23

LR 1.56 32.54 4.72 0.09 20.85 6.89 11.96

OB 0.48 23.15 3.58 0.05 48.23 6.47 14.81

AT 0.00 32.94 5.01 0.00 32.94 6.57 10.49

PO Seeds 0.39 27.26 3.98 0.12 69.89 6.85 10.2

*Trace amounts of N may be found after purification

Table 4. Observed FT-IR bands and their assignments

SA 3432 2929 1618 1421 1350 1250 1074 1042 891 645, 500 2154 AN 3410 2932 1626 1460 1421 1377 1253 1037 850 600 2127, 1736 MP 3359 2920 1605 1420 1377 1246 1047 896 620, 500 POH 3400 2926 1650 1460 1420 1377 1250 1150 1000 890 600, 500 2170 AM 3422 2936 1620 1460 1421 1377 1250 1037 850 600 2120, 1736 LR 3384 2923 1648 1421 1377 1244 1153 1059, 1035 896 668, 618 OB 3368 2920 1638 1460 1422 1376 1153 1057 910 618 1720 POS 3463 2926 1655 1462 1043 880, 849 616 LR 3384 2923 1648 1421 1377 1244 1153 1059, 1035 896 668, 618 OB 3368 2920 1638 1460 1422 1376 1153 1057 910 618 1720 POS 3463 2926 1655 1462 1043 880, 849 616

3.2.4. Thermal analysis

Thermal behavior of the isolated polysaccharides was studied by TGA and DSC from ambient to

600 C. TGA of all the materials, except AT, exhibited an endothermic weight loss of 8–20% in the 80–120 C range, which was due to the loss of trapped moisture[166-169] (Fig 11-19). The major weight loss (18–36%) occurred in the range 225–325 C (Fig. 20a), which was due to degradation of the polysaccharide structure. This step was associated with a wide exothermic enthalpy change as shown in the DSC scan (Fig. 20b). The mean comprehensive index of thermal stability (ITS) and integral procedural decomposition temperature (IPDT) values were found to be in the range 0.33–0.43 and 213–270 C, respectively (Table 5), which are indicative of good thermal stability of the materials. In case of AT a rapid weight loss of about 90% occurred in the ambient – 95 C, which is due to loss of water (Fig. 20c) and 100% weight loss is due to negligible ash in the material. The isolated polysaccharides could be classified on the basis of their thermal behavior by use of HCA as shown in Fig. 21. It can be seen that LR and SP polysaccharides with

ITS values 0.35 and 0.33, respectively, are on the lower side and they appear to form one major group while others form the second major group containing small groups at various similarity levels. As the IPDT and ITS are calculated from the area under the TGA curve, the LR and SP depicted lower values due to higher moisture contents (SA = 20%, AT = 21%) in them (Fig. 20a).

Flynn–Wall–Ozawa analysis

The apparent Ea values for major stage of decomposition were calculated by FWO method at different conversions ( = 0.1 - 0.90 with 0.1 increment). Typical -T and FWO plots for LR are

a b

c d

Fig. 11. TGA and DSC scans of the polymer isolated from AT at a 5 C, b 10 C, c 15 C and d 20 C

a b

c d

Fig. 12. TGA and DSC scans of the polymer isolated from AN at a 5 C, b 10 C, c 15 C and d 20 C

a b

c d

Fig. 13. TGA and DSC scans of the polymer isolated from AM at a 5 C, b 10 C, c 15 C d

20 C

b a c

c d c c

Fig. 14. TGA and DSC scans of the polymer isolated from PO husk at a 5 C, b 10 C, c 15 C and d 20 C

b a

c d

Fig. 15. TGA and DSC scans of the polymer isolated from PO seed at a 5 C, b 10 C, c 15 C and d 20 C

a b

c d

Fig. 16. TGA and DSC scans of the polymer isolated from SP at a 5 C, b 10 C, c 15 C and d 20 C

a b

d c

Fig. 17. TGA and DSC scans of the polymer isolated from LR at a 5 C, b 10 C, c 15 C and d 20 C

a b

c d

Fig. 18. TGA and DSC scans of the polymer isolated from MP at a 5 C, b 10 C, c 15 C and d 20 C

a b

c d

Fig.19. TGA and DSC scans of the polymer isolated from OB at a 5 C, b 10 C, c 15 C and d 20 C

Fig. 20. a) TGA curves of the polymers under study at heating rate of 5 C min-1. MP; PO seed;

SP; AN; PO husk; AM; LR; AT; OB .

Fig. 20. b) Representative TGA, DTG and DSC curves for AT at heating rate of 5 C min-1

Fig. 20. c) TGA curves of AT at different heating rates

Fig. 21. Thermal classification of the polymers: Dendrogram showing similarity levels of

thermograms for the polymers shown in Fig. 22 a and b respectively. The Ea- curves (22c) indicated the dependence of Ea on the degree of conversion . The polysaccharides from MP, SP and PO seed showed strong dependence of Ea on suggesting a multistep degradation pattern for these materials. The Ea values of OB, LR and AT remained almost constant indicating that these polysaccharides may be decomposed in one step. The Ea for PO husk, AN and AM varies with , which suggests a multistep degradation of these materials [145]. The multistep decomposition may be attributed to the diversity of sugar content of the material. The average activation energies are given in Table 5.

Since FWO method does not provide a direct estimate of the pre-exponential factor A, this factor was calculated by use of the compensation effect relationship [145] according to Eq. (10).

lnA = a + bEa (10) where a and b are the compensation parameters. This relationship suggests that any change in lnA shall be accompanied by a corresponding change, in a linear fashion, in Ea as calculated by use of

Coats–Redfern equation, Eq. (11). The a and b were determined by model-fitting approach using this equation

where T is the average experimental temperature. The g( ) models used for solving the Eq. (11) in the present work are listed in Table 6. From the ln (g( )/T2) vs 1/T plots values of A and Ea were determined, which were used in Eq. (10) to obtain the values of a and b.

Fig. 22. a) Representative –T curve for AT, b) Representative FWO plot for LR to calculate Ea., c) Dependence of Ea on for polysaccharides

Table 5. Thermal and compensation effect parameters for polysaccharides

Compensation equation Parameters

Sample a b |r| Ea (kJ InA IPDT ITS ΔH ΔS ΔG Code mol-1) (oC) AT -2.410 0.222 0.999 187.0 43.8 241 0.38 182.7 114.6 122.9 AN -2.203 0.214 0.999 157.5 35.8 254 0.40 152.8 47.6 126.1 SP -2.395 0.219 0.999 157.6 36.9 226 0.35 153.2 57.1 123.0 AM -2.414 0.218 0.999 132.6 31.3 269 0.42 128.1 10.4 122.5 MP -2.199 0.208 0.999 165.0 36.6 27 0.43 160.6 54.6 131.6 OB -2.444 0.213 0.999 164.7 37.6 261 0.41 160.2 62.7 126.0 PO -2.414 0.212 0.999 154.9 35.2 247 039 150.2 42.5 126.4 Seeds LR -2.343 0.223 0.999 169.6 40.2 213 0.33 165.3 84.7 121.4 PO Husk -1.437 0.212 0.999 175.4 38.7 262 0.41 170.7 71.5 130.2

*Mean Ea, IPDT and ITS value are reported from different heating rates upto 600oC. InA is calculated from mean Ea

The lnA values calculated from this equation using average FWO activation energies are listed in

Table 5. The best model was selected on the basis of: i) the correleation coefficient and ii) the closeness of the activation energy with that determined by FWO method. Thus the first order model

(F1) was found to be the best of the ten for most of the polysaccharides. The AN, SP, AM, MP, PO seed and PO husk polysaccharides exhibited a multistep decomposition with first order kinetics, whereas OB, LR and AT were found to be single step decompositions. This finding supports the general practice of using Broido method for determination of kinetic parameters of polysaccharides where the reaction mechanism is assumed to be of first order. In case of PO husk the data also fits well in A2 model. Similarly in case of other materials, the diffusion models D1 and D3 also provide a good fit (Table 6). All the polysaccharides under investigation exhibited very high stability (life time > 20 years) at 40 C, except those from PO seeds and AM (life time about 1 month), as predicted by the model-free analysis (Fig. 23). The isocoversional method provides more accurate values of Ea and A than the single-heating rate method. By use of this method relatively more reliable values of S , H and G are being reported here (Table 5).

3.2.5. Electron microscopy

SEM images of the polysaccharides were used to study their morphology and surface topography.

As can be seen (Fig. 24) these polymers contain voids (a – d, f, h and i) or layers (e and g), so their structures are suitable for dispersion of drug particles in them.

3.2.6. Atomic force microscopy

The AFM images of the polymers under investigation are shown in Fig. 25. The polymers

Table 6. Kinetic parameters for polysaccharides determined by model-fitting approach using Coats–Redfern equation a.

AT AN SP AM MP OB PO seeds LR PO husk

Co Ea InA |r| Ea In |r| Ea In |r| Ea InA |r| Ea In |r| Ea InA |r| Ea InA |r| Ea In |r| Ea InA |r| -de g(a) A A A A 1/4 α Power P1 13 0.23 0.849 26 2.83 0.977 11 0.13 0.78 12 0.12 0.898 8 0.60 0.72 14 0.34 0.837 17 0.79 0.888 0.19 0.669 72 13.31 0.984 11 Law Power P2 1/3 20 1.63 0.885 38 5.50 0.981 18 1.15 0.83 18 1.22 0.926 14 0.22 0.80 22 1.91 0.902 26 2.67 0.911 17 1.01 0.738 99 19.3 0.985 α Law Power P3 1/2 34 4.99 0.910 61 10.8 0.984 32 4.24 0.87 32 4.35 0.944 25 2.56 0.85 38 5.44 0.923 43 6.59 0.928 30 4.01 0.790 154 31.14 0.986 α Law Power P4 3/2 121 24.65 0.932 202 41.4 0.987 113 22.6 0.91 114 22.8 0.960 95 17.8 0.90 132 26.03 0.942 148 29.34 0.944 107 21.9 0.840 480 100.9 0.987 α Law One D1 2 164 34.25 0.935 273 56.4 0.987 153 31.5 0.91 155 31.9 0.962 129 25.2 0.90 179 36.08 0.944 201 40.48 0.946 146 30.6 0.845 643 135.5 0.987 dimensio α -nal diffusion First F1 -ln(1-α) 114 24.00 0.977 186 38.6 0.999 108 22.3 0.96 107 21.9 0.991 90 17.7 0.95 124 25.19 0.982 139 28.23 0.980 105 22.2 0.911 440 93.08 0.993 order Avrami A1 [-ln(1-α) ] 1/4 22 2.28 0.960 40 6.10 0.999 20 1.83 0.93 20 1.75 0.984 16 0.71 0.91 24 2.56 0.970 28 3.34 0.968 20 1.81 0.853 103 20.27 0.992 Erofeyev Avrami A2 [-ln(1-α) ] 1/3 32 4.75 0.967 56 9.83 0.999 30 4.13 0.94 30 4.02 0.987 24 2.51 0.93 35 5.14 0.975 40 6.19 0.973 29 4.11 0.877 140 28.49 0.992 Erofeyev Avrami A3 [-ln(1-α) ] 1/2 53 9.66 0.973 88 17.2 0.999 49 8.76 0.95 49 8.58 0.990 41 6.36 0.94 58 10.25 0.979 65 11.81 0.977 48 8.72 0.896 215 44.77 0.993 Erofeyev 210 43.11 0.966 340 69.5 0.998 197 40 0.95 197 39.7 0.985 167 32 0.94 228 45.28 0.973 255 50.56 0.971 191 39.5 0.895 797 166.6 0.993 Three D3 [-ln(1-α) ] 2 dimensio 1/3 -nal diffusion Contracti CS 1-(1-α) 1/3 100 19.48 0.963 166 32.9 0.997 94 17.9 0.94 94 17.7 0.983 79 13.8 0.94 109 20.57 0.970 123 23.29 0.969 91 17.6 0.886 394 81.83 0.993 -ng sphere Contracti - CC 1-(1-α) 1/2 94 18.33 0.956 156 31.1 0.996 88 16.8 0.93 88 16.7 0.978 74 12.9 0.93 103 19.37 0.963 115 21.96 0.963 85 16.5 0.873 372 77.61 0.992 ng cylinder

Fig. 23. Life time prediction of the polymers at different temperatures and 5%degree of conversions for the polymers

(a ) ( b) (c )

(d ) (e ) (f )

(g ) ( h) (i )

Fig. 24. SEM images of films of : a) OB, b) LR, c) PO seeds, d) SP, e) AN, f) MP, g) AM, h) PO husk and i) AT

) (i)

(i)

( c ) ( a) ( b )

( d) ( e) ( f )

( g)

Fig. 25. AFM images of films of : a) SP, b) MP, c) PO seeds, d) OB, e) LR, f) AN and g) AM appeared to consist of nanostructures. The particle size of the nanostructure varied from polymer to polymer (Fig. 26). but the size of particles in each material was different as in AN the size ranged from 90-110 nm, in AM it was 160-240 nm, in MP it was 120-220nm, PO seed contained the smallest particle size of 10-70 nm, OB was 100-220 nm, LR contained a large variety of particle size from 100-650nm and SP particle size ranged from230-320 nm. The roughness parameters of the materials under investigation are recorded in Table 7. A nanocarpet type surface with roughness ranging from 4.3 (AN) to 196.1 nm (LR) was observed in the AFM images of these polymers.

3.2.7. Monosaccharide analysis by HPLC

The results of monosaccharide analysis by HPLC are given in Table 8. Monosaccharide content of

AN and PO were found to be similar to those already reported [161,170 - 171]. Based on the monosaccharide contents the polymers were characterized as: SP, rhamnoxylan; AN, galactoarabinan; MP, glucoxylan; PO, arabinoxylan; AM, rhanogalactoarabinan; LR, xylogalactorhamnoarabinoglucan and OB, galactorhamnoarabinoxyloglucan.

3.2.8. Protein analysis

The method used for determination of protein produced a good (R2 = 0.986) calibration curve as shown in Fig. 27. This curve was used for determination of protein cont of SP, AN, AM, PO seeds

and MP; the content of other polymers could not be determined as they were insoluble. The results are given in Table 8. The content varied from 0.1 % to 0.6 % in AN; a similar result has

35 90 80 b a 30 70 25 60 50 20 40 15 30 20 10 10 5 0 0 90-97 98-110 nm nm

Fig. 26. Nanostructure in a) AN, b) AM, c) MP, d) OB, e) PO seeds, f) LR and g) SP

e 25

30 g 25 20 15 10 5 0

nm nm

Fig. 26. (continued) d) OB, e) PO seeds, f) LR and g) SP Table 7. Roughness parameters of the polymers

Material RMS Ave Mean Ht Median Peak Valley Volume Surface Projected roughness roughness (nm) Ht (nm) (nm) ( m2) area area (nm) (nm) (nm) ( m2) ( m2) AN 31.15 4.349 973.5 973.8 294.3 -973.5 24.34 29.96 25

AM 22.88 17.07 199.8 201.6 110.1 -199.8 4.995 26.49 25

MP 84.37 65.37 264 260.7 233.5 -264 6.601 26.82 25

OB 59.18 46.85 159.8 154.8 217.7 -159.8 3.996 26.70 25

PO seeds 10.18 7.424 25.21 23.36 46.26 -25.21 0.6302 25.06 25

LR 225.8 196.1 624.1 635.7 500.2 -624.1 15.60 29.96 25 SP 34.07 26.42 267.4 267 195.5 -267.4 6.686 36.59 25

Table 8. Monosaccharide and protein analysis

Monosaccharide content (% of total monosaccharides) Protein (%) Sample Ara Gal Glc Xyl Rha SP (S) - - - 100 - 0.41 SP (M) - - - 99.32 0.68 AN (S) 74.17 25.83 - - - 0.09 AN (M) 75.74 24.26 - - - MP (S) - - 30.89 69.11 - 0.26 MP (M) - - - 100 - PO husk (S) 23.11 - - 76.89 - - PO husk (M) 21.37 - - 78.63 - AM (S) 68.09 30.11 - - 1.79 0.13 AM (M) 67.88 29.99 - - 2.13 LR (S) 16.39 7.55 63.90 1.19 10.97 - LR (M) 29.14 1.28 - - 69.59 OB (S) 9.82 5.59 55.84 19.10 9.66 - OB (M) 20.39 11.67 21.35 23.31 23.27 PO seeds (S) 21.90 - - 78.10 - 0.34 PO seeds (M) 18.99 - - 81.01 - AT(S) - 58.12 - - 41.88 - AT(M) - 55.92 - - 44.08 -

Fig. 27. Calibration curve for protein analysis

been reported earlier [170]. It can be seen that these polymers do not contain significant amounts of proteins (0.09 – 0.41 %), therefore, they can be categorized as pure polysaccharides.

3.2.9. NMR study

1 AN and AM, could be characterized by NMR analysis in this work. HNMR spectra were in D2O and the 13CNMR spectra were recorded in solid state. The 1HNMR spectra were complex and proton splitting patterns were not obvious. Therefore, the assignments were made by comparing the spectra with those reported for similar materials [86, 172-177]. In the 13CNMR spectra two major absorptions, AN/AM: = 100.5/103.5 ppm due to C-1 of branched pyranose Gal (Galp) and =

109.6/109.6 ppm due to C-1 of furanose Ara (Araf) of the main chain [86, 172] were observed in the anomeric region (Table 9, Fig.28). In the spectrum of AM a small signal at = 98.6 ppm due to C-1 of branched Rhap, present in very small amount (<2%), was also observed. The resonances of the carbons in glycosidic linkages were observed at =

109.6 ppm (C-1 of 1,5-linked Araf), 100-103.5 ppm (C-1 of 1,3- linked Galp), 80.5 ppm (C-3 of 3- linked Galp), 82 ppm (C-2 of 2-linked Araf) and 65.1 ppm (C-5 of 5- linked Araf). The other signals at = 175 and 17.1 ppm were due to C-6 of Galp and Rhap residues. Based on these observations the polymers were characterized as branched structures.

1 The HNMR spectra are shown in Fig. 29 and 30. In these spectra multiplets due to H1-5 (Araf), and

H1-6 (Galp) were observed at = 5-5.5 ppm. The anomeric proton signals were well resolved and appeared at = 5.27 ppm due to H-1 of Rhap, = 5.16 ppm due to H-1 of Araf, = 5.05 ppm

Table 9. 13C and 1H NMR data of AN and AM

Glycosyl Chemical shift /ppm residue

C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6 L-Araf 109.6 82 73.0 74.5 65.1

5.16 4.4 4.1 4.2 3.7 68.5 80.5 78.0 76.6 -D-Galp 103.5(AM) 175.6 (AM)

100.5 (AN) 178.1(AN) 5.05 4.05 4.10 4.20 4.17

Fig. 28. 13C NMR of AN and AM

Fig. 29. 1H spectrum of AN having several sharp lines

Fig. 30. HSQC plots showing superposition for AN (red) and AM ( blue)

due to H-1 of Galp and = 4.40 ppm due to H-2 of Araf (Table 9). The CH3 (on C-6 of Rhap) signal was observed at = 1.19 ppm.

The assignments of two dimensional 13C-1H NMR (HSQC) spectra (Fig. 30) of AN and AM polysaccharide are given in Table 9. The HSQC plots showed that each cross peak has coordinates corresponding to the respective chemical shift of a 13C and its directly bonded proton.

The anomeric protons resonating at = 5.16, 5.05 and 5.27 ppm correlated with carbon signals at

= 109.6 (AN and AM), 100.5 (AN), 103.5 (AM) and 99.5 ppm (AN and AM). These chemical shifts are characteristic of anomeric carbons of Araf, Galp and Rhap residues [173]. The two broad proton signals at = 5.05 and 4.49 ppm correlated with the anomeric carbon shifts at = 100.5 (AN) and

103.5 ppm (AM) due to C-1 of Galp residues. The cross peaks in the high magnetic field at = 1.19

(CH3) and at = 17.43 (CH3) confirmed the presence of Rhap units in the polysaccharide [173].

13 The C signals with very low intensities at = 17.1 ppm (CH3 on Rhap) and 175 in AM and 178 ppm in AN (COOH on GalA) were also observed. t appears that a small amount of the H 2OH on

C-5 of Galp has been oxidized to COOH [86]. The peak at =

17.1 ppm also corresponds with the monosaccharide analysis (Table 8), where Rhap is present in

AM (2%) and absent in AN.

Appearance of 13C signals due to C-1 and C-5 in Araf at relatively higher values than expected for the monosaccharide suggests an -(1,5) linkage of L-arabinose in the main chain [176].

Similarly the appearance of peaks at = 100.5 (AN) and 103.5 (AM) due to C-1 of Galp and at 80.5 ppm for C-3 of Galp suggest a -(1,3) linkage of D-galactose in the side chain. At few points arabinose appears to be connected at C-2 of Araf (Fig. 31).

Fig. 31. Structure suggested on the basis of NMR and literature 86, 172-177.

3.2.10. Rheology

Rheology is the study of flow and deformation of a material. Rheology study is an important aspect in characterization of polymers. Polysaccharides behave as flexible coils in dilute solution [178].

The process of coating different materials, such as pharmaceutical tablets, with polymers is dependent on viscosity and elasticity of the polymer. Therefore, it was relevant per se to study rheology of the polysaccharides under investigation with a view to assess their potential as filmcoating, viscosity enhancing and suspending agents.

The polymers were subjected to rheological measurement, at different concentrations and pH (Table

10), in shear rate region of 0.01 to 1000 s-1. AT could not be studied because it did not form a homogeneous solution required for the study. The rheograms are shown in Fig.32. AN and AM exhibited Newtonian flow, whereas other materials showed non-Newtonian behavior. In case of SP,

PO seeds, PO husk, OB, LR, MP no significant change in structure was observed as indicated by the repetition of the reverse rheogram on the same line.

The viscosity decreased with an increase in shear rate. A cursory view of the viscosity data at 1% concentration level and shear rate 10 s-1 (Table 10) shows that PO husk possesses the highest viscosity and AN the lowest. It can be seen that the viscosity of PO husk is about two times that produced by PO seed, whereas the latter resembles LR in this respect. The trend in viscosity was found to be: PO husk > LR > POseeds > SP > AM > MP > OB > AN. It appears that AN and AM polymers can be used for applications where Newtonian flow is required.

3.2.11. Determination of molar mass

Three different techniques, including intrinsic viscosity data, size exclusion chromatography and Table 10. Concentration (%) and pH of polymer solutions used for rheological studies at 24 C

Name of sample Concentration(%) pH of polymer Viscosity of 1% solutions solution at shear rate 10 s-1 SP 1.15 7.04 0.017

AN 25.9 4.41 0.002

MP 1.16 6.63 0.004

PO husk 6.68 0.16 0.86

AM 24.15 4.29 0.005

0.19 LR 5.16 0.079

0.29 OB 6.78 0.003

PO seed 1.7299 6.03 0.075

Fig. 32. Graph of shear rate vs viscosity ultracentrifugation, were employed to determine the molar mass of the polymers under investigation. AN and AM being water soluble were analyzed by all the three techniques, whereas

PO husk and SP could be analyzed only by size exclusion chromatography. The molar mass of LR

(3.65×106 g/mole) is reported in literature [61] whereas other materials under study, being insoluble in common solvents, could not be analyzed.

Calculation of molar mass from rheology data by Mark-Houwick equation involved step-wise calculation using ɳ (from reduced and inherent viscosity plots against concentration (Fig. 33)), K and a parameters (from SEC-MALS data). The results are given in Tables 11 and 12.

The average molar masses of the polymers under investigation ranged from 9.28 × 105 to 3.92 × 106

Daltons (Table 11(b)). The molar masses of AN and AM as determined from rheology data and

SEC-MALS were found to be similar. The mass of AN was comparable with that already reported

[179]. The most abundant high molar masses were 1.31 × 106 (AN) and 1.22 × 106

Daltons (AM). Shapes of AN and AM as determined by viscosity data (Fig. 34 (a) and (b)) by Ellipse

1 software [180] resembled those observed physically. This validates all the viscosity measurements.

Different fractions of AN and AM having distinct molar masses were fractionated by ultracentrifugation of various concentrations of the polymers. The results are given in Table 11. It can be seen that with dilution fragmentation increases. As the branched chains are usually more vulnerable to hydrolysis these results suggest that the polymers are branched in a complex manner.

The most abundant mass found was 6.2 × 105 Dalton from AN (96%) and AM (97.5%), which is relatively lower than those determined by rheology data and SEC-MALS. This result suggests slight polydispersity of the polymers. GPC analysis showed three polymeric

Fig. 33. Reduced and inherent viscosity plots against concentration of a) AN and b) AM in water at

250C

Table 11(a). Viscosities of AN and AM AN

C gcm-3 [Ƞ]Pa.s Ƞr Ƞsp Ƞsp/c lnȠr lnȠr/c 0.14 0.0076 7.58 6.58 47 2.0255 14.4679 0.07 0.00303 3.024 2.024 28.9143 1.1065 15.8071 0.04 0.00175 1.746 0.746 18.65 0.5576 13.94 0.018 0.00132 1.317 0.317 17.6111 0.2756 15.3111 AM

0.28 0.278 277.44 276.44 987.286 5.6256 20.0914 0.14 0.0326 32.53 31.53 225.214 3.48216 17.4108 0.07 0.00904 9.022 8.022 114.6 2.19967 21.9967 0.04 0.00409 4.082 3.082 77.05 1.40659 58.0412

Table 11(b). Comparative molar masses M (from SEC-MALS)

Most abundantAverage of most K a 1/a M = ([ ]/K) species abundant and high molecular-mass species AN 2.709 10-2 0.462 9.06 × 105 9.28 × 105 1.31 × 106 AM 6.038 10-6 1.059 3.92 × 106 1.20 × 106 1.22 × 106

Table 12. Molar masses of different fractions from ultracentifugation

Conc. Mr(% of all fractions) (% m/v) AN AM 1.00 6.2 × 105 (96), 2.84 × 106 (4) 6.24 × 105 (97.6), 2.84 × 106 (2.4) 0.50 1.30 × 106 (93.7), 3.75 × 106 1.30× 106 (94.1), 3.75 × 106 (2.7), (2.9), 4.82 × 106 (3.4) 4.82× 106 (3.2)

0.25 9.64 × 104 (1.9), 5.72 × 105 9.64 × 104 (1.8), 5.72 × 105 (24), 8.32 (27.1), 8.32 × 105 (17.7), 1.04 × × 105 (16.8), 1.04 × 106 (16.9), 1.37 × 106 (17.9), 1.37 × 106 (17.7), 106 (16.8), 1.74 × 106 (9.3), 2.31 × 106 1.74× 106 (9.8), 2.31× 106 (15.2) (15.1)

a- b-

Fig. 34. Shapes of a) AN and b) AM as determined by Eclipse I (software) components in the isolated fraction (Table 13a) with weight-average molar masses 9.3544 ˣ 106 g mol-1 (fraction 1), 5.0087 ˣ 101 g mol-

1 (fraction 2) and 1.2671 ˣ 103 g mol-1 (fraction 3). This result suggests the presence of three distinct polysaccharides in the water-extracted gel of PO husk as reported earlier [181-183]. The mass of fraction I is in the characteristic range of the arabinoxylan of the hull-less barley [161]. For an ideal monodisperse polymer, the molar mass averages are equal i.e. Mn=Mw=Mz. However, for a polydispersed system the relationship is Mn

GPC analysis of SP (Table 13b) showed four polymeric components in the isolated fraction with weight-average molar masses 7.0496 ˣ 106 g mol-1 (fraction 1), 4.7331 ˣ 101 g mol-1 (fraction 2),

1.0862 ˣ 103 g mol-1 (fraction 3) and 9.1631 ˣ 103 g mol-1 (fraction 4). This result suggests the presence of four distinct polysaccharides in the water-extracted gel of SP. The fraction I, 2, 3 and

4 with PDI values of 1.18, 1.57, 1.13 and 1.28, respectively, appear to be almost monodispersed polymers.

3.2.12. Mechanical strength

Mechanical strength is an important parameter to be determined for film forming polymers due to

their potential application in film coating of tablets and as biocompatible and biodegradable

packaging material for food items. All the polysaccharides under investigation were found to be

capable of forming strong films. The results are given in Table 14 and Fig. 35. These materials

exhibited diverse strengths ranging from 0.47 to 19.68 Nmm-2, which reflects a diversity in their

structures. Three of the materials, LR, PO seeds and husk showed higher

Table 13a. GPC data of the peak of higher molar mass for the PO husk Parameters Fraction 1 Fraction 2 Fraction 3

Mn 7.7959 ˣ 106 3.7394 ˣ 101 1.0928 ˣ 103

Mw 9.3544 ˣ 106 5.0087 ˣ 101 1.2671 ˣ 103

Mz 1.0946 ˣ 107 6.2953 ˣ 101 1.4601 ˣ 103

Mp 9.5387 ˣ 106 4.2629 ˣ 101 1.1509 ˣ 103 Vp 1.5077 ˣ 101 9.7632 8.2798

Table 13b. GPC data of the peak of higher molar mass for the SP

Parameters Fraction 1 Fraction 2 Fraction 3 Fraction 4

Mn 5.9822 ˣ 106 3.0450 ˣ 101 9.6436 ˣ 102 7.1549 ˣ 103

Mw 7.0496 ˣ 106 4.7331 ˣ 101 1.0862 ˣ 103 9.1631 ˣ 103

Mz 8.1507 ˣ 106 6.4473 ˣ 101 1.2107 ˣ 103 1.1470 ˣ 103

Mp 6.9195 ˣ 106 4.2629 ˣ 101 1.1144 ˣ 103 7.2444 ˣ 103

Vp 1.5422 ˣ 101 9.7632 8.2943 7.4518

Table 14. Mechanical strength Tensile Material Thickness(mm) Width(mm) Max.force N strength N/mm2 SP 0.24 15.17 12.58 3.45 AN 0.25 11.78 10.00 3.40 MP 0.10 16.65 6.075 3.66 PO husk 0.07 14.15 12.12 12.24 AM 0.22 12.46 16.38 5.98 LR 0.15 9.15 27.35 19.68 OB 0.15 15.19 0.825 0.47 PO Seeds 0.15 16.18 39.38 16.61

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MP SP AN

PO husk AM LR

OB PO seeds

Fig. 35. Mechanical strength of SP, AN, MP, PO husk, AM, LR, OB and PO seeds. strengths than carboxy methyl cellulose (~11 Nmm-2) [184, 185], gelatine (~6 Nmm-2) [184] and hydroxyl propyl cellulose (~14

Nmm-2) [185] , whereas the value for AM was comparable with that of gelatin. SP, AN and MP exhibited similar moderate strengths. The overall trend was recorded as: LR > PO seeds > PO husk > AM > MP > SP

> AN > OB.

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3.2.13 Swelling index

The swelling index of the polymers ranged from4.32% (AT) to 40.49% (PO husk) table 15. The high swelling characteristics of these materials make them good candidates for fabrication of delivery devices. From these polymers release of drug can be controlled by the water content and pore size. For rapid drug release high water content and large pore size may be used [186].

3.2.14. Water retention

The results (Table 15) indicate that MP possesses the greatest capacity to hold water with an average retention of 79% whereas in case of other materials the water retention value was in the range 4

- 48.8%. SP and AM dissolved in water, so their values could not be determined reproducibly. Thus

MP, PO and OB having very high to moderately high values can be considered as suitable materials for formulation of ophthalmic solutions or suspensions.

3.3 Evaluation of polysaccharides as drug carriers

The polymers under investigation were evaluated for their potential as drug carriers. In this regard the drug-loaded polymer films and synthetic matrix-tablets were subjected to electron microscopy,

ToF-SIMS and dissolution studies, the results are discussed as follows.

Table 15. Swelling index and water retention value Swelling WRV(%) Sample code Index(%) SP Dissolved Dissolved AN 14.4 14.88 MP 37.5 79.45 PO husk 40.49 48.83 AM Dissolved Dissolved

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LR 16.5 29.5 OB 30.33 30.44 PO seeds 17.20 41.35 TG 4.32 4.32

3.3.1. Electron microscopy

SEM is a useful technique to study drug loading. SEM images (Fig. 36 - 44) shows that polysaccharides under study had voids and layered structures, therefore are suitable for encapsulation of drugs molecules. SEM images provided clear evidence of the presence of drug substances in the polymer matrices.

103

3.3.2. ToF-SIMS

ToF-SIMS is a powerful technique for surface analysis with the potential of depth profiling [187] and mapping of encapsulated substances in polymeric materials. In this work this technique has been successfully employed to study drug loading and distribution in the polymers under investigation. This technique provided important information on molecular specificity with good sensitivity and lateral resolution [188-192].

Some of these polymers have already been studied for formulation of sustained release tablets of some drug molecules [193-195] but these studies lack the verification of uniformity of content therein, which is an essential requirement for mass production of a pharmaceutical product.

ToFSIMS spectra and images were obtained in respect of caffeine and diclofenac loaded polymers under investigation. The spectra of the drugs, polymers (blank) and drug-loaded polymers are

+ + + shown in Fig. 46. The m/z peaks at 22.9932, 39.0225 and 195.09 due to Na ,C3H3 and C8H11O2N4

, respectively were considered as signatures of caffeine. Similarly, the peaks at 22.9932, 39.0225

+ + + and 339.92 due to Na , C3H3 and C14H10Cl2NO2Na2 respectively (Fig. 45) were considered as signatures of diclofenac sodium. The results showed a uniform dispersion of

104

a- b - c - a-

b - c - a-

b - c-

Fig. 36. SEM images of OB a) without drug, b) with CAF and c with DS

Fig. 37. SEM images of LR a) without drug, b) with CAF and c with DS

Fig. 38. SEM images of PO seeds a) without drug, b) with CAF and c with DS

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a- b- c - a-

b - c -

Fig. 39. SEM images of SP a) without drug, b) with CAF and c with DS

Fig. 40. SEM images of AN a) without drug, b) with CAF and c with DS

a- b - c -

Fig. 41. SEM images of MP a) without drug, b) with CAF and c with DS

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a- b - c - a-

b - c- a-

b - c-

Fig. 42. SEM images of AM a) without drug, b) with CAF and c with DS

Fig. 43. SEM images of PO husk a) without drug b) with CAF c with DS

Fig. 44. SEM images of AT a) without drug b) with CAF c with DS

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Fig. 45. Mass spectra (TOF-SIMS) of diclofenac sodium and caffeine

caffeine particles in the polymer matrix of SP, AN, AM, MP, PO seeds and husk (Fig. 46), while the dispersion was relatively less uniform in LR and OB. In SP and AM diclofenac sodium dispersed

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more uniformly than others. On the other hand the pattern of drug uptake was different in all the polymers (Fig. 47.). This appears to depend upon solubility and hydrophilicity of the drug molecules, and the polymer‟s structure. n the present list the best uptake was shown by OB for both the drugs.

3.3.3. Dissolution study

It can be seen that there is no derth of drug substances available in the market. A number of drug molecules are available for a treatment. The research in discovery of new molecules has reached a level of saturation in some indications. There are drugs with excellent efficacy but these are generally associated with several adverse effects. It is therefore, now being felt that it is more desirable to have drugs with lower toxicity profiles. One way to reduce the toxic side effects is to protect the body from over exposure of drugs by way of targeted delivery or controlled release.

Therefore, the focus is now shifting from synthesis of new drug entities to the targeted or controlled delivery of existing drug substances.

Currently, most of the targeted or controlled drug delivery systems involves the use of synthetic polymers as the matrix for drug release. Synthetic polymers release toxic degradation products in vivo. So, the synthetic polymers are being discouraged for their use in drug delivery. The best alternative being looked into is the potential use of natural polymeric materials for these applications. Preliminary studies on the natural polymers which are carbohydrate polymers obtained from plant material, under investigation suggested them to be good candidate for drug

109

110

Fig. 46. a) TOF-SIMS of caffeine loaded samples

111

112

Fig. 46. b) TOF-SIMS of diclofenac sodium loaded samples

113

Fig. 47. Uptake of caffeine by different materials

Fig. 48. Uptake of diclofenac sodium by different materials encapsulation and delivery [195].

In the present work two different formulations (drug-load films and matrix tablets) were prepared and their release was studied by the USP dissolution methods. The drug-loaded polymers would swell when in contact with body fluids and deliver the encapsulated drug in a controlled manner.

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The release mechanism depends on the polymer structure and nature of drug molecule. In order to study the kinetics and mechanism of release different models were applied. Drug release was studied from the drug-loaded films and direct-compressed tablets containing a polymer as an adjuvant. The results are discussed as follows.

3.3.3.1. Release profile of diclofenac sodium loaded polymer films in phosphate buffer

Mathematical models describe the release of drug as a function of time. A number of models have been put forward to explain the release mechanism of the drugs from swellable systems. However, none of the methods is successful enough to explain the release mechanism from all types of systems. In the present study release profile of diclofenac sodium drug from prepared films of SP,

AN, MP, PO husk, AM, LR, OB and PO seeds was studied. The release profiles of diclofenac sodium-loaded polymer films are shown in Fig. 49. and Table 16. As compared with the solubility curve of naked drugs the polymers produced sustained release up to about 30 h. Drug release study of these polymers were carried out in phosphate buffer of pH 6.8. Release data was fitted into

1/2 equations: M=k0 t (Zero order equation), lnM = k1 t (First order equation), M = kH t (Higuchi

1/3 1/3 equation), Mo – Mt = kHC t (Hixon-Crowell cube root law) and lnMt /M = Inkp + nInt (Power law equation). The R2value, MSC and n values for different models are recorded in

Table 16. For diclofenac sodium films MP (Fig. 53), OB (Fig. 55) and PO husk (Fig. 56) followed Higuchi model, AM (Fig.51), AN (Fig.50) and SP (Fig.57) followed Power law, LR (Fig.54) followed first order and

PO seeds (Fig.52) followed zero order. As far as release mechanisms are concerned, AM, AN, MP, LR, OB,

SP showed non-Fickian, i. e., diffusion and swelling controlled and PO seeds and husk exhibited complex mechanism involving diffusion, swelling and erosion,

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The suggested mechanism was based on the n value according to the criteria: 0.45 (Fickian), 0.45

< n < 0.89 (non-Fickian) and n > 0.89 (super case-II). Thus these polymers appear to be suitable for formulation of various types of ophthalmic solutions or suspensions.

3.3.3.2 Release profile of diclofenac sodium loaded polymer films in 0.1 N HCl

The films of diclofenac sodium drug prepared from SP, AN, MP, PO husk, AM, LR, OB and PO seeds polysaccharides showed no release in 0.1 N HCl. The Absorbance spectra of DS-polymer films in 0.1M HCl are shown in Fig. 58.

3.3.3.3 Release profile of caffeine loaded polymer films in distilled water

The release profiles of caffeine-loaded polymer films are shown in Fig. 59. As compared with the solubility curve of naked drugs the polymers produced sustained release for about 30 h. The release data of all the polymers (SP (Fig. 66), AN (Fig. 60), MP (Fig. 62), PO husk (Fig. 65), AM (Fig. 61),

LR (Fig. 63),OB (Fig. 64) and PO seeds (Fig. 67)) for caffeine fitted well in Higuchi model followed by Power law (Table 16). Best linearity was found in Higuchi equation for all the polymers (Table

17) indicating the release of drug from matrix as a square root of time dependent process. However, first order model gave best fit release model for MP (Fig. 62, Table 17). As far as release mechanisms are concerned, they were: non-Fickian, i. e., diffusion and swelling controlled (AM,

AN, MP, PO husk, SP for caffeine) and complex mechanism involving diffusion, swelling and erosion (LR, OB for caffeine). Whereas PO seeds exhibited nearly Fickian, i. e., only diffusion controlled mechanism for caffeine. The suggested mechanism was based on the n value according to the criteria: 0.45 (Fickian), 0.45 < n < 0.89 (non-Fickian) and n > 0.89 (super caseII). Thus these

116

polymers also appear to be suitable for formulation of various types of ophthalmic solutions or suspensions.

117

min

Fig. 49. Release profiles of the polymer films in phosphate buffer pH 6.8 (DS)

118

Table 16. Fitness of release data of diclofenac sodium in phosphate buffer from different films to various mathematical models Material/Model AM AN PO seeds MP LR OB PO husk SP

Zero order R2 0.733 0.761 0.783 0.899 0.623 0.92 0.852 0.860 MSC 1.014 1.125 3.444 1.985 0.6689 2.126 1.605 1.659 First order R2 0.909 0.918 0.858 0.976 0.818 0.953 0.949 0.961 MSC 2.093 2.204 1.553 3.429 2.563 2.674 2.682 2.943 Higuchi R2 0.908 0.930 0.882 0.993 0.824 0.980 0.973 0.980 MSC 2.083 0.396 1.739 4.666 1.432 3.547 5.017 3.640 Power law R2 0.905 0.954 0.860 0.955 0.884 0.959 0.995 0.987 MSC 2.329 2.790 1.567 2.809 1.849 2.798 4.989 4.105 n 0.621 0.484 0.381 0.630 0.494 0.451 0.259 0.472 Hixon R2 0.733 0.761 0.783 0.899 0.623 0.92 0.852 0.860 crowell MSC 1.014 1.125 1.128 1.985 0.669 2.126 1.605 1.389 % release 360 360 300 920 180 420 420 720 (t min50)

102

0.5 Power law 0.0003 Zero order 0 0.00025 -0.5 0 5 10 0.0002 -1 0.00015 y = 1E -07 x + 8E- 05 R² = 0.7613 -1.5 0.0001 -2 0.00005 y = 0.4848x - 3.628 -2.5 R² = 0.9548 0 0 500 1000 1500 2000 -3 In t t ( min )

100 0 80 -2 0 1000 2000 -4 60 y = -0.0008 x - 8.3421 -6 40 R² = 0.9188 -8 y = 1.8598x + 9.0628 20 R² = 0.9305 -10 -12 0 t 0 50First order Higuchi model t 1/2 0.0001 Hixson - crowell model 0.00008 0.00006 0.00004 y = 4E - 08 x + 3E -05 0.00002 R² = 0.7613 0 0 500 1000 1500 2000 t

Fig. 50. Typical model fitting plots for AN-diclofenac sodium film in phosphate buffer of (pH 6.8) at 37±0.1°C

121

Zero order 1 Power law 0.0003 0.00025 0 0 5 10 0.0002 -1 0.00015 -2 0.0001 y = 1E- 07 x + 7E - 05 y = 0.6214x - 4.4896 0.00005 -3 R² = 0.7333 R² = 0.9054 0 -4 In t 0 1000 2000 t (min)

100 0 80 -2 0 1000 2000 -4 60 -6 y = - 0.0008 x - 8.3125 40 R² = 0.9094 -8 y = 1.9495x + 5.8645 20 -10 R² = 0.9084 -12 0 0 20 40 60First order Higuchi model t t 1/2 0.00012 Hixson - crowell model 0.0001 0.00008 y = - 4 E -08 x + 8E - 05 0.00006 R² = 0.7333 0.00004 0.00002 0 0 1000 2000 t

Fig. 51. Typical model fitting plots for AM-diclofenac sodium film in phosphate buffer of (pH

6.8) at 37±0.1°C Power law Zero order

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0 0.0002 0 5 10 -0.5 0.00015

-1 0.0001 -1.5 0.00005 y = 3E -07 x + 8E - 05 y = 0.3811x - 2.8107 R² = 0.7831 -2 R² = 0.8601 0 -2.5 0 200 400 600 In t t (min) First order 70 Higuchi model -8 60 0 200 400 600 50 -8.2 40 -8.4 y = - 0.0014 x - 8.3457 30 R² = 0.8581 20 y = 2.1602x + 13.242 -8.6 R² = 0.8822 10 -8.8 0 -9 0 20 40

t t 1/2

Hixson-crowell model 0.00007 0.00006 0.00005 0.00004 0.00003 y = 9E -08 x + 3E - 05 0.00002 R² = 0.7831 0.00001 0 0 500 t

Fig. 52. Typical model fitting plots for PO seeds-diclofenac sodium film in phosphate buffer of (pH

6.8) at 37±0.1°C

Power law 0.0003 Zero order

123

0 0 5 10 0.00025 -1 0.0002 0.00015 -2 0.0001 y = 1E- 07 x + 4E - 05 -3 0.00005 R² = 0.899 y = 0.6309x - 4.869 0 -4 R² = 0.9557 In t 0 1000 2000 t ( min)

First order 80 Higuchi model -8 70 0 1000 2000 60 -8.2 50 -8.4 40 -8.6 30 -8.8 20 y = 1.7288x - 0.9928 R² = 0.9931 -9 10 y = - 0.0006 x - 8.1865 0 -9.2 R² = 0.9762 0 20 40 60 -9.4

t t 1/2

Hixson-crowell model 0.0001 0.00008 0.00006 0.00004 y = 4E -08 x + 1E - 05 0.00002 R² = 0.899 0 0 1000 2000 t

Fig. 53. Typical model fitting plots for MP-diclofenac sodium film in phosphate buffer of (pH

6.8) at 37±0.1°C

124

1 0.0003 Zero order Power law 0.00025 0 0.0002 0 5 10 0.00015 -1 0.0001 y = 1E - 07 x + 1E -04 R² = 0.6234 -2 0.00005 y = 0.4948x - 3.6037 0 R² = 0.8843 -3 0 1000 2000 In t t (min)

First order 100 Higuchi model 0 80 -2 0 1000 2000 -4 60 -6 y = - 0.0007 x - 8.4353 40 y = 1.7743x + 14.203 R² = 0.8186 -8 20 R² = 0.8244 -10 0 -12 0 20 40 60 t 1 / 2 t

0.0001 Hixson-crowell model

0.00008 0.00006

0.00004 y = 3E - 08 x + 3E -05 R² = 0.6234 0.00002 0 0 1000 2000 t

Fig. 54. Typical model fitting plots for LR-diclofenac sodium film in phosphate buffer of (pH 6.8) at 37±0.1°C

125

Power law 0 -0.5 0 5 10 -1 -1.5

-2 y = 0.4515x - 3.4728 -2.5 R² = 0.9592 -3 In t 0.0002 Zero order

0.00015

0.0001

y = 3E -07 x + 5E -05 0.00005 R² = 0.92 0 0 200 400 600 t ( min ) 50 Higuchi model First order -8.1 40 -8.2 0 200 400 600 30 -8.3 -8.4 20 y = 2.041x + 4.3795 -8.5 R² = 0.9807 10 -8.6 -8.7 y = -0.0011 x - 8.2183 0 R² = 0.9538 1 / 2 -8.8 0 10 t 20 30 t

126

0.00006 Hixson - crowell model 0.00005 0.00004 0.00003 0.00002 y = 8E - 08 x + 2E -05 0.00001 R² = 0.92 0 0 200 400 600 t

Fig. 55. Typical model fitting plots for OB-diclofenac sodium film in phosphate buffer of (pH

6.8) at 37±0.1°C 0.0003 0 0 5 10 0.00025 -0.5 0.0002 0.00015 -1 0.0001 y = 8E -08 x + 0.0001 R² = 0.8522 0.00005 -1.5 y = 0.2598x - 2.2489 0 R² = 0.995 -2 0 1000 2000 Power law Zero order In t t (min)

100 Higuchi model First order -8.2 80 -8.4 0 1000 2000 60 -8.6 40 -8.8 y = 1.3345x + 20.843 R² = 0.9732 -9 20 -9.2 y = -0.0006 x - 8.4372 -9.4 0 R² = 0.9497 -9.6 0 20 40 60

t t 1/2

Hixson-crowell model

127

0.0001 0.00008 0.00006 0.00004 y = 3E - 08 x + 3E -05 0.00002 R² = 0.8522 0 0 1000 2000 t

Fig. 56. Typical model fitting plots for PO husk-diclofenac sodium film in phosphate buffer of

(pH 6.8) at 37±0.1°C

0 0.0003 -0.5 0 5 10 0.00025 -1 0.0002 -1.5 0.00015 0.0001 y = 1E- 07 x + 6E - 05 -2 y = 0.4723x - 3.7275 0.00005 R² = 0.8601 -2.5 R² = 0.9879 0 -3 Power law Zero order

100 Higuchi model 80 First order -8 60 0 1000 2000 -8.5 40 In t y = 1.7172x + 5.093 0 20 -9 R² = 0.9807 1000 y = -0.0007 x - 8.262 0 2000 -9.5 R² = 0.9613 t (min) -10

t 0 20 t 1/2 40 60

128

Hixson-crowell model 0.0001 0.00008 0.00006

0.00004 y = 4E -08 x + 2E - 05 R² = 0.8601 0.00002 0 0 1000 2000 t

Fig. 57. Typical model fitting plots for SP-diclofenac sodium film in phosphate buffer of (pH 6.8) at 37±0.1°C

Fig. 58. Release profiles of DS-polymer films in 0.1M HCl

129

Fig. 59. Release profiles of the polymer films in distilled water (caffeine)

130

Table 17. Fitness of release data of caffeine in distilled water from different films to various mathematical models Material/Model AM AN PO seeds MP LR OB PO husk SP

Zero order R2 0.950 0.945 0.931 0.954 0.982 0.958 0.903 0.970 MSC 2.695 2.599 1.362 2.771 3.712 3.347 2.032 3.221 First order R2 0.989 0.988 0.983 0.986 0.942 0.957 0.975 0.970 MSC 4.229 4.18 3.780 3.972 3.558 2.853 3.406 3.222 Higuchi R2 0.996 0.997 0.993 0.985 0.984 0.991 0.982 0.992 MSC 6.136 5.677 4.658 3.894 3.836 4.435 4.790 4.599 Power law R2 0.997 0.995 0.991 0.974 0.964 0.988 0.979 0.983 MSC 6.111 5.188 4.418 3.343 3.030 4.186 3.560 3.819 n 0.496 0.550 0.425 0.530 0.363 0.372 0.482 0.694 Hixon R2 0.950 0.945 0.931 0.954 0.982 0.958 0.903 0.970 crowell MSC 2.694 2.599 2.374 2.771 3.712 2.869 2.032 3.221 % release (t min50) 920 1080 920 1120 680 680 720 740

112

Power law 0.0005 Zero order 0 -0.5 0 5 10 0.0004 -1 0.0003 -1.5 0.0002 -2 y = 2E -07 x + 7E- 05 -2.5 y = 0.5503x - 4.3922 0.0001 R² = 0.9454 R² = 0.9959 -3 0 -3.5 0 1000 2000 In t t ( min )

First order 80 Higuchi model -7.4 70 -7.6 0 1000 2000 60 -7.8 50 -8 40 -8.2 30 -8.4 20 y = 1.7713x - 1.6602 R² = 0.9975 -8.6 10 y = - 0.0007 x - 7.5738 -8.8 R² = 0.9889 0 -9 0 20 40 60 t t 1 / 2

0.00016 Hixson - crowell model 0.00014 0.00012 0.0001 0.00008 0.00006 0.00004 y = 7E -08 x + 2E - 05 0.00002 R² = 0.9454 0 0 1000 2000

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0 0

Fig. 60. Typical model fitting plots for AN-caffeine film in distilled water at 37±0.1°C. Power law Zero order

0.0005 -0.5 5 10 0.0004 -1 0.0003 -1.5 0.0002 y = 2E -07 x + 8E- 05 -2 0.0001 R² = 0.9503 y = 0.4967x - 4.1071 -2.5 0 R² = 0.9978 -3 0 1000 2000 In t t (min)

First order Higuchi model -7.4 80 -7.6 0 1000 2000 70 -7.8 60 50 -8 40 -8.2 30 y = 1.611x + 0.0316 -8.4 20 10 R² = 0.9966 y = -0.0006 x - 7.5873 -8.6 0 R² = 0.9893 -8.8 0 20 40 60 t t 1/2

Hixson-crowell model 0.00016

134

Power law

0.00014 y = 6E - 08 x + 3E -05 0.00012 R² = 0.9503 0.0001 0.00008 0.00006 0.00004 0.00002 0 0 1000 2000 t

Fig. 61. Typical model fitting plots for AM-caffeine film in distilled water at 37±0.1°C Zero order 0 0.0005 -0.5 0 5 y = 2E -07 x + 7E- 05 0.0004 R² = 0.954 -1 -1.5 0.0003 -2 0.0002 -2.5 0.0001 -3 y = 0.5306x - 4.3435 -3.5 R² = 0.974 0 In t 0 1000 2000 t (min)

First order Higuchi model

135

0 0

-7.4 80 -7.6 0 1000 2000 60 -7.8 -8 40 -8.2 20 -8.4 y = 1.6202x - 0.975 y = -0.0006 x - 7.5726 -8.6 R² = 0.985 R² = 0.9861 0 -8.8 0 20 40 60 t t 1/2

Hixson-crowell model

y = 6E -08 x + 2E -05 R² = 0.954

0.00016 0.00014 0.00012 0.0001 0.00008 0.00006 0.00004 0.00002 0 0 1000 2000 t

Fig. 62. Typical model fitting plots for MP-caffeine film in distilled water at 37±0.1°C

136

Power law

0 0 10 Zero order 0.0006 y = 2E- 07 x + 0.0001 0.0005 -0.5 5 R² = 0.982 0.0004 -1 0.0003 -1.5 0.0002 0.0001 -2 y = 0.3631x - 2.9703 R² = 0.9645 0 -2.5 In t 0 1000 2000 t

First order Higuchi model 0 100 0 1000 2000 -2 80 -4 y = - 0.0013 x - 7.5694 60 -6 R² = 0.9426 40 -8 y = 1.9667x + 5.5415 20 -10 R² = 0.9841 0 -12 t 0 20 40 60 t 1 / 2

Hixson-crowell model 0.0006 0.0005 0.0004 0.0003 0.0002 0.0001 y = 8E - 08 x + 0.0004 R² = 0.982 0 0 1000 2000

137

0 0 10 t

Fig. 63: Typical model fitting plots for LR -caffeine film in distilled water at 37±0.1°C

0 0.0006 0 5 0.0005 -0.5 0.0004 -1 0.0003 -1.5 0.0002 y = 0.3723x - 2.9932 y = 2E -07 x + 0.0001 -2 R² = 0.9888 0.0001 R² = 0.9583 -2.5 0 0 1000 2000 In t Zero order

t (min)

138

Power law

10 Higuchi model 0 100 -2 0 1000 2000 80 -4 60 y = -0.0012 x - 7.6227 -6 40 R² = 0.9576 -8 y = 1.9069x + 7.4657 20 R² = 0.9913 -10 0 -12 t 0 20 40 60 1 / 2 t First order

Hixson-crowell model 0.0002

0.00015

0.0001

0.00005 y = 8E -08 x + 4E -05 R² = 0.9583 0 0 1000 2000

139

0 0 10 Fig. 64. Typical model fitting plots for OB-caffeine film in distilled water at 37±0.1°C Power law Zero order 0.0006 -0.5 5 0.0005 -1 0.0004 -1.5 0.0003 0.0002 -2 y = 2E - 07 x + 0.0001 0.0001 R² = 0.9036 -2.5 y = 0.4824x - 3.7626 R² = 0.9791 0 -3 0 1000 2000 In t t (min)

First order Higuchi model 0 100 0 1000 2000 -2 80 60 -4 40 -6 y = - 0.0009 x - 7.6327 R² = 0.9756 20 y = 1.8594x + 3.6615 -8 0 R² = 0.9821 -10 0 20 40 60 t 1/2 t 1/2

0.0002

0.00015

0.0001

y = 7E - 08 x + 4E -05 0.00005 R² = 0.9036

0 0 500 1000 1500 2000

140

Power law

10 t

Fig. 65. Typical model fitting plots for PO husk-caffeine film in distilled water at 37±0.1°C

141

0 0.0006 y = 3E - 07 x + 6E -05 0 2 4 6 8 0.0005 R² = 0.9707 -1 y = 0.6946x - 5.2334 0.0004 R² = 0.9839 0.0003 -2 0.0002 -3 0.0001 0 -4 0 1000 2000 In t

t ( min) 0 0 500 1000 1500 2000 100 -2 y = 2.2186x - 7.9351 80 -4 R² = 0.9926 60 -6 y = -0.0012 x - 7.4694 R² = 0.9707 40 -8 20 -10 0 -12 t 1 / 2 0 20 40 60 1 / 2 t

0.0002 y = 9E - 08 x + 2E -05 0.00015 R² = 0.9707

0.0001

0.00005

0 0 500 1000 1500 2000 t

Fig. 66. Typical model fitting plots for SP-caffeine film in distilled water at 37±0.1°C Power law Zero order

142

0 0.0005 y = 2E - 07 x + 0.0001 0 5 10 -0.5 0.0004 R² = 0.9315 y = 0.425x - 3.5103 -1 R² = 0.9914 0.0003 -1.5 0.0002 -2 0.0001 0 1000 2000 -2.5 0 -3 In t t (min)

First order Higuchi model

-7.4 80 y = 1.6272x + 4.6697 -7.6 0 1000 2000 R² = 0.993 -7.8 -8 -8.2 -8.4 70 -8.6y = - 0.0007 x - 7.6402 -8.8 R² = 0.9832 60 -9 50 0 20 40 60 t t 1 / 2 40 30 20 10 0

Hixson- crowell model 0.00016

143

y = 6E -08 x + 3E -05 R² = 0.9315

0.00014 0.00012 0.0001 0 1000 2000 0.00008 0.00006 0.00004 0.00002 0

Fig. 67. Typical model fitting plots for PO seeds-caffeine film in distilled water at 37±0.1°C

3.3.3.4. Release profile of diclofenac sodium loaded polymer tablets in phosphate buffer The release profiles of the prepared tablets are shown in Fig.68. It can be seen that all the polymers are imparting a sustained release effect. The materials exhibited following trend in sustained release:

MP > OB ≈ SP > PO husk > PO seeds ≈ AN > AM > LR > Control (for diclofenac sodium). MP exhibited the best sustained release for diclofenac sodium. Generally the data fitted well (R2: 0.838

– 0.998, MSC: 1.24 – 8.343) in all the release models (Table 19, Fig. 69-78). PO seeds (Fig. 72),

MP (Fig. 69), OB (Fig. 74) and AN (Fig. 70) exhibited best fit to first order and SP (Fig.76) to

Higuchi model. A good fit to the first order model and Higuchi equation showed that the drug release decreases slowly with time (Table 19). The best fit models were selected by MSC analysis.

Zero order model showed a good fit for LR and AT which means the release was constant over the

144

time. AM and PO husk exhibited power law. The release data of diclofenac sodium fitted well in power law and first order. The power law also showed a good fit to the data. A cursory view of the data indicates that all the materials under investigation exhibit zero-order release kinetics for at least first 120 min. The values of n for diclofenac sodium from the power law (Table 19) suggest non-Fickian release mechanism by diffusion and swelling for

AN, PO seeds, LR, OB, MP, SP and AM exhibit time-independent, super case-II tansport (Table 19) release mechanisms. In case of tablets prepared from diclofenac sodium + PO husk , the n values were significantly less than 0.45, which indicates that the release occurs through a complex mechanism where other factors like erosion in addition to diffusion and swelling are playing a role.

3.3.3.5 Release profile of diclofenac sodium loaded polymer tablets in 0.1 N HCl

It is desirable for diclofenac sodium not to be released in stomach rather to be delivered in the intestine, therefore, the release of diclofenac sodium was studied at pH 6.8 and in 0.1M HCl. The results show that its release is sustained in phosphate buffer pH 6.8 and absorbance spectra of

DSpolymer tablets show negligible release in 0.1M HCl as it is insoluble in acidic medium (Fig.79).

3.3.3.6 Release profile of caffeine loaded polymer tablets in distilled water

The release profiles of the prepared tablets are shown in Fig. 80. It can be seen that all the polymers are imparting a sustained release effect. Caffeine solubility is pH independent so its release was studied only in distilled water.

145

The materials exhibited following trend in sustained release: MP > AM ≈ SP > AN > PO seeds >

PO husk ≈ LR > OB > Control (for caffeine). MP exhibited the best sustained release for caffeine.

Generally the data fitted well (R2: 0.817 – 0.993, MSC: 1.24 – 8.576) in all the release models

(Table 19, Fig. 80). In caffeine the data fitted well in power law (AN, LR and OB), first order (AM,

PO husk and MP) and Higuchi (PO seeds and SP) models (Fig. 81-90). A cursory view of the data indicates that all the materials under investigation exhibit zero-order release kinetics for at least first 120 min. The values of n for caffeine non-Fickian (AM, AN, MP, PO husk and SP) and Fickian

(PO seeds) mechanisms were exhibited. In case of tablets prepared from caffeine + LR and caffeine

+ OB the n values were significantly less than 0.45, which indicates that the release occurs through a complex mechanism where other factors like erosion in addition to diffusion and swelling are playing a role.

146

Fig. 68. Release profiles of tablets in phosphate buffer pH 6.8 (DS)

147

Table 19: Fitness of release data of diclofenac sodium in phosphate buffer from different material tablets to various mathematical models Material/Model AM AN PO seeds MP LR OB PO husk SP AT Control

Zero order R2 0.954 0.962 0.963 0.912 0.998 0.901 0.838 0.943 0.959 0.925 MSC 2.525 2.605 2.303 1.869 5.787 1.817 1.020 3.724 2.506 1.925 First order R2 0.986 0.993 0.998 0.955 0.968 0.975 0.999 0.971 0.935 0.978 MSC 3.698 4.432 5.350 2.536 2.654 3.190 1.459 3.046 2.082 3.152 Higuchi R2 0.987 0.989 0.991 0.898 0.979 0.966 0.992 0.988 0.926 0.963 MSC 3.782 3.908 3.735 1.967 3.080 2.884 1.778 3.958 1.329 2.645 Power law R2 0.992 0.992 0.980 0.954 0.997 0.946 0.964 0.978 0.845 0.964 MSC 4.030 4.167 2.937 2.329 5.222 2.433 2.526 3.357 1.370 2.671 n 0.916 0.673 0.842 0.570 0.874 0.471 0.238 0.457 0.050 0.813 Hixon R2 0.954 0.962 0.963 0.898 0.998 0.901 0.838 0.943 0.959 0.924 crowell MSC 2.525 2.605 2.303 1.967 5.787 1.817 1.020 2.378 2.506 1.922 % release (t min50) 115 102 35 140 75 60 20 110 <15 70

124

Power law Zero order y = 8E - 07 x + 3E - 05 R² = 0.912

0 0.00025 0 100 200 300

First order Higuchi model 0 2 4 6 0.0002 -0.5 0.00015 -1 y = 0.5704x - 3.3225 0.0001 R² = 0.9542 0.00005 -1.5 0 -2 ln t t (min)

-8 100 0 100 200 300 80 -8.5 60 40 -9 y = 0.252x + 20.48 20 R² = 0.898 y = - 0.005 x - 8.221 -9.5 R² = 0.955 0 0 200 400 1 / 2 -10 t t

Hixson - crowell model

150

0.0001 0.00008 0.00006 0.00004 y = 3E - 07 x + 2E - 05 0.00002 R² = 0.898 0 0 200 400 t (min)

Fig. 69. Typical model fitting plots for MP-diclofenac sodium tablets in phosphate buffer of (pH

6.8) at 37±0.1°C

0 Power law Zero order 0 2 4 6 0.0003 y = 1E - 06 x + 4E -05 -0.5 y = 0.673x - 3.707 0.00025 R² = 0.9621 R² = 0.992 0.0002 -1 0.00015 -1.5 0.0001 0.00005 -2 0 In t 0 100 200 t (min) First order Higuchi model -8 100 -8.2 0 100 200 y = 6.7674x - 12.203 -8.4 80 R² = 0.9897 -8.6 -8.8 60 -9 -9.2 40 -9.4 20 -9.6 y = -0.008 x - 8.1056 -9.8 R² = 0.9939 0 t 1 / 2 0 5 t 10 15

151

Hixson crowell model 0.00009 0.00008 0.00007 0.00006 0.00005 0.00004 0.00003 0.00002 y = 4E - 07 x + 1E -05 0.00001 R² = 0.9621 0 0 100 200 t (min)

Fig. 70. Typical model fitting plots for AN-diclofenac sodium tablets in phosphate buffer of (pH

6.8) at 37±0.1°C

0.5 Power law 0.00035 Zero order 0 0.0003 y = 1E -06 x + 2E- 05 0 5 10 R² = 0.9548 -0.5 0.00025 -1 0.0002 -1.5 0.00015 -2 0.0001 y = 0.916x - 4.9856 -2.5 0.00005 R² = 0.9824 -3 0 In t 0 100 200 300 t (min) First order Higuchi model 0 100 -2 0 1 00 200 300 y = 7.3803x - 23.241 80 R² = 0.9871 -4 60 -6 y = -0.009 x - 7.9635 40 -8 R² = 0.986 20 -10 0 -12 t 0 10 20 t 1 / 2

Hixson crowell model

152

0.00012 0.0001 0.00008 0.00006 0.00004 y = 4E -07 x + 8E- 06 0.00002 R² = 0.9548 0 0 200 400 t (min)

Fig. 71. Typical model fitting plots for AM-diclofenac sodium tablets in phosphate buffer of (pH

6.8) at 37±0.1°C

Power law Zero order 0 0.0003 0 5 0.00025 -0.5 0.0002 -1 0.00015 0.0001 y = 3E- 06 x + 3E - 05 0.00005 -1.5 R² = 0.9632 y = 0.8425x - 3.6978 0 -2 R² = 0.9805 0 In t 50 100 t (min)

153

First order 80 Higuchi model -8.2 y = 12.981x - 25.619 -8.4 0 50 100 60 R² = 0.9912 -8.6 40 -8.8 -9 y = - 0.0233 x - 7.9958 20 R² = 0.9983 -9.2 0 -9.4 0 5 10

-9.6 t t 1/2

Hixson crowell model 0.0001 0.00008 0.00006 0.00004 y = 1E -06 x + 1E - 05 0.00002 R² = 0.9632 0 0 50 100 t (min)

Fig. 72. Typical model fitting plots for PO seeds-diclofenac sodium tablets in phosphate buffer of

(pH 6.8) at 37±0.1°C

Power law

0 2 4 6 -0.5 0.0002

-1 y = 2E - 06 x + 1E -05 0.0001 -1.5 R² = 0.9986 y = 0.8748x - 4.39 -2 0 R² = 0.9976 0 100 200 -2.5 In t t ( min)

0 0.0003 Zero order

154

First order Higuchi model 0 100 -2 0 50 100 150 80 y = 9.9342x - 29.279 R² = 0.9794 -4 60 y = - 0.0159 x - 7.8457 -6 R² = 0.9684 40 -8 20 -10 0 -12 t 0 5 10 15 1 / 2 t

Hixson-crowell model 0.0001 y = 7E -07 x + 4E - 06 0.00008 R² = 0.9986 0.00006 0.00004 0.00002 0 0 50 100 150 t (min)

Fig.73. Typical model fitting plots for LR-diclofenac sodium tablets in phosphate buffer of (pH 6.8) at 37±0.1°C

Power law 0.0003 Zero order 0 0.00025 0 5 10 -0.5 0.0002 0.00015 -1 0.0001 y = 6E -07 x + 8E- 05 R² = 0.9015 -1.5 y = 0.4711x - 2.853 7 0.00005 R² = 0.9467 0 -2 0 200 400 In t t (min)

155

First order -8 100 Higuchi Model 0 200 400 80 -8.5 60 -9 40 y = 4.2878x + 7.1741 -9.5 20 R² = 0.9661 y = -0.0045 x - 8.3101 R² = 0.975 0 -10 t 0 10 20 t (min) 0.0001 Hixson- crowell model 0.00008 0.00006 0.00004 y = 2E- 07 x + 3E -05 0.00002 R² = 0.9015 0 0 200 400

t (min)

Fig.74. Typical model fitting plots for OB-diclofenac sodium tablets in phosphate buffer of (pH 6.8) at 37±0.1°C

Power law 0 0.0003 Zero order 0 2 4 6 0.00025 -0.2 0.0002 y = 0.2382x - 1.3697 0.00015 -0.4 R² = 0.9641 0.0001 y = 8E- 07 x + 0.0002 0.00005 -0.6 R² = 0.838 0 -0.8 0 50 100 150 In t t ( min)

156

First order 100 Higuchi model -8.6 -8.8 0 50 100 150 80 -9 60 -9.2 40 -9.4 y = 4.1816x + 34.034 20 R² = 0.9241 -9.6 y = - 0.0076 x - 8.6926 0 -9.8 R² = 0.8955 0 5 10 15 t t 1/2 0.0001 Hixson- crowell model 0.00008 0.00006 0.00004 y = 3E -07 x + 5E -05 0.00002 R² = 0.838 0 0 50 100 150 t (min)

Fig.75. Typical model fitting plots for PO husk-diclofenac sodium tablets in phosphate buffer of

(pH 6.8) at 37±0.1°C

Power law 0.0004 Zero order

0 2 4 6 0.0003 -0.5 y = 0.4571x - 2.698 0.0002 R² = 0.9789 -1 y = 7E - 07 x + 9E -05 0.0001 R² = 0.9437 -1.5 0 0 200 400 -2 In t t ( min ) 0

157

First order Higuchi model 0 100 -2 0 200 400 80 -4 60 -6 y = - 0.0064 x - 8.2459 40 -8 R² = 0.9711 20 y = 4.8015x + 6.6868 R² = 0.9884 -10 0 -12 0 10 20 t t 1/2

0.00012 Hixson- crowell model 0.0001 0.00008 0.00006 0.00004 y = 2E -07 x + 3E- 05 0.00002 R² = 0.9437 0 0 200 400 t (min) Power law 0.00028 Zero order 0 0.00027 Fig. 76. -0.05 0 2 4 6 0.00026 -0.1 Typical 0.00025 -0.15 y = 0.0501x - 0.4709 y = 1E - 07 x + 0.000 2 0.00024 -0.2 R² = 0.8459 R² = 0.9593 model -0.25 0.00023 -0.3 0.00022 fitting plots -0.35 -0.4 for SP- In t diclofenac sodium tablets in phosphate buffer of (pH 6.8) at 37±0.1°C

0 200 400 t (min)

158

90 Higuchi model 85 80

75 y = 0.8989x + 68.851 R² = 0.9269 70 First order -9.2 -9.4 0 200 400 -9.6 -9.8 y = - 0.0021 x - 9.3 374 -10 R² = 0.9354 -10.2 t 0 5 10 15 20 t 1/2

Hixson-crowell model

y = 4E- 08 x + 8E - 0 5 R² = 0.9593 0.000095 0.00009 0.000085 0.00008 0.000075 0 200 t 400 (min) Fig. 77. Typical model fitting plots for AT-diclofenac sodium tablets in phosphate buffer of (pH 6.8) at 37±0.1°C

159

0.5 Power law 0.0004 0 0.0003 -0.5 0 2 4 6 -1 0.0002 -1.5 0.0001 y = 2E - 06 x + 4E - 05 -2 y = 0.813x - 4.102 R² = 0.9251 R² = 0.9645 0 -2.5 0 100 200 In t Zero order t (min)

First order 0 120 -2 0 100 200 100 -4 80 -6 60 -8 40 -10 20 y = 9.7987x - 25.626 -12 R² = 0.9635 y = -0.0282 x - 7.4277 0 -14 R² = 0.978 t 0 5 10 15 Higuchi model t 1/2 0.00014 Hixson- crowell model 0.00012 0.0001 0.00008 y = 6E- 07 x + 1E - 05 0.00006 R² = 0.9249 0.00004 0.00002 0 0 100 200 t (min)

Fig. 78. Typical model fitting plots for control tablets in phosphate buffer of (pH 6.8) at 37±0.1°C

160

Fig. 79. Absorbance spectra of DS-polymer tablets in 0.1M HCl

Fig. 80. Release profiles of polymer tablets in distilled water (caffeine)

161

Table 19. Fitness of release data of caffeine in distilled water from different material tablets to various mathematical models

Material/Model AM AN PO MP LR OB PO husk SP AT Control seeds

Zero order R2 0.936 0.899 0.926 0.852 0.908 0.922 0.912 0.967 0.836 0.920 MSC 3.383 1.898 2.106 1.240 1.995 1.988 3.481 3.027 5.527 2.129 First order R2 0.971 0.940 0.971 0.906 0.969 0.975 0.943 0.977 0.817 0.989 MSC 8.170 2.428 3.041 6.622 3.099 3.124 8.343 3.411 8.576 4.187 Higuchi R2 0.989 0.974 0.982 0.944 0.978 0.981 0.965 0.988 0.817 0.983 MSC 5.184 3.241 3.527 2.212 3.447 3.409 4.411 4.085 5.570 3.706 Power law R2 0.993 0.985 0.981 0.954 0.991 0.992 0.983 0.980 0.849 0.990 MSC 5.397 3.832 3.514 3.572 4.417 4.282 2.771 3.528 1.392 4.242 n 0.480 0.553 0.436 0.525 0.273 0.353 0.584 0.750 0.110 0.564 Hixon crowell R2 0.936 0.899 0.926 0.852 0.908 0.922 0.912 0.967 0.836 0.920

MSC 3.383 1.899 2.106 1.240 1.995 1.987 3.481 3.027 5.527 2.129 % release (t min50) 240 210 170 300 110 90 110 240 15 110

136

Power law 0 0.0005 Zero order -0.2 0 5 10 0.0004 -0.4 0.0003 -0.6 0.0002 -0.8 y = 6E - 07 x + 0.0002 0.0001 R² = 0.9089 -1 y = 0.2734x - 1.9024 -1.2 R² = 0.9919 0 0 200 400 600 -1.4 In t t ( min)

First order 100 Higuchi model -7.6 -7.8 0 200 400 600 80 -8 -8.2 60 -8.4 40 y = 2.7011x + 23.862 -8.6 R² = 0.9787 -8.8 20 -9 y = -0.0026 x - 7.9139 0 -9.2 R² = 0.9698 0 10 20 30 t t 1/2

0.0006 Hixson - crowell model 0.0005 0.0004 0.0003 y = 2E -07 x + 0.0004 0.0002 R² = 0.9089 0.0001 0 0 200 400 600 t (min)

Fig. 81. Typical model fitting plots for LR-caffeine tablets in distilled water at 37±0.1°C

164

Power law 0.0004 Zero order

0 2 4 6 8 -0.5 0.0003 -1 0.0002

-1.5 0.0001 y = 7E - 07 x + 1E -04 y = 0.4793x - 3.3556 R² = 0.9365 -2 R² = 0.9934 0 -2.5 0 500 In t t (min ) 0 First order -7.4 80 Higuchi model 0 200 400 600 -7.6 60 -7.8 40 -8 y = 2.9174x + 2.5209 20 -8.2 R² = 0.9895 -8.4 y = - 0.0019 x - 7.6426 0 -8.6 R² = 0.9718 0 10 20 30 t 1 / 2 t

0.0002 Hixson - crowell model 0.00015 0.0001 y = 3E - 07 x + 4E -05 0.00005 R² = 0.9365

0 0 200 400 600 t (min)

Fig. 82. Typical model fitting plots for AM-caffeine tablets in distilled water at 37±0.1°C

165

Zero order 0 5 10 0.0004 -0.5 0.0003 y = 7E - 07 x + 9E -05 -1 0.0002 R² = 0.8996 -1.5 0.0001 y = 0.5537x - 3.7178 -2 R² = 0.9855 0 0 200 400 600 -2.5 t ( min) In t Power law 0.0005 0

First order 70 Higuchi model -7.4 60 -7.6 0 200 400 600 50 -7.8 40 -8 30 -8.2 20 y = 3.2415x - 0.46 -8.4 10 R² = 0.9743 y = -0.0021 x - 7.6321 -8.6 R² = 0.9409 0 t 0 10 20 30 1 / 2 t

166

0.00014 Hixson - crowell model 0.00012 0.0001 0.00008 0.00006 0.00004 y = 2E - 07 x + 3E -05 0.00002 R² = 0.8996 0 0 200 400 600 t (min)

Fig. 83. Typical model fitting plots for AN-caffeine tablets in distilled water at 37±0.1°C Power law

0 5

y = 9E -07 x + 0.0001 y = 0.4366x - 2.8597 R² = 0.9262 R² = 0.9819 00.0005 Zero order

10 0.0004 -0.5 0.0003 -10.0002 0.0001 -1.50 0 200 400 -2 In t t (min)

167

First order Higuchi model -7.6 80 0 200 400 -7.8 60 -8 40 -8.2 y = 3.5739x + 6.4961 -8.4 20 R² = 0.9822 -8.6 y = - 0.003 x - 7.701 0 R² = 0.971 -8.8 0 10 20 t 1 / 2 t

0.00015 Hixson - crowell model

0.0001

0.00005 y = 3E- 07 x + 4E - 05 R² = 0.9262 0 0 200 400 t (min)

Fig. 86. Typical model fitting plots for POseeds-caffeine tablets in distilled water at 37±0.1°C 0 0.0004 Zero order 0 5 10 -0.5 0.0003

-1 0.0002 -1.5 y = 6E - 07 x + 1E -04 0.0001 y = 0.5255x - 3.6127 R² = 0.8521 -2 R² = 0.9547 0 -2.5 0 500 In t t (min) Power law

168

80 -7.4 -7.6 0 200 400 600 60 -7.8 40 -8 -8.2 20 y = 2.8453x + 2.7068 R² = 0.9447 y = - 0.0018 x - 7.6586 -8.4 0 R² = 0.906 -8.6 0 10 20 30 First order Higuchi model t t 1/2

0.00012 0.0001 0.00008 0.00006 y = 2E - 07 x + 3E -05 0.00004 R² = 0.8521 0.00002 0 0.00014 Hixson-crowell model 0 200 400 600 t (min)

Fig. 87. Typical model fitting plots for MP-caffeine tablets in distilled water at 37±0.1°C

Power law 0.0005 Zero order

169

0 2 4 6 0.0004 0.0003 -0.5 y = 0.353x - 2.2107 0.0002 y = 1E- 06 x + 0.0002 R² = 0.9922 0.0001 -1 R² = 0.9226 0 0 100 200 300 -1.5 In t t (min ) 0 First order Higuchi model -7.6 80 -7.8 0 100 200 300 60 -8 -8.2 40 y = 3.9112x + 15.448 -8.4 R² = 0.9813 20 -8.6 -8.8 y = - 0.0044 x - 7.8136 0 R² = 0.9752 -9 0 10 20

t t 1/2 0.0002 Hixson - crowell model

0.00015

0.0001

0.00005 y = 4E -07 x + 6E - 05 R² = 0.9226 0 0 100 200 300 t (min)

Fig. 86. Typical model fitting plots for OB-caffeine tablets in distilled water at 37±0.1°C

170

Zero order 0 0.0004 0 2 4 6 -0.5 0.0003 y = 0.5848x - 3.4707 y = 1E -06 x + 1E - 04 R² = 0.9835 0.0002 -1 R² = 0.912 0.0001 -1.5 0 -2 0 100 200 300 In t t (min ) Power law 0.0005

First order 80 Higuchi model -7.4 70 -7.6 0 100 200 300 60 -7.8 50 -8 40 -8.2 30 -8.4 20 y = 5.132x - 4.7416 -8.6 R² = 0.9653 y = -0.0049 x - 7.6097 10 -8.8 R² = 0.9436 0 -9 0 10 20 t 1 / 2 t

0.0002 Hixson - crowell model

0.00015

0.0001

0.00005 y = 5E -07 x + 3E -05 R² = 0.912 0 0 100 200 300 t (min)

Fig. 87. Typical model fitting plots for PO husk-caffeine tablets in distilled water at 37±0.1°C

171

Power law 0.0005 Zero order 0 -0.5 0 5 10 0.0004 -1 0.0003 -1.5 0.0002 y = 9E -07 x + 5E - 05 -2 0.0001 R² = 0.9675 -2.5 y = 0.7505x - 4.7623 0 R² = 0.9803 -3 0 200 400 600 -3.5 In t t (min)

First order -7.4 80 Higuchi model -7.6 0 200 400 600 -7.8 60 -8 -8.2 40 -8.4 y = 4.0802x - 10.771 20 -8.6 R² = 0.9887 -8.8 y = -0.003 x - 7.5048 R² = 0.9779 0 -9 t 0 20 40 t 1/2

Hixson-crowell model 0.00016

y = 3E- 07 x + 2E - 05 R² = 0.9675 0.00014 0.00012 0.0001 0.00008 0.00006 0.00004 0.00002 0 0 200 400 600

t (min)

172

Fig. 88. Typical model fitting plots for SP-caffeine tablets in distilled water at 37±0.1°C Power law 0.0005 Zero order

-0.1 0 2 4 6 0.0004 -0.2 0.0003 -0.3 0.0002 -0.4 -0.5 y = 0.1104x - 0.9893 0.0001 y = 4E - 07 x + 0.0003 R² = 0.8493 R² = 0.8367 -0.6 0 -0.7 0 200 400 -0.8 In t t (min) 0 First order 80 Higuchi model -8 0 200 400 -8.2 60 -8.4 40 -8.6 20 y = 1.485x + 45.837 R² = 0.8437 -8.8 y = -0.0019 x - 8.196 0 -9 R² = 0.8172 t 0 10 20 t 1 / 2

0.0002 Hixson - crowell model 0.00015 0.0001 y = 1E -07 x + 1E -04 0.00005 R² = 0.8367 0 0 200 400 t (min)

Fig. 89. Typical model fitting plots for AT-caffeine tablets in distilled water at 37±0.1°C

173

0.5 Zero order Power law 0.0007 0 0.0006 0 2 4 6 8 0.0005 -0.5 0.0004 -1 0.0003 0.0002 y = 1E -06 x + 0.0001 -1.5 y = 0.5643x - 3.3523 0.0001 R² = 0.9203 R² = 0.9904 -2 0 0 200 400 600 -2.5 In t t (min)

First order Higuchi model 0 120 100 -2 0 200 400 600 80 -4 60 -6 y = - 0.0068 x - 7.5242 40 -8 R² = 0.9898 20 y = 5.0745x - 2.3627 -10 R² = 0.9835 0 -12 0 10 20 30 t t 1 / 2

0.00025 Hixson- crowell model 0.0002 0.00015 0.0001 y = 4E - 07 x + 4E -05 0.00005 R² = 0.9203 0 0 500 t (min)

Fig. 90. Typical model fitting plots for control tablets in distilled water at 37±0.1°C

174

3.3.4 Targeted delivery

All of the polymers under investigation were found to be insoluble in acidic medium therefore,

the drug-loaded polymers are expected to deliver the drugs in the intestine.

3.3.5 Disintegration study

When administered orally the disintegration time of the prepared tablets from caffeine and

diclofenac sodium ranged from 8 min to 169 min (Table 20).

3.4 Evaluation as binders in tablets

The direct compressed tablets incorporating the polymer as binder and acetaminophen as an active

pharmaceutical ingredient were subjected to hardness testing The hardness of LR is 11.5 (table 21)

which is approximately the same as methyl cellulose and HPMC (range from 11-12 kg/cm2 ) [196]

which are already in use as commercial binders. Whereas the other polymers possesed less

hardness. The trend of hardness was MP< SP=AM < AN< PO seeds = AT < PO husk< OB< LR.

3.5 Evaluation as suspending agents

Sedimentation of the prepared suspensions of acetaminophen suspensions incorporating the

polymers under investigation were recorded according to the standard method. The results are

shown in Fig. 89 as bar chart. The suspensions prepared from SP and OB remained stable for more

than 2 months time. The trend of stability was found to be: SP OB AN AM PO seeds TG

PO husk LR MP. The results were compared with a standard preparation. It can be Table

20. Disintegration time of caffeine and Diclofenac sodium

175

Materials Disintegration time (min) Caffeine Diclofenac sodium AM 45 35 LR 60 22 PO seeds 78 36 AT 05 08 OB 43 21 PO husk 145 97 SP 169 43 AN 68 120 MP 44 14 Voltral 36

Table 21. Hardness of tablets Material Hardness (kgcm-2) SP 1.6 AN 2.1 MP 1.2 POH 3.6 AM 1.6 LR 11.5 OB 4.1 POS 3.8 AT 3.8

176

Fig. 91. Suspensions of paracetamol: a) sedimentation bar chart

177

Fig. 91. (Continued) Suspensions of paracetamol: b) pictures

149

seen that all the polymers produced more stable suspension than the standard.

3.6 Evaluation as thickening agents

Thickening agents increases the viscosity without significantly altering its properties. As observed from table 10 the trend in viscosity was found to be: PO husk > LR > POseeds > SP > AM > MP >

OB > AN at 1% concentration level and shear rate 10 s-1. It shows PO husk is the best as thickener and can increase viscosity even if added in very small amount.

3.7 Evaluation as film coating materials

Film coating is an important and versatile step in the manufacture of solid dosage forms of drug product in the pharmaceutical industry. The film coat applied helps to protect the active ingredient inside the tablet from environment (air or light). It mask the taste, colour and odour and make the tablet palatable or to determine controlled release dosage form [197]. Film coating agents also play a vital role in drug delivery by making the tablets either for immediate release or for modified release. Film coating was performed without the use of a platicizer and rupture of film coating was checked with drop test [198]. The pictures of the coated tablets are shown in Fig. 92. In this test one drop of water is placed on the outer surface of tablet with the help of a micropipette and the surface was studied after 0, 10 and 20 second. The time of film rupture was

OB SP AN AM PO seeds PO husk LR MP. Even in the absence of plasticizer LR and MP exhibited a fairly good coating ability and there is a chance of further improvement with the use of plasticizer.

180

SP AN

MP PO husk

Fig. 92. Coated tablets

181

AM LR

OB PO seeds

g - h -

Fig. 92. (Continued) Coated tablets

182

3.8 Concluding remarks

Polysaccharides studied in this work came out to be low cost, easily available nonhazardous and environment friendly. They can be used as biocompatible and biodegradable materials in pharmaceutical formulations as tablet coating agents, suspending and thickening agents, binders in tablets, fabrication of capsule shells, filling of pharmaceutical capsules, contact lenses, targeted and controlled drug delivery devices.

These materials have a great potential for their use in formulation of ophthalmic solutions and suspensions due to their very high water retention, drug-loaded capacity and sustained release characteristics. Future work on evaluation of their use as the materials for medicated contact lenses, non-gelatin capsule shells and biomedical scaffolds would provide interesting results.

183

3.9 Research publications by the author from this work

A) Papers published in international journal

1- Mohammad S. Iqbal, Shazma Massey, Jamshed Akbar, Chaudhury M. Ashraf, Rashid Masih

(2013) ; Thermal analysis of some natural polysaccharide materials by isoconversional method.

Food Chemistry, 140(1-2): 178-82. (Impact Factor =3.334)

2- Jamshed Akbar, Mohammad S. Iqbal, Shazma Massey, Rashid Masih (2012) ; Kinetics and

mechanism of thermal degradation of pentose and hexose-based polysaccharides. Carbohydrate

polymers, 90(3): 1386-93. (Impact Factor =3.479)

3- Shazma Massey, Mohammad S. Iqbal, Bettina Wolf, Irfana Mariam, Shumaila Rao (2016);

Comparative drug loading and release study on some carbohydrate polymers. Latin American

Journal of Pharmacy, 35(1): 146-155.

B- Oral presentation

1- In the conference organized by Department of Chemistry, Forman Christian College (A

Chartered University) Lahore, Pakistan on Exploring New Avenues in Medicinal Chemistry,

Opportunities & Challenges from January 21-23, 2015.

2- Paper accepted for oral presentation in 251st ACS National Meeting to be held in San Diego,

California, March 13-17, 2016. PAPER ID: 2394304, PAPER T TLE: “ solation,

characterization and pharmaceutical applications of polysaccharides from plants”

184

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