30
Chapter-8 Caffeine Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 186 Introduction – Caffeine is a pharmacologically active substance and depending on the dose, can be a mild central nervous system stimulant. Caffeine does not accumulate in the body over the course of time and is normally excreted within several hours of consumption [1-2]. In humans, caffeine acts as a central nervous system stimulant, temporarily warding off drowsiness and restoring alertness. It is the world's most widely consumed psychoactive drug, but, unlike many other psychoactive substances, it is both legal and unregulated in nearly all parts of the world [3]. Structure - IUPAC Name - 3, 7-dihydro-1, 3, 7-trimethyl-1H-purine-2,6-dione. - 1, 3, 7,-Trimethylxanthine. - 1, 3, 7-Trimethyl-4, 6-dioxopurine Formula - C 8 H 10 N 4 O 2 Solubility - Freely soluble in chloroform, methanol and in boiling water; sparingly soluble in water and in ethanol (95 per sent) Mol. Wt. - 194.19 Brand Name - Vivarin-200 mg [A] - Stay awake-200 mg [B] - Ra stay awake-200 mg [C] Identification - Identification of pure drug is performed by FT-IR (Shimadzu 8400s) and compared with standard one [4].

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 186

Introduction – Caffeine is a pharmacologically active substance and

depending on the dose, can be a mild central nervous system

stimulant. Caffeine does not accumulate in the body over the course

of time and is normally excreted within several hours of consumption

[1-2].

In humans, caffeine acts as a central nervous system stimulant,

temporarily warding off drowsiness and restoring alertness. It is the

world's most widely consumed psychoactive drug, but, unlike many

other psychoactive substances, it is both legal and unregulated in

nearly all parts of the world [3].

Structure -

IUPAC Name - 3, 7-dihydro-1, 3, 7-trimethyl-1H-purine-2,6-dione.

- 1, 3, 7,-Trimethylxanthine.

- 1, 3, 7-Trimethyl-4, 6-dioxopurine

Formula - C8H10N4O2

Solubility - Freely soluble in chloroform, methanol and in

boiling water; sparingly soluble in water and in

ethanol (95 per sent)

Mol. Wt. - 194.19

Brand Name - Vivarin-200 mg [A]

- Stay awake-200 mg [B]

- Ra stay awake-200 mg [C]

Identification - Identification of pure drug is performed by FT-IR

(Shimadzu 8400s) and compared with standard

one [4].

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 187

Wave number (cm-1)

Fig. 8.1: Reference IR Spectrum of CFI

Fig. 8.2: IR Spectrum of pure CFI

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 188

Table 8.1: Characteristics absorption frequencies for identification of

pure CFI

S. No. Types of Vibrations Frequency (cm-1) 1. Ar. C – H Stretching 3113.21

2. Ar. C – H Bending 758.05

3. C – N Stretching 1359.86

4. C = O Stretching 1697.41

5. C = N 1599.04

6. Ar. C - C Stretching 1604.83

Bioavailability - 99%

Protein binding - 17% to 36%

Metabolism - Caffeine is metabolized in the liver into three primary

metabolites, paraxanthine (84%), theobromine (12%) and theophylline

(4%). Caffeine from coffee or other beverages is absorbed by the small

intestine within 45 minutes of ingestion and then distributed

throughout all tissues of the body [5]. Peak blood concentration is

reached within one hour [6]. Caffeine can also be absorbed rectally,

evidenced by the formulation of suppositories of ergotamine tartrate

and caffeine (for the relief of migraine) [7], chlorobutanol and caffeine

(for the treatment of hyperemesis) [8]. Caffeine is metabolized in

the liver by the cytochrome P450 oxidase enzyme system (to be specific,

the isozyme) into three metabolic dimethylxanthines [9].

Half – Life - 5 hrs

Excretion - urine

History - Caffeine was first isolated from coffee in 1820 by the German

chemist Friedlieb Ferdinand Runge, and then independently in 1821

by French chemists Pierre Robiquet, Pierre Pelletier, and Joseph

Caventou. Pelletier coined the word "caffeine" from the French word

for coffee (café), and this term became the English word "caffeine".

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 189

The history of coffeine has been recorded as far back as the

ninth century. During that time, coffee beans were available only in

their place of origin, Ethiopia. Legends trace the discovery of coffee

either to a Sufi dervish named Omar, or to a goatherder named Kaldi,

who observed goats become elated and sleepless at night after grazing

on coffee shrubs and, upon trying the berries the goats had been

eating, experienced the same vitality [10].

In 1819, the German chemist Friedlieb Ferdinand Runge isolated

relatively pure caffeine for the first time; he called it "Kaffebase" (i.e.,

a base that exists in coffee) [11].

The structure of caffeine was elucidated near the end of the 19th

century by Hermann Emil Fischer, who was also the first to achieve

its total synthesis. This was part of the work for which Fischer was

awarded the Nobel Prize in 1902 [12].

Use - Caffeine which is found in tea and coffee imparts bitterness and

also acts as a flavor constituent. It is a mild nervous stimulant

towards drowsiness and fatigue. Caffeine is used as a drug on the

basis of its effect on respiratory, cardiovascular and the central

nervous system. It is included with ergotamine in some anti-migraine

preparations, the object being to produce a mildly agreeable sense of

alertness. Caffeine is administered in the treatment of mild respiratory

depression caused by central nervous system depressants such as

narcotic.

Adverse effect - Common adverse effects include drowsiness,

headaches, migraines, confusion, Incontinence and liver damage.

Bio–Analytical methods -

Several methods have been reported for the determination of

CFI standards and in pharmaceutical preparations.

B. B. Fredholm et al., have described the actions of caffeine in

the brain with special reference to factors that contribute to its

widespread use [13].

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 190

A. Astrup et al., have studies caffeine a double-blind, placebo

controlled study of its thermogenic, metabolic, and cardiovascular

effects in healthy volunteers [14].

J. M. Kalmar et al., have introduced the effects of caffeine on

neuromuscular function [15].

E. Hogervorst et al., have described the caffeine improves

physical and cognitive performance during exhaustive exercise [16].

I. Hindmarch, et al., have studied the effects of black tea and

other beverages on aspects of cognition and psychomotor performance

[17].

M. Johnson-Kozlow, et al., have described the coffee consumption

and cognitive function among older adults [18].

M. Kivipelto, et al, introduced the midlife vascular risk factors

and Alzheimer's disease in later life: longitudinal, population based

study [19].

C. A. Manning, et al., have studied the glucose enhancement of

24-h memory retrieval in healthy elderly humans [20].

P. J. Mitchell, et al., have described the effects of caffeine, time

of day and user history on study-related performance [21].

Caffeine an alkaloid of the methylxanthine family is a naturally

occurring substance found in the leaves, seeds or fruits of over 63

plants species worldwide. The most commonly known sources of

caffeine are coffee, cocoa beans, cola nuts and tea leaves. In its pure

state, it is an intensely bitter white powder. Various manufacturers

market caffeine tablets, claiming that using caffeine of pharmaceutical

quality improves mental alertness. These effects have been borne out

by research that shows that caffeine use results in decreased fatigue

and increased attentiveness [22].

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 191

Determination of λmax using solvent

The pure form of CFI was accurately weighed 10 mg and

dissolved in 100 mL of medium (PEG 4000 9× 10-5 M). The Stock

solution 100 μg/mL was further diluted 1.0 mL in 100 mL media to

give a concentration of 1 μg/mL, the absorption spectra were obtained

with Elico 164 UV-Visible double beam spectrophotometer a scan

range of 200-400 nm and determine the maximum absorbance of drug

at λmax 270 nm. (Fig. 8.3.)

Fig. 8.3: Determination of λmax

using solvent

Determination of calibration curve using solvent

The stock solution (100 μg/mL) of CFI was prepared by

dissolving 10 mg of CFI in 100 mL volumetric flask and made upto

100 mL by aqueous methanol.

Aliquots of 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, 1.2 mL, 1.4

mL and 1.6 mL, of the stock solution were pipetted out into 100 mL

volumetric flask. The volume was made up to the mark with aqueous

methanol. The absorbance of prepared solutions of CFI was

measurement at λmax

270 nm using against aqueous methanol blank.

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 192

y = 0.054x + 0.002R² = 0.999

0.000.100.200.300.400.500.600.700.800.901.00

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0

Abso

rban

ce

Conc. (µg /mL)

Fig. 8.4: Determination of calibration curve by using solvent

Solubility measurements:

The solubility of CBZ was measured in different media such as

distilled water, CTAB, PEG- 400, mixed CTAB + PEG 400, PEG- 4000,

PVP 44000, mixed PEG 4000 + PVP 400O. An excess amount of drug

(25 mg) was then added to 50 mL of each fluid in conical flask. The

mixture was stirred on a magnetic stirrer for half an h. 5.0 mL aliquot

was withdrawn at 10 min. interval and filter immediately using a 0.45

μm syringe filter, diluted with water and then assayed

spectrophotometrically at λmax

270 nm. Shaking continued until two

consecutive estimations are the same.

Table 8.2: Solubility of CFI in different media

S. No.

Sample (each fluid at their

CMC values)

Wt. of drug (mg)

Overall volume

(mL)

Abs. at λ

max

270 nm

Solubility increase in fold

1. CBZ + Distilled water 25 50 0.092 1.00 2. CBZ + CTAB 25 50 0.392 4.26 3. CBZ + PEG 400 25 50 0.561 6.09 4. CBZ + CTAB+PEG 400 25 50 0.442 4.80 5. CBZ + PEG 4000 25 50 0.998 10.84 6. CBZ + PVP 44000 25 50 0.720 7.82

7. CBZ +PEG 4000+ PVP44000

25 50 0.572 6.21

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 193

Polymeric surfactant PEG-4000 (9× 10-5 M) grandly increases

the solubility of CFI. At CMC, the suability of CFI was increased by

10.84 fold. Among the different solvent tested, PEG 4000 shows the

highest increase in the solubility.

Micellization of biologically active substances is a rather general

phenomenon, since it is believed that the increase in the

bioavailability of a lipophilic drug upon oral administration is caused

by drug solubilization in the gut by naturally occurring biliary lipid

fatty acid-containing micelles produced by the organism as a result of

the digestion of dietary fat. On the other hand, surfactant micelles are

widely used as adjuvant and drug carrier systems in many areas of

pharmaceutical technology and controlled drug delivery research

[23-25].

Fig. 8.5: Solubility enhancement by micellization

The ‘ideal’ pharmaceutical micelle should possess a suitable size

(from 10 to 100 nm), demonstrate sufficiently high stability both in

vitro and in vivo (i.e. have a good combination of reasonably low CMC

value and reasonably high kinetic stability), be able to stay in the body

long enough and still eventually disintegrate into bio-inert and non-

toxic unimers that should be easily cleared from the body, and carry a

substantial quantity of a micelle-incorporated pharmaceutical agent.

To meet these ‘net’ requirements, the core compartment should

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 194

demonstrate high loading capacity, controlled release profile for the

incorporated drug, and good compatibility between the core-forming

block and incorporated drug.

The kinetic stability (the actual rate of micelle dissociation below

CMC) depends on many factors including the physical state of the

micelle core contents of a solvent inside the core the size of a

hydrophobic block and the hydrophobic/hydrophilic ratio [26-28].

The process of solubilization of water sparingly soluble drugs by

micelle forming amphiphilic block copolymers was investigated both

as a theoretical and practical problem [29]. It was shown that the

solubilization process strongly depends on the type and efficacy of the

interactions between a solubilized drug and micelle core-forming

hydrophobic block of a copolymer. However, the interactions between

a drurg to be solubilized and the hydrophilic corona forming block as

well as interfacial interactions between drug and solvent (methanol)

may also influence the solubilization process. Recent mathematical

simulation of the solubilization process [30], demonstrated that the

initial solubilization proceeds via the displacement of solvent

(methanol) molecules from the micelle core, and later a solubilized

drug begins to accumulate in the very center of the micelle core

pushing hydrophobic blocks away from this area. Extensive

solubilization may result in some increase of micelle size due to the

expansion of' its core with a solubilized drug. The compatibility

between the loaded drug and core-forming component determines the

efficacy of drug incorporation [31].

Determination of λmax using medium (PEG-4000 at CMC 9× 10-5 M)

The pure form of CFI was accurately weighed 10 mg and

dissolved in 100 mL of medium (PEG 4000 at CMC 9× 10-5 M). Stock

solution 100 μg/mL. The stock solution was further diluted 1.0 mL

(above solution) in 100 mL medium to obtain a concentration of 1

μg/mL. The absorption spectra were obtained with Elico 164 UV-

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 195

Visible double beam spectrophotometer, a scan range of 200-400 nm

and determine the maximum absorbance of drug at λmax 272 nm.

Fig 8.6: Determination of λ

max using medium

Preparation of stock solution from standard solution:

The stock solution was prepared from the standard solution.

Aliquots of 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, 1.2 mL, 1.4 mL,

1.6 mL and 1.8 mL of the standard solution were pipette out into 10

ml volumetric flask. The volume was make up to the mark with

medium. To obtain a concentrations of 2, 4, 6, 8, 10, 12, 14, 16, and

18 µg/mL.

The absorbance of prepared solutions of CFI was measured at

λmax 272 nm using against medium blank. Averages of such eight set

of values were taken for standard calibration curve.

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 196

y = 0.050x + 0.001R² = 0.999

0.000.100.200.300.400.500.600.700.800.901.00

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0

Abso

rban

ce

Conc. (µg /mL)

Fig 8.7: Determination of calibration curve by using medium

Table 8.3: Quality Control Parameters

S.

No. Parameters

Solvent

(methanol)

Media (PEG-4000

- 9× 10-5 M)

1. λmax (nm) 272 272

2. Beer’s Range (µg /mL) 2-16 2-18

3. Molar Absorptivity (L mol-1 cm-1) 1.03×104 1.05×104

4. Sandell’s Sensitivity (µg cm-2) 1.871 0.018

5. Regression equation 0.999 0.999

6. Intercept 0.002 0.001

7. Slope 0.054 0.050

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 197

In Vitro dissolution study:

1. Apparatus: Electrolab TDT – 08L USP apparatus.

2. Dissolution Media: PEG- 4000 at CMC 9× 10-5 M

3. Rotation speed: 100 rpm.

4. Preparation of CFI standard solution: 10 mg CFI standard was

weighed precisely, put in 100 mL volumetric flask and made up

to the mark with dissolution media.

5. Test preparation: Dissolution testing was performed on tablets

containing 10 mg CFI in 9× 10-5 M PEG 4000 (37°C ± 0.5°C) using

paddle method (USP apparatus- II) at 100 rpm. Sample of 5 mL

were withdrawn at regular time intervals, replaced by fresh medium

and spectro-photometrically analyzed at λmax 272 nm after

filtration through 0.45μm syringe filter. All dissolution tests were

performed in triplicate.

6. Time point: Dissolution amount was measured separately at 05,

10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 minutes.

The percentage of drug released obtained by using by following

formula -

Sample Absorbance Standard weight Dilution Medium × × × × Potency Standard Absorbance Dilution 1 Label Claim

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 198

Table8.4: Sample absorbance at different time intervals

S. No. Time (min) Absorbance

Brand- A Brand- B Brand- C 1 5 0.123 0.136 0.111

2. 10 0.235 0.263 0.193

3. 15 0.333 0.369 0.299

4. 20 0.397 0.433 0.374

5. 25 0.451 0.466 0.438

6. 30 0.485 0.498 0.460

7. 35 0.515 0.526 0.498

8. 40 0.538 0.544 0.527

9. 45 0.549 0.565 0.546

10 60 0.571 0.577 0.569

Standard Abs. 0.585

Table 8.5: % drug release of various formulations in PEG 4000 at

different time

S. No. Time (min) % Drug release

Brand- A Brand- B Brand- C 1 05 21.09 23.32 19.04

2. 10 40.31 45.11 33.10

3. 15 57.12 63.29 51.29

4. 20 68.10 74.27 64.15

5. 25 77.36 79.93 75.13

6. 30 83.19 85.42 78.90

7. 35 88.34 90.22 85.42

8. 40 91.94 93.31 90.40

9. 45 94.17 96.91 93.66

10 60 97.94 98.97 97.60

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 199

Table 8.6: log time, square root of time and log % of drug relea

S. No.

Time (min)

log time

Square root of time

log % drug release

Brand-A Brand-B Brand-C

1 05 0.69 2.23 1.32 1.36 1.27

2. 10 1.00 3.16 1.60 1.65 1.51

3. 15 1.17 3.87 1.75 1.80 1.71

4. 20 1.30 4.47 1.83 1.87 1.80

5. 25 1.39 5.00 1.88 1.90 1.87

6. 30 1.47 5.47 1.92 1.93 1.89

7. 35 1.54 5.91 1.94 1.95 1.93

8. 40 1.60 6.32 1.96 1.96 1.95

9. 45 1.65 6.70 1.97 1.98 1.97

10 60 1.77 7.74 1.99 1.99 1.98

Kinetics of drug release

The dissolution data of three brands of CFI tablets (200 mg)

were applied to Zero order, First order, Higuchi model and Korsmeyer

–Peppas models.

Table 8.7: Kinetic parameters for Zero-Order

% Drug release k x mole min-1

Time

[min] Brand-A Brand-B Brand-C Brand-A Brand-B Brand-C

0 0.00 0.00 0.00 0.0000 0.0000 0.0000 5 21.09 23.32 19.04 4.2180 4.6640 3.8080 10 40.31 45.11 33.10 4.0310 4.5110 3.3100 15 57.12 63.29 51.29 3.8080 4.2193 3.4193 20 68.10 74.27 64.15 3.4050 3.7135 3.2075 25 77.36 79.93 75.13 3.0944 3.1972 3.0052 30 83.19 85.42 78.90 2.7730 2.8473 2.6300 35 88.34 90.22 85.42 2.5240 2.5777 2.4406 40 91.94 93.31 90.40 2.2985 2.3328 2.2600 45 94.17 96.91 93.66 2.0927 2.1536 2.0813 60 97.94 98.97 97.60 1.6323 1.6495 1.6267

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 200

Fig. 8.8: Dissolution profile (n=3) of three commercial products of CFI

in polymeric micellar media (Zero order plot)

Fig. 8.9: Regression plot for zero order

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 201

Table 8.8: First - Order rate constant

log % Drug release kx10-1min-1

Time

[min] Brand-A Brand-B Brand- C Brand- A Brand- B Brand- C

0 0.00 0.00 0.00 0.0000 0.0000 0.00000

5 1.32 1.36 1.27 3.7983 3.7985 2.4018

10 1.60 1.65 1.51 1.8992 1.8993 1.9370

15 1.75 1.80 1.71 1.1602 1.1627 1.1403

20 1.83 1.87 1.80 0.8321 0.8396 0.8124

25 1.88 1.90 1.87 0.6480 0.6617 0.6256

30 1.92 1.93 1.89 0.5287 0.5424 0.5158

35 1.94 1.95 1.93 0.4485 0.4590 0.4330

40 1.96 1.96 1.95 0.3885 0.4003 0.3750

45 1.97 1.98 1.97 0.3435 0.3522 0.3299

60 1.99 1.99 1.98 0.2551 0.2629 0.2462

Fig.8.10: First order plot

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 202

Fig.8.11: Regression plot for first order

Table 8.9: Higuchi rate constant

log % Drug Release kxt1/2 min-1/2

Time [min]

Min1/2 t1/2 Brand-A Brand-B Brand- C Brand- A Brand-B Brand-C

0 0.00 0.00 0.00 0.00 0.0000 0.0000 0.0000

5 2.23 21.09 23.32 19.04 9.4574 10.4574 8.5381

10 3.16 40.31 45.11 33.10 12.7563 14.2753 10.4747

15 3.87 57.12 63.29 51.29 14.7597 16.3540 13.2532

20 4.47 68.10 74.27 64.15 15.2349 16.6152 14.3512

25 5.00 77.36 79.93 75.13 15.4720 15.9860 15.0260

30 5.47 83.19 85.42 78.90 15.2084 15.6161 14.4241

35 5.91 88.34 90.22 85.42 14.9475 15.2657 14.4535

40 6.32 91.94 93.31 90.40 14.5475 14.7642 14.3038

45 6.70 94.17 96.91 93.66 14.0552 14.4642 13.9791

60 7.74 97.94 98.97 97.60 12.6537 12.7868 12.6098

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 203

Fig. 8.12: Higuchi plot

Fig. 8.13: Regression plot for Higuchi model

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 204

Table 8.10: Korsmeyer-Peppas rate constant

log % drug release kx10-1min-1

Time

[min]

log

time Brand-A Brand-B Brand-C Brand-A Brand-B Brand-C

0 0.00 0.00 0.00 0.00 0.0000 0.0000 0.0000

5 0.69 1.32 1.36 1.27 1.8437 1.9593 1.7044

10 1.00 1.60 1.65 1.51 0.7713 0.8196 0.6991

15 1.17 1.75 1.80 1.71 0.4796 0.5086 0.4511

20 1.30 1.83 1.87 1.80 0.3386 0.3566 0.3205

25 1.39 1.88 1.90 1.87 0.2602 0.2708 0.2492

30 1.47 1.92 1.93 1.89 0.2094 0.2169 0.1984

35 1.54 1.94 1.95 1.93 0.1347 0.1398 0.1289

40 1.60 1.96 1.96 1.95 0.0982 0.1013 0.0940

45 1.65 1.97 1.98 1.97 0.0638 0.0661 0.0614

60 1.77 1.99 1.99 1.98 0.0451 0.0465 0.0432

Fig. 8.14: Korsmeyer Plot

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Fig. 8.15: Regression plot for Korsmeyer model

Table 8.11: Regression (R2) value of kinetic models

S.

No Brands

Zero

order

First

order

Higuchi

model

Korsmeyar

model ‘n’exponent

1. A 0.828 0.479 0.992 0.900 0.48

2. B 0.795 0.451 0.993 0.881 0.49

3. C 0.862 0.513 0.994 0.921 0.46

Characterization of nano particles

Particle size

The volume mean diameter (VMD) of particles was determined

using a Mastersizer (Malvern instruments UK). The results obtained

were analyzed by the Fraunhofer model and are represented as VMD

(µm).

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Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 206

Fig.8.16: Size distribution of CFI Scanning electron microscopy

In SEM, a source of electrons is focused in vacuum into a fine

probe that is rastered over the surface of the specimen. The electron

beam passes through scan coils and objective lens that deflect

horizontally and vertically so that the beam scans the surface of the

sample. The ultimate resolution of the SEM levels out near 0.6nm at

5kV. In Scanning Trasmission Electron Microscopy in which internal

microstructure images of thin specimens are obtained, achieved

resolution is up to 1.5nm at 30Kv [32].

Transmission electron microsoopy

Transimission Electron Microscopy (TEM) is a technique where

an electron beam interacts and passes through a specimen. The

electrons are emitted by a source and are focused and magnified by a

system of magnetic lenses. The electron beam is confined by the two

condensers lenses which also control the brightness of the beam,

passes the condenser aperture and “hits” the sample surface [33].

The operation of TEM requires an ultra high vacuum and a high

voltage. The first step is to find the electron beam, so the lights of the

room must be turned off. Through a sequence of buttons and

adjustments of focus and brightness of the beam, we can adjust the

settings of the microscope so that by shifting the sample holder find

the thin area of the sample. Then tilting of the sample begins by

rotating the holder. This is a way to observe as much areas as we can,

so we can obtain as much information [34].

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Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 207

Fig. 8.17: SEM Image of CFI

Fig. 8.18: TEM Image of CFI

220 nm

130 nm

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Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 208

Results and discussion

The solubility of CFI was determined at 37ºC ± 0.5ºC in different

fluids is listed in Table 8.2. PEG 4000 greatly increases the solubility

of CFI. The surprising increase in solubility of CFI by nearly 10.84 fold

is found in PEG - 4000.

Mechanism and kinetics of drug release - To know the mechanism

of drug release from these formulations, the data were treated

according to zero-order (cumulative amount of % drug release vs. time

and its regression plots are shown in Fig. 8.8 and 8.9), first order (log

cumulative % of drug release vs time and its regression plot in Fig.

8.10 and 8.11), Higuchi’s (cumulative % of drug release vs. square

root of time and its regression plot in Fig. 8.12 and 8.13), Korsmeyer

plot (log cumulative % of drug release vs log time and its regression

plots in Fig.8.14 and 8.15) and equations. Diffusion is related to

transport of drug from the dosage matrix in to the in vitro study fluids

depending on the concentration. As gradient varies, the drug is

released, and the distance for diffusion increases, which is referred as

Higuchi’s kinetics. In our experiment, the in vitro release profile of CFI

from all the brands could be best expressed by Higuchi’s equation, as

the plot showed high linearity (r2 > 0.994). The slope of the regression

line from Higuchi’s plot indicates the rate of drug release. The

comparative Higuchi’s release rates for different brands are presented

in Table 8.9, Fig. 8.12 and Fig.8.13 reveal that the k obtained from

Higuchi’s model shows better result with high correlation coefficient

(r2 =0.994).

The Korsmeyer-Peppas model is used to analyze drug release

from pharmaceutical dosage forms when the release mechanism is not

well known or when more than one type of release phenomena is

involved. The exponent, termed the release exponent “n”, was studied

by Peppas and coworkers to characterize different drug release

mechanisms from thin films. They noted that profile with n = 0.5

exhibited a drug release mechanisms controlled by Fickian diffusion,

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Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 209

while drug release rate was independent of time and controlled by a

swelling mechanism when n = 1. In the current study, the value of

release rate exponent (n), ranged between 0.46–0.49. Values of n

between 0.5 and 1.0 were regarded as an indicator for the

superposition of both phenomena, and the drug release mechanism

follow was Fickian diffustion.

Polymer micelles have been extensively worked as drug carriers

[35]. Conjugating to ligands such as antibodies can enhance targeting

potential of micelles. micelles is one such novel approach in which

antibody conjugated polymeric micelles containing anti-stimulant

drugs was prepared and results demonstrated effective delivery at

BBB site [36]. Targeting has also been achieved in other drugs with

reduced toxicity [37]. Novel polymeric micelles with targetability and

stimuli sensitivity have emerged as promising carriers in gene and

drug delivery, and can potentially establish landmarks in the future of

drug delivery systems [38].

Micelles as drug carries provide a set of advantage- they

physically entrap sparingly soluble pharmaceutical and deliver them

to the described site of action at concentration that can exceed their

intrinsic water solubility and thus bioavailability. The stability of the

drug is also increased through micelle incorporation. Further more,

undesirable side effects are lessened, as contact of the drug with

inactivating species, such as enzymes present in biological fluids, are

minimized, in comparison with free drug [39-41]. They can be

prepared in large quantities easily and reproducibly [42-43]. The most

important feature of micellar delivery systems, which distinguish them

from other particulate drug carriers, line in their small size (1-100 nm)

and the narrow size distribution [44].

Micelles made of polymeric surfactant are widely used as

adjuvant and drug carrier system in many areas of pharmaceutical

technology and controlled drug delivery [45-50].

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Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 210

Particle size, charge determination and morphology of final

dissoluted drug were examined by Zeta sizer show result 150.6 nm

and TEM show the result 130 nm. As shown in the Fig. 8.16 and 8.18.

Scanning Electron Microscopy (SEM) to examine the surface

topography morphology of fractured of sectioned surface, to analyze

the surface of polymeric drug delivery system that can provide

important information about the SEM analysis. The result for

scanning microscope 220 nm is shown in the Fig.8.17. These images

indicate the smooth regular and spherical surfaces of all

nanoparticles, As seen in the photomicrographs, nanoparicles seen

smaller than the particle size determined by DLS.

As previously discussed, the property of nanoparticle

formulations that make this approach highly beneficial is related to

the surface properties imparted on nanometer-sized entities. Although

in recent years, tremendous emphasis and focus have been placed on

nanotechnology research, as early as 1906, Ostwald published “The

World of the Neglected Dimensions,” wherein colloidal nanoparticles

exhibited special properties that resided between the molecular and

the material sciences. In practice, applying NanoCrystal Technology or

one of the alternative nanoparticle formulation approaches to the

many formulation and performance issues associated with sparingly

water-soluble compounds in the pharmaceutical industry provide

many benefits. These benefits can be categorized into three major

areas: formulation-performance improvements related to enhanced

dissolution, safer and more patient-compliant dosage forms, and the

potential for dose escalation for improvements in efficacy.

CNS drugs represent one of the largest segments of the total

drug market, and it constitutes the segment with the greatest

potential for substantial growth in the years ahead, largely because of

the rapidly increasing numbers of individuals with CNS disorders.

However, most CNS disorders are not treated well, if at all, and the

time taken for CNS drugs to get to market is longer than other

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Chapter-8 Caffeine

Department of Chemistry, Dr. Hari Singh Gour Central University, Sagar (M.P.) 211

therapeutic areas, and the probability of getting to market is lower.

Thus, there is a clear need for CNS drug discovery to become more

efficient and more effective in order to meet the burgeoning need for

CNS therapeutics. A number of bottlenecks have been identified; one

of the key factors is the failure to pay sufficient attention to the

prediction and assessment of the ability of compounds to cross the

BBB.

There is a great hope that this problem can be tackled through

applications nanobiotechnology. Nanoplatforms can be used for

incorporation of multiple drugs, which means patient compliance

against continuous administration of drugs can be minimized. To

transport nano drugs across BBB. There are still many questions to

resolve before the translation of the research in nanocarriers to

clinical reality. However more research needed to be done regarding

the safety, immunogenicity and toxicity of the solid polymeric

nanoparticles as a whole after chronic systemic administration and

can be accomplished in future with interdisciplinary research

collaborations approaches between academia and Pharma -industry

will hopefully advance nanocarrier systems which can smartly and

clinically deliver any molecular neuro-therapeutic to the human brain.

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