CHAPTER- 03

MATERIALS AND METHODS

3.1.Selection, Collection & Authentication of Plant Samples 58

3.2. Standardization of Plant Samples 58

3.2.1. Botanical 58

3.2.2. Physicochemical Evaluation 61

3.2.3. Microbial Contamination 62

3.2.4. Toxic Heavy Metal Analysis 63

3.3.Comparison of Antimicrobial Activity 64

3.4.Column Fractional Separation &Purification of the Potent Antimicrobial

Compound from Selected Extract 65

3.4.1.Purification And Characterization of the Isolated Compound 66

3.5.Development and Evaluation of Chrysophanol Gel Formulation 71

3.5.1.Preparation of Gels 71

3.5.2.Evaluation of Chrysophanol Gel 72

3.6.Comparative Pharmacodynamic Evaluation 78

3.6.1.Animal Model 78

3.6.2.Wound Infection and Dressing 79

3.6.3.Wound Healing Rate 80

3.6.4.Bacteriological Examination of Granulated Tissue 80

3.6.5. Analysis of Data 81

3.1. SELECTION, COLLECTION & AUTHENTICATION OF PLANT

SAMPLES

Five medicinal plants Allium sativum, Azadiracta indica, Cassia fistula,

Curcuma longa and Wrightia tinctoria were utilized for the present study. These

plants have previously been reported to have antimicrobial activity against different

microbial strains, and used in various skin infection conditions in Ayurveda. Plants

were selected in consultation with the Ayurvedic experts and literature review, both

Ayurvedic and other journals. All plants were collected randomly from tropical -

humid regions of Kerala, India, under the guidance of an expert of Indian herbs.

And All the specimens were identified, authenticated with the help of Taxonomist,

voucher specimens were prepared and stored for future reference. Plant materials

were dried in the shade without the exposure of sunlight at room temperature and

stored for future use.

3.2. STANDARDISATION OF PLANT SAMPLES

3.2.1. Botanical

3.2.1.1. Determination of foreign matter99

Plant material was weighed (For leaf drugs 250 g and for rhizomes and bulb

500 g) and spread in a thin layer. The foreign matter was sorted into groups either by

visual inspection or by using a magnifying lens (6x or 10x), or with the help of a

suitable sieve, according to the requirements for the specific plant material. The

remainder of the sample was sifted through a No. 250 sieve; dust is regarded as

mineral admixture. The content of each group (Foreign Plants/ Foreign Animals/

Foreign Mineral) was calculated in grams per 100g of air-dried sample

3.2.1.2. Sensory evaluation99

Size

A graduated ruler in millimetres was used for the measurement of the length,

width and thickness of the crude materials.

Colour

The untreated sample was examined under diffuse daylight. If necessary, an

artificial light source with wavelengths similar to those of daylight was used. The

colour of the sample was compared with that of a reference sample.

Surface characteristics, texture and fracture characteristics

The untreated sample was examined, if necessary by using, a magnifying lens

(6x to 10x). Wetting with water as required, may be necessary to observe the

characteristics of a cut surface, material was touched to determine if it is soft or hard.

It was bent and ruptured to obtain information regarding brittleness and the

appearance of the fracture plane (whether it is fibrous, smooth, rough, granular, etc.).

3.2.1.3. Microscopy 100-106

The required samples of different organs were cut and removed from the

plant and fixed in FAA (Farmalin-5ml + Acetic acid-5ml +70% Ethyl alcohol-90ml).

After 24 hrs of fixing, the specimens were dehydrated with graded series of tertiary-

Butyl alcohol as per the schedule given by Sass106

.Infilration of the specimens was

carried by gradual addition of paraffin wax (melting point 58-60 C) until TBA

solution attained super saturation. The specimens were cast into paraffin blocks.

Sectioning

The paraffin embedded specimens were sectioned with the help of Rotary

Microtome .The thickness of the sections was 10-12 μm. Dewaxing of the sections

was done by adopting customary procedure104

. The sections were stained with

Toluidine blue as per the method published by O‟Brien et al105

. Since Toluidine

blue is a polychromatic stain, the staining results were remarkably good; and

cytochemical reactions were also obtained. The dye imparted pink colour to the

cellulose walls, blue colour to the lignified cells, dark green colour to suberin, violet

colour to the mucilage and blue colour to the protein bodies . Wherever necessary

sections were also stained with Safranin and Fast-green and Iodine solution (for

starch).

For studying the stomatal morphology, venation pattern and of trichomes

distribution clearing of the leaf with 5% sodium hydroxide and epidermal peeling

after partial maceration in Jeffrey‟s maceration fluid 106

was done. The peelings were

mounted in glycerine. Powdered materials of different parts were cleared with NaOH

and mounted in glycerin medium after staining. Different cell component were

studied and their dimensions were measured.

Photomicrographs

Photomicrographs of different magnifications were taken with Nikon

Labphot 2 Microscopic Unit. For normal observations bright field was used. For the

study of crystals, starch grains, and lignified cells polarized light was employed.

Since these structures have birefringent property, under polarized light they appear

bright against dark background. Magnifications of the figures are indicated by the

scale bars. Descriptive terms of the anatomical features used were as given in the

standard Plant Anatomy books100

.

3.2.2. Physicochemical evaluation

3.2.2.1. Determination of Ash Values99

Total ash

About 2-4g of the ground air-dried material was accurately weighed, in a

previously ignited and tared silica crucible. The material was spread in an even layer

and ignite by gradually increasing the heat to 500-600°C in a muffle furnance until

the ash is white, indicating the absence of carbon. The crucible was cooled in a

desiccator and weighed.

Acid-insoluble ash

To the crucible containing the total ash, 25 ml of hydrochloric acid TS (70g/l)

was added. The crucible was covered with a watch-glass and boiled gently for 5

minutes. The watch-glass was rinsed with 5 ml of hot water and the washing was

added to the crucible. The contents of the crucible was filtered using ashless filter

paper. The insoluble matter on an ashless filter paper was washed with hot water

until the filtrate was neutral. The filter-paper containing the insoluble matter was

transferred to the original crucible, heated to dryness on a hot-plate and ignited to

constant weight .The residue was allowed to cool in a suitable desiccator for 30

minutes and then weighed without delay. The content of acid-insoluble ash was

calculated in mg per g of air-dried material.

3.2.2.2. Determination of extractable matter99

(Water and Ethanol soluble

extractable matter)

About 4.0g of coarsely powdered air-dried material was accurately weighed

and placed in a glass-stoppered conical flask. It was macerated with 100ml of the

solvent specified for the plant material concerned for 6 hours, shaking frequently,

then allowed to stand for 18 hours. The product was rapidly filtered taking care not

to lose any solvent. 25 ml of the filtrate was transferred to a tared flat-bottomed dish,

evaporated to dryness on a water-bath, cooled in a desiccator for 30 minutes and

weighed without delay.The content of extractable matter in mg per g of air-dried

material was calculated.

For ethanol-soluble extractable matter, ethanol was used as solvent and for

water-soluble extractable matter, water was used as the solvent.

3.2.2.3. Determination of water Content (Loss on drying by gravimetric

method)99

About 2-5g of the prepared air-dried material, or the quantity specified in the

test procedure for the plant material concerned was accurately weighed in a

previously dried and tared flat weighing bottle. The sample was dried in a Hot Air-

Oven (KOS-3) at 100-105°C until two consecutive weighings did not differ by more

than 5mg, unless otherwise specified in the test procedure. The loss of weight in mg

per g of air-dried material was calculated.

3.2.3. Microbial Contamination99

Preparation of Plant Material for the Test

Ten grams of each of the samples was mechanically homogenized in 100ml

of buffered lactone broth for evaluation of E.coli, Salmonella spp. & total aerobic

count. Ten grams of each of the samples was mechanically homogenized in 100ml of

buffered sodium chloride and peptone solution for evaluation of Pseudomonas

aeruginosa and Staphylococcus aureus.

Evaluation Of Microbial Contamination99

Inoculated into Mac.conkey broth and incubated at 43-450c for 18 24hrs for

evaluation of E.coli. Salmonella spp was inoculated into Mac.conkey broth and

incubate at 35-370c for 5-24hrs for, Pseudomonas aeruginosa and Staphylococcus

aureus were inoculated into 100ml of soyabean-casein digest medium and incubated

at 35-370c for 24-48hrs. The plates were placed on a colony counter and the number

of colony forming units was determined.

Total viable Aerobic Count99

10ml of samples solution was transferred on to each of two membrane filters

and filtered immediately. The membrane was washed three times with buffered

sodium chloride –peptone solution. Each of the membrane filter was transferred to

the surface of a plate with casein soybean digest agar, incubate the plates were

inoculated for five days at 30-350 c for the detection of bacteria. The number of

colonies formed were counted and the number of microorganisams per gram of the

material tested was calculated. The microbial content was taken as the mean of the

duplicate determinations.

3.2.4. Toxic Heavy Metal Analysis107

(Cadmiun, Mercury and Lead

Determination)

The protocol used to determine the metals in the plant material is a

modification of the method proposed by Chow et al. 107

. In brief, 2 g of the sample

was transferred to a 100 ml Nessler tube, 15 ml of 10% HNO3 v/v was added and left

in water bath at 100 0C for 3 h. For the analysis of mercury, the digested solution, is

analyzed by cold vapour AAS after reduction with NaBH4. For cadmium and lead,

the digested sample solutions were treated twice under reflux with concentrated

HNO3 before determination of the metals by flame AAS. Each sample was analyzed

in duplicate. The limits of quantification will be 2 mg/g for lead and 0.2 mg/g for

cadmium.

3.3. COMPARISON OF ANTIMICROBIAL ACTIVITY OF SELECTED

PLANT SAMPLES

3.3.1. Preparation of plant extract

Selected plant samples were extracted by soxhlet extraction, the solvent was

distilled under reduced pressure in a rotary evaporator until the extract became

completely dry.The percentage yield for each extract was determined108

.

3.3.2. Determination of Antimicrobial Action

3.3.2.1. Microorganisms used

The test organisms used were Staphylococus aureus(ATCC29737),

Escherichia coli(ATCC2068), Pseudomonas aeruginosa(ATCC9027), GroupA.

Streptococus & Candida albicans(ATCC10231)

3.3.2.2. Inoculum

The microorganisms were inoculated into Soybean Casein Broth( SBCB )and

incubated at 35 ± 2°C for 4 h. The resultant turbid suspension was diluted with

SBCB to match with 1 McFarland turbidity standard. This level of turbidity was

equivalent to approximately 3.0 × 108

CFU/ml109

.

3.3.2.3. Agar diffusion assay

The modified agar well diffusion method of Perez et al110

was employed.

Mueller-Hinton agar plates were inoculated by streaking the swab over the entire

sterile agar surface. This procedure was repeated by streaking 2 more times, rotating

the plate approximately 60° each time to ensure even distribution of the inoculum.

As a final step, the rim of the agar was also swabbed. After allowing the inoculum to

dry at room temperature, 6-mm-diameter wells were bored in the agar .Each extract

was checked for antimicrobial activity by introducing 100 μL of 4000μg/ml

concentration into triplicate wells. Simultaneously, gentamicin sulfate (S. aureus, P.

aeruginosa, and E. coli), and nystatin (C. albicans) were used as positive controls at

a concentration of 1.0 μg/ml and the dilution medium for the positive controls was

sterile distilled water, ethanol and petroleum ether. The plates were allowed to stand

at room temperature for 1hr for the extract to diffuse into the agar and then they

were incubated at 35 ± 2°C for 24 h, except C. albicans which was incubated at 29 ±

2°C.

3.4. COLUMN FRACTIONAL SEPARATION &PURIFICATION OF THE

POTENT ANTIMICROBIAL- COMPOUND FROM SELECTED

EXTRACT

In the primary comparative antimicrobial study the methanol extract was

found to be very active (Table 4.5). Therefore, the methanol extract was applied on

silica gel column chromatography was carried out. The column was successfully

eluted with stepwise gradient of chloroform, methanol (2:1&1:1). The elution was

monitored by thin layer chromatography and antimicrobial assay by modified agar

well diffusion method of Perez et al110

.The test organisms used were Staphylococus

aureus (ATCC29737), Escherichia coli(ATCC2068), Pseudomonas

aeruginosa(ATCC9027), GroupA Streptococus. .Similar fractions were pooled

together, concentrated and dried under vacume.The 4th

&5th

fractions eluted with

chloroform: methanol (1:1) gave the highest antibacterial activity.

3.4.1. Purification and Characterization of The Isolated Compound

The fractions were further purified by using silica gel Colum

chromatography, which gave orange crystals with melting point of 1950C and single

spot on TLC. The chemistry of the compound was elucidated on the basis of UV,IR

& NMR spectral data and referring to reported literature.

3.4.1.1 TLC

Analytical TLC were performed on aluminium sheets coated with silica gel

60 F254 (E.M.Merck, 0.20 mm thickness). Chromatograms were examined under

UV light using a UV-viewing cabinet and visualized by spraying with a 4%

phosphomolybdic acid solution containing a trace of ceric sulfate in 5% H2SO4.

3.4.1.2. Melting point

The melting point is one of physical properties of a substance that is useful

for characterizing and identifying the substance. Melting points were determined by

melting point apparatus.

3.4.1.3. Infrared spectroscopy (IR)

IR spectra may be recorded on plant substances in an automatic recording IR

spectrophotometer (Thermo-Nicolet Avtar 370 FT-IR Instrument). in the solid state

mixed with potassium bromide. The range of measurements from 4000 to 667 cm-1

showed spectral bands due to the vibration of individual bonds or functional groups

in the molecule under examination. The functional groups can be identified by their

characteristic vibration frequencies that makes the IR spectrum the simplest and most

reliable mechanism for assigning a compound to its class.

3.4.1.4. Nuclear Magnetic Resonance Spectroscopy (NMR)

Proton NMR spectroscopy provides a means of determining the structure of

an organic compound by measuring the magnetic moment of its hydrogen atoms. In

most compounds, hydrogen atoms are attached to different groups and the proton

NMR spectrum provides a record of the number of hydrogen atoms in these different

positions. The sample is placed in solution, in an inert solvent, (DMSO) between

the poles of a powerful magnet and the protons undergo different chemical shifts

according to their molecular environments within the molecule. These are measured

in the NMR spectroscope (Bruker NRC-IISC) relative to a inert standard,

Teramethylsilane (TMS).

3.4.1.5. Ultra Violet spectroscopy (UV)

The absorption spectra of plant constituents can be measured in very dilute

solution against a solvent blank (Methanol) using an automatic recording UV-VIS

spectrophotometer (Systronics Double Beam UV-VIS Spectrophotomerer 2201).

Mesurments are made in the range of 200-400nm. The wavelength of maxima and

minima of the absorption spectrum so obtained are recorded in nm.

3.4.1.6. Determination of Minimal Inhibitory Concentration (MIC) and

Minimal Bactericidal Concentration (MBC) of Chrysophanol

The MIC was determined by micro-broth dilution methods109

. The

reconstituted drug was serially diluted two fold in Mueller-Hinton broth medium.

Duplicate tubes of dilution ranging from 0.025 µg/ml to 25.6 µg/ml were inoculated

with 5 x 105cells (cfu) of the test bacterial strain and cultures incubated at 37 °C for

18 h and for Multidrug resistant (MDR) organisms duplicate tubes of dilution

ranging from 0.025 µg/ml to 1024 µg/ml were inoculated with 5 x 105cells (cfu) of

the test bacterial strain and cultures incubated at 37 °C for 18 h. MIC was taken as

the highest dilution (least concentration) of the drug showing no detectable growth.

MBC was determined by sub-culturing the test dilution in a fresh drug-free solid

medium and incubating further for 18-24 h. The highest dilution that yielded no

single bacterial colony on a solid medium was taken as MBC

3.4.1.7. Development and Validation of Spectrophotometric Method for

Chrysophanol in Gel Formulations

3.4.1.7.1. Preparation of Standard Stock Solution

1mg Chrysophanol was accurately weighed, transferred to 10 ml volumetric

flask and dissolved in 10ml of methanol to give a standard solution of 100µg/ml ( if

necessary, the volumetric flask may be placed in an ultrasonic bath to effect

dissolution )

3.4.1.7.2. Determination of λ max.

1ml of standard solution (100µg/ml) was pipetted out into 10ml volumetric

flask and made up the volume by adding appropriate quantity of methanol. The

absorbance of the resultant solution was scanned in UV range (200-400nm) for

maximum absorbance after enabling blank correction for methanol in the above

region.

3.4.1.7.3. Procedure for Plotting Calibration Curve

The standard solution were prepared by proper dilutions of the primary stock

solution with methanol to obtain working standards in the concentration range of 1-

10 µg/ml The absorbance was measured at 225nm against a solvent blank and the

calibration curve was plotted. Similarly absorbance of sample solution was measured

and the amount of chrysophanol was determined by referring to the calibration curve.

3.4.1.7.4. Estimation of Chrysophanol in Gel Formulation.

For the estimation of the drug in gels, chrysophanol was extracted from 1gm

of each gel formulation with 50ml of methanol for 30 min and the resultant extract

was filtered through membrane filter (pore size 0.45µm). From this 2.5ml was

pipetted out and made up to 10ml. Then 1ml of the resultant solution was again

diluted to 10 ml and the absorbance of the sample was determined

spectrophotometrically at 225nm. The concentration of Chrysophanol was estimated

from the regression equation of calibration curve. It is also estimated using HPLC89

3.4.1.7.5. Validation of the Proposed Analytical Method111-113

3.4.1.7.5.1. Linearity

To establish linearity of the proposed method, six separate series of solutions

of Chrysophanol (1–10 μg/ml in methanol) were prepared from the stock solution

and analyzed. Least square regression analysis was performed on the obtained data

(Appendix-C).

3.4.1.7.5.2. Accuracy

The accuracy of the method is the closeness of the measured value of the true

value for the sample. To determine the accuracy of the proposed method, three

different levels of drug concentrations were prepared from independent stock

solutions and analyzed (n = 6). Accuracy was assessed as the percentage relative

error and mean % recovery. To provide an additional support to the accuracy of the

developed assay method, a standard addition method was employed, a known

preanalyzed formulation sample and the total concentration of Chrysophanol was

determined using the proposed methods (n = 6). The % recovery of the added pure

drug was calculated as % recovery = [(Ct–Cs)/Ca] x 100, where Ct is the total drug

concentration measured after standard addition; Cs,drug concentration in the

formulation sample; Ca, drug concentration added to formulation. The absorbance of

each solution was measured at 225 nm with methanol as blank

3.4.1.7.5.3. Repeatability / Precision

Repeatability expresses the precision under same operating conditions over a

short interval of time. It is also termed as intra-assay precision. The precision of an

analytical procedure is usually expressed as the closeness of agreement between a

series of measurements obtained from multiple sampling of the same homogenous

sample under the prescribed conditions. The precision of an analytical procedure is

usually expressed as the standard deviation, variance, or coefficient of variance of a

series of measurements.

3.4.1.7.5.4. Stability Profile

Stability of absorbance is of major importance in Spectrophotometric

measurements. The period over which absorbance value at 225nm of Chrysophanol

in methanol remained stable was investigated using three different concentration 4,

5,and 6µg/ml. The absorbance values were measured at 15 min intervals for a period

of 1 hour.

3.4.1.7.5.5. Limit of Detection (LOD) and Limit of Quantification (LOQ)

The LOD and LOQ for Chrysophanol by the proposed method were

determined using calibration standards. LOD and LOQ were calculated as 3.3 σ/S

and 10 σ/S, respectively, where S is the slope of the calibration curve and σ is the

standard deviation of the lowest standard concentration (n = 6) .

3.4.1.7.5.6. Range

The range of an analytical procedure is the interval between the upper and

lower concentrations of analyte in the sample for which it has been demonstrated that

the analytical procedure has a suitable level of precision, accuracy and linearity.

3.5. DEVELOPMENT AND EVALUATION OF CHRYSOPHANOL GEL

FORMULATION

3.5.1. Preparation of Gels

Hydrogels of Chrysophanol (0.1%, w/w) were prepared using Carbopol 940

NF50,114

(Pena, 1990; 50.Wade and Weller, 1994). The Carbopol resin was soaked in

mixture of water and glycerine and preservative for 2 h and then dispersed by

agitating at approximately 600 rpm with aid of mechanical stirrer to get a smooth

dispersion. The stirring was stopped and liquid was allowed to stand so that any

entrained air could escape. To this prepared dispersion aqueous solution of

triethanolamine was added with slow speed agitation. At this stage, Chrysophanol

and the enhancers were dissolved in ethanol and incorporated into the prepared base.

In addition, glycerine and purified water are added to the gel to make up the weight

of the product to 10gm. Any entrapped air in the gel was allowed to escape by

allowing the gels to stand overnight. Table 1 shows the formulae of the topical

preparations used in the study.

Table 3.1: Composition of the Batches of Gels

Composition C1

1

C1

2

C1

3

C1

4

C2

1

C2

2

C2

3

C2

4

C3

1

C3

2

C3

3

C3

4

Chrysophanol(

g) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

Carbopol( g) 1 1 1 1 1.2

5

1.2

5

1.2

5

1.2

5 1.5 1.5 1.5 1.5

DMF% V/W - 15 - - - 15 - - - 15 - -

DMSO% V/W - - 15 - - - 15 - - - 15 -

PEG 400%

V/W - - - 15 - - - 15 - - - 15

Glycerine

%V/W 10 10 10 10 10 10 10 10 10 10 10 10

Triethanolamin

e(ml) 2 2 2 2 2.5 2.5 2.5 2.5 3 3 3 3

Methyl

paraben

(W/V)ml

2 2 2 2 2 2 2 2 2 2 2 2

Propyl Paraben

(W/V)ml 1 1 1 1 1 1 1 1 1 1 1 1

Deionized

water to

produce(gms) 10 10 10 10 10 10 10 10 10 10 10 10

3.5.2. Evaluation of Chrysophanol Gel

3.5.2.1 Physico-Chemical Evaluation of Gels

3.5.2.1.1 pH

Direct measurements were made using a digital pH meter (MK-IV

SYSTRONICS).

3.5.2.1.2 Determination of Viscosity

Viscosities of the gels prepared were determined in a cone and plate

viscometer (Digital Rheometer model DV11, Brookfield). A spindle (no. 7) was

rotated at 10 rpm. Samples of the gels (0.5gm) were left to settle over 30 min at the

assay temperature (37°C) before measurements were taken. Viscosities were noted in

cps as mean of triplicate

3.5.2.1.3 Spreadability115

Spreadability was determined by using a spreadability apparatus (131). After

applying weight, time in seconds required to separate the slides was noted.

Spreadability of each formulation was reported in seconds. Spreadability was then

calculated by using the formula:

S = M.L/ T

Where, S = Spreadability,

M = Weight tide to upper slide,

L = Length of glass slide and

T = Time taken to separate the slide completely from each other.

3.5.2.1.4 Extrudability116

Extrudability was determined by using an extrudability apparatus (132). A

closed collapsible tube containing formulation was pressed firmly at the crimped

end. When the cap was removed, formulation extruded until the pressure dissipated.

Weight in grams required to extrude a 0.5cm ribbon of the formulation in 10 seconds

was determined. The average extrusion pressure in grams was reported.

3.5.2.1.5 Test for Homogeneity

The formulations were tested for their homogenicity by visual appearance

after the gels have set in the container. Apart from this, a small quantity of each gel

is pressed between the thumb and the index finger and the consistency of the gel was

assessed as to whether the gel is homogeneous or not.

3.5.2.1.6 Analysis of Drug Content117

For the estimation of the drug in gels, Chrysophanol was extracted from 1gm

of each gel formulations with 50ml of methanol for 30min and the resultant extract

was filtered through membrane filter (pore size 0.45µm). From this 2.5ml was

pipette out and made up to 10ml. Then 1ml was diluted to 10 ml, and the absorbance

of the sample was determined spectrophotometrically at 225nm. The concentration of

Chrysophanol was estimated from the regression equation of calibration curve.

3.5.2.1.7 Primary Skin Irritancy Test118,119

The protocol of the study was approved by the Animal Ethical Committee of

Amrita Institute of Medical Sciences and Research Centre (AIMS), Kochi

(Appendix-B). Six healthy, previously unused New Zealand White rabbits of either

sex or which free from skin irritation or other dermatological lesions (weighing 2.0–

2.5 kg) from Experimental Animal Centre of AIMS, Kochi, India were chosen for

this study. The animals were individually housed in stainless steel cages. The dorsal

side of each animal was clipped free of fur with an electric clipper at least four hours

but no more than 24 hours before testing. A 50mg portion of Chrysophanol gel was

applied to two sites (one site intact, and the other site abraded) on each rabbit under a

double layer of gauze in an area of skin approximately 1 × 1 inch square. After 24

hour exposure, the gauze was removed. The test sites were gently sponged with

deionized water to remove any remaining test sample residue. The test sites were

evaluated after 24 hours and 72 hours following removal of the gauze. Each test site

was examined for dermal reaction in accordance with the Draize scoring criteria. The

primary irritation index (the sum of the scored reactions divided by a factor

representing the number of scoring intervals multiplied by the number of test

parameters multiplied by the number of rabbits) was calculated following completion

of the test .

3.5.2.1.8 Evaluation of Stability

Stability studies were performed for 6 months. Samples (packed in glass

vials) were prepared in triplicates and were kept at two stability testing conditions,

viz. 4–8ｏ

C serving as control and 30ｏ

C/70%RH (Stability Chamber, Remi-2K)

serving as test condition as per ICH Guidelines. Stability of samples were evaluated

for drug content, pH, homogeneity and viscosity at each sampling point (0, 3 and 6

months).

3.5.2.2. In vitro Drug Release Studies

The in vitro drug release from gel formulations was studied across cellulose

membranes using Franz-type diffusion cells with effective diffusional surface area of

1.54 cm2

The cellulose acetate membrane (cellophane membrane) having a pore size

0.45μ was mounted between the donor and receptor compartment of the diffusion

cell 120-122

.The receiver compartment was filled with 15 ml of phosphate buffer of

pH 7.4 to ensure sink condition. The donor compartment of the cell was filled with

1g vehicle containing the test drug. The system was maintained at 370 C±0.5

0 C by a

water bath circulator and a jacket surrounding the cell, with stirring at 600rpm

throughout the experiment. 1 ml samples were withdrawn at intervals of one hour for

a period of 12 hrs and each time equal volume was replaced with drug free receptor

fluid123,124

. All the samples were analyzed by UV spectrophotometer at 225nm117

.

The experiment was carried out in triplicate, and the mean cumulative percentage

release from three batches were calculated.

3.5.2.3. Ex vivo Permeation Studies

Male Wistar rats weighing 200±20 g were purchased from Experimental

Animal Center of Amrita University (Kochin, India) for the Ex vivo permeation

studies. Skin from the abdominal region was excised after hair was removed with a

depilatory, and examined for integrity using a lamp-inspecting method. The

subcutaneous fat and connective tissue were carefully removed. The skin thus

obtained was rinsed with physiological saline125

.The skin samples were mounted

carefully on Franz-type diffusion cells with the stratum corneum126

side up and an

effective diffusion area of 1.54cm2 . The receiver compartment was filled with 14 ml

of phosphate buffer pH 7.4 to ensure sink condition. The system was maintained at

37±0.5◦C by a water bath circulator and a jacket surrounding the cell, with stirring at

600 rpm throughout the experiment. The skin samples were equilibrated for 30 min

before being dosed127

. The donor compartment of the cell was filled with 1 g vehicle

containing the test drug. 1ml samples were withdrawn at intervals of one hour for a

period of 12 hrs and each time equal volume was replaced with drug free receptor

fluid123,124

. All the samples were and analyzed by UV spectrophotometer at

225nm117

. The experiment was carried out in triplicate and the mean cumulative

percentage release from three batches was calculated.

3.5.2.3. Data Analysis

To analyze the in vitro release data, various kinetic models were used. The

zero order rate Equation (1) describes the systems where the drug release rate is

independent of its concentration128

. Higuchi-1963129

described the release of drugs

from insoluble matrix as a square root of time dependent process based on Fickian

diffusionEquation (2).

C = K 0t (1)

Where, K0 is zero-order rate constant expressed in units of concentration/time and t

is the time.

Q= 2 Cd (Dt ⁄ π )1⁄2 (2)

Higuchi diffusion equation (Eqn 2) 129

which depicts the release of drugs from

insoluble matrix as a square root of time dependent process based on Fickian

diffusion. where Q is the amount of drug released into the receptor phase per unit

area of exposure, Cd denotes the initial drug concentration in the vehicle, D is the

apparent diffusion coefficient of drug, and t represents the time elapsed since the

start of drug release. To know the additional mechanisms involved in drug release

from gel matrix Peppas equation was applied. It has been extensively used to analyze

release behavior of various pharmaceutical systems.

Mt /M∞= Ktn (3)

Log Mt/ M∞ = log k + n log t

In Eq. (3) Mt/M∞ is the fraction of drug released at each time point (t), k the

kinetic constant and n is the diffusion exponent for drug release. According to the

value calculated for the diffusion exponent n, we can derive information about the

solute release mechanism; Fickian diffusion occurs when n is between 0.43 and 0.50

(depending on the matrix geometry), while when n ≥ 1 the release follows case-II

transport, or anomalous transport. The polymer relaxation and diffusion are coupled

leading to a complex transport behavior, known as anomalous transport130,131

.

The following Pharmacokinetic parameters are calculated

Cumulative quantity of Chrysophanol collected in the receiver (µg/cm2) was

plotted as a function of time. The flux value (Jss, µg/cm2/h) for each experiment was

obtained from the slope (steady-state portion) of the linear portion of the data fitted

by regression analysis132

. For release data analysis, cumulative permeation (µg/cm2)

was plotted as a function of square root of time, where linearity is indicative of

diffusion controlled drug release129

. Release rate was estimated as the slope of such

plots (µg/cm2/t

0.5). Penetration-enhancing activities were expressed as enhancement

ratios (ER), i.e., the ratio of the flux value with enhancer to that obtained without

enhancer. Study was designed in a systematic pattern similar to 3X3 Latin Square

design, so that data was subjected to statistical analysis employing two way analysis

of variance (ANOVA) followed by post-hoc test (Scheff‟s test) using Graph pad

(Appendix-D).

3.6. COMPARATIVE PHARMACODYNAMIC EVALUATION OF GELS

3.6.1. Animal Model

Healthy Wistar rats (male and female),weighing 150–200 g obtained from the

Center for Laboratory Animals, Amrita Institute of Medical Sciences and Research

Centre (AIMS), Kochi were used in the evaluation of the wound healing properties

of the tested agents (Appendix-B).The animals were randomly distributed into three

treatment groups (Control treated with DMSO alone, Test treated with

Chrysophanol gel, Standard treated with Neosporin ointment) and were maintained

in accordance with the recommendations in the University Guidelines for the Care

and Use of Laboratory Animals and approved by Animal Ethical Committee

(Appendix-B).The rats were placed in individual cages where they had free access to

food and clean drinking water throughout the experimental period. The animals were

rehabilitated following experimentation.

3.6.2. Wound Infection and Dressing

Bacteria Tested

The test organisms used were Staphylococus aureus (ATCC29737),

Pseudomonas aeruginosa (ATCC9027) and Group A-Streptococcus.

Inoculum

The microorganisms were inoculated into Soybean Casein Broth( SBCB)

and incubated at 35 ± 2°C for 4 h. The resultant turbidity suspension was diluted

with SBCB to match with 1 McFarland turbidity standard. This level of turbidity is

equivalent to approximately 3.0 × 108 CFU/ml

An excision wound model was used for this study. Albino rats (Wistar strain)

of both sexes weighing between 150–200 g were randomly divided into 3 groups of

six animals each. Full thickness wounds (1.5 X1.5 cm) were created on the shaved

dorsal side of rats using sterile surgical blade. Wounds were inoculated with the test

organisms at 108 CFU (0.1 ml) between thin skin muscle and paraspinus muscle and

allowed to infect for 24 h133

. All surgical procedures were carried out under Xylazin

and ketamine 1:3 (40 mg/kg intramuscularly). The treatment groups were dressed

daily with the respective treatments (test treated with Chrysophanol gel, Standard

treated with Neosporin ointment), while the control group was dressed with gel base

containing same quantity of DMSO only.

3.6.3. Wound Healing Rate

The percentage of wound closure was calculated as follows by using the

initial and final area drawn on glass slides during the experiments134

% of wound closure =

3.6.4. Bacteriological Examination of Granulated Tissue

The Granulated tissues from the Test, Standard and control groups were

collected prior to application of ointment formulation on day 4, 8,12, and 16 using

sterile scissors and cotton plug. The collected tissues were rinsed with sterile saline,

vortexed for few minutes with 10 ml of sterile saline, and the total bacterial count

was determined by serial dilution method.

3.6.5. Analysis of Data

X 100 Wound area on day 0 – Wound area on day n

Wound area on day 0

All data are presented as the mean ± the standard deviation (S.D). Data were

analyzed by Kruskal–Wallis one-way analysis of variance using the SPSS statistical

package version 11. If a significant difference (p < 0.05) between the respective

outcome parameters was obtained or the H value was close to significance,

comparison was performed by multiple range test.