CHAPTER- 03 MATERIALS AND METHODSshodhganga.inflibnet.ac.in/bitstream/10603/4310/14/14_chapter...
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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.