113

MATERIALS AND METHODS

114

MATERIALS

Absolute alcohol Hayman,England Acetone, AR Rankem, New Delhi

Acetonitrile Rankem, New Delhi

Ammonia, AR E.Merk (I) Ltd., Mumbai Calcium Carbonate SD Fine Chemicals, Mumbai Calcium chloride, AR SD Fine Chemicals, Mumbai Chitosan Central Institute of Fisheries (Indian

Fisheries Institute, Coachin, India)

Chloroform Rankem, New Delhi

Chloroform, AR Rankem, New Delhi

Cholesterol estimation Kits Spans Diagnostcs Ltd., India Disodium hydrogen orthophodphate, AR

Rankem, New Delhi

D-Sphingosine Sigma Chemicals, Co., USA Dulbecco modified eagle medium

(DMEM)

Invitrogen life sciences, USA

Ethanol, AR Loba Chemicals, Mumbai Ethyl acetate Qualigens fine chemical, Mumbai Fetal bovine serum (FBS) Invitrogen life sciences, USA

Formaldehyde SD Fine Chemicals, Mumbai Formic acid Loba Chemie Pvt. Ltd., Mumbai

Human normal skin keratinocyte cell

line, (HaCaT)

Provided by Dr. Sudhir Krishna

(National Centre for Biological Sciences,

Bangalore, India)

Hydrochloric acid Rankem, New Delhi

Iso-amyl alcohol Loba Chemie Pvt. Ltd., Mumbai

Isobutanol Merk Ltd., Mumbai

Methanol, AR Rankem, New Delhi

n-Butanol, AR Rankem, New Delhi

Octanol Loba Chemie Pvt. Ltd., Mumbai

O-ohthaldehyde SRL, Mumbai

O-phosphoric acid, AR SD Fine Chemicals, Mumbai

Penicillin Invitrogen life sciences, USA

115

Polyethylene glycol 400, LR Rankem, New Delhi

Potassium Chloride, AR E. Merk (I) Ltd., Mumbai

Potassium dihydrogen phosphate,

AR

Rankem, New Delhi

Propylene glycol, AR E. Merk (I) Ltd., Mumbai

Repaglinide

Sodium azide, AR CDH, Ltd., New Delhi

Sodium Chloride E. Merk (I) Ltd., Mumbai

Sodium hydroxide, AR Rankem, New Delhi

Sodium Hyroxide Rankem, New Delhi

Streptomycin Invitrogen life sciences, USA

Streptozotocin Sigma Chemicals, Co., USA Sulphuric acid

Rankem, New Delhi

Triglyceride estimation Kits

(Enzokits)

Ranbaxy Laboratories Ltd., Gurgaon

116

EQUIPMENTS

12 well plate Cat No.-150628, Denmark

1ml/1ml pipette Cellstar, greiner bio-one

60mm dish

Becton Dickinson (USA) - 35 3002

Aqua check

(Roche Diagnostics, Germany).

Centifuge Remi Equipments, Mumbai

Centrifuge eppendroffs 5415R

Centrifuge eppendroffs 5415R

Confocal Lasar Scanning Microscope Zeiss Axioplan fluorescence microscope

Differential scanning calorimetry Mettler toledo, 821E, switzerland

Diffusion cell apparatus Fabricated by USIC, Punjabi University, Patiala

Electronic weighing balance

Afcoset (ER 182A)

Falcon (15 and 50 ml) Tarson

Gel Doch Uvipro Platinum(made in UK)

High performance liquid chromatography Water’s HPLC system with waters 600 controller pump, water’s inline degasser, Waters HPLC system equipped with 515 binary pump, degasser and a 2487 dual wavelength ultraviolet detector (UV), rheodyne manual injector and RP C-18 column (250 × 4.6 mm, particle size 5 µm) and Empower-2 software.

Homogenizer Remi Equipments, Mumbai

HPTLC

Camag, linomat IV sample applicator fitted with 100 µl syringe (hamilton, Bonaduz, Switzerland), Camag

117

winCATS planar chromatography manager software (version 4)

Incubator

Narang scientific woks Pvt. Ltd., Ambala

Incubator Forma Scientific

Incubator Forma Scientific

Nunclon (6 well plate) Cat No.-140685, Denmark

Optical microscope Olympus

Orbital shaking incubator

C-24, Remi Equipment, Mumbai

pH meter

Esico,Model-1012, Ambala

Rotary evaporator Narang scientific woks Pvt.

Ltd., Ambala Scanning electron microscope Leo 435 svp, Cambridge,

UK Sonicator

Transsonic T570/H Elma, Germany

Soxhlet assembly Perfit India Pvt. Ltd

Spectrofluorimeter SL 174, Elico India T25 Cat No.-156367, Denmark

T75 Cat No.-156499, Denmark

Tewameter (TM 210) Courage and Khazaka Electronic GmbH, Koln, Germany

Transmission electron microscope Morgagni 268 D, Sei, Netherlands

UV-VIS Spectrophotometer Beckman, USA (DU 640B)

Vaccum desiccator Tarsons

Vorter shaker Popular Traders, Ambala Cantt.

Water bath

Narang scientific woks Pvt. Ltd., Ambala

118

METHODS

4.1 Preliminary studies on repaglinide (RGE)

4.1.1. Identification of RGE

Waters system equipped with 515 binary pump, degasser and a 2487

dual wavelength ultraviolet detector (UV), rheodyne manual injector and RP

C-18 column (250 mm × 4.6 mm, particle size 5 ìm) was used for HPLC

analysis. The mobile phase consisted of ammonium acetate: acetonitrile,

30:70 v/v. The drug solution (20 µl) was injected into the column (flow rate 1

ml/min) and the peak area and retention time was recorded.The output signal

was monitored and processed using Empower-2 software.

4.1.2. Analytical method validation of RGE

Stock solution of RGE was made by dissolving RGE in methanol and

further dilutions were made with phosphate buffer (PB) I.P pH 7.4 containing

polyethylene glycol 400 (10% v/v) and sodium azide (0.05%w/v). Polyethylene

glycol 400 and sodium azide were used in the receptor fluid, respectively, for

maintaining sink condition and as preservation of excised skin samples. All

solutions were stored at 4ºC and proctected from light. The stability of this

RGE solution was determined in presence as well as absence of light at 37ºC.

Spectrophotmetric conditions

All absorbances were measured at 243 nm wavelength

spectrophotometrically.

Spectrofluorimetric conditions

Solution of RGE prepared in different solvents were scanned

spectrofluorimeterically from 200-700 nm using SL-174 spectroflourimeter

(ELICO, India Ltd.). All fluorescence measurements were made using 20 nm

excitation and emission windows at a sensitivity of 450 nm. The fluorescence

spectra was recorded at a rate of 240 nm /min.

Chromatograhic conditions

Working standard solutions of RGE were prepared by suitable dilution

of the stock solution with the mobile phase (ammonium acetate: acetonitrile,

30:70 v/v). Each drug solution (20 µl) was injected into the column and the

119

peak area and retention time was recorded by HPLC method. The system

configuration was the same as detailed for identification purpose.

4.1.3. Standard Plot of RGE

In vitro standard plot of RGE using spectrophotometer/

spectrofluorimeter

The solvents used were methanol, phosphate buffer pH 7.4 (PB), PB

containing sodium azide and PEG 400 or PB containing constituents leached

from excised epidermis. Scans were performed in these systems with the

objective of ensuring that these materials did not interfere with excitation or

emission maxima of repaglinide. Fluorescence of different concentrations of

RGE solutions was read on both emission wavelength for each excitation

wavelength during pre excitation and pre emission scanning. Solution of RGE

prepared in different solvents were scanned spectrofluorimeterically from 200-

700 nm using SL-174 spectroflourimeter (ELICO, India Ltd.). Aliquotes of the

stock solution was serially diluted with buffer to obtain solutions ranging from

5 to160 µg/ml concentration of RGE. The fluorescence intensity of these

solutions was measured by employing emission wavelength of 379 nm and

excitation wavelength of 282 nm using PB containing 10% v/v PEG 400 and

0.05% sodium azide as blank. All fluorescence measurements were made

using 20 nm excitation and emission windows at a sensitivity of 450 nm. The

fluorescence spectra was recorded at a rate of 240 nm /min.

The influence of pH on fluorimetric attributes of RGE was assessed by

preparing solutions containing 50 µg/ml RGE in phosphate buffer (PB) in the

range of pH 1.2-12.2. Their spectra were recorded against a blank prepared

with the same reagent concentrations without RGE. The suitability of

fluorimetric analysis for estimating RGE during in vitro permeation

experiments across skin was assessed. Aliquots of stock solution of RGE

(1000 µgml-1) prepared in methanol were transferred to volumetric flasks and

volume adjusted with PB (pH 7.4), PB containing 10% v/v PEG and

(0.05%w/v) sodium azide or PB (pH 7.4) containing constituents leached

from excised epidermis. All solutions were scanned for excitation and

emission maxima.

120

In vivo standard plot of RGE using HPLC

The plasma samples were stored in the deep freezer. The samples

were thawn at room temperature before processing. The mobile phase

composed of acetonitrile–ammonium formate (pH 2.7; 0.01 M) (60:40, v/v).

The formate buffer was prepared fresh daily with double-distilled deionized

water. All solutions were filtered through a 0.45µm membrane (Sartorius,

Germany) prior to use. The flow rate was 1 ml/min and the column

temperature was maintained at 25 ºC. The volume of injection was 20 µl. The

column was equilibrated for at least 20 min with the mobile phase prior to

sample injection. Stock solutions of RGE (100 µg/ml) and indomethacin (100

µg/ml) were prepared by dissolving 1mg of each drug in 10 ml ethanol. The

standard solutions were stored at 4 ºC in a clear glass volumetric flask and

covered with aluminium foil. The data was aquired at 244 nm.

Calibration curve

The calibration curve of RGE in plasma was constructed in the range of

20-200 ng/ml to encompass the expected concentrations in measured

samples. Repaglinide concentrations in the working solution chosen for the

calibration curves were 20, 30, 60, 80, 100 and 200 ng/ml. Quality control

(QC) samples (of low, medium and high concentration) at 40, 70 and 150

ng/ml were prepared in the same way as the calibration standards. These

working solutions were prepared fresh daily by making further dilutions of the

stock solution in ethanol. Working standard solution of internal standard

indomethacin was prepared by diluting the stock solution prepared in ethanol

to obtain a final concentration of 500ng/ml. The internal standard

(indomethacin) and RGE solutions were added to blank plasma samples in

round bottom glass tubes. 1.0 ml extraction buffer (0.1 mol/l potassium

dihydrogen orthophosphate (KH2PO4, pH 5.9) was added to the tubes. The

mixture was vortexed. 5ml ethylacetate and 50µl isoamylalcohol were added

and the pH adjusted to 7.4 with 2MNaOH. The tubes were shaken on a rotater

for 10 min. This was followed by centrifugation at 3000 rpm for 30 min. After

centrifugation, the ethylacetate phase was transferred into V-tubes and

evaporated to dryness under a stream of nitrogen at 45 ºC. The dried extract

121

was reconstituted with 70µl of mobile phase, vortex and transferred to a clean

autosampler vial. 20µl of this solution was injected into the HPLC system.

Estimation of RGE in tablets by HPLC and spectrofluorimetric method

According to USP (2007) eight tablets were weighed to obtain the

average tablet weight and were then powdered. A sample of the powdered

tablets was dissolved in methanol and diluted with methanol:phosphate buffer

pH 4.0 (7:3) to obtain a solution having a concentration of 80 µg/ml. The

solution was filtered through a 0.45 um membrane filter and then injected for

HPLC analysis. The same samples were subjected to spectrofluorimetric

analysis.

4.1.4. Analytical Method Validation studies on RGE

Calibration curves

Calibration curves were prepared and assayed in triplicate on three

different days to evaluate linearity, precision, accuracy, limit of quantitation

(LOQ), limit of detection (LOD) and selectivity. The calibration curve was

constructed in the concentration range of 5-160 µg / ml for spectrofluorimeter

and spectrophotometer. The relative fluorescence intensity of solution was

measured against blank.

Precision and accuracy

The precision and accuracy of the fluorimetric assay were determined

by using solutions containing low (20 ng/ml), medium (40 ng/ml) and high (80

ng/ml) concentrations of RGE. The concentrations of RGE used for HPLC

method validation were 0.1µg/ml, 0.6 µg/ml, 1.2 µg/ml.

Limit of detection

Limit of detection (DL) is defined as the lowest amount of analyte in a

sample which can be detected but not necessarily quantitated as an exact

value. The limit of detection was calculated DL=3.3 σ/S

Limit of quantification

The limit of quantification was (DQ) calculated by using the

following formulas:

DQ=10 σ/S

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Where, ‘σ’ was obtained from standard deviation of intercepts of the

regressed lines and ‘S’ was the average slope of the regressed line.

Linearity

The calibration curves were obtained by least square linear regression

analysis. The range of drug concentration over which calibration curves

showed linearity was noted.

Intra-assay Precision and Accuracy

For intraassay precision and accuracy, fluorescence of six replicates of

samples were observed and they were analyzed on three consecutive days

along with standard calibration curves.

Inter-assay Precision and Accuracy

Inter-assay precision and accuracy was evaluated by processing a set of

calibration and quality control samples (three levels analyzed twice) in six

separate batches.

4.1.5. Stability of RGE in receptor fluid (PB)

The stability of repaglinide was investigated in PB as well as in buffer

containing sodium azide (0.05%w/v) as preservative both in presence and

absence of light as well as in cold conditions. Samples were withdrawn at

different time intervals and scanned for excitation and emission spectra to

further determine the fluorescence. The study was continued through 48 h.

4.1.6. Dose designing of RGE

To achieve an effective plasma concentration of repaglinide, the

required release rate of RGE was calculated by using the following formula

CpxVdxKe, where,Cp is the effective plasma concentration,Vd is the volume

of distribution and Ke is the elimination rate constant.

4.2 Extraction of alkaloids and vanilloids

Various plants containing alkaloids and vanilloids were surveyed from

literature. Piper nigrum and Zingiber officinale were selected on the basis of

their high content of alkaloid and vanilloid, respectively. Piper nigrum contains

piperine (5-10%) and Zingiber officinale contains gingerol (similar to vanilloid

moiety).

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4.2.1. Extraction of Piper nigrum

The dried fruits of piper nigrum (200 g) were coarsely ground to a fine

and extracted using Soxhlet Apparatus with 95% ethanol for 4 hours. The

extracted solution was cooled and filtered through fine filter paper. The

solution was concentrated under vacuum on water bath to remove ethanol. 10

mL of 10% w/v alcoholic KOH was added to the residue and left undisturbed

for 1h. The solution was decanted from the insoluble residue. The alcoholic

solution was allowed to stand undisturbed overnight to obtain long yellow

needles of piperine. The yellow needle shaped cryatals were separated and

washed by vacuum filtration and wash with minimum volume of 95% ethanol.

Finally the crystals were air dried and weighed.

4.2.2. Extraction method of Zingiber officinale

The dried rhizome (250g) was powdered mechanically and extracted

by cold percolation with 95% ethanol for 24 h. The extract was recovered and

95% ethanol was further added to the plant material and the extraction

continued. The process was repeated three times. The three extracts were

pooled together and the combined extract was concentrated under reduced

pressure (22–26 mmHg) at 45– 60°C. Solvent free extract (30 g) was

obtained.

4.3. Extraction of coumarins and furanocoumarins

4.3.1. Extraction method of Angelica archangelica

The dried roots of Angelica archangelica (200 g) was pulverized into

coarse powder. One-eighth of the coarse powder was further ground and

passed through a 200-mesh sieve (sample a). Seven-eighth of coarse powder

was orderly extracted by petroleum ether (bp 60–90 ◦C), 95% ethanol and

water under reflux for three times, respectively. The petroleum ether extract

was condensed under vacuum as yellow oil (3.50 g). The alcoholic extract

and aqueous extract were condensed under vacuum and dried to obtain

cream (19.25 g and sample d, 85.75 g), respectively. The material residue

was dried, ground and then passed through a 200-mesh sieve (59.50 g). All

extracts were frozen drying except the petroleum ether extract.

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4.3.1. Extraction method of Melilotus officinalis

The freshly flowering tops were sorted, dried at room temperature

(about 20–25ºC) to constant weight and ground to obtain a homogenous drug

powder. The material was stored in stoppered glass jars protected from

sunlight. The powder was extracted with ethanol 95 %v/v for 8 h using 250 ml

ethanol. The solvent was then evaporated to obtain a dark green powder.

4.4. Standardization of alkaloids and vanniloids present in Piper nigrum

and Zingiber officinale as well as of coumarins and furanocoumarin

present in Angelica archangelica and Melilotus officinalis using HPTLC

IR spectroscopy and HPTLC analysis were done for characterizing the

purified sample of piperine where as for other plants HPTLC was done. The

samples were spotted in the form of band (6mm) with a camag microlitre

syring on precoated silica gel aluminium plate 60F-254 (20 cm x 10cm x 0.2

mm) Camag Linomat IV (Switzerland). The plate was prewashed by methanol

and activated at 60ºC for 5 minutes prior to chromatography. A constant

Application rate of 15 s/ul was used and space between two band was 7 mm.

The slit dimension was kept at 6 mm x 0.2 mm and 100 mm/s scanned thrice

and baseline correction was used. 10 ml of mobile phase was used per

chromatography. Linear ascending development was carried out in 10 cm x

10 cm twin through glass chamber (Camag Switzerland). Densitometric

scanning was performed on Camag TLC scanner III in the reflectance-

absorbance mode and operated by CATS software (V 4.06, Camag).

4.5. Solubility of RGE

The solubility of RGE in PB, PB containing 10% v/v PEG 400 and

0.05% w/v sodium azide or in PB solutions containing different concentrations

of piperine (0.004-0.1% w/v), Zingiber officinale extract (ZO-E) (0.025% w/v-

0.1 w/v), Angelica archengelica extract (AA-E) (1-6% w/v) or Melilotus

officinalis extract (MO-E) (1-6 % w/v) was determined. Excess RGE was

added and stirred at 37±2⁰C in shaker incubator for 24 h. The solutions were

filtered through G-4 filter and the filtrates were immediately analyzed for RGE

spectrophotometrically at 243 nm when piperine or ZO-E was used. AA-E and

MO-E was analyzed spectrofluorimetrically using excitation and emission

125

wavelength respectively, at 282nm and 379nm. Each experiment was

performed in triplicate.

4.6. Partition coefficient of RGE

The partition coeffcient (KO/PB) was determined by adding RGE (40mg)

to a 1: 4 saturated mixture of octanol (1ml) and PB (5ml) containing PEG 400

(10% w/v) in absence or presence of different concentrations of piperine, ZO-

E and AA-E and MO-E. The mixture was stirred at 37±2ºC in shaker incubator

for 24 h and analyzed spectrophotometrically at 243 nm when piperine or ZO-

E was used. AA-E and MO-E was analyzed spectrofluorimetrically using

excitation and emission wavelength respectively, at 282nm and 379nm. The

concentration of RGE partitioned into octanol was calculated by difference.

The ratio of amount of RGE in octanol to that in PB was calculated. All

experiments were performed in triplicate.

4.7. Dose dependent influence of extracts on skin cholesterol,

triglycerides and sphingosine content in excised rat epidermis.

4.7.1. Skin preparation

Excised dorsal skin obtained from Wistar rats (either sex) was used for

the study. Dorsal hair was removed with mechanical clipper followed by

shaving with electric razor. The animal was sacrificed after 24 h by spinal cord

dislocation and the dorsal skin excised.The skin was wiped clean with

physiological saline. The whole skin was soaked in water at 60ºC for 45 sec,

followed by careful removal of dermis by gentle scrApping to obtain epidermal

sheet (Kligman and Christophers, 1963). Freshly obtained epidermal skin was

used in all experiments.

4.7.2. Total lipid extraction

Epidermis obtained from excised skin after treatments with different

cocentrations of piperine, ZO-E, AA-E, MO-E, chitosan (CTN) (1% w/v), CTN

and selected concentration of herbal extracts was cut into small pieces and

dried to constant weight at 50°C. Extraction of the skin lipids was done by

Folch method (1957). Skin was homogenized in methanol (10ml) for 1 min,

followed by homogenization in chloroform (20 ml) for additional 2 in. The

mixture was filtered and the residue was again homogenized in a solution (30

126

ml) of chloroform-methanol (2:1) for 3 min. The solid obtained after filteration

of the mixture was washed once each with chloroform (20 ml) and methanol

(10 ml). These filtrates were combained and potassium chloride (0.88% w/v)

equal to one fourth the total volume of filtrate was added with stirring. After

allowing it to settle, the upper layer was aspirated and discarded after allowing

the mixture to settle. The chloroform layer obtained after evaporation was

used for determination of cholesterol, triglycerides or sphingosine content

using respective methods as reported earlier (Babita et al., 2005). The

process was repeated for determining the effect of these extracts on viable rat

skin.

4.7.3. Cholesterol (CHOL) estimation

The cholesterol content was determined by following the method

outlined by Ranbaxy Laboratories Ltd. (Enzokits). Briefly, three samples

blank, standard and test was prepared. The test sample obtained 0.025 ml of

lower layer obtained after the extraction process explained above along with

CHOL reagent (5ml). CHOL reagent (5ml). The standard contained CHOL

reagent (5ml) and 0.025 ml of working cholesterol standard. All the samples

were placed immediately I boiling water for 90 sec and then cooled. The

optical density (O.D) of all the three samples was measured at 560 nm. CHOL

content was calculated by using the formula (O.D of test sample / O.D of

standard sample) X 200.

4.7.4. Triglycerides (TGL) estimation

The TGL content was determined by following the method outlined by

Ranbaxy Laboratories Ltd. (Enzokits). Briefly, three samples blank, standard

and test was prepared. The test sample contained 10 µl of lower layer

obtained after the extraction process explained above along with working

reagent (1 ml). The blank contained only working reagent (1 ml). The standard

contained working reagent (1 ml) and 10 µl of triglycerides standard. The

respective solution was mixed and incubated at 37 ºC for 5 min. The

absorbance of test (AT), standard (AS) and blank (AS) were measured at 546

nm. The TGL content was calculated by using formula: [(AT- AS) ÷ (AS-AB)] X

100.

127

4.7.5. Sphingosine (SPE) estimation in excised skin

For SPE estimation, the residue obtained after evaporation in above

section was derivatized with o-phthalaldehyde solution (0.5 mg/ml containing

3% boric acid, pH 10.5 0.05% 2-mercaptoethanol). SPE content was

estimated spectroflouometrically by utilizing excitation wavelength of 340 nm

and emission wavelength 455 nm (Sabbadini et al., 1993). A standard plot of

D-sphingosine (50-400 µg/ml) was prepared spectroflourimetrically after its

derivatization with o- phthalaldehyde.

4.8. Cholesterol, triglyceride and sphingosine estimation in epidermis

obtained from piperine, ZOE or AA-E, MOE treated viable rat epidermis

Two patches were prepared on dorsal side of rat by shaving with

mechanical clipper. One patch was treated with PG: Alcohol (7:3) that served

as control. The second patch received tretment with CTN (1% w/v) or

piperine, ZOE or AA-E, MOE or a mixture containing CTN (1% w/v) or selectd

concentration of respective alkaloid/vanniloid or comarin or furanocoumarin.

Surface area exposed to treatment was kept constant. The animals were

sacrificed after (4 h, 12 h, 24 h, or 48 h). The treated skin patches were

excised and epidermis separated. These epidermal sheets were dried to

constant weight and total lipids were extracted respectively by Folch method.

CHOL, TGL and SPE content in these extracts were determined by using

methods described above. Percentage of CHOL / TGL / SPE extracted was

calculated by using the formula: [1 − (content remaining in skin after treatment

÷ content in normal skin) × 100]. All the experiments wer carried out in

triplicate.

4.9. Thermotropic studies on piperine, ZOE or AA-E, MOE treated

excised skin

The skin was excised, adhering fat and visceral debris removed and

washed with physiological saline solution. Epidermis was separated and

treated with different concentrations of piperine, ZO-E or AA-E, MO- E, CTN

(1% w/v) or selected dose of extract–CTN mixture for 48 h. After washing, it

was dried to get constant weight and then stored in a chamber containing a

saturated solution of sodium chloride (75% RH at 25°C) for 3–4 days. DSC

128

analysis of these samples were performed using heating rate of 1°C/min over

ambient to 120°C. All DSC curves were evaluated especially with regard to

the phase-transition enthalpies (peak areas) and maximum temperatures

(Tm) of lipid phase transitions. The percentage reduction in enthalpy (H) of

endothermic transitions (j/g) was evaluated by using the formula:

of lipid endotherm due to treatment100 *100

of lipid endotherm in untreated epidermis

H

H

∆−

∆

All the experiments were carried out in triplicate.

4.10. In vitro permeation of RGE

4.10.1. Selection of Donor vehicle

Permeation of RGE was studied across epidermis obtained from

excised full thickness rat skin. The epidermal sheets were stabilized in

receptor fluid for 4 hr. RGE was dispersed in ethanol, propylene glycol:

ethanol (7:3), ethanol: propylene glycol (3:7) or octanol: ethanol (9.5:0.5) and

loaded in donor compartment. The receptor fluid (25 ml) consisted of PB,

sodium azide (0.05% w/v) as preservative and polyethylene glycol 400 (10%

v/v) for maintaining sink condition. The stirring rate was at 120 rpm. The entire

cell assembly was securely positioned in a thermostatically controlled water

bath maintained at 37±2ºC and was protected from light. Samples (1ml) were

withdrawn repeatedly through 48h and the samples were analyzed for the

amount of RGE permeated by spectrophotometer at λmax 243 nm as well as

by spectrofluorimetric analysis using 282 nm as excitation and 379 nm as

emission wavelengths. An equal amount of PB containing sodium azide

(0.05% w/v) and polyethylene glycol 400 (10% v/v) maintained at 37±2ºC was

replaced into receptor compartment after each sampling.

4.10.2. In vitro permeation of RGE across treated excised rat epidermis

Excised dorsal skin obtained from Wistar rats (either sex) was used for

the study. Dorsal hairs were removed with mechanical clipper followed by

shaving with electric razor. The skin was wiped clean with physiological

saline. The whole skin was soaked in water at 60 ºC for 45 sec, followed by

careful removal of dermis by gentle scrapping to obtain epidermal sheet

129

(Kligman and Christophers, 1963). . Freshly obtained epidermal sheets were

mounted between the donor and receptor compartments. Different

concentrations of piperine, ZO-E, AA-E or MO-E were admixed with RGE

dispersed in 5 ml of PG: EtOH (7:3) solution. The donor compartment was

sealed using a parafilm. Receptor fluid contained phosphate buffer saline (pH

7.4), sodium azide (0.05%) and PEG 400 (10% v/v). The receptor fluid

samples were withdrawn repeatedly through 48 h and analyzed

spectrophometrically at 243 nm when piperine or ZO-E were used as

enhancers and spectrofluorimetrically for amount of RGE permeated into the

receptor compartment employing excitation and emission wavelength of,

respectively, 282 nm and 379 nm when AA-E or MO-E was used as

enhancer.

4.10.3. In vitro permeation of RGE across epidermis obtained from

treated viable rat epidermis

One patch was prepared on dorsal side of rat by shaving with

mechanical clipper. The patch received treatment with selected dose of

piperine, ZO-E, AA-E or MO-E mixture for different studies. Surface area

exposed to treatment was kept constant. The animals were sacrificed after (4

h, 8h, 12 h, 24 h, 36 h and 48 h). The treated skin patches were excised;

epidermis separated and used for in vitro permeation studies as outlined

earlier. Experiments were carried out in triplicate.

4.11. Microscopic studies

4.11.1. Transmission electron micrograph (TEM)

TEM investigations were carried out to visualize the change in ultra

structure of rat epidermis. Selected concentrations of extracts, CTN or mixture

of CTN and selected concentrations of extracts were treated for 48 h when

applied to excised epidermis for different time intervals (4 h, 8 h, 12 h, 24 h,

48 h) to viable skin. The treated samples were fixed in Karnovosky’s fixative

(2% paraformaldehyde and 1% (w/v) glutaraldehyde in 0.1M phosphate buffer

(pH 7.4) and processed (Van den Bergh et al., 1997). Ultra-thin (60–90 ° A)

sections were cut and double stained with uranyl acetate and lead citrate for

TEM observation (Morgani-268).

130

4.11.2. Scanning electron microscopy (SEM)

The epidermal samples (as obtained for TEM studies) were fixed for 4

h at 4°C in a 2.5% w/v gluteraldehyde in 0.1 M cacodylate buffer (pH 7.2). The

samples were washed three times in 0.1 M cacodylate buffer (pH 7.2) for 10

min each to remove excess fixative. Post fixation was performed for 1 h at

4°C in 1% w/v osmium tetraoxide solution in 0.1 M cacodylate buffer (pH 7.2).

After rinsing two times in buffer, these specimens were dehydrated by placing

into graded ethanol solutions and critically dried in a critical point drier. The

samples were then mounted on a clean aluminum stubs with silver PAG-915

and coated with gold palladium alloy (160 angstrom thickness) on a sputter

coater (Singh et al., 1998). Specimens were then observed under scanning

electron microscope (LEO 435VP) and photographed.

4.12. Transeidermal water loss measurements:

Wistar rats (Approximately 250 g) were anesthetized. The hair of the

dorsal skin was shaved with electric clippers. Two patches (7 cm2), one on

either side of spinal cord, were prepared by shaving with mechanical clipper

and left undisturbed for 24 h. One patch was left utreated and served as

control. The other patch received treatment with selected dose of extract,

CTN (1% w/v) or solution containing mixture of CTN (1% w/v) and selected

concentrations of extracts. The laboratory temperature and humidity were

maintained at, respectively, 25± 2°C; 55 ± 5% RH. The control and treatment

sites were marked as circular area (~7 cm2) with a felt tip marker on the dorsal

surface of the rat. Surface area exposed to treatment was kept constant.The

rats were anaesthetized during the measurements of TEWL. TEWL was

recorded on each site after (4 h, 8h, 12 h, 24 h, 36 h or 48 h) after removal of

the patch by using Tewameter TM 210 (Courage + Khazaka Electronic

GmbH, Koln, Germany). Data was stored and analyzed by the

TEWAMETERÒ computer software. The probe of the Tewameter was placed

perpendicular to the surface of the skin and a stable reading of TEWL was

reached in about 60 s. The results were expressed in g/hm2. Each experiment

was replicated on three skin patches prepared on different rats.

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4.13. Draize Test:

The study employed albino rabbits (1.2-2.5 kg) of either sex (n=5)

for testing skin irritation. The procedure adopted for this study was in

accordance to that described earlier. The area on the back of each rabbit

was shaved so as to accommodate two patches each of two square inch

area. Patch made from two layers of light gauze was dipped in crude

extracts solutions prepared in PG: EtOH (7:3). The animals were

immobilized in the special holder during the 24-hour patch exposure.

Upon removal of the patches the animals were observed for any sign of

erythema or oedema for a period of 72 hours and scored as reported by

Mullerdecker et al. (1994). The observations were repeated after 72

hours.

4.14. Evaluation of pharmacokinetic parameters of RGE in animals:

Wistar rats of either sex (230-250g) were used for the bioavailability

studies. The animals were selected after superficial examination of the skin

surface for abnormalities. The Wistar rats were housed under controlled

environmental conditions (temperature, 25± 2°C; humidity, 55 ± 5%) and fed

with a commercial diet with free access to water. About 10 cm2 of skin was

shaved on the dorsal side. Rats were observed for 24 hours for any untoward

effect of shaving. The rats were divided into the following groups each

comprising of 6 rats.

Group I : Oral administration of RGE (1 ug/kg) after overnight fasting.

Group II : Application of transdermal patch containing RGE + piperine

(0.008% w/v) mixture dispersed in donor vehicle.

Group III : Application of transdermal patch containing RGE + piperine

(0.008% w/v) - CTN (1% w/v) mixture dispersed in donor

vehicle.

Group IV : Application of transdermal patch containing RGE + ZO-E

(0.05%w/v) mixture dispersed in donor vehicle.

Group V : Application of transdermal patch containing RGE + ZO-E

(0.05%w/v) - CTN (1% w/v) mixture dispersed in donor vehicle.

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Group VI : Application of transdermal patch containing RGE + AA-E

(5%w/v) mixture dispersed in donor vehicle.

Group VII : Application of transdermal patch containing RGE + AA-E

(5%w/v) - CTN (1% w/v) mixture dispersed in donor vehicle.

Group VIII : Application of transdermal patch containing RGE + MO-E

(3%w/v) mixture dispersed in donor vehicle.

Group IX : Application of transdermal patch containing RGE + MO-E

3%w/v) - CTN (1% w/v) mixture dispersed in donor vehicle.

Blood sample (0.3 ml) via the post-orbital venous plexus veins was

collected in heparinized tubes at 1, 2, 4, 6, 8, 12, 24, 28, 36, 42 and 48 h. The

protocol for the study was approved by Animal Ethics Committee of the

Department of Pharmaceutical Sciences and Drug Research Punjabi

University, Patiala. The blood samples were transferred to a heparinized

eppendoff tube, mixed gently, and then centrifuged (4000 rpm, 5 min) to

obtain 200µl plasma, which was kept at −20°C until analysis. The

concentration of RGE in the plasma was determined by HPLC analysis. The

plasma samples (1 ml) were extracted using extraction buffer (0.1 mol/l

potassium dihydrogen orthophosphate (KH2PO4, pH 5.9) 1 ml, ethylacetate 5

ml and isoamylalcohol 50µl. The pH was adjusted to 7.4 with NaOH 2M. The

tubes were vortexed for 10 min. This was followed by centrifugation at 3000

rpm for 30 min. After centrifugation, the ethylacetate phase was transferred

into vials and evaporated to dryness under a stream of nitrogen at 45°C. The

dried extract was reconstituted with 70 µl of mobile phase, vortex-mixed and

transferred to a clean autosampler vial. This solution was injected into the

HPLC system for analyzing RGE.

4.15. Confocal laser scanning microscopy

4.15 .1. Cells, media and culture conditions

Human normal skin keratinocyte cell line (HaCaT), provided by Dr.

Sudhir Krishna (National Centre for Biological Sciences, Bangalore, India)

was maintained in Dulbecco modified eagle medium (DMEM) with 10% fetal

bovine serum (FBS), 100 units/ml penicillin and 100 µg/ml streptomycin

(Invitrogen life sciences, USA). The FBS for culturing HaCaT cells was heat

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inactivated for 30 mins at 55°C. HaCaT cells were cultivated according to the

method described by Brandner (2000). The cells were maintained at 37°C in a

humified atmosphere with 5% CO2. Second or third passage HaCaT cells

were plated on coverslips in a 12 well plate at a density of about 5,000 cells/

cm2. After overnight incubation at 37°C in a humified 5% CO2 incubator, cells

were supplemented with fresh DMEM medium containing 10% FBS. The cells

were incubated untill they were approximately 50-60% confluent. Cells were

made serum free 16-24 hrs prior fixation and treated with solutions containing

different concentrations of piperine, ZO-E, AA-E or MO-E for 6 h. Extract in

%age w/v were prepared in PG: EtOH (7:3) solution and the solution was

sterilized by filtering (Sartolab. vl15, pore size 0.2 lam), followed by serial

dilution in DMEM to obtain solutions. The 100% confluent cells in 12 well plate

were treated with solutions containing concentrations of extracts. A control

without addition of different extracts was also prepared. The cells were

cultured under the conditions described above for 6 h. The growth of cells was

monitored on an inverted-phase Nikon TMS light microscope. All experiments

were repeated three times.

4.15.2. Immunoflourescence

Fixation was performed by immersing the cells into freshly prepared

4% paraformaldehyde for 10 minutes. Immediately after fixation, cells were

permeabilized with 0.1% TritonX-100 (sigma) for 5 minutes. Cells were then

rinsed with phosphate buffer saline (PBS) for 3 minutes and blocked in 10%

FBS in PBS for 1hr. This was followed by incubation with primary anti-TJP1

(ZO-1) antibody (1:100 dilution) for 2hr at room temperature (or overnight at

4°C). After three rinses in PBS for 10 minutes each, coverslips were

incubated with FITC-conjugated anti rabbit IgG at 10ug/ml in PBS containing

2% FBS for 2h. Slides were then rinsed three times in PBS and coverslips

were mounted in antifade mounting medium (Vector laboratories). Nucleus

was stained using propidium iodide (5 µg / ml). Control for antibody specificity

was prepared by omitting the primary antibody. The control demonstrated

minimal or no staining. For microscopic examination, a Zeiss Axioplan

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fluorescence microscope (Zeiss) equipped with a confocal scanning unit

MRC-600 (Bio-rad) and an argon-Krypton laser were used.

4.16 Efficacy of transdermal patches in hyperglycemic rats

Wistar rats, weighing 150–200 g, fasted overnight were used for

induction of diabetes. The experimental protocol was approved by Institutional

Animal Ethics Committee of the Punjabi University, Patiala and the care and

handling of the animals was in accordance with the National Institutes of

Health guidelines. The animals were allowed to acclimatize for 2 weeks

before the experiment. The animals were housed in polypropylene cages

inside a well-ventilated room. Each cage consists of not more than 3 rats.

They were maintained under standard laboratory conditions of temperature,

25± 2°C; humidity, 55 ± 5% and 12 hour light/dark cycle. They were fed a

standard commercial pellet diet and water ad libitum. The diet consisted of

71% carbohydrate, 18% protein, 7% fat, 4% salt mixture and adequate

minerals and vitamins.

4.16.1Experimental groups and protocol:

Diabetes was induced in wistar rats by i.p injection of STZ (60 mg/kg).

STZ was dissolved in 0.01 M sodium citrate buffer, pH 4.5 to overnight fasted

rats and always prepared freshly. During the first 24 h of diabetes induction,

STZ-treated animals were allowed to drink 5% glucose solution to overcome

drug-induced hypoglycemia. Forty-eight hours after STZ administration,

diabetes was confirmed by the presence of hyperglycemia. The blood glucose

concentration was measured every week from the day of STZ injection.

Rats were divided into 11 groups (n=6). Animals were placed into

different groups after 7 days of induction of diabetes. The hair on the backside

of the rats was removed with an electric hair clipper on the previous day of the

experiment. The rats were treated as following:

Group I (control): Citrate buffer

Group II : Oral administration of RGE (1µg/g body weight) after

overnight fasting.

Group III : Application of transdermal patch containing RGE + PG:

EtOH (7:3).

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Group IV : Application of transdermal patch containing RGE + piperine

(0.008% w/v) mixture dispersed in donor vehicle.

Group V : Application of transdermal patch containing RGE + piperine

(0.008% w/v) - CTN (1% w/v) mixture dispersed in donor

vehicle.

Group VI : Application of transdermal patch containing RGE + ZOE

(0.05%w/v) mixture dispersed in donor vehicle.

Group VII : Application of transdermal patch containing RGE + ZO-E

(0.05%w/v) - CTN (1% w/v) mixture dispersed in donor

vehicle.

Group VIII : Application of transdermal patch containing RGE + AA-E

(5%w/v) mixture dispersed in donor vehicle.

Group IX : Application of transdermal patch containing RGE + AA-E

(5%w/v) - CTN (1% w/v) mixture dispersed in donor

vehicle.

Group X : Application of transdermal patch containing RGE + MO-E

(3%w/v) mixture dispersed in donor vehicle.

Group XI : Application of transdermal patch containing RGE + MO-E

(3%w/v) - CTN (1% w/v) mixture dispersed in donor

vehicle.

Blood samples via the post-orbital venous plexus veins was

collected and blood glucose levels were determined using Aqua check

(Roche Diagnostics, Germany).

4.17 Data and statistical analysis:

Castesian plots with a cumulative amount of RGE present in the

receptor compartment Vs. time were plotted. The RGE cocentration of

the receptor compartment was corrected for sample removal by use of

the equatonderived by Hayton and Chen (1982):

C1n=Cn (VT/VT-VS) (C1

n-1/Cn-1)

Where C1n and Cn are the corrected and measured concentrations

of RGE in the nth sample, respectively.

VT is the total volume of the receptor compartment.

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VS is the total volume of the sample withdrawn.

C1n-1 and Cn-1 are the corrected and measured concentrations of

RGE in the (n-1)th sample, respectively.

The cumultive amount of RGE permeated per unit surface area

was plotted against the time and slope of the linear portion of the

graph was estimated as steady state flux (JSS).

Enhancement ratio of RGE after vrious tretments (ER) was

calculated as:

ER=Ptreatment / Pcontrol

Where, Ptreatment is the flux of RGE through enhancer treated

epidermis and Pcontrol is the flux of RGE through untreated

epidermis.

ANOVA was followed by Dunnett’s test when data of treated

groups were compared with control group. However, Turkey test

was employed after ANOVA for all pair-wise multiple comparisons

among groups. The levelf significance was fixed at P<0.05.