5. materials and methods 5.1 materials -...

Materials and Methods

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5. MATERIALS AND METHODS

5.1 Materials

Table 6. List of Materials

S. No. Materials/Chemicals Manufacturer/Supplier

1 Donepezil Dr. Reddy’s Laboratories, Hyderabad, India

2 Curcumin Sami Laboratories, Bangalore, India

4 Acetonitrile( HPLC grade) Sigma chemical Ltd., USA

5 Triethylamine Sigma chemical Ltd., USA

6 Ammonium acetate Rankem Ltd., Delhi, India

7 Isopropyl alcohol Rankem Ltd., Delhi, India

8 Capmul MCM Abitec, USA

9 Captex 8000 Abitec, USA



12 Glyceryl monostearate (Capmul GMS-50K) Abitec, USA

13 Plurol oleique CC 497 Gattefosse, Mumbai, India

14 Labrafac lipophile WL 1349 Gattefosse, Mumbai, India

15 Labrasol Gattefosse, Mumbai, India

16 Compritol 888 ATO (Glyceryl dibehenate) Gattefosse, Mumbai, India

17 Precirol ATO 5 (Glyceryle distearate) Gattefosse, Mumbai, India

18 Cremophor RH 40 BASF, Mumbai, India

19 Medium chain triglyceride Lipoid GmbH, Ludwigshafen, Germany

20 Stearic acid S.D Fine chemicals Ltd. Mumbai, India

21 Tween 80 S.D Fine chemicals Ltd. Mumbai, India

22 Tween 20 S.D Fine chemicals Ltd., Mumbai, India

23 Ethanol S.D Fine chemicals Ltd., Mumbai, India

24 Potassium bromide (IR Grade) S.D Fine Chemicals Ltd., Mumbai, India

25 Lactose S.D Fine Chemicals Ltd., Mumbai, India

26 Sucrose S.D Fine Chemicals Ltd., Mumbai, India

27 Dextrose S.D Fine Chemicals Ltd., Mumbai, India

28 Mannitol S.D Fine Chemicals Ltd., Mumbai, India

29 Orthophosphoric acid S.D Fine Chemicals Ltd., Mumbai, India

30 Sodium chloride S.D Fine Chemicals Ltd., Mumbai, India

31 Sodium carbonate S.D Fine Chemicals Ltd., Mumbai, India

32 Sodium bicarbonate S.D Fine Chemicals Ltd., Mumbai, India

33 Sodium hydroxide S.D Fine Chemicals Ltd., Mumbai, India

34 Potassium chloride S.D Fine Chemicals Ltd., Mumbai, India

35 Polyethylene glycol 400 S.D Fine Chemicals Ltd., Mumbai, India

36 Polyethylene glycol 200 S.D Fine Chemicals Ltd., Mumbai, India

37 Potassium dihydrogen orthophosphate S.D Fine Chemicals Ltd., Mumbai, India

38 Isopropyl alcohol S.D Fine Chemicals Ltd., Mumbai, India

39 Acetic acid S.D Fine Chemicals Ltd., Mumbai, India

40 Formaldehyde S.D Fine Chemicals Ltd., Mumbai, India

41 Calcium chloride Merck Pvt Ltd., Mumbai, India

42 Tripalmitin Sigma Aldrich, St Louis, USA

43 Pluronic F68 (Polaxamer 188) Sigma Aldrich, St Louis, USA


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S. No. Materials/Chemicals Manufacturer/Supplier

44 Pluronic F127 Sigma Aldrich, St Louis, USA

45 Triton X 100 Sigma Aldrich, St Louis, USA

46 Cremophor EL Sigma Aldrich, St Louis, USA

47 Acetylthiocholine Sigma Aldrich, St Louis, USA

48 5,5ɂ-dithiobis(2-nitrobenzoic acid) Sigma Aldrich, St Louis, USA

49 Chitosan Sigma Aldrich, St Louis, USA

50 Dialysis bag (MWCO-12,000-14,000 g/mL) Himedia labs, Mumbai, India

51 Hydrochloric acid Fischer Chemicals Ltd., Mumbai, India

52 Dimethyl sulfoxide Qualigens, Fine Chemicals, Mumbai, India

53 Hydrogen peroxide Loba Chemie Pvt Ltd., Mumbai, India

54 Thiobarbituric acid Loba Chemie Pvt Ltd., Mumbai, India

55 Sodium dodecyl sulfate Loba Chemie Pvt Ltd., Mumbai, India

56 Ethylene diamine tetraacetic acid Loba Chemie Pvt Ltd., Mumbai, India

57 Sodium lauryl sulphate Loba Chemie Pvt Ltd., Mumbai, India

58 Monosodium phosphate anhydrous Loba Chemie Pvt Ltd., Mumbai, India

59 Disodium phosphate anhydrous Loba Chemie Pvt Ltd., Mumbai, India

60 Magnesium chloride Loba Chemie Pvt Ltd., Mumbai, India

61 Diethyl ether Rankem, Delhi, India


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5.2 Equipments

Table 7. List of Equipments

S. No. Equipment Manufacturer/Supplier

1 UV/Visible spectrophotometer UV-1700 series, Shimadzu, Japan

2 High performance liquid chromatography Shimadzu LC 2010A HT, Japan

3 Fourier transform infrared spectrophotometer Shimadzu, Japan

4 Differential Scanning Calorimeter DSC Q 200 TA Instruments, USA

5 Powder X-Ray Diffractometer Bruker AXS D8, USA

6 Zetasizer Malvern ZS 90, UK

7 Transmission electron microscope Topcon 002B, USA

8 Scanning electron microscope FEI Quanta 200F SEM/EDAX, UK

9 Franz diffusion cells Kovai Glass Works, Coimbatore, India

10 Dissolution apparatus Labindia, Mumbai, India

11 Deep freezer Labline Instruments, Kochi, India

12 Freeze dryer Christ alpha 2-4 LD plus, Germany

13 Brookfield DV II Ultra+Viscometer Brookfield Engineering Laboratories, Inc., USA

14 Electroconductometer (Conductivity meter 305)

Systronic, Mumbai

15 Centrifuge Remi Instruments, Mumbai, India

16 Digital pH meter Eutech Instruments, Mumbai, India

17 Digital electronic balance Sartorius, Bangalore, India

18 Magnetic stirrer Remi Equipments, Mumbai

19 Blade stirrer with speed regulator Remi Instruments, Mumbai, India

20 Vortex mixer Yorco Instruments, Delhi, India

21 Sonicator Bandelin RK 100 H, Germany

22 Isothermal shaker IKA® KS 400I, Germany

23 Dissolution apparatus Electro lab, Mumbai

24 Water bath Scientec, Mumbai

25 Abbe’s Refractometer Bausch and Lomb Optical Company, NY, USA

26 Stereotaxic apparatus Stoelting Co., IL, USA

27 Light microscope Motic, USA


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5.3 Drug Profile

5.3.1 Donepezil

Donepezil (DZP), marketed under the trade name Aricept by its developer Eisai and

partner Pfizer is a centrally acting reversible acetylcholinesterase inhibitor. It is the

second drug approved by US FDA for the treatment of mild to moderate AD. It is a new

class of AChE inhibitor having an N-benzylpiperidine and an indanone moiety that

shows longer and more selective action. Donepezil, a piperidine, is a highly selective

inhibitor of the enzyme AChE that is chemically unique from other AChE inhibitors. In

vitro and preclinical studies have demonstrated that DZP is approximately 1200 times

more selective for AChE in the brain than for BuChE in the periphery (Asiri and

Mostafa, 2010).

Molecular structure

Physico-chemical Properties

Molecular formula: C24H29NO3 (Donepezil); C24H29NO3.HCl (Donepezil hydrochloride)

Molecular weight: 379.5 (Donepezil); 415.95 (Donepezil hydrochloride)

Melting point: 229.85°C

Occurrence: white to off-white solid

Log P: 3.6

Dissociation constant: pKa 8.90

Solubility: Donepezil HCl is a white powder and is freely soluble in water, soluble in

chloroform, sparingly soluble in glacial acetic acid and in ethanol, slightly soluble in

acetonitrile, very slightly soluble in ethyl acetate, and insoluble in n-hexane.

Pharmacokinetics

Pharmacokinetics of DZP are linear over a dose range of 1-10 mg given once daily. The

rate and extent of absorption of Aricept tablets are not influenced by food. Based on

population pharmacokinetic analysis of plasma DZP concentrations measured in patients

with AD, following oral dosing, peak plasma concentration is achieved for Aricept 23 mg

tablets in approximately 8 hours, compared with 3 hours for Aricept 10 mg tablets. Peak

plasma concentrations were almost 2-fold higher for Aricept 23 mg tablets than Aricept


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10 mg tablets. Aricept ODT 5 mg and 10 mg are bioequivalent to Aricept 5 mg and 10

mg tablets, respectively. A food effect study has not been conducted with Aricept oral

dispersible tablet (ODT); however, the effect of food with Aricept ODT is expected to be

minimal. Aricept ODT can be taken without regard to meals.

Following multiple dose administration, DZP accumulates in plasma by 4-7 fold,

and steady state is reached within 15 days. The steady state volume of distribution is 12 -

16 L/kg. DZP is approximately 96% bound to human plasma proteins, mainly to

albumins (about 75%) and alpha1-acid glycoprotein (about 21%) over the concentration

range of 2-1000 ng/mL. DZP is both excreted in the urine intact and extensively

metabolized to four major metabolites, two of which are known to be active, and a

number of minor metabolites, not all of which have been identified. DZP is metabolized

by CYP 450 isoenzymes 2D6 and 3A4 and undergoes glucuronidation.

Half life: 70h

Protein binding: 96%

Bioavailability: l00%

Toxicity: In an acute dose neurotoxicity study in female rats, oral administration of

donepezil and memantine in combination resulted in increased incidence, severity, and

distribution of neurodegeneration compared with memantine alone. The no-effect levels

of the combination were associated with clinically relevant plasma donepezil and

memantine levels. The relevance of this finding to humans is unknown.

5.3.2 Curcumin

Source and chemical nature: It is a hydrophobic polyphenol isolated from the rhizomes

of turmeric (Curcuma longa L.) and related species (family: Zingiberaceae), has been used

traditionally as an Indian spice. It is a bis-α, β-unsaturated β-diketone.

Physico-chemical Properties

Molecular formula: C21H20O6

Molecular weight: 368.38

Melting point: 179-183°C

Occurrence: bright yellow crystalline solid

Log P: 2.85

pKa: 7.8, 8.5 and 9.0


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Solubility: Sparingly soluble in water and ether; readily soluble in dimethylsulfoxide,

dimethyl formamide, glacial acetic acid, ethanol or acetone. The solubility of drug in

these solvents is ~1mg/ml, and in acetone it is ~20mg/ml.

Pharmacokinetics: CUR shows low systemic bioavailability after oral dosing, probably

due to rapid first-pass metabolism and some degree of intestinal pre-metabolism. Its

metabolism on the one hand involves, successive reductions, which transform CUR to

hexahydrocurcuminol and hexahydrocurcumin (probably through the intermediates

dihydrocurcumin and tetrahydrocurcumin), and, on the other hand, rapid molecular

modification by conjugation, mostly in the liver, to glucuronide, sulfate and glucuronide-

sulfate forms. Moreover, when given orally, 40% of the drug is excreted unchanged in the

feces. It also undergoes extensive enterohepatic recirculation, resulting in its rapid

elimination in bile and urine.

Half life: 1.39h

Protein binding: 60%

Bioavailability: low oral bioavailability (<0.1%)

Pharmacological properties: A large number of in vitro and in vivo studies in both

animals and humans have indicated that CUR exhibits promising pharmacological

activities including anti-oxidant, anti-inflammatory, anti-angiogenic, anti-spasmodic,

anti-microbial and anti-plasmodial activities.

Toxicity: Even at 8g/day, no toxicity is reported from humans and animals.

Stability: Under physiological conditions, CUR can exist in both an enol and a bis-keto

form, which coexist in equilibrium. In acidic and neutral solutions as well as in the solid

state, the keto form predominates, and CUR acts as a potent donor of H-atoms. In

contrast, under alkaline conditions (≥pH 8), the enolic form predominates, and the

phenolic part of the molecule plays the principal role as an electron donor. In solution, it

has been demonstrated that 90% of CUR degrades to trans-6-(4’-hydroxy-3’-

methoxyphenyl)-2,4-dioxo-5-hexanal,vanillin, feruloylmethane, and ferulic acid within

30min. CUR is similarly unstable at basic pH. Yellow CUR changes to dark red colour at

alkaline pH and under physiological conditions the λ max for CUR is observed at 420nm.

Aqueous solution of CUR should not be stored for more than 12h. It should be kept in a

tightly closed container, protected from light and stored in a cool place.


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5.4 Analytical Method Development

Analytical methods were developed for estimation of DZP and CUR using RP-HPLC.

5.4.1 Selection of detection wavelength

100µg/mL of DZP and CUR solutions were prepared separately in acetonitrile (ACN).

The solutions were scanned in the UV-Visible region of 200-800nm and the spectrum was

recorded using photodiode array (PDA).

5.4.2 Preparation of standard stock solutions

10mg of DZP and CUR working standards were accurately weighed and transferred into

a 10mL volumetric flask separately and dissolved in ACN and made up to the volume

with the same solvent to produce a 1mg/mL (1,000µg/mL) of DZP and CUR stock

solutions respectively. The stock solutions were further diluted to 100µg/mL by taking

10 mL of respective drug solution (1mg/mL) and diluting upto 100mL in volumetric

flask with ACN. These solutions were then stored in the refrigerator at 5oC ± 3oC until

analysis.

5.4.3 Linearity and range of DZP

Linearity and range were analyzed by preparing calibration curves using different

concentrations of the standard solution. The calibration curve was plotted using mean

peak area (x) versus the respective concentration (y) of the standard drug solutions.

Linearity for DZP was established over the range of 1-100µg/mL using the weighted

least square regression analysis. From the stock solution (100µg/mL), aliquots of 0.1,

1.0, 2.0, 4.0, 6.0, 8.0 and 10 mL were pipetted out in 10mL volumetric flasks and made

up to the volume with ACN to obtain the concentration of 1-100µg/mL and analyzed at

230nm by RP-HPLC. Calibration curve data was subjected to linear regression analysis

to obtain intercept, slope and regression equation. All measurements were made in

triplicate and mean±S.D. was recorded.

5.4.4 Linearity and range of CUR

Linearity and range were analyzed by preparing calibration curves using different

concentrations of the standard solution. The calibration curve was plotted using mean peak

area (x) versus the respective concentration (y) of the standard drug solutions. Linearity for

CUR was established over the range of 1-100 µg/mL using the weighted least square

regression analysis. From the stock solution (100µg/mL), aliquots of 0.1, 1.0, 2.0, 4.0, 6.0,

8.0 and 10 mL were pipetted out in 10mL volumetric flasks and made up to the volume

with ACN to obtain the concentration of 1-100µg/mL and analyzed at 425nm by RP-

HPLC. Calibration curve data was subjected to linear regression analysis to obtain


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intercept, slope and regression equation. All measurements were made in triplicate and

mean±S.D. was recorded.

5.4.5 Mobile phase preparation

A weighed quantity (1.7 gm) of potassium dihydrogen orthophosphate was dissolved in

500mL of milli Q water and the pH was adjusted to 4.5 with ortho-phosphoric acid and

triethylamine. The resultant buffer solution was filtered through 0.45μm nylon

membrane filter and degassed. The mobile phase used was ACN and 25mM potassium

dihydrogen orthophosphate buffer (pH 4.5) in the ratio of 75:25 v/v.

5.4.6 Optimized Chromatographic Conditions

Stationary phase Hibar C18 (250 x 4.6mm i.d., 5m)

Mobile Phase ACN: Potassium dihydrogen orthophosphate

Mobile phase ratio 75:25

Flow rate 1.0mL/min

Sample volume 20ml using Rheodyne 7725i injector

Detection λ 230nm (DZP) and 425nm (CUR)

pH 4.5

Buffer strength 25mM

Data station LC-20AD (PDA)

Retention time of DZP 2.51±0.1min

Retention time of CUR 4.49±0.1min

Run time for DZP 4.0 min

Run time for CUR 6.0 min

5.4.7 Analytical method validation

The RP-HPLC method was validated according to the ICH guidelines, Q2 (R1) (ICH,

2005). Validation of optimized HPLC method was done with respect to following

parameters:

Specificity

Specificity is the ability of a method to discriminate between the intended analyte and

other components in the sample. A method is said to be specific when it produces a

response only for a single analyte in the presence of other interferences. The specificity of

the method is to analyze unequivocally the analyte in the presence of other endogenous

compounds (degradants, excipients, impurities). It was carried out by comparing the

standard retention time spectra and the sample retention time spectra.


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Accuracy

Accuracy is expressed as the closeness of agreement of trueness. It was determined by

standard addition method. For this purpose, known quantities of DZP and CUR were

supplemented to the sample solution previously analyzed. The results of this solution

were compared with the true results. This experiment was carried out by analyzing

replicates (n=6) at three quality control (QC) levels. The mean, standard deviation (S.D.)

and percentage relative standard deviation (% R.S.D.) was calculated. Accuracy was

calculated by comparing the averaged measured concentration to the actual

concentration, and was expressed in percentage nominal.

%Nominal = (Measured concentration /Actual concentration) x 100

Precision

Precision was measured by inter-day (day to day precision, on 3 different days) and intra-

day (repeatability on the same day) variations by analyzing six replicates over three

different concentration of DZP (40, 50, 60 µg/mL) and CUR (40, 50, 60 µg/mL) at same

optimized chromatographic conditions. Precision was evaluated by calculating the

R.S.D. of measured concentrations at each sample based on linearity plots. In all

situations, R.S.D. values were less than 5%, which was considered to be acceptable.

%R.S.D. = (Standard Deviation /Mean) x 100

Limit of detection and quantitation

Limit of detection (LOD) and quantitation (LOQ) of the method were estimated by

injecting a series of dilute solutions with known concentration by visual observation and

signal-to-noise ratio.

LOD = 3.3 σ/s; LOQ = 10 σ/s

where, σ is the standard deviation of response and s is slope of calibration curve

Robustness

Robustness of the method was studied by injecting the standard solutions with slight

variations in the optimized conditions ±4% in the ratio of ACN in mobile phase, ±0.1

mL of the flow rate and ±0.1 in the pH value.

5.5 Bioanalytical Method Development

Bioanalytical methods were developed for estimation of DZP and CUR in rat plasma by

RP-HPLC.


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5.5.1 Preparation of standard stock solutions

10mg of DZP and CUR working standards were accurately weighed and transferred into

a 10mL volumetric flask separately and dissolved in ACN and made up to the volume

with the same solvent to produce a 1mg/mL (1,000µg/mL) of DZP and CUR stock

solutions respectively. The stock solutions were further diluted to 100µg/mL by taking

10 mL of respective drug solution (1mg/mL) and diluting upto 100mL in volumetric

flask with ACN. These solutions were then stored in the refrigerator at -200C ± 20C until

analysis.

5.5.2 Linearity and range for DZP

Eight point calibration curve was prepared by serial dilution of DZP stock solution

(100µg/mL) in the range of 50-2000ng/mL. The concentrations were corrected for

potency and amount weighed. Calibration standards were prepared daily by spiking

0.2mL of blank plasma with 200µL of the appropriate working solution resulting in

concentrations of 50, 100, 200, 250, 500, 1000 and 2000 ng of DZP per mL plasma.

200µL of CUR was added as an internal standard (IS) at concentration of 2µg/mL. To

the resulting solution 200µL of precipitating agent (10% perchloric acid) was added. The

mixture was vortexed for 5 min and centrifuged at 4000 rpm for 10 min. The supernatant

layer was separated and analyzed. A plot with the resulting peak area ratios of DZP to IS

(response factor) was obtained against the concentrations. QC samples (low quality

control (LQC), 50ng/mL; medium quality control (MQC), 500ng/mL; high quality

control (HQC), 750ng/mL; were prepared by spiking 200µL aliquot of blank plasma with

200µL of spiking solution of drug as well as the IS. All solutions were stored in the

refrigerator at 5.0±3.0 oC. The bulk spiked calibration and QC samples were stored at -20

oC.

5.5.3 Linearity and range of CUR

Eight point calibration curve was prepared by serial dilution of CUR stock solution

(100µg/mL) in the range of 50-2000ng/mL. The concentrations were corrected for

potency and amount weighed. Calibration standards were prepared daily by spiking

0.2mL of blank plasma with 200µL of the appropriate working solution resulting in

concentrations of 50, 100, 200, 250, 500, 1000 and 2000 ng of CUR per mL plasma.

200µL of DZP was added as an internal standard (IS) at concentration of 1µg/mL. To

the resulting solution 200µL of precipitating agent (10% perchloric acid) was added. The

mixture was vortexed for 5 min and centrifuged at 4000 rpm for 10 min. A plot with the

resulting peak area ratios of CUR to IS (response factor) was obtained against the

concentrations. Quality control (QC) samples (low quality control (LQC), 50ng/mL;

medium quality control (MQC), 500ng/mL; high quality control (HQC), 750ng/mL;


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were prepared by spiking 200µL aliquot of blank plasma with 200µL of spiking solution

of drug as well as the IS. All solutions were stored in the refrigerator at 5.0±3.0 oC. The

bulk spiked calibration and QC samples were stored at -20 oC.

5.5.4 Preparation of blank plasma

Blank plasma (200µL) was transferred into 2.0 mL centrifuge tube and 200µL of

precipitating agent (10% perchloric acid) were added. The resulting solution was

vortexed for 5 min and centrifuged at 4000 rpm for 10 min. The supernatant layer was

separated and analyzed.

5.5.5 Mobile phase preparation

A weighed quantity (1.7 gm) of potassium dihydrogen orthophosphate was dissolved in

500mL of milli Q water and the pH was adjusted to 6.5 with orthophosphoric acid and

triethylamine. The resultant buffer solution was filtered through 0.45μm nylon

membrane filter and degassed. The mobile phase used was ACN and 25mM potassium

dihydrogen orthophosphate buffer (pH 6.5) in the ratio of 50:50 v/v.

5.5.6 Optimized Chromatographic Conditions

Stationary phase Hibar C18 (250 x 4.6mm i.d., 5m)

Mobile Phase ACN: Potassium dihydrogen orthophosphate

Mobile phase ratio 50:50

Flow rate 1.0mL/min

Sample volume 20ml using Rheodyne 7725i injector

Detection λ 230nm (DZP) and 425nm (CUR)

Ph 6.5

Buffer strength 25mM

Data station LC-20AD (PDA)

Retention time of DZP 5.17±0.1min

Retention time of CUR 12.70±0.1min

Run time 15.0 min

5.5.7 Bioanalytical method validation

The RP-HPLC method was validated according to the ICH guidelines (ICH, 2005).

Validation of optimized HPLC method was done with respect to following parameters:

Specificity

Specificity is the ability of a method to discriminate between the intended analyte and

other components in the sample. A method is said to be specific when it produces a

response only for a single analyte in the presence of other interferences. The specificity of

the method is to analyze unequivocally the analyte in the presence of other endogenous


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compounds (degradants, excipients, impurities). It was carried out by comparing the

standard retention time spectra and the sample retention time spectra.

Accuracy

It was determined by analyzing the percentage recovery of DZP and CUR in plasma

samples. For this purpose, known quantities of DZP and CUR were supplemented to the

blank plasma samples. This experiment was carried out by analyzing replicates (n=6) at

three QC levels (LQC, MQC and HQC). The mean, SD and % RSD were calculated.

Accuracy was calculated by comparing the averaged measured concentration to the

actual concentration, and was expressed in percentage recovery.

% Recovery = (Measured concentration /Actual concentration) x 100

Precision

Precision was measured by inter-day (day to day precision, on 3 different days) and intra-

day (repeatability on the same day) variations by analyzing six replicates over three QC

levels of DZP (50, 500, 750 ng/mL) and CUR (50, 500, 750 ng/mL) at same optimized

chromatographic conditions. Precision was evaluated by calculating the relative standard

deviation (RSD) of measured concentrations at each sample based on linearity plots. In

all situations, RSD values were less than 5%, which was considered to be acceptable.

%RSD = (Standard Deviation /Mean) x 100

Limit of detection and quantitation

LOD and LOQ of the method were estimated by injecting a series of dilute solutions

with known concentration by visual observation and signal-to-noise ratio.

LOD = 3.3 σ/s; LOQ = 10 σ/s

where, σ is the standard deviation of response and s is slope of calibration curve

Stability in rat plasma

The stability of spiked samples of DZP and CUR was assessed at different storage

conditions. Short-term (at room temperature over 4 h), long-term (at -20oC for 30 days),

and two freeze-thaw cycles stability was evaluated by analyzing replicates (n=6) at three

QC levels.

5.6 Solubility studies

The solubility of drugs was determined in different oils, oil mixtures, surfactants and co-

surfactants separately by adding an excess amount of drugs (in increments of 1mg till the

drug saturation was achieved) to 2 mL of each excipients (as shown in Table 8) in 5mL


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capacity stoppered vials, and mixed using a vortex mixer (Yorco Instruments, Delhi,

India) (Jain et al., 2013a). The vials were kept at 25±0.5oC in an isothermal shaker

(IKA® KS 4000i, Germany) for 72h to reach equilibrium. The equilibrated vials were

removed from the shaker and centrifuged at 4,000rpm for 15min using centrifuge (Remi

Instruments, Mumbai, India). The supernatant was taken and filtered through a 0.45µ

membrane filter (Sartorius, Germany). The concentration of drugs was determined after

suitable dilution using HPLC (n=3).

Table 8. List of excipients used for solubility studies

Oils

Capmul MCM (MCM), Captex 8000 (CTX 8000), Captex 500 (CTX

500), Captex 300 (CTX 300), Labrafac lipophile WL 1349 (Labrafac), Medium chain triglyceride (MCT)

Oil mixtures CTX 500+MCT(1:1), CTX 500+MCM(1:1), CTX 500+Labrafac (1:1)

Surfactants Cremophor EL, Labrasol, Tween 80, Tween 20

Co-surfactants Polyethylene glycol 400 (PEG 400), Polyethylene glycol 200

(PEG 200), Plurol oleique, Ethanol

5.7 Partition coefficient

The partition coefficient (PC) of drugs was determined in various solid lipids viz. stearic

acid (SA), glyceryl monostearate (GMS), compritol, precirol and tripalmitin (TP) by

isothermal shaker method as reported previously (Sood et al., 2013). Ten mg of drug was

dispersed in a blend of melted lipid (1g) and hot phosphate buffer (PB) (1mL) pH 7.4,

and shaken for 30min over a hot water bath shaker maintained at 70oC. Aqueous phase

was then separated from lipid by centrifugation at a speed of 10,000 rpm for 20 min. The

clear supernatant obtained was suitably diluted with acetonitrile and drug content was

estimated using HPLC (n=3). PC was calculated as:

PC = (Ci - C) / C (1)

where, Ci = the initial amount of drug added (10mg);

C = the concentration of drug in pH 7.4 PB

5.8 Solubility and Stability in Simulated Nasal Fluid

Saturation solubility of both the drugs was performed in simulated nasal fluid (SNF) pH

6.4 containing 1% w/v sodium lauryl sulphate (SLS) at 25±0.5oC in isothermal shaker.

SNF consisted of monosodium phosphate anhydrous (447mg), disodium phosphate

anhydrous (210mg), sodium chloride (4.4g), potassium chloride (1.5g) and calcium

chloride dehydrate (368mg) dissolved and made upto 500mL with distilled water having

final pH of 6.4. The drug was added in increments of 1mg till the saturation was

achieved. Drug solubility was determined by HPLC at the end of 72h (n=3). For stability


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studies, standard solutions of drug (5-25µg/mL) were prepared in SNF pH 6.4 containing

1% w/v SLS and stored at 37±0.5oC for 72h. The samples were assayed for drug content

using HPLC method at 0, 24, 48 and 72h (n=3).

5.9 Compatibility studies

Compatibility of drug and lipid was studied using Fourier transform infrared

spectroscopy (FTIR) and differential scanning calorimetry (DSC).

5.9.1 Procedure for FTIR

A physical mixture of drug and lipid (either alone or in combination) was prepared and

mixed with anhydrous potassium bromide (KBr) in 1:4 ratio. About 100mg of this

mixture was ground into fine powder using mortar and pestle followed by compression to

form a transparent KBr pellet using a Beckmann hydraulic press (Beckman Instruments

Inc., Fullerton, USA) set at 15 tons pressure. Each KBr pellet was scanned at 4mm/s at a

resolution of 2cm over a wave number region from 4000 to 400 cm-1 in a FTIR

spectrophotometer (Shimadzu, Japan). The IR spectrum of the physical mixture (1:1)

was compared with those of pure drug and lipid and IR peak matching was done to

detect any appearance or disappearance of peaks. FTIR was also carried out for

optimized batch of lyophilized drug loaded NLC formulations.

5.9.2 Procedure for DSC

DSC analysis was performed using DSC Q200 (TA Instruments, USA). The instrument

was calibrated for temperature and heat flow using high purity indium standard. Briefly,

about 5mg of samples were accurately weighed in non-hermetically sealed aluminum

pans and crimped. The DSC thermograms covered the range from 20-250oC and 20-

200oC for DZP and CUR respectively, at a heating rate of 10oC/min under constant

purging of nitrogen at a flow rate of 50mL/min. An empty pan, sealed in the same way

as that of the sample, was used as a reference. DSC thermograms were analysed for pure

drug, lipid and its physical mixture (1:1) using TA Universal Analysis software.

5.10 Purity of drugs

Purity of DZP and CUR was determined using DSC as per procedure described in

section 5.8.2. The thermograms of the drugs were subjected to purity analysis using Vant

Hoff’s equation (Eq.2) that measures the fraction melted as a function of temperature.

(2)

where, Ts = Sample temperature

To = Theoretical melting point for 100% pure crystalline compound

R = Gas constant

X = Total mole fraction impurity

F = Fraction melted at Ts

F

1 )

H

T X R( - T T

2o

os

fD=


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5.11 Formulation of nanoemulsion

5.11.1 Pseudoternary phase diagrams

The nanoemulsions were prepared using spontaneous nanoemulsification method

(Fig. 12) and phase behavior was studied using pseudoternary phase diagrams

(Shafiq et al., 2007).

Fig.12. Formulation of nanoemulsion by spontaneous nanoemulsification method and

photographs of donepezil and curcumin nanoemulsions (NE)

Pseudoternary phase diagrams were constructed using aqueous titration of oil, surfactant

and co-surfactant mixture. The oil phase was heated gently at 45-50 oC for 5 min.

Surfactant and co-surfactant (smix) were mixed together in different volume ratios (1:0,

1:1, 1:2, 1:3, 2:1, 3:1) and heated at same temperature. These smix ratios were chosen to

reflect the increasing concentration of co-surfactant with respect to surfactant and

increasing concentration of surfactant with respect to co-surfactant for the detailed study

of the phase diagrams for the formulation of NE. Oil and smix were mixed in different

volume ratios (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1) to form homogenous isotropic

mixtures and were slowly titrated with aqueous phase. The amount of aqueous phase

added was at interval of 5% v/v. After each addition of aqueous phase, visual

observations were made as clear nanoemulsions, nanoemulsion gels, emulsions or

emulsion gels. Oil, surfactants and cosurfactants were grouped in three different

combinations for phase studies (Table 9).


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Table 9. Oil, surfactants and cosurfactants grouped in different combinations

Group Oil Surfactant Co-surfactant

I CTX 500+ Capmul MCM Tween 80 PEG 400

II CTX 500+ Capmul MCM Tween 80 + Cremophor EL PEG 400

III CTX 500+ Capmul MCM Tween 80 + Cremophor EL PEG 400 + Transcutol

Pseudoternary phase diagrams were constructed using Chemix Software Ver.3.50 (MN,

USA). The calculation for the addition of aqueous phase was done by calculating the

percentage of each component of the nanoemulsion at each addition. The calculation of

percentage of components for construction of phase diagrams is shown in Table 10-18.

The physical state was plotted on a pseudo-three-component phase diagram with one

axis representing the aqueous phase, the second representing the oil phase, and third

representing a mixture of surfactant and cosurfactant (smix) at a fixed volume ratio. For

each smix ratio, a separate phase diagram was constructed. In the phase diagrams, only

NE points were plotted (shaded area), so that there is no overcrowding of the phases in

the diagram, as for formulation development only the nanoemulsion area is of interest.

Table 10. Calculation for percentage of oil, surfactant, and water used in the construction of phase

diagram (Oil:Smix ratio 1:9)

Oil (µL) Smix (µL) Water (µL) Water added (µL) Total (µL) Oil (%) Surfactant (%) Water (%)

10 90 5 -- 105 9.52 85.72 4.76

10 90 12 7 112 8.92 80.37 10.71

10 90 18 6 118 8.47 76.28 15.25

10 90 25 7 125 8.00 72.00 20.00

10 90 34 9 134 7.46 67.17 25.37

10 90 44 10 144 6.94 62.51 30.55

10 90 54 10 154 6.49 58.45 35.06

10 90 67 13 167 5.98 53.91 40.11

10 90 82 15 182 5.49 49.46 45.05

10 90 100 18 200 5.00 45.00 50.00

10 90 122 22 222 4.50 40.55 54.95

10 90 150 28 250 4.00 36.00 60.00

10 90 187 37 287 3.48 31.37 65.15

10 90 235 48 335 2.98 26.88 70.14

10 90 300 65 400 2.50 22.50 75.00

10 90 400 100 500 2.00 18.00 80.00

10 90 550 150 650 1.53 13.86 84.61

10 90 900 350 1000 1.00 9.00 90.00

10 90 1900 1000 2000 0.50 4.50 95.00


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20 80 5 -- 105 19.04 76.20 4.76

20 80 12 7 112 17.85 71.44 10.71

20 80 18 6 118 16.94 67.81 15.25

20 80 25 7 125 16.00 64.00 20.00

20 80 34 9 134 14.92 59.71 25.37

20 80 44 10 144 13.88 55.57 30.55

20 80 54 10 154 12.98 51.96 35.06

20 80 67 13 167 11.97 47.92 40.11

20 80 82 15 182 10.98 43.97 45.05

20 80 100 18 200 10.00 40.00 50.00

20 80 122 22 222 9.00 36.05 54.95

20 80 150 28 250 8.00 32.00 60.00

20 80 187 37 287 6.96 27.89 65.15

20 80 235 48 335 5.97 23.89 70.14

20 80 300 65 400 5.00 20.00 75.00

20 80 400 100 500 4.00 16.00 80.00

20 80 550 150 650 3.07 12.32 84.61

20 80 900 350 1000 2.00 8.00 90.00

20 80 1900 1000 2000 1.00 4.00 95.00




30 70 5 -- 105 28.57 66.66 4.76

30 70 12 7 112 26.78 62.50 10.71

30 70 18 6 118 25.42 59.32 15.25

30 70 25 7 125 24.00 56.00 20.00

30 70 34 9 134 22.38 52.24 25.37

30 70 44 10 144 20.83 48.61 30.55

30 70 54 10 154 19.48 45.45 35.06

30 70 67 13 167 17.96 41.92 40.11

30 70 82 15 182 16.48 38.46 45.05

30 70 100 18 200 15.00 35.00 50.00

30 70 122 22 222 13.51 31.53 54.95

30 70 150 28 250 12.00 28.00 60.00

30 70 187 37 287 10.45 24.39 65.15

30 70 235 48 335 8.95 20.89 70.14

30 70 300 65 400 7.50 17.50 75.00

30 70 400 100 500 6.00 14.00 80.00

30 70 550 150 650 4.61 10.77 84.61

30 70 900 350 1000 3.00 7.00 90.00

30 70 1900 1000 2000 1.50 3.50 95.00




40 60 5 -- 105 38.09 57.14 4.76

40 60 12 7 112 35.71 53.57 10.71

40 60 18 6 118 33.89 50.85 15.25

40 60 25 7 125 32.00 48.00 20.00

40 60 34 9 134 29.85 44.77 25.37

40 60 44 10 144 27.77 41.67 30.55

40 60 54 10 154 25.97 38.96 35.06

40 60 67 13 167 23.95 35.93 40.11

40 60 82 15 182 21.97 32.97 45.05

40 60 100 18 200 20.00 30.00 50.00

40 60 122 22 222 18.01 27.03 54.95

40 60 150 28 250 16.00 24.00 60.00

40 60 187 37 287 13.93 20.91 65.15

40 60 235 48 335 11.94 17.90 70.14

40 60 300 65 400 10.00 15.00 75.00

40 60 400 100 500 8.00 12.00 80.00

40 60 550 150 650 6.15 9.23 84.61

40 60 900 350 1000 4.00 6.00 90.00

40 60 1900 1000 2000 2.00 3.00 95.00


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50 50 5 -- 105 47.62 47.62 4.76

50 50 12 7 112 44.64 44.65 10.71

50 50 18 6 118 42.37 42.38 15.25

50 50 25 7 125 40.00 40.00 20.00

50 50 34 9 134 37.31 37.32 25.37

50 50 44 10 144 34.72 34.73 30.55

50 50 54 10 154 32.47 32.47 35.06

50 50 67 13 167 29.94 29.95 40.11

50 50 82 15 182 27.47 27.48 45.05

50 50 100 18 200 25.00 25.00 50.00

50 50 122 22 222 22.52 22.53 54.95

50 50 150 28 250 20.00 20.00 60.00

50 50 187 37 287 17.42 17.43 65.15

50 50 235 48 335 14.93 14.93 70.14

50 50 300 65 400 12.50 12.50 75.00

50 50 400 100 500 10.00 10.00 80.00

50 50 550 150 650 7.69 7.70 84.61

50 50 900 350 1000 5.00 5.00 90.00

50 50 1900 1000 2000 2.50 2.50 95.00




60 40 5 -- 105 57.14 38.09 4.76

60 40 12 7 112 53.57 35.71 10.71

60 40 18 6 118 50.84 33.90 15.25

60 40 25 7 125 48.00 32.00 20.00

60 40 34 9 134 44.77 29.85 25.37

60 40 44 10 144 41.66 27.78 30.55

60 40 54 10 154 38.96 25.97 35.06

60 40 67 13 167 35.92 23.96 40.11

60 40 82 15 182 32.96 21.98 45.05

60 40 100 18 200 30.00 20.00 50.00

60 40 122 22 222 27.02 18.02 54.95

60 40 150 28 250 24.00 16.00 60.00

60 40 187 37 287 20.90 13.94 65.15

60 40 235 48 335 17.91 11.93 70.14

60 40 300 65 400 15.00 10.00 75.00

60 40 400 100 500 12.00 8.00 80.00

60 40 550 150 650 9.23 6.15 84.61

60 40 900 350 1000 6.00 4.00 90.00

60 40 1900 1000 2000 3.00 2.00 95.00




70 30 5 -- 105 66.66 28.58 4.76

70 30 12 7 112 62.50 26.79 10.71

70 30 18 6 118 59.32 25.43 15.25

70 30 25 7 125 56.00 24.00 20.00

70 30 34 9 134 52.23 22.40 25.37

70 30 44 10 144 48.61 20.84 30.55

70 30 54 10 154 45.45 19.49 35.06

70 30 67 13 167 41.91 17.98 40.11

70 30 82 15 182 38.46 16.49 45.05

70 30 100 18 200 35.00 15.00 50.00

70 30 122 22 222 31.53 13.52 54.95

70 30 150 28 250 28.00 12.00 60.00

70 30 187 37 287 24.39 10.46 65.15

70 30 235 48 335 20.89 8.97 70.14

70 30 300 65 400 17.50 7.50 75.00

70 30 400 100 500 14.00 6.00 80.00

70 30 550 150 650 10.76 4.63 84.61

70 30 900 350 1000 7.00 3.00 90.00

70 30 1900 1000 2000 3.50 1.5 95.00


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80 20 5 -- 105 76.19 19.05 4.76

80 20 12 7 112 71.42 17.87 10.71

80 20 18 6 118 67.79 16.96 15.25

80 20 25 7 125 64.00 16.00 20.00

80 20 34 9 134 59.70 14.93 25.37

80 20 44 10 144 55.55 13.90 30.55

80 20 54 10 154 51.94 13.00 35.06

80 20 67 13 167 47.90 11.99 40.11

80 20 82 15 182 43.95 11.00 45.05

80 20 100 18 200 40.00 10.00 50.00

80 20 122 22 222 36.03 9.02 54.95

80 20 150 28 250 32.00 8.00 60.00

80 20 187 37 287 27.87 6.98 65.15

80 20 235 48 335 23.88 5.98 70.14

80 20 300 65 400 20.00 5.00 75.00

80 20 400 100 500 16.00 4.00 80.00

80 20 550 150 650 12.30 3.09 84.61

80 20 900 350 1000 8.00 2.00 90.00

80 20 1900 1000 2000 4.00 1.00 95.00




90 10 5 -- 105 85.71 9.53 4.76

90 10 12 7 112 80.35 8.94 10.71

90 10 18 6 118 76.27 8.48 15.25

90 10 25 7 125 72.00 8.00 20.00

90 10 34 9 134 67.16 7.47 25.37

90 10 44 10 144 62.50 6.95 30.55

90 10 54 10 154 58.44 6.50 35.06

90 10 67 13 167 53.89 6.00 40.11

90 10 82 15 182 49.45 5.50 45.05

90 10 100 18 200 45.00 5.00 50.00

90 10 122 22 222 40.54 4.51 54.95

90 10 150 28 250 36.00 4.00 60.00

90 10 187 37 287 31.35 3.50 65.15

90 10 235 48 335 26.86 3.00 70.14

90 10 300 65 400 22.50 2.50 75.00

90 10 400 100 500 18.00 2.00 80.00

90 10 550 150 650 13.84 1.55 84.61

90 10 900 350 1000 9.00 1.00 90.00

90 10 1900 1000 2000 4.50 0.50 95.00

5.11.2 Optimization of nanoemulsion using Box-Behnken design

Based on initial screening, concentration of oil, surfactant and co-surfactant were

optimized from NE region of pseudoternary phase diagrams. Box-Behnken design (BBD)

was used statistically to optimize the effect of three independent variables namely

concentration of oil (%), surfactant (%) and co-surfactant (%) and evaluate the main

effects, interaction effects and quadratic effects of these formulation ingredients on the

dependent variables namely globule size (GS) and zeta potential (ZP). Design-Expert®

software (Version 8.0.7.1, M/s Stat-Ease, Minneapolis, USA) was used to conduct the

study. Seventeen experiments with five centre points were designed by the software (in

order to allow the estimation of pure error) and experiments were run in random order.

ranges were selected. Experiments were established based on a BBD with three factors at


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three levels and each independent variable were coded at three levels between −1, 0 and

+1 as shown in Table 19.

Table 19. Factor level for Box-Behnken design for optimization of nanoemulsion

Independent factors Design level

Uncoded Coded Uncoded level Coded level

Concentration of Oil (%) A

10.00 -1

14.00 0

18.00 +1

Concentration of Surfactant (%) B

25.00 -1

30.00 0

35.00 +1

Concentration of Co-surfactant (%) C

10.00 -1

15.00 0

20.00 +1

5.11.3 Optimization of mucoadhesive nanoemulsion using full factorial design

Further to study the effect of chitosan on GS and ZP, a 24

factorial design was studied

using concentration of oil (10-14%), surfactant (30-35%), co-surfactant (15-20%) and

chitosan (0.10-0.25%) as independent factors. A total of 16 experiments were by the

software and experiments were run in random order. Table 20 shows the coded and

uncoded independent variables.

Table 20. Factor level for 24 design for optimization of mucoadhesive nanoemulsions



Concentration of Oil (%) A 10.00 -1

14.00 +1

Concentration of Surfactant (%) B 30.00 -1

35.00 +1

Concentration of Co-surfactant (%) C 15.00 -1

20.00 +1

Concentration of Chitosan (%) D 0.10 -1

0.25 +1

Mucoadhesive nanoemulsions (MNEs) were prepared by initially preparing

nanoemulsion of drug using minimum volume of external phase and then adding


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chitosan solution (1%, w/v) so that final concentration of chitosan in nanoemulsion was

achieved (Kumar et al., 2009).

5.12 Formulation of nanostructured lipid carriers

Drug-loaded DZP and CUR nanostructured lipid carriers (NLCs) were prepared by a

microemulsion method as reported earlier (Sood et al., 2013). The chosen solid lipid and

liquid lipid were melted at 75oC, to which drug was added under continuous stirring for

5min. Twenty milliliter of hydrophilic surfactant solution heated to same temperature

was added to the melted lipid as a continuous phase, with mechanical stirring for 15min.

A clear warm oil-in-water (o/w) microemulsion was formed under stirring at a

temperature above the melting point of the lipid used. The warm o/w microemulsion

constitutes the internal phase. NLC dispersions were obtained by dispersing the warm

o/w microemulsion dropwise into ice cold distilled water (external phase) maintained at

(3-4oC) in a beaker under continuous stirring using triple blade stirrer. The dispersion was

centrifuged at 10,000rpm for 20min and supernatant was discarded. The resulting NLC

pellet was redispersed using millipore water and centrifuged again. The procedure was

repeated twice to remove both free surfactant and free drug molecules and the resultant

dispersion was finally lyophilized. In a similar manner, blank NLC formulation was also

prepared without incorporating the drug to lipid matrix.

Fig. 13. Representation of preparation of nanostructured lipid carriers using microemulsion

technique


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5.12.1 Study on the effect of formulation/process variables

The effect of formulation/process variables such as stirring time, stirring speed, lipid

concentration, type and concentration of surfactant on the particle size was studied. To

investigate the effect of formulation/process variables, each time one parameter was

varied, keeping the others as constant. From the results obtained, optimum level of

variables was selected and kept constant in the subsequent evaluations.

Effect of stirring time

Four different batches of lipid nanoparticles were prepared corresponding to 1, 2, 3 and

4h stirring time, keeping the following parameters as constant,

Lipid concentration : 1% w/v

Stirring speed : 2000rpm

Surfactant concentration (Pluronic F 68) : 2% w/v

Internal:External phase ratio : 1:10

Effect of stirring speed

Four different batches of lipid nanoparticles were prepared corresponding to 1000, 1500,

2000 and 2500rpm stirring speed, keeping the following parameters as constant,

Lipid concentration : 1% w/v

Stirring time : 2h



Effect of lipid concentration

Four different batches of lipid nanoparticles were prepared corresponding to 0.5%, 1%,

1.5% and 2% w/v lipid concentration, keeping the following parameters as constant,


Stirring time : 2h



Effect of surfactant type

Five different batches of lipid nanoparticles were prepared using different type of

surfactants viz. Tween 20, Tween 80, Cremophor EL, Pluronic F68 and Pluronic F127,

keeping the following parameters as constant,

Stirring time : 3h


Lipid concentration : 1.5 % w/v

Surfactant concentration : 2% w/v



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Effect of surfactant concentration

Five different batches of lipid nanoparticles were prepared corresponding to 1, 1.5, 2.0,

2.5 and 3% w/v surfactant concentration, keeping the following parameters as constant,


Stirring time : 2h



Effect of ratio of internal phase to external aqueous phase

Four different batches of lipid nanoparticles were prepared and corresponding to different

ratios of internal to external aqueous phase (1:1, 1:5, 1:10, 1:20), keeping the following

parameters as constant,


Stirring time : 2h


Surfactant concentration : 2.5 % w/v

5.12.2 Optimization of nanostructured lipid carriers using Box-Behnken design

Based on number of factors and their level, Box-Behnken design (BBD) was used for

formulation optimization of donepezil NLC (D-NLC) and curcumin (C-NLC). The

coded and uncoded independent variables for both D-NLC and C-NLC are given in

Table 21. A total of 29 experiments were carried out using Design expert® software.

Table 21. Factor level for Box-Behnken design for optimization of donepezil and curcumin

nanostructured lipid carriers



Lipid concentration (% w/v) A

0.25 -1

0.88 0

1.50 +1

Liquid lipid to total lipid ratio B

0.05 -1

0.17 0

0.30 +1

Drug to lipid ratio C

0.10 -1

0.15 0

0.20 +1

Surfactant concentration (% w/v) D

1.00 -1

1.75 0

2.50 +1


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The effect of four independent variables viz. concentration of lipid (0.25-1.50% w/v),

liquid lipid to total lipid ratio (0.05-0.30), drug to lipid ratio (0.10-0.20) and surfactant

concentration (1.0-2.5% w/v) on dependent variables viz. particle size (PS), drug loading

(DL) and entrapment efficiency (EE) were studied at three different levels, coded as –1, 0

and 1. BBD is an independent, rotatable quadratic design with no embedded factorial or

fractional factorial points where the variable combinations are at the midpoints of the

edges of variable space and at the center.

5.12.3 Optimization of cryoprotectant concentration

Lyophilization of the NLC dispersion was carried out by using 1%, 2% and 5% (w/v) of

mannitol, sucrose, lactose and dextrose as cryoprotectants. The NLC dispersions were

frozen in aqueous cryoprotectant solution at -20oC for about 24h and then the samples

were transferred to the freeze-dryer (Christ, Alpha 2-4 LD plus, Germany) operated at -

40oC and pressure of 0.001bar for 72h to obtained the NLC powders for further

experiments. Freeze dried NLC were characterized for PS, PDI and redispersibility. The

Sf/Si ratio of freeze dried NLC was calculated where Sf and Si indicates particle size

after and before freeze drying respectively. To assess the redispersibility of lyophilized

NLCs, 50mg of product was redispersed in 1 mL of millipore water and visually assessed

using following grading system:

Grade A: Readily redispersible (<15 sec, clear solution)

Grade B: Moderately redispersible (>15 sec, clear to translucent solution)

Grade C: Poorly redispersible ( does not redisperse, presence of large particles)

5.13 Evaluation

5.13.1 Particle /globule size and zeta potential analysis

The mean particle/globule size and ZP of NLC and NE/MNE were determined using a

zetasizer ZS 90 (Malvern Instruments, UK). The mean particle/globule size was

measured based on photon correlation spectroscopy technique that analyzes the

fluctuations in dynamic light scattering due to brownian motion of the particles. The

mean diameter was obtained at an angle of 90° in 10 mm diameter cells at 25°C. The ZP,

reflecting the electric charge on the particle surface, is a very useful way of evaluating the

physical stability of any colloidal system. It was determined based on an electrophoretic

light scattering technique (Jain et al., 2013b). All size and ZP measurements were carried

out at 25oC using disposable polystyrene cells and disposable plain folded capillary zeta

cells, respectively, after appropriate dilution of all samples with original dispersion


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medium (Jain et al., 2013a). Three replicate analysis was performed for each

formulation, and data presented as mean±S.D.

5.13.2 Thermodynamic stability tests

Optimized drug loaded NE/MNE formulations were subjected to different

thermodynamic stability tests (Shafiq et al., 2007).

Centrifugation test: The samples were centrifuged at 3,500rpm for 30min.

Heating cooling cycle test: The samples were subjected to 6 cycles between refrigerator

temperature 4oC and 45oC with storage at each temperature for 48h.

Freeze thaw cycle test: The samples were subjected to 3 cycles between -21oC and +25oC

with storage at each temperature for 48h.

5.13.3 Drug content

The NE/MNE formulations were diluted to required concentration using acetonitrile

as solvent and drug content was estimated using HPLC method. The drug content

(n=3) was calculated as:

Drug content (%) = Analyzed content/Theoretical content x 100

5.13.4 Refractive index and percent transmittance

The refractive index of NE/MNE was measured by Abbe’s refractometer (Bausch and

Lomb optical company, NY, USA) by placing a drop of NE/MNE formulation onto the

slide (n=3). The percent transmittance of NE/MNE was measured at 650nm using UV

spectrophotometer (Shimadzu, Japan) keeping distilled water as blank (n=3) (Ghosh et

al., 2006).

5.13.5 Viscosity determination

The viscosity of the NE/MNE formulations (0.5g) were determined as such without

dilution using Brookfield DV-II ultra+viscometer (Brookfield Engineering Laboratories,

Inc., Middleboro, MA, USA) with spindle # CPE 40 at 25±0.5ºC (n=3). The software

used for the calculations was Rheocalc V2.6. In the small volume adapter, the NE/MNE

was filled and the angular velocity was increased gradually from 10, 20, 50 and 100rpm.

The hierarchy of the angular velocity was reversed (Jain et al., 2013a).

5.13.6 Conductivity study

To determine the nature of the continuous phase and to detect the phase inversion

phenomenon, the electrical conductivity measurement of the NE/MNE formulation was

carried out by an electroconductometer (Conductivity meter 305, Systronic) by inserting

the probe in NE/MNE taken in a beaker (n=3) (Ghosh et al., 2006). The tested


96 | P a g e

NE/MNE was prepared with a 0.01N aqueous solution of sodium chloride (NaCl)

instead of distilled water.

5.13.7 Determination of pH

The pH of the NE/MNE formulations was measured using digital pH meter (n=3)

(Eutech instruments, Mumbai).

5.13.8 Determination of entrapment efficiency and drug loading percentage

Entrapment efficiency (EE) and drug loading (DL) percentage of lyophilized NLCs were

determined according to the procedure described earlier (Sood et al., 2013). Weighed

quantity of lyophilized drug loaded NLC (10mg) were suspended in hydroalcholic

solution (ethanol and water in 50:50) under water bath 75-80oC for 30min. This ensures

melting of NLC and release of entrapped drug in media. The solution is allowed to cool

at room temperature to preferentially precipitate the lipid. The amount of drug in the

supernatant after centrifugation (10,000rpm for 30min) was determined by HPLC (n=3).

Entrapment efficiency (%) = Wdrug/ Wtotal x 100 (3)

Drug loading (%) = Wdrug/ Wlipid x 100 (4)

Wdrug; analyzed amount of drug in the supernatant,

Wtotal; total amount of drug used in formulation,

Wlipid; weight of lyophilized NLC formulation.

5.13.9 Scanning electron microscopy (SEM)

External surface morphology of lyophilized drug loaded NLC was recorded using SEM

(FEI QUANTA 200 SEM/EDAX, UK) at 20kV as an accelerating voltage (Sood et al.,

2013). Weighed amount of samples (5-7mg) were mounted on an aluminium stub with

double sided adhesive tape. The tape was firmly attached to the stub and lyophilized

sample was scattered carefully over its surface. The stub with the sample was then sputter

coated with a thin layer of gold to make the sample conductive. Processed sample was

subjected to SEM analysis. The images were captured under magnification of 10,000-

15,000x and recorded.

5.13.10 Transmission electron microscopy (TEM)

The shape and morphology of drug loaded NLC dispersion and drug loaded NE were

analyzed using TEM (TOPCON 002B, USA) at an accelerating voltage of 200kV (Jain et

al., 2013a). Prior to the analysis, the samples were diluted 100 times with double distilled

water and a drop (5-10µL) was placed onto carbon-coated 200-mesh copper grids to

create a thin film. Before the film dried on the grid, the samples were negatively stained

with 2% w/v phosphotungstic acid by adding a drop of the staining solution to the film


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for 30s; any excess droplets were drained off with a filter paper. The grid was allowed to

air-dry under room temperature. Digital micrograph and soft imaging viewer software

were used to capture the image of samples.

5.13.11 Differential Scanning Calorimetry (DSC)

The crystalline behavior of both drug and lipid was studied by DSC. Thermograms were

recorded for pure drug, lipid and lyophilized drug loaded NLC. DSC was carried out as

per the procedure described earlier. Degree of crystallinity of lyophilized drug loaded

NLC was calculated by comparing the enthalpy of NLC with enthalpy of bulk lipid

(Freitas and Müller, 1999). The melting enthalpy of bulk lipid was used as a reference

(100%) to calculate the percentage of crystallinity of NLC.

(5)

where, ∆H NLC indicates enthalpy of freeze dried NLC

∆Hbulk indicates enthalpy of bulk lipid.

5.13.12 Powder X-Ray Diffraction (PXRD)

PXRD patterns were recorded for pure drug, lipid, physical mixture (drug and lipid in

1:1) and lyophilized drug loaded NLC. PXRD pattern of samples were collected using a

Bruker AXS D8 Advance powder diffractometer, USA. The samples were exposed to Cu

Kα radiation generated at 40kV, 35mA and scanned from 3o to 80o, 2θ at a step size of

0.020o and step time of 31.2s.

5.13.13 In vitro Release Studies

The release of drug from developed formulations (NLC and NE/MNE) and solution was

performed in SNF pH 6.4 containing 1% SLS using the dialysis bag method (Sood et al.,

2013). For both the drugs, solution was prepared by dissolving 80 mg of DZP and 120

mg of CUR in a mixture of 1mL ethanol and 2mL propylene glycol and finally volume

was made to 10mL with distilled water separately to produce concentration of 8mg/mL

for DZP and 12mg/mL for CUR. Dialysis membrane having pore size of 2.4nm and

molecular weight cut off 12,000-14,000 (Dialysis membrane-150, HiMedia, Mumbai,

India) was used. The bags were soaked in distilled water for 24h before use. Drug

solution, lyophilized drug loaded NLC and drug loaded NE/MNE were placed in

dialysis bags separately and sealed at both the ends. The bags were placed in baskets

(USP Dissolution apparatus Type I, LabIndia, Mumbai) and immersed in 500mL of

dissolution medium maintained at 37±0.5oC and stirred at 100rpm. Aliquots of the


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samples were withdrawn from dissolution medium at regular time intervals and same

volume of fresh dissolution medium was replaced to maintain a constant volume. The

samples were analyzed for drug content by HPLC (n=3). The drug release profile was

constructed by plotting the cumulative percent drug release versus time (h). The kinetic

analysis of the release data were fitted to various kinetic models such as zero order, first

order and Higuchi’s equation (Costa and Lobo, 2001).

5.13.14 Ex vivo Permeation Studies

To investigate the permeation efficacy of drug from NLC, NE/MNE and solution across

the freshly excised sheep nasal mucosa, ex vivo permeation studies were performed using

the Franz diffusion cell with surface area of 1.79cm2 and volume of 25mL (Kovai Glass

Works, Coimbatore, India) (Seju et al., 2011). The freshly excised sheep nasal mucosa

was collected from the slaughter house in PBS, pH 6.4. Excised superior nasal membrane

was cut to an appropriate size and thickness (0.2mm), made free from adhered tissues

and mounted between the donor and receptor compartment of the Franz diffusion cell,

with mucosal side facing the donor compartment. The mounted tissue was allowed to

stabilize and stirred under SNF pH 6.4 containing 1% SLS (diffusion medium) for 15min

on a magnetic stirrer. The diffusion cell was thermostated at 37±0.5oC. Solution from

both the compartments was removed after 15min, and the receptor compartment was

freshly filled with diffusion medium. The mounting of nasal membrane was done on the

rim of the receptor compartment; the donor compartment of diffusion cell was placed

over it and secured with a clamp to avoid the leakage of diffusion media. Permeation

studies of pure drug solution, lyophilized drug loaded NLC reconstituted with SNF and

drug loaded NE/MNE were carried out by placing 1mL onto stabilized sheep nasal

membrane on donor compartment and continuously magnetic stirred at 600rpm. Aliquot

(0.5mL) of media were withdrawn from the receptor compartment at predetermined time

intervals, filtered through 0.45µm nylon filter paper and analyzed for drug content using

HPLC. Each removed sample was replaced immediately by an equal volume of fresh

diffusion media maintained at 37±0.5oC to maintain the constant volume at each time

interval. Each study was carried out for a period of 6h, during which the amount of drug

permeated across the sheep nasal mucosal membrane was determined at each sampling

point using HPLC (n=3). The permeation profile was constructed by plotting the amount

of drug permeated per unit skin surface area (µg/cm2) versus time (h). The steady state

flux (Jss, µg /cm2.h) was calculated from slope of the plot using linear regression analysis.

The kinetic analysis of the release data were fitted to various kinetic models such as zero

order, first order and Higuchi’s equation (Costa and Lobo, 2001).


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5.14 Toxicity assessment

The toxicological assessment of the developed lipid based nano formulations (NE and

NLC) were carried out using in vitro cytotoxicity assay on SK-N-SH cell line, in vitro

hemolytic toxicity on rat erythrocytes and nasal ciliotoxicity study on sheep nasal

mucosa stained with H and E stain to assess the safety of the developed formulations to

brain cells, blood and for nasal epithelium respectively.

5.14.1 In vitro cytotoxicity assay

The toxicity studies of the blank and drug loaded formulations (NE/MNE and NLC)

were carried out in SK-N-SH cells, a human neuroblastoma cell line. The cells were

maintained in minimum essential medium (MEM), supplemented with 10% v/v FBS,

penicillin (100 IU/mL), streptomycin (100μg/mL) and amphotericin B (5μg/mL) in a

humidified atmosphere of 5% CO2 at 37°C until confluent. The cells were then seeded in

multiwall culture plates for experimental procedure. The cytotoxicity assay was carried

out using cell suspension, containing 5,000 cells seeded in each well of a 96 well

microtiter plate (Nunc and Tarsons, Kolkata, India) and incubated for 24h at 37°C. Cells

were treated with 250-2,000μg/mL concentration of nanoformulations. Control cells

were incubated without the test compound and with MEM. The microtiter plates were

incubated at 37°C in a humidified incubator with 5% CO2 for a period of 72h.

Morphological changes in the cells were inspected daily and observed for microscopically

detectable alterations, i.e., loss of monolayer, granulation and vaculation in the

cytoplasm. The cytopathic effect was observed. A plot of % growth inhibition versus

concentration was plotted to determine the IC50 value (Concentration of the drug that

produces 50% inhibition of the cells) by SRB (Sulphorhodamine B) assay (Skehan et al.,

1990).

5.14.2 In vitro hemolytic toxicity

The hemolytic activity has been suggested as a toxicity screen in vitro and it also serves

as a simple and reliable measure for estimating the membrane damage caused by

formulation in vivo. The in vitro hemolytic potential of the formulations was studied by

using the method proposed by Jumaa et al. (Jumaa et al., 1999). The Fresh blood from

rats was collected in a vial containing ethylene diamine tetraacetic acid (EDTA) as an

anticoagulant. Blood was centrifuged at 3000rpm for 20min to remove white blood cells

(WBC) debris and suspended RBCs were taken out. The RBCs were washed 3 times with

isotonic saline solution (0.15M NaCl and pH 7.4) before diluting with buffer to prepare

erythrocyte stock dispersion. The RBC cell suspension was adjusted to 50% hematocrit.

Hemolysis experiments were carried out for both blank and drug loaded formulations of

NE and NLC at a concentration of 0, 5, 10, 25, 50, 100, 250 and 500μg/mL. Incubation


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was carried at 37oC for a period of 1h. After incubation under shaking, debris and intact

RBCs was removed by centrifugation and 100µL of resulting supernatant was dissolved

in 2mL of an ethanol/hydrochloric acid (HCl) mixture (39:1, 99% ethanol and HCl,

w/v). This mixture dissolves all components and avoids the precipitation of hemoglobin.

The absorbance of the mixture was determined at 398nm by spectrophotometer

monitoring against a blank sample. Control sample of 0% lysis (in buffer) and 100% lysis

(in Triton X 100) was employed in the experiment. The % hemolysis caused by the test

sample (n=3) was calculated by following equation:

% Hemolysis = Absorbance of Test - Absorbance at 0% x 100 (6)

Absorbance at 100% lysis - Absorbance at 0%

5.14.3 Nasal ciliotoxicity studies on sheep nasal mucosa

Nasal ciliotoxicity studies were carried out using the freshly isolated sheep nasal mucosa

collected from a slaughter house in a phosphate buffered saline (PBS) pH 6.4 (Seju et al.,

2011). Each piece was treated with drug solution in PBS pH 6.4, blank NLC, lyophilized

drug loaded NLC, blank NE, drug loaded NE/MNE, PBS pH 6.4 (as negative control)

and isopropyl alcohol (IPA) (nasal mucociliary toxicity agent used as a positive control),

respectively. After treatment for 2h, all the samples were washed properly with distilled

water and were preserved with 10% formalin until further analysis. Each sample was

sectioned and stained with H and E. The mucosa was then dissected out, and the

mucocilia was examined on an optical microscope by a pathologist.

5.15 Stability studies

The optimized batch of lyophilized drug loaded NLC and drug loaded NE/MNE were

evaluated for long term stability for 6 months at 4±1oC and 25±1oC (n=3). Samples were

transferred in amber colored glass vials, sealed and were stored upright. NE/MNE

formulations samples were withdrawn at specified time intervals (0, 1, 3 and 6 months of

storage) and assessed for any change in GS, ZP, refractive index, drug content, viscosity

and pH in addition to physical appearance (clarity and phase separation). The clarity and

phase separation of the NE/MNE formulations were determined by visual examination

under light alternatively against white and black backgrounds. NLC formulations were

rehydrated with ultrapure water and evaluated for any change in PS, ZP, DL and EE at

specified time intervals (0, 1, 3 and 6 months of storage). Further, polymorphic transition

of lipid in NLC formulations upon storage at 25±1oC was assessed using DSC and

thermograms were recorded at 0 and 6 months.


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5.16 Pharmacokinetic and brain uptake studies

The animal experiments were carried out with approval from institutional animal ethical

committee (IAEC), J.S.S College of Pharmacy, Udhagamandalam, India (Proposal no.

JSSCP/IAEC/Ph.D/Ph.Ceutics/01/2013-14). Pharmacokinetic and brain uptake

studies were carried out using male wistar rats for CUR and DZP loaded NLC

formulations (reconstituted with 1 mL of saline), NE, MNE and drug solution (Haque et

al., 2012).

Table 22. Grouping of animals for pharmacokinetic studies

Group Formulations Dose

I Curcumin solution, CS (IV)

1mg/kg

II Curcumin solution, CS (IN)

III Curcumin NLC, CNLC (IN)

IV Curcumin NE, CNE (IN)

V Curcumin mucoadhesive nanoemulsion, CMNE (IN)

Group Formulations Dose

I Donepezil solution, DS (IV)

0.3mg/kg

II Donepezil solution, DS (IV)

III Donepezil NLC, DNLC (IN)

IV Donepezil NE, DNE (IN)

V Donepezil mucoadhesive nanoemulsion, DMNE (IN)

Rats were housed in cages and had free access to standard laboratory diet (Lipton feed,

Mumbai, India) and water ad libitum. Animals were maintained at 22±1oC and 65±5%

relative humidity. The efficacy of IN route was compared with that of IV route. Before

nasal administration, the rats were lightly anesthetized by exhaling diethyl ether.

Intranasal administration was carried out with help of micropipette attached to low

density polyethylene tube having 0.1mm internal diameter. Animals (n=3) were

sacrificed by cervical dislocation and blood samples of approximately 0.25mL were

collected by cardiac puncture at the following time points: 0.25, 0.5, 0.75, 1, 2, 4 and 6h.

Blood samples were placed into tubes containing 0.3mL of anticoagulant solution and

centrifuged at 3,000rpm for 15min. After centrifugation, the plasma obtained was stored

at -20oC until analysis. The brain samples were collected by cutting open the skull, rinsed

with saline solution and blotted with filter paper to remove the blood taint and blood

vessels. Brain samples were homogenized in PBS (pH 7.4) to determine the amount of


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drug in the brain tissue. The homogenate was centrifuged at 6,000rpm for 15min at 4oC;

supernatant was collected and frozen at -20oC until further analysis by HPLC.

Pharmacokinetic parameters like elimination rate constant (Ke), half life (t1/2) and area

under curve (AUC). Cmax (peak plasma concentration) and Tmax (time of peak plasma

concentration) were obtained directly from the plasma concentration-time profile and

brain concentration-time profile. All data were expressed as the mean ± S.E.M. (standard

error mean).

Further, drug targeting efficiency (DTE%) that represents time average partitioning ratio

was calculated as follows:

Drug targeting efficiency (DTE%)= (AUCbrain/AUCblood) IN × 100 (7)

(AUCbrain/AUCblood) IV

Nose to brain direct transport percentage (DTP%) was calculated as follows:

Direct transport percentage (DTP%)= BIN – Bx × 100 (8)

BIN

where Bx = (BIV/PIV) x PIN,

Bx is the brain AUC fraction contributed by systemic circulation through the BBB

following intranasal administration;

BIV is the AUC0-6 (brain) following intravenous administration;

PIV is the AUC0-6 (blood) following intravenous administration;

BIN is the AUC0-6 (brain) following intranasal administration;

PIN is the AUC0-6 (blood) following intranasal administration

Pharmacokinetic data was analyzed by one way ANOVA followed by Bonferroni post

test. Values are said to be significant when p < 0.05.

5.17 Pharmacodynamic studies

5.17.1 Animals

Three-month old male Wistar rats weighing 250-300g were obtained from the central

animal facility of JSS College of Pharmacy, Udhagamandalam, Tamilnadu, India. The

animals were acclimatized to laboratory conditions ten days prior to the commencement

of the experiment in order to adapt to the environmental conditions of light-dark cycle

(12:12 h), relative humidity (55-65%) and temperature (21±10oC). The animals were

housed in polypropylene cages with sterilized paddy husk bedding. Food and water were

available ad libitum throughout the experimental period. All the experiments were

carried out with prior approval of the institutional animal ethical committee (IAEC),


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J.S.S. College of Pharmacy, Udhagamandalam, India (Proposal no. JSSCP/IAEC/

Ph.D/Ph.Ceutics/03/2012-13).

5.17.2 Experimental induction of AD

Animals were anesthetized using ketamine hydrochloride (70 mg/kg, IP) and secured in

a stereotactic frame. A midline sagittal incision on the scalp was made and burr holes

were drilled on the both sides of the skull with coordinates at 0.8 mm posterior to bregma

and 1.5 mm lateral to the sagittal suture. Forty eight animals were injected with 20 µL of

sSTZ dissolved in artificial cerebrospinal fluid (aCSF) at a dose of 3 mg/kg (Ishrat et al.,

2009; Sharma and Gupta, 2001) bilaterally into the lateral ventricles by placing the

Hamilton syringe needle 3.6 mm beneath the surface of the brain. The procedure was

repeated 48 h later. The sham operated group consisting of 8 animals were treated

following a similar procedure but were administered aCSF (aCSF:147mM NaCl; 2.9mM

KCl; 1.6mM MgCl2; 1.7mM CaCl2 and 2.2mM dextrose). Animals were then sutured, an

antiseptic (Betadiene ointment and Neosporin) was applied to the wound and they were

injected with saline (1 mL, subcutaneously) to prevent dehydration. Animals were

transferred to a thermo-regulated chamber to maintain normal body temperature until

recovery and then transferred to their home cages. Animals were divided into 7 groups

consisting 8 in each and assigned as shown in Table 23.

Table 23. Grouping of animals for pharmacodynamic studies

5.17.3 Behavioural studies

5.17.3.1 Radial arm maze (RAM) task

RAM is used to assess reference and working memory in animals (Bhagya et al., 2011).

The RAM apparatus consists of eight identical arms radiating from the centre of the

platform (Fig.14). The apparatus was elevated to a height of 28 cm from the ground and

Group Treatment Dose

1 Sham ---

2 Negative control (NC) ---

3 DZP Oral 3mg/kg

4 CUR Oral 50mg/kg

5 DMNE IN 0.3mg/kg

6 CMNE IN 1mg/kg

7 D+CMNE IN 0.3mg/kg+1mg/kg


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placed in a small room containing a number of visual clues. On 12th day of 1st STZ

injection, animals received training for 2 days.

Fig. 14. Radial Arm Maze (RAM) Apparatus

Training

Prior to the acquisition, all the arms were baited and rats were allowed to explore the

maze for 10 min and were subjected to two such acclimatization sessions on consecutive

days (Day 12-13).

Fig. 15. Scheme of experimental procedure


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Acquisition

Rats were given two trials a day for 30 days (Day 14-43). At the beginning of each trial,

the maze was thoroughly cleaned with 70% ethanol and four of the arms (2, 3, 6, and 8)

were baited with food reinforcement (Kellogg’s Planets and Stars™, Kellogg India Ltd.,

Mumbai, India). The rat was placed in the center of the octagon and was allowed a free

choice. An arm choice was recorded when a rat ate a bait or reached the end of an arm.

The maze arms were not rebaited, so only the first entry into the baited arm was recorded

as a correct choice. The trial continued until the rat entered all the four baited arms or 5

min had elapsed. At the end of the trial, the rat was returned to the home cage and was

given the second trial after an intertrial interval of 1 h. Training was continued until the

rats attained the criteria of 80% correct choice (at least 4 correct entries out of 5 entries).

The scheme of experimental procedure is given in Fig. 15.

5.17.3.2 Statistical analysis

Behavioural parameters were analysed by two-way ANOVA followed by Tukey post-

test. Values are said to be significant when p < 0.05.

5.17.4 Biochemical parameters

5.17.4.1 Drugs and Chemicals

20% Acetic acid solution (pH 3.5)

20mL of acetic acid (99%) dissolved in 80 mL of water and the pH was adjusted to 3.5

with 0.1 N NaOH solution.

Acetylthiocholine

216.7 mg of Acetylthiocholine dissolved in 10 mL of distilled water.

DTNB (5,5ɂ-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent)

39.6 mg of DTNB dissolved in 10 mL of phosphate buffer pH 7.0 containing 15mg

sodium bicarbonate.

Ethylene diamine tetraacetic acid (EDTA)

37.22 mg of EDTA was dissolved in 100 mL of water.

Hydrogen peroxide solution (7.5mM)

21µL of commercial H2O2 solution was made upto 100mL with distilled water.

Potassium phosphate buffer (65mM pH 7.8)

2.2g of potassium dihydrogen phosphate and 11.23g of dipotassium hydrogen phosphate

were dissolved in 250mL and 1L of water respectively and then mixed together. The pH

was adjusted to 7.8 with potassium dihydrogen phosphate solution.


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Sodium carbonate buffer (0.05mM pH 10.2)

5.3g of sodium carbonate and 4.2g of sodium bicarbonate were dissolved separately in 1

L of distilled water which served as stock solution. Buffer was prepared by mixing 64mL

of sodium carbonate and 70mL sodium bicarbonate solutions. The pH of the buffer was

adjusted to 10.2 using above stock solutions.

8% Sodium dodecyl sulfate (SDS)

8g of SDS was dissolved in 100 mL of water.

Sucrose solution

10.95g of sucrose was dissolved in 100mL of distilled water.

10% Trichloroacetic acid solution

10g of trichloroacetic acid was dissolved in 100mL of distilled water.

Thiobarbituric acid(0.9%)

0.9g of thiobarbituric acid was dissolved in 100mL of distilled water. The ph was

adjusted to 7.4 with 0.1 N NaOH/0.1N HCl solutions.

5.17.4.2 Tissue preparation

At the end of 30 days treatment following behavioural studies, animals were

anaesthetized with ether. The animals were decapitated, brains extracted and cleaned

with chilled ice cold saline. The cerebral hemispheres were separated and the left

hemisphere was fixed in neutral buffered paraformaldehyde (10% v/v) for histological

examination. The hippocampus and frontal cortex were dissected from the right

hemisphere and weighed. A 10% (w/v) homogenate of brain samples (0.03 M sodium

phosphate buffer, pH-7.4) was prepared by using teflon glass homogenizer. The

homogenate was centrifuged at 1000 rpm 4oC for 3 min and the supernatant divided into

two portions, one of which was used for measurement thiobarbituric acid reactive

substance (TBAR). The remaining supernatant was again centrifuged at 12,000 rpm at

4oC for 15 min and used for the measurement of AChE, superoxide dismutase (SOD),

catalase (CAT), and glutathione (GSH). Protein and homogenized used for estimation of

biochemical parameters like AChE activity, lipid peroxidation, glutathione, superoxide

dismutase and catalase.

5.17.4.3 Estimation of AChE

Cholinergic dysfunction was assessed in terms of acetylcholinesterase (AChE) activity.

The quantitative measurement of AChE levels in brain were performed according to the

method of (Ellman and Courtney, 1961). The assay mixture contained 0.05 mL of


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supernatant, 3mL of 0.01M sodium phosphate buffer (pH 8), and 0.10 mL DTNB and

absorbance was measured at 412nm until absorbance stopped increasing and finally set to

zero. About 0.10 mL of acetylthiocholine iodide was added and the change in

absorbance was measured at 412 nm for 5 min. The enzyme activity was expressed as

micromoles/min/mg of protein. Results were calculated using molar extinction

coefficient of chromophore (1.36 x 10-4M-1cm-1) and expressed as percentage of control.

The equation describing absorbance is A = ε*c*l (also known as Beer-Lambert law),

where:

A – absorbance measured in OD (Optical Density units)

ε – epsilon, the molar extinction coefficient

c – concentration measured in molarity

l – light path length measured in centimeters

5.17.4.4 Estimation of TBARS

The extent of lipid peroxidation in the brain was determined quantitatively by the

method by (Wills, 1966). The incubation mixture consists of 0.5mL of supernatant,

0.2mL sodium dodecyl sulphate (8%), 1.5mL acetic acid solution (20%, pH 3.5), 1.5mL

of thiobarbituric acid (0.9%, pH 7.4). The incubation mixture was made upto 5 mL with

double distilled water and then heated in boiling water bath for 30 min. After cooling, the

red chromogen was extracted into 5 mL mixture of n-butanol and pyridine (15:1 v/v)

centrifuged at 4000 rpm for 10 min, the organic layer was separated and its absorbance

measured at 532nm. The values were calculated using the molar extinction coefficient of

chromophore (1.56 x 10-5M-1cm-1).

5.17.4.5 Estimation of SOD

Superoxide dismutase measurement was carried out by the ability of SOD to inhibit

spontaneous oxidation of adrenaline to adrenochrome (Saggu et al., 1989). A mixture of

2.80 mL of sodium carbonate (0.05mM) buffer (pH 10.2), 100µL of EDTA (1.0mM) and

20µL of brain homogenate or sucrose (blank) were incubated at 30oC for 45 min.

Thereafter, reaction was initiated by adding 100µL of adrenaline solution (9.0mM). The

change in the absorbance was recorded at 480nm for 8-12 min. Similarly, SOD

calibration curve was prepared by taking 10units/mL of standard solution. One unit of

SOD produced approximately 50% inhibition of auto-oxidation of adrenaline. The

results are expressed as SOD µmol/min/mg of protein.


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5.17.4.6 Estimation of CAT

Catalase measurement was carried out by the ability of CAT to oxidize hydrogen

peroxide (H2O2). 2.25 mL of potassium phosphate buffer (65mM, pH 7.8) and 100µL of

the brain homogenate were incubated at 25oC for 30 min. A 650µL H2O2 (7.5mM) was

added to the brain homogenate to initiate the reaction. The change in absorption was

measured at 240nm for 2–3 min and the results were expressed as CAT µmol/min/mg of

protein (Beer and Seizer, 1952).

5.17.4.7 Estimation of GSH

Glutathione was measured according to the method of Ellman (Ellman, 1959). The equal

quantity of brain homogenate was mixed with 10% trichloroacetic acid and centrifuged

to separate the proteins. To 0.1 mL of this supernatant, 2mL of phosphate buffer (pH

8.4), 0.5 mL of DTNB and 0.4 mL of double distilled water were added. The mixture

was vortexed and absorbance read at 412nm within 15 min. The concentration of

reduced glutathione was expressed as nmol/mg of protein.

5.17.4.8 Statistical analysis

Biochemical parameters were analysed using one-way ANOVA followed by Tukey

multiple comparison tests. Values are said to be significant when p < 0.05.

5.17.5 Histology and neuronal count

Hippocampus and frontal cortex was dissected from the left hemisphere of the animals

and fixed in neutral buffered formaldehyde (10% v/v). The hippocampus and frontal

cortex was sectioned at 3 mm thickness and 5-10 mm slices, using a Leica RM 2135

microtome and stained with H and E. Another batch of samples were stained with cresyl

violet (CV) acetate (MP Biomedicals, Mumbai, India) and CV positive neurons were

counted using Motic microscope fitted with camera according to a procedure described

earlier (Park et al., 2012). The number of cresyl violet positive neurons in all groups was

counted in a 250×250 μm square, applied approximately at the medial, center and lateral

parts of the CA1 region in the stratum pyramidale. Cell counts were obtained by

averaging the counts from the sections taken from each animal. The number of CV

positive neurons was compared to those in the each sham group.

5. materials and methods 5.1 materials -...

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