5.1 materials 5.1.1. list of chemicals chemicals...
TRANSCRIPT
Experimental
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5.1 Materials
5.1.1. List of Chemicals
Chemicals Supplier
Sulphuric acid Spectrochem
Hydrocholric acid Spectrochem
Methanol (A.R grade) SD Fine chemicals
Ethyl acetate (A.R grade) SD Fine chemicals
Petroleum ether (60-80) (A.R grade) SD Fine chemicals
Diethyl ether (A.R grade) SD Fine chemicals
Methanol (HPLC grade) Sigma aldrich
Acetonitrile (HPLC grade) Sigma aldrich
Petroleum ether (60-80) Sigma aldrich
Diethyl ether (HPLC grade) Sigma aldrich
HPLC grade water Sigma aldrich
α-naphthol Rankem
Benedict's reagent Rankem
Fehling's A and Fehling's B solution Rankem
Barfoed’s reagent Rankem
Selewinoff’s reagent Rankem
Sodium hydroxide SD Fine chemicals
Experimental
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Chemicals Supplier
Acetic anhydride Rankem
Sodium nitroprusside SD Fine chemicals
Ferric chloride SD Fine chemicals
Sodium chloride Rankem
Gelatin SD Fine chemicals
Lead acetate SD Fine chemicals
Diphenyl-2-picrylhydrazyl Sigma Aldrich
Dimethyl Sulfoxide SD Fine chemicals
Sulfanilamide SD Fine chemicals
Phosphoric acid (H3PO4) SD Fine chemicals
Napthylethylenediaminedihydrochloride SD Fine chemicals
Nutrient agar Himedia
Sabouraud dextrose agar Himedia
Ketamine HCl Pfizer
Formaldehyde Spectrochem
L-hydroxyproline Sigma Aldrich
D(+) glucosamine HCl Merck
Olive oil SD Fine chemicals
Experimental
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Chemicals Supplier
Cetosteryl alcohol Sigma Aldrich
Glyceryl monostearate SD Fine chemicals
Glycerine SD Fine chemicals
Carbopol 940 Sigma Aldrich
Triethanolamine SD Fine chemicals
Formalin SD Fine chemicals
Alloxan monohydrate SD Fine chemicals
Bovine hyaluronidase Sigma Aldrich
Calcium chloride SD Fine chemicals
Sodium hyaluronate Merck
p-dimethyl amino benzaldehyde Sigma Aldrich
Bovine collagen Sigma Aldrich
TES buffer Spectrochem
Porcine pancreatic elastase Sigma Aldrich
N- Succ-(Ala)-nitroanilide Merck
Tyrosine hydroxylase Sigma Aldrich
Tyrosine Sigma Aldrich
Thiobarbituric Acid Himedia
Experimental
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Chemicals Supplier
Tricarboxcylic acid Himedia
Acetic acid SD Fine chemicals
Ethanol SD Fine chemicals
Chloramphenicol Sigma Aldrich
Fluconazole GSK
Folin-Ciocalteu reagent Rankem
Aluminium trichloride Spectrochem
Formic acid Rankem
L-Ascorbic acid SD fine chemicals
Rutin Sigma Aldrich
Ferric chloride Rankem
5.1.2. Instruments
Instruments Supplier
Muffle furnace Thermolab
Hot air oven Fourtech
Electronic balance (LCT-203-B) High precision
Digital weighing balance (AB 204) Mettler
Experimental
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Instruments Supplier
Digital pH meter (EQ-610) Equip-tronics
Incubator Thermolab
Tissue homogenizer Remi motors
Refrigerated centrifuge (MP400R) Estek centrifuge
Laboratory centrifuge (R4C) Remi motors
Temperature controlled water bath Subzero
Rotary vacuum pump Equitron
HPLC system Younglin SK
Sonicator Lab Enterprises
UV spectrophotometer (V-630) Jasco
Deep freezer Remi motors
Franz diffusion cell Electrolab, India
Tensiometer Lab made
Spreadability apparatus Lab made
Accucheck active glucometer Roche Diagnostics
Experimental
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5.2 SELECTION, PROCUREMENT AND AUTHENTICATION OF PLANT
MATERIAL
The plant materials selected for the study are given in table 5.1
Table 5.1 Plant materials selected for the study
Sr. No. Plant Plant Part/Plant material
1 Mimusops elengi Bark and fruits
2 Rosa damascena Flower petals
Authenticated powdered bark of Mimusops elengi and powdered flower petals of Rosa
damascena were procured from Amsar Private Limited.
The unripe fruits of Mimusops elengi were collected from S.N.D.T University campus,
Juhu Road, Mumbai and sent for authentication to Agarkhar Research institution, Pune,
Maharashtra.
5.3 STANDARDIZATION OF PLANT MATERIAL191, 192
Standardization of plant material is essential in order to assess the quality and purity of
drugs. Standardization of plant materials were carried out using following parameters:
a) Organoleptic characterization
The organoleptic characters - the color, odor, taste, shape, size and texture of the plant
material were estimated by visual and sensory evaluation.
b) Physicochemical analysis
1. Loss on drying
The loss on drying test is designed to measure the amount of water and volatile matter in
plant material under specified conditions. An excess of water in medicinal plant materials
will encourage microbial growth, the presence of fungi or insects, and deterioration of
phytoconstituents following hydrolysis. Limits for water content should therefore be set
for plant material.
Procedure
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About 5g of the finely grounded plant material was weighed in flat and thin porcelain
dish. It was placed in a hot air oven and was heated at about 100°C-105°C for 5 hrs.
The plant material was dried to a constant weight and after the drying was completed,
it was allowed to cool in a desiccator before weighing.
The drying was continued until two consecutive weights do not differ by more than
5mg, unless otherwise specified in the test procedure.
The porcelain dish and the contents were weighed and the loss of weight was
estimated in mg per g of plant material.
2. Determination of ash values
The residue remaining after incineration is the ash content of the drug, which represents
inorganic salts, naturally occurring in drug or adhering to it or deliberately added to it as
form of adulteration. Ash value is criteria to test the identity or purity of drug.
Procedure
Plant material was dried at temperature not exceeding 60oC and pulverized in the
electrical mixer. The powdered plant material was used for determination of ash value.
i. Total Ash value
The powdered plant material (2g) was weighed accurately in a tared silica crucible
and heated with a burner till vapors almost cease to be evolved, previously ignited,
cooled and weighed.
The resultant ash in the crucible was incinerated by gradually increasing the heat, not
exceeding 450°C, until free from carbon; cool. It was then allowed to cool in a
desiccator and later weighed.
In order to obtain carbon free ash, crucible was cooled and the residue was moistened
initially with hot water. The residue was collected on an ashless filter paper and
incinerated with filter paper at a temperature not exceeding 450˚C.
Again the residue was moistened with alcohol and above procedure was followed.
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The residue was allowed to cool in a suitable desiccator for 30 min, and then weighed
without delay.
The percentage of total ash was calculated with reference to the air dried sample of
the crude drug (plant material).
ii. Acid-insoluble ash
About 1g of the total ash obtained was boiled with 25 ml of dilute hydrochloric acid
for 5 min.
The insoluble matter was collected on an ashless filter paper and washed with hot
water until the filtrate was neutral.
The filter paper containing the insoluble matter was transferred to the crucible, and
heated gently until vapors cease to be evolved and ignited at a temperature not
exceeding 450˚C in a muffle furnace.
The residue was allowed to cool in a desiccator for 30 min and weighed immediately.
The content of acid insoluble ash with reference to the air-dried drug was calculated.
iii. Water soluble ash
About 1g of the total ash obtained was boiled with 25 ml of water for 5 min.
The insoluble matter was collected on an ashless filter paper, washed with hot water,
and ignited at a temperature not exceeding 450˚C in a muffle furnace for 15 min.
The weight of the insoluble matter was subtracted from the weight of the ash, and the
difference in weight represented the water-soluble ash.
The percentage of water-soluble ash was calculated with respect to the air-dried drug.
An average of three readings was determined.
iv. Sulphated ash
A silica crucible was heated to redness for 10 min, and allowed to cool in a desiccator
and weighed. About 2g of the powdered plant material was accurately weighed,
placed into the crucible, and ignited until the substance was thoroughly charred.
The crucible was then cooled, and the residue was moistened with 1ml of sulphuric
acid.
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The crucible was then heated again until white fumes no longer evolved and the
residue was ignited at 800oC ± 25
oC until all black particles disappeared.
The crucible was allowed to cool, and few drops of sulphuric acid were added to it
and heated. The ignition procedure was repeated as before, until two successive
weighing did not differ by more than 0.5mg.
3. Determination of extractive values
Determination of extractive values reveals the amount of active constituents extracted
with solvents from a given amount of plant material.
i. Alcohol soluble extractive value
About 5g of the powdered plant material was macerated with 100ml of alcohol in a
closed flask for 24 hrs, shaking frequently during 6 hrs and allowed to stand for
eighteen hours.
The contents were filtered and from the total volume of solvent, 25ml of the filtrate
was evaporated to dryness in a tared flat bottomed shallow dish, and dried at 105oC,
to constant weight.
The dish was then weighed and the percent of alcohol soluble extractive with
reference to the air-dried crude drug was calculated.
ii. Water soluble extractive value
The procedure performed for the determination of water soluble extractive was same
as that of alcohol-soluble extractive, except for the solvent used was chloroform-
water instead of ethanol.
iii. Ether soluble extractive value
About 100g of the air dried, coarsely powdered drug was transferred to an extraction
thimble and extracted with 500ml of solvent ether in a continuous extraction
apparatus (Soxhlet extractor) for 6 hrs.
The extract was filtered and a 10ml of the extract was transferred to a tared
evaporating dish. The solvent was evaporated off on a water bath and the residue
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was dried at 105oC to constant weight. The percentage of ether soluble extractive
with reference to the air-dried drug was calculated.
5.4 EXTRACTION OF PLANT MATERIAL
A. Mimusops elengi bark and fruit
1. Preparation of methanol, ethyl acetate and petroleum ether extracts of bark and
fruit:
The dried and coarsely powdered drug was extracted with different solvents (methanol,
ethyl acetate and pet ether) in the ratio of (1:5) for a period of 18 hrs using Soxhlet
extraction method. The temperature range for extraction was 40-45º C using a calibrated
heating mantle for heating. After the extraction period, the resultant solution was filtered.
The marc was discarded and the filtrate was concentrated on a rotary evaporator under
vacuum. The extracts were further dried in vacuum dessicator. The percentage yield of
extract was calculated. The extracts were stored in amber colored bottles at 2-4°C until
further use.
2. Preparation of aqueous (water) extracts of bark and fruit:
The dried and coarsely powdered drug was extracted at 40-45ºC in a round bottom flask
with distilled water as the solvent for extraction. The drug: solvent ratio of 1:5, was used
to obtain the maximum extractive yield. The drug was continuously extracted for a period
of 3 hrs and the resultant solution was filtered through muslin cloth and then through
filter paper to avoid any suspended particles in the extract. The marc was discarded and
the filtrate was concentrated on a rotary evaporator under vacuum. The extracts were
further dried in vacuum dessicator. The percentage yield of extract was calculated. The
extracts were stored in amber colored bottles at 2-4°C until further use.
Experimental
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B. Rosa damascena flower petals
1. Preparation of methanol extract:
The dried and coarsely powdered drug was continuously extracted with methanol, in the
ratio of (1:5) for a period of 18 hrs using Soxhlet extraction method. The temperature
range for extraction was 40-45º C using a calibrated heating mantle. After extraction, the
resultant solution was filtered. The marc was discarded and the filtrate was concentrated
on a rotary evaporator under vacuum. The extracts were further dried in vacuum
dessicator. The percentage yield of extract was calculated. The extracts were stored in
amber colored bottles at 2-4°C until further use.
2. Preparation of aqueous (water) extract:
The dried and coarsely powdered drug was extracted at 40-45ºC in a round bottom flask
with distilled water as the solvent for extraction. The drug: solvent ratio of 1:5, was used
to obtain the maximum extractive yield. The drug was continuously extracted for a
period of 3 hrs and the resultant solution was filtered through muslin cloth and then
through filter paper to avoid any suspended particles in the extract. The marc was
discarded and the filtrate was concentrated on a rotary evaporator under vacuum. The
extracts were further dried in vacuum dessicator. The percentage yield of extract was
calculated. The extracts were stored in amber colored bottles at 2-4°C until further use.
Table. 5.2 Codes for prepared extracts from plant materials
Extract Mimusops elengi
bark
Mimusops elengi
fruits
Rosa damascena
flower petals
Aqueous MEB-AE MEF-AE RD-AE
Methanol MEB-ME MEF-ME RD- ME
Ethyl acetate MEB- EA MEF- EA --
Petroleum
ether MEB- PE MEF- PE --
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5.5 PHYSIOCHEMICAL CHARACTERIZATION AND PRELIMINARY
PHYTOCHEMICAL SCREENING OF THE PLANT EXTRACTS192, 193, 194
5.5.1. Physiochemical characterization
The plant extracts were evaluated with respect to their physicochemical parameters such
as color, consistency and percent yield (% w/w).
5.5.2. Phytochemical Screening of Extracts
One gram of each extracts of Bakul bark and fruit and Rose flower petals was dissolved
in 100 ml of respective solvents used for extraction to obtain a stock of concentration 1%
(v/v). The extracts thus obtained were subjected to preliminary phytochemical screening
following the methodology described below.
1. Test for Carbohydrates
a. Molisch's test
The test solution is treated with few drops of alcoholic solution of alpha-naphthol. About
0.2 ml of conc. sulfuric acid was slowly added through the sides of the test tube.
Formation of violet ring indicates the presence of carbohydrates.
b. Benedict's test
The test solution is treated with few drops of Benedict's reagent (alkaline solution
containing cupric citrate complex) and boiled on water bath, to check the presence of
reducing sugars.
c. Fehling's test
Equal volume of Fehling's A (Copper sulfate in distilled water) and Fehling's B
(Potassium tartarate and Sodium hydroxide in distilled water) reagents are mixed and few
drops of sample are added and boiled. A brick red precipitate of cuprous oxide forms, if
reducing sugars are present.
d. Barfoed’s test
Equal volumes of Barfoed’s reagent and test solution are mixed. The solution is heated in
a boiling water bath for 1-2 min and cooled. Red precipitate indicates the presence of
monosaccharides.
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e. Seliwinoff’s test
About 1 ml of the test solution is added to 3 ml of Seliwinoff’s reagent and boiled in a
boiling water bath for 1-2 min. Fructose gives red color within half min. The test is
sensitive to 5.5 mmol/liter if glucose is absent, but if glucose is presents, it is less
sensitive and in addition of large amount of glucose can give similar color.
f. Tests for non-reducing polysaccharides
About 3 ml of the test solution is mixed with few drops of dilute iodine solution. A blue
color disappears on boiling and develops on cooling indicating the presence of starch.
2. Test for proteins:
a. Biuret test (General test):
To 3 ml extract solution, 4% sodium hydroxide and few drops of 1% copper sulfate
solution were added. The appearance of violet or pink color indicates the presence of
proteins.
3. Tests for amino acids:
a. Ninhydrin test (General test):
The extract and 3 drops of 5% Ninhydrin solution were heated in a boiling water bath for
10min. Purple or bluish color indicates the presence of amino acids.
b. Millon’s reagent:
The extract was heated with 3 drops of Millon’s reagent. The dark red color solution
confirms the presence of tyrosine.
4. Test for glycosides:
A small portion of the extract was hydrolyzed by boiling with dilute hydrochloric acid for
few minutes and hydrolysate was subjected to following tests:
a. Libermann-Burchard test:
Chloroform solution of hydrolysate was treated with acetic anhydride and sulphuric acid.
Formation of blue or blue-green color indicates the presence of steroidal saponins
whereas red, pink or violet color indicates the presence of triterpenoid saponins.
Experimental
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b. Legal’s test:
The hydrolysate was dissolved in pyridine and solution of sodium nitroprusside was
added to it and made alkaline. Formation of pink or red color indicates the presence of
cardiac glycosides.
c. Borntrager’s test:
An organic solvent like ether or chloroform was added to the hydrolysate and the
contents were shaken. The organic layer was shaken and treated with solution of
ammonia. The development of pink color indicates the presence of anthraquinone
glycosides.
5. Test for Saponin Glycosides:
a. Foam test:
About 1ml of extract was diluted with water to 20 ml and shaken in a graduated cylinder
for 15 min. A 1 cm layer of stable foam indicates presence of saponins.
6. Test for flavonoids:
a. Shinoda test:
A small piece of magnesium ribbon was added to the alcoholic solution of the extract
followed by drop wise addition of concentrated hydrochloric acid. The green blue color
indicates the test is positive.
7. Test for alkaloids:
A small portion of solvent free extract was stirred with few drops of dilute hydro
alcoholic acid and filtered. The filtrate was tested with following reagents:
a. Dragendorff reagent (Potassium bismuth iodide):
To 2-3ml filtrate, few drops of the reagent was added. Orange brown precipitate is
formed.
b. (Mercury potassium iodide):
To 2-3ml filtrate, few drops of the reagent added gives cream precipitate.
c. Hager’s reagent (Saturated picric acid):
With 2-3ml of filtrate the reagent gives yellow precipitate.
Experimental
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d. Wagner’s reagent (Iodine reagent):
With 2-3ml of filtrate the reagent gives reddish brown precipitate.
8. Test for Tannins and Phenolic Compounds
a. Ferric chloride test:
To the test solution few drops of 5% FeCl3 solution are added. The development of blue
black color indicates the presence of tannins and phenolics.
9. Test for Fats and Fixed Oils
a. Stain test
A small quantity of extract is pressed between two filter papers. If the filter paper is
stained then it indicates the presence of fixed oils.
b. Saponification test
Few drops of 0.5N of alcoholic potassium hydroxide is added to small quantities of
various extracts along with a drop of phenolphthalein separately. The mixture is heated
on a water bath for 1-2 hrs. The formation of soap or partial neutralization of alkali
indicates the presence of fixed oils and fats.
5.6 IN VITRO EVALUATION OF ANTIOXIDANT ACTIVITY
5.6.1 Background
Free radicals are molecules with one or more unpaired electrons. They are fundamental to
any biochemical process and represent an essential part of aerobic life and our
metabolism. They are continuously produced by the body’s normal use of oxygen such as
respiration and some cell mediated immune functions. The oxygen consumption inherent
to cell growth leads to the generation of a series of reactive oxygen species (ROS). ROS
which include free radicals such as superoxide anion radicals (O2), hydroxyl radicals
(OH∙) and non free radical species such as hydrogen peroxide (H2O2) and singlet oxygen
(1O2) are various forms of activated oxygen. ROS are continuously produced during
normal physiologic events and can easily initiate the peroxidation of membrane lipids,
leading to the accumulation of lipid peroxides.195
Experimental
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There have been a number of methods developed to measure the efficiency of
antioxidants as pure compounds or in extracts. These methods focus on different
mechanisms of the antioxidant such as scavenging of oxygen, nitrite and hydroxyl
radicals, reduction of lipid peroxyl radicals, chelation of metal ions or inhibition of lipid
peroxidation.
In our research work, the antioxidant capacity of extracts was determined by DPPH free
radical scavenging activity and Nitric Oxide radical scavenging activity.
5.6.1.1. DPPH free radical scavenging activity196
Principle: DPPH (di-phenyl picryl hydrazyl) is a stable free radical that can accept an
electron or hydrogen radical to become a stable diamagnetic molecule. Due to its odd
electron, the methanolic solution of DPPH shows a strong absorption band at 517 nm.
When the solution of DPPH is mixed with a suitable reducing agent, the electrons
become paired off. This gives rise to a reduced form of DPPH as shown in fig 5.1. The
solution loses color stiochometrically with the number of electrons taken up. Such
reactivity has been widely used to test the ability of compounds/ plant extracts to act as
free radical scavenger. Reduction of DPPH radicals can be observed by the decrease in
absorbance at 517 nm.
DPPH relatively stable- DPPH reduced to 1,1- Diphenyl-2-Picryl - radical
deep violet colour Hydrazine in presence of anti-oxidant-
Yellow colour
Figure 5.1 Reduction of DPPH radical to 1, 1- Diphenyl-2- Picryl Hydrazine197
Experimental
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Procedure: 0.1mM solution of DPPH in methanol was prepared. 1.5ml of DPPH
solution was added to 1.5ml of extract solution at different concentrations. The mixtures
were shaken vigorously and incubated in the dark for 30 min. Thirty minutes later, the
absorbance was measured at 517nm. Ascorbic acid was used as standard. Lower
absorbance of the reaction mixture indicates higher free radical scavenging activity.
Assay was done in triplicates. The capability to scavenge the DPPH radical was
calculated using the following equation:
% inhibition= (A control – A test/std / A control) × 100
5.6.1.2. Nitric Oxide radical scavenging activity198
Principle: NO is very unstable in biological systems and has a physiological half life of
only 1–40 s. Sodium nitroprusside in aqueous solution at physiological pH spontaneously
generates nitric oxide which interacts with oxygen to produce nitrite ions. After reduction
to nitrite, samples are reacted with the Griess reagent consisting of equal volumes of
sulfanilamide solution and N-(1- napthyl)ethylenediamine (NED) solution (fig 5.2).
Figure. 5.2 Schematic diagram representing the Griess reaction principle199
Procedure: Sodium nitroprusside (10mM, 1.5ml) in phosphate –buffered saline (PBS)
was mixed with 0.5ml of different concentrations of the extract dissolved in the suitable
Experimental
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solvent systems and incubated at 250C for 150 min. The sample mixtures were then
reacted with 1ml Griess reagent for 15 min (1% sulphanilamide, 2% H3PO4 and 0.1%
napthylethylenediamine dihydrochloride). The absorbance of the chromophore formed
during the diazotization of nitrite with sulphanilamide and subsequent coupling with
napthylethylenediamine was read at 546nm with reference to the absorbance of standard
solutions of ascorbic acid, treated in the same way with Griess reagent.
The % of NO scavenging activity is calculated as follows:
Scavenging Effect (%) = (A cont − A test)/A cont × 100
5.7 EVALUATION OF ANTIMICROBIAL ACTIVITY
5.7.1 Background
Antimicrobials are substances or mixtures of substances used to destroy or suppress the
growth of harmful microorganisms. There is need to accurately determine the microbial
susceptibility to antimicrobial agents. The use of plant extracts and phytochemicals, both
with known antimicrobial properties, are of great significance to therapeutic treatments.
The goal of invitro antimicrobial susceptibility testing is to provide a reliable predictor of
how a microorganism is likely to respond to antimicrobial therapy in the infected host.200
Invitro antimicrobial susceptibility testing can be performed using a variety of methods,
the most common being agar disc diffusion and agar dilution techniques.
5.7.2 Methodology
Two techniques were used to test the antibacterial activity of Rosa damascena and
Mimusops elengi extracts viz; agar ditch plate diffusion technique and agar cup plate
diffusion technique against bacterial and fungal strains.
Microorganisms tested
The test organisms used for evaluation of antimicrobial activity were based on their
known pathogenic effects on wounds. The organisms were obtained from National
Chemical Laboratory, Pune, Maharashtra.
Experimental
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Gram positive- Staphylococcus aureus (NCIM- 5022), Sterptococcus pyogenes (NCIM-
2608), Clostridium perfringens (NCIM- 2677).
Gram negative- Pseudomonas aeruginosa (NCIM- 2200), Escherichia coli (NCIM-
2065), Klebsiella pneumoniae (NCIM- 5082), Klebsiella aerogenes (NCIM- 2239)
Fungi- Candida albicans (NCIM- 3471), Aspergillus niger (NCIM- 1196)
Preparation of Inoculums
Authentic pure cultures of bacteria were cultivated in Nutrient broth (NB) at 37 ± 0.2°C.
Fungi and yeasts were cultured on Sabouraud dextrose agar at 25 ± 0.2°C. The cultures
of bacteria and fungi were maintained in their appropriate agar slants at 4°C throughout
the study and used as stock cultures. Microbial cultures were suspended in a saline
solution (0.85% NaCl) and adjusted to a turbidity of 0.5 MacFarland standard (108
CFU/ml).
Sample preparation
Methanol, ethyl acetate and petroleum ether (60-80°C) extracts were dissolved in
Dimethyl sulfoxide (DMSO) while aqueous extract was dissolved in sterile distilled
water to obtain the required concentrations for the study.
5.7.2.1 Agar ditch plate diffusion technique201
Agar ditch plate diffusion technique is used for primary screening of extracts against
various organisms. The antimicrobial susceptibity of each organism to the test extracts is
tested in this method.
In this technique 30ml of molten agar (nutrient agar/sabouraud dextrose agar) was poured
into a sterile petri plate. The molten agar was allowed to set and harden at room
temperature. Ditch (1cm×3cm) was made in the agar plate using a sterile scalpal.
Solution of each extract (1ml solution containing 50mg of extract) was placed in ditch. A
loopful of inoculum of each organism (gram positive, gram negative and fungi) was
streaked across the agar at right angle to the ditch. All the petri plates containing bacterial
Experimental
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cultures were incubated at 37º C for 24 hrs. Plates containing fungal cultures were
incubated at 25º C for 72 hrs.
5.7.2.2 Agar well diffusion technique202
The extracts were then evaluated for antimicrobial activity using agar well diffusion
method. Nutrient agar plates were seeded with 1ml of bacterial suspension and sabouroud
dextrose agar plates with fungal strain (equivalent to 108 cfu/ml). The seeded plates were
allowed to set. A sterile cork borer of 11mm diameter was then used to cut equidistant
wells on the surface of the agar. The wells were filled with 0.2ml solution of each extract
at various concentrations (50, 100 and 200µg/ml). The plates were incubated at 37°C for
24hrs and 25ºC for 72hrs after which the diameter of zones of inhibition were measured.
Chloramphenicol (100 g) for bacteria and Fluconazole for fungi (15 g) were used as
positive control. All the experiments were done in triplicates.
Statistical analysis
All the test analyses were run in triplicate and values were expressed as Mean ± SEM.
5.8 QUANTITATIVE PHYTOCHEMICAL ANALYSIS OF BIOACTIVE
EXTRACT/S.
Extracts of Rosa damascena flower petals and Mimusops elengi bark were analyzed
quantitatively for tannins and phenols. Fruits of Mimusops elengi exhibited poor
antioxidant and antimicrobial activities hence were not taken for further studies.
5.8.1 Total phenolics and tannins by Folin-Ciocalteu method203
Principle:
The Folin-Ciocalteu reagent (FCR) or Folin's phenol reagent or Folin-Denis reagent, also
called the Gallic Acid Equivalence method (GAE), is a mixture phosphotungstate used
for the colorimetric assay of phosphomolybdate and phenolic and polyphenolic
antioxidants. It works by measuring the amount of the substance being tested needed to
inhibit the oxidation of the reagent. The reaction forms a blue chromophore constituted
Experimental
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by a phosphotungstic- phosphomolybdenum complex, where the maximum absorption of
the chromophores depends on the alkaline solution and the concentration of phenolic
compounds.
Procedure
The amount of total phenolic and tannins in the plant extract was determined
calorimetrically with the Folin-Ciocalteu reagent (FCR). The reaction mixture contained
50µl of the extract (1mg/ml) in methanol, 250µl of FCR, 750µl of sodium carbonate
solution. The volume was made upto 5ml with distill water and was incubated in dark
under ambient conditions for 2 hrs to complete the reaction. In the control tube, the
extract volume was replaced by methanol.
The absorbance of the resulting solution was measured at 760nm in a UV
spectrophotometer. The concentration of total phenolics and tannins was expressed as mg
of gallic acid equivalents (GAE) per g of dried extract, using a standard curve of gallic
acid. All the measurements were carried out in triplicates.
Total phenolic content was calculated using the following formula:
C=c×V/ m, where
C= total content of phenolic compounds in mg/g plant extract in GAE or mg GAE/g
extract
c= the concentration of gallic acid established from the calibration curve in mg/ml
V= the volume of extract in ml; m= the weight of plant extract in g
5.8.2 Total flavonoids by aluminium chloride method204
Principle:
The aluminium ion (Al3+)
is reacted with the flavonoids in the sample to form the stable
flavonoid-Al3+
complex, which has a yellow colour and intensity proportional to the
flavonoid concentration. This reaction causes a bathochromic shift and intensification in
the absorption, which can be measured without influence from other phenolic compounds
present in the sample.
Experimental
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Procedure:
The flavonoids content in the plant extract was determined by aluminium trichloride
method using rutin as a reference compound. The 100µl of plant extract (10mg/ml) in
methanol was mixed 100µl with 20% aluminium trichloride in methanol and a drop of
acetic acid, and then diluted with methanol to 5ml. The absorption at 415nm was read
after 40 min. Blank consists of 100μl of extract, a drop of acetic acid and adjusted to 5ml
with methanol. The absorption of standard rutin solution (0.5mg/ml) in methanol was
measured under the same conditions. All measurements were carried out in triplicates.
The amount of flavonoids in the extract in rutin equivalents (RE) was calculated using
following formula-.
X = (A. mo)/(Ao.m)
Where, X = flavonoid content of extract in mg/g extract in RE or mg RE/g extract,
A= absorption of plant extract solution, A0 = absorption of standard rutin solution,
m = weight of extract in mg and mo = weight of rutin in the solution in mg.
5.9 PHARMACOLOGICAL INVESTIGATIONS
Preparation of animals, house and feeding conditions for pharmacological studies
Rats and mice of albino wistar strain were procured from Haffkine’s Research Centre,
Parel and Bharat Serum and Vaccines Pvt. Ltd, Thane, Mumbai and housed in animal
house of C. U. Shah College of Pharmacy. Animals were acclimatized to the
experimental room for one week and conditioned at room temperature and natural
photoperiods. Animals were caged in polypropylene cages containing paddy husk as
bedding with maximum of three animals in each cage. Animals were provided with free
access to standard food pellets as basal diet and water ad libitum. Study was conducted
after obtaining ethical committee clearance from the Institutional Animal Ethical
Committee (IAEC) of C. U .Shah College of Pharmacy, S. N. D. T University, Mumbai.
Experimental
83
5.9.1 WOUND HEALING STUDIES OF EXTRACTS
The wound healing activity of extracts was evaluated using excision, incision and dead
space wound models.
5.9.1.1 Experimental design
Materials and reagents: Depilator (Veet hair remover cream), ketamine HCl, sterilized
absorbant and non- absorbant cotton, sterilized scissors, sterilized forceps, sterilized
scalpel blade, black surgical thread, curved needle (no.19), sterilized cotton pellets,
Animals: Male Wistar rats of 2-3 month old weighing 180-250g
Animal housing and feeding conditions during the experiment:
The animals were housed in polypropylene cages, maintained under standard conditions
(12h/12h light and dark) at 25±3ºC and 35%-60% humidity. They were fed with standard
rat pellet diet and water ad libitum. Experimental animals were housed individually in
single cage to avoid contamination of wound from each other. Measures were taken to
avoid minimal contamination. Cages were washed and sterilized daily with dilute alcohol
solution and bedded with filter paper instead of paddy husk. The filter papers were
changed daily twice.
Study groups
Group I: Negative control group (untreated)
Group II: Rosa damascena - aqueous extract (0.5 gm, topically).
Group III: Rosa damascena - methanol extract (0.5 gm, topically).
Group IV: Mimusops elengi bark - aqueous extract (0.5 gm, topically).
Group V: Mimusops elengi bark - methanol extract (0.5 gm, topically).
Group VI: Mimusops elengi bark - ethyl acetate extract (0.5 gm, topically).
Group VII: Mimusops elengi bark - petroleum ether extract (0.5 gm, topically).
Group VIII: Mimusops elengi fruit - aqueous extract (0.5 gm, topically).
Group IX: Mimusops elengi fruit - methanol extract (0.5 gm, topically).
Group X: Mimusops elengi fruit - ethyl acetate extract (0.5 gm, topically).
Experimental
84
Group XI: Mimusops elengi fruit - petroleum ether extract (0.5 gm, topically).
Group XII: Positive control group (Mupirocin cream, 0.5 gm, topically)
5.9.1.2 Excision wound model205
The hair on the back of the rats was depilated with depilator cream (Veet) one day prior
of the study. An excision wound was inflicted by cutting away full thickness of skin of
area 500mm2 from depilated skin on the back of the rats. Hemostasis was achieved by
blotting the wound with a cotton swab soaked in normal saline. The wound was left
undressed to the open environment. Extracts were applied topically once a day till
complete epithelization; starting from the day of operation. The parameters studied are
wound closure and period of epithelization.
% Wound contraction
The rate of wound contraction was measured as percentage reduction of wound sizes
every 2 day interval. Progressive decrease in the wound size was monitored periodically
using transparency paper and a marker, and the wound area was assessed graphically to
monitor the percentage of wound closure, which indicates the formation of new epithelial
tissue to cover the wound. Wound contraction was expressed as reduction in percentage
of the original wound size.
% wound contraction = wound area on day 0 – wound area on day n X 100
wound area on day 0
Period of epithelization
Falling of eschar without any raw wound area was considered as complete healing of
wound and the number of days required for falling of eschar without any residual raw
wound was calculated as a period of epithelialisation.
Experimental
85
5.9.1.3 Incision wound model205, 206
Animals were anaesthetized with ketamine HCl (0.2ml, i.p.). Two longitudinal
paraventral incision of 6 cm were made through the skin and cutaneous muscles on the
depilated back of the rat using sterile scalpel blade. After the incision was made the
parted skin was kept together and stitched and closed with interrupted sutures (black
surgical thread and curved needle no.19) 1 cm apart. Extracts were applied topically once
a day. The sutures were then removed on the 8th
post – wounding day and the tensile
strength of 10-day old wound was measured by tensiometer.
Measurement of tensile strength
Tensile strength is the resistance to breaking under tension. It indicates how much the
repaired tissue resists to breaking under tension and may indicate in part the quality of
repaired tissue.
Sutures were removed on the day 9, the newly formed tissue including scar was excised
and tensile strength was measured with the help of tensiometer. The clamps were
carefully attached to the skin on the opposite sides of the wound at a distance of 0.5 cm
away from the wound. Weights were added on the metal pan gradually until the wound
split. The values of total weights on the pan were considered as an indirect measure of the
tensile strength of the wound. The mean determination of tensile strength on the two
paravertebral incisions on both sides of the animals was taken as the measures of the
tensile strength of the wound for an individual animal.
Histopathological evaluation
Wound tissue specimens from treated and untreated rats were collected. The tissues were
then fixed in 10% formaldehyde solution and after the usual processing 6 mm-thick
sections were cut and stained with haematoxylin and eosin. Sections were qualitatively
assessed under the light microscope and graded in respect of, oedema, infiltration of
polymorphonuclear leukocytes and monocytes, necrosis, fibroblast proliferation, collagen
formation, angiogenesis and epithelisation.
Experimental
86
5.9.1.4 Dead space wound model206
Animals were anaesthetized with ketamine HCl (0.2 ml, i.p.). A longitudinal paraventral
incision was made through the skin on the depilated back of the rat using sterile scalpel
blade. Dead space wounds were inflicted by implanting two sterilized cotton pellets (10
mg), one on either side of the lumbar region on the ventral surface of each rat. The parted
skin were kept together and stitched and closed with interrupted sutures (black surgical
thread & curved needle no.19) 1cm apart. On the 10th
post wounding day, the granulation
tissue formed on the implanted cotton pellet was carefully removed. The wet weight of
the granulation tissue was noted. The granulation tissue was further processed for the
estimation of lipid peroxides and collagen tissue parameters (hydroxyproline and
hexosamine)
The granulation tissues were dried at 60°C for 12 hrs, and weighed, and the dry weight
was recorded. The dried tissue were then hydrolyzed with 5ml 6 N HCl and kept at
110°C for 24 hrs in a sealed glass tubes. This acid hydrolysate was used for estimation of
hydroxyproline and hexosamine content.
Estimation of granulation tissue lipid peroxides by Thiobarbituric Acid Assay207
1ml of 10% of granulation tissue homogenate prepared in 50 mM phosphate buffer saline
(pH 7) was combined with 2ml of TCA-TBA-HCI and mix thoroughly. The solution was
heated for 15mins in a boiling water bath. After cooling, the flocculent precipitate was
removed by centrifugation at 1000 rpm for 10mins. The absorbance of the sample was
determined at 535nm against a blank that contains all the reagents minus the tissue
homogenate. The malondialdehyde (MDA) concentration of the sample was calculated
using an extinction coefficient of 1.56 × 105 M
-1 cm
-1and reported as µmol/g of wet
tissue.
Determination of Hydroxyproline content208
The above acid hydrolysate was neutralized to pH 7. The hydrolysate was neutralized to
pH 7.0.The samples were mixed with 1ml of 0.01M CuSO4 followed by the addition of
Experimental
87
1ml of 2.5 N NaOH and then 1ml of 6% H2O2. The solution was mixed and shaken
occasionally for 5 min. All the tubes were incubated at 80 C for 5 min with frequent
vigorous shaking. Upon cooling, 4ml of 3N H2SO4 was added with agitation. Finally, 2
ml of 5 % p-dimethylaminobenzaldehyde was added. The samples were incubated at
70ºC for 16 min, cooled by placing the tubes in water at 20 ºC, and the absorbance was
measured at 500 nm using spectrophotometer. The amount of hydroxyproline in the
samples was calculated using a standard curve prepared with pure L-hydroxyproline at
the same time.
Determination of Hexosamine content209
Acid hydrolyzed fraction (0.05ml) was diluted to 0.5ml with distilled water. To this was
added 0.5ml of acetyl acetone reagent and heated in boiling water bath for 20 min then
cooled under tap water. To this 1.5ml of 95% alcohol was added, followed by an addition
of 0.5ml of Ehrlichs reagent. The reaction was allowed for 30 min to complete. Color
intensity was measured at 530nm against the blank. Hexosamine content of the samples
was determined from the standard curve prepared with D (+) glucosamine hydrochloride
5.10 FORMULATION DEVELOPMENT AND ITS EVALUATION
5.10.1 Topical Drug Delivery system
Over the last decades the treatment of illness has been accomplished by administrating
drugs to human body via various routes namely oral, sublingual, rectal, parental, topical,
inhalation etc. Topical delivery can be defined as the application of a drug containing
formulation to the skin to directly treat cutaneous disorders (e.g. wounds, acne) or the
cutaneous manifestations of a general disease (e.g. psoriasis) with the intent of containing
the pharmacological or other effect of the drug to the surface of the skin or within the
skin. Semi-solid formulation in all their diversity dominate the system for topical
delivery, but foams, spray, medicated powders, solution, and even medicated adhesive
systems are in use.210
Experimental
88
Topical delivery includes two basic types of product:
External topicals that are spread, sprayed, or otherwise dispersed on to cutaneous
tissues to cover the affected area.
Internal topicals that are applied to the mucous membrane orally, vaginally or on
anorectal tissues for local activity.211
For the most part topical preparations are used for the localized effects at the site of their
application by virtue of drug penetration into the underlying layers of skin or mucous
membranes. Although some unintended drug absorption may occur, it is sub therapeutics
quantities and generally of minor concern.
Advantages of Topical Drug Delivery Systems:
Avoidance of first pass metabolism.
Convenient and easy to apply.
Avoidance of the risks and inconveniences of intravenous therapy and of the varied
conditions of absorption, like pH changes, presence of enzymes, gastric emptying
time etc.
Achievement of efficacy with lower total daily dosage of drug by continuous drug
input.
Avoids fluctuation in drug levels, inter- and intrapatient variations.
Ability to easily terminate the medications, when needed.
A relatively large area of application in comparison with buccal or nasal cavity
Ability to deliver drug more selectively to a specific site.
Avoidance of gastro-intestinal incompatibility.
Providing utilization of drugs with short biological half-life, narrow therapeutic
window.
Experimental
89
Improving physiological and pharmacological response.
Improve patient compliance.
Provide suitability for self-medication.
Disadvantages of Topical Drug Delivery Systems:
Skin irritation of contact dermatitis may occur due to the drug and/or excipients.
Poor permeability of some drugs through the skin.
Possibility of allergenic reactions.
Can be used only for drugs which require very small plasma concentration for action
Enzyme in epidermis may denature the drugs
Drugs of larger particle size not easy to absorb through the skin212,213
Classification of Topical Drug Delivery Systems211
Table no. 5.3 Classification of Topical Drug Delivery Systems based
on physical state
Solid Liquid Semi-solid
Powder Lotion Ointment
Aerosol Liniment Cream
Plaster Solution Paste
Emulsion Gel
Suspension Jelly
Aerosol Suppository
Experimental
90
5.11 DEVELOPMENT AND EVALUATION OF TOPICAL FORMULATIONS
CONTAINING METHANOL EXTRACT OF MIMUSOPS ELENGI AND ROSA
DAMASCENA
5.11.1 Background
Topical formulations are preferred choice for healing dermal wounds as they are locally
well absorbed to produce pharmacodynamic action effectively. In addition, this approach
is in tandem with the traditional use of Mimusops elengi and Rosa damascena involved in
topical application and is cited to be effective in various skin ailments like cuts, wounds,
inflammation. Several studies have reported that the use of herbal extracts incorporated in
topical dosage forms such as creams, ointment and gel exhibited better wound healing
potential than the crude extracts.214,215
From the invivo pharmacological activity, it was observed that, MEB-ME and RD-ME
showed promising wound healing activity. Hence an attempt was made to evaluate the
wound healing and antiaging activity of MEB-ME and RD-ME in a pharmaceutically
accepted topical dosage forms such as gel and cream. Topical cream and gel formulations
containing varying concentrations of MEB-ME and RD-ME were successfully
developed.
5.11.2 Methodology
Extracts used: Methanol extract of Mimusops elengi bark (MEB-ME) and methanol
extract of Rosa damascena flower petals (RD-ME).
Concentration/dose of extracts: 0.1%, 0.25%, 0.5% and 1% of both MEB-ME and RD-
ME were incorporated in preparation of cream and gel formulations.
5.11.2.1 Development and evaluation of cream formulation
RD-ME extract was dissolved in water whereas MEB-ME extract was dissolved in
ethanol.
Experimental
91
Two formulae 1 and 2 of cream were prepared, containing different concentrations of
mineral oil (olive oil) and cetosteryl alcohol.
Phase I was prepared by melting mineral oil, cetosteryl alcohol and glyceryl
monostearate at 70ºC with constant stirring. Phase II was prepared by mixing RD-ME
extract solution, MEB-ME extract solution, glycerine and water. Both the phases were
heated to 70ºC. Phase I was added to phase II with constant stirring using an overhead
stirrer for 20 -25 mins until a uniform cream was obtained.
Table no 5.4 Formulation of batches of cream (%w/w)
Ingredients Formula 1 Formula 2
F1C1 F1C2 F1C3 F1C4 F2C1 F2C2 F2C3 F2C4
RD-ME extract 0.1 0.25 0.5 1 0.1 0.25 0.5 1
MEB-ME extract 0.1 0.25 0.5 1 0.1 0.25 0.5 1
Mineral oil (olive
oil) 3 3 3 3 2 2 2 2
Cetosteryl
alcohol 5 5 5 5 3 3 3 3
Glyceryl
monostearate 15 15 15 15 15 15 15 15
Glycerine 10 10 10 10 10 10 10 10
Water q.s to
100%
q.s to
100%
q.s to
100%
q.s to
100%
q.s to
100%
q.s to
100%
q.s to
100%
q.s to
100%
Physical Evaluation of cream formulations
The prepared creams were evaluated for physical parameters like homogeneity and
consistency.
From the physical evaluation, it was observed that creams prepared with formula 2, gave
consistent cream. Creams prepared with formula 1 were poor in consistency, and hence
unsuitable for application. Formulations prepared with formula 2 were selected for
further characterization.
Experimental
92
5.11.2.2 Development and evaluation of Gel formulation
Carbopol 940 polymer was used as gelling agent for preparation of gel. Three formulae 1,
2 and 3 of gels containing varying concentrations of carbopol 940 (0.5, 1 and 1.5%) were
prepared.
Carbolpol 940 was soaked in distilled water overnight. RD-ME extract was dissolved in
water whereas MEB-ME extract was dissolved in ethanol. Extracts were then added to
the soaked polymer and stirred using an overhead stirrer for 1-1.5 hrs. Alcohol and
glycerin were added to the above mixture and stirred till a uniform suspension was
obtained. After the addition was complete, gels were spontaneously formed with addition
of TEA and adjustment of pH to 7. Methyl paraben (0.1%) was added to the final
preparation as a preservative.
Table 5.5 Formulation of batches of Gel formulations (%w/w)
Ingredient Formula 1 Formula 2 Formula 3
F1
G1
F1
G2
F1
G3
F1
G4
F2
G1
F2
G2
F2
G3
F2
G4
F3
G1
F3
G2
F3
G3
F3
G4
RD-ME
extract 0.1 0.25 0.5 1 0.1 0.25 0.5 1 0.1 0.25 0.5 1
MEB-ME
extract 0.1 0.25 0.5 1 0.1 0.25 0.5 1 0.1 0.25 0.5 1
Carbopol
940 0.5 0.5 0.5 0.5 1 1 1 1 1.5 1.5 1.5 1.5
Glycerin 1 1 1 1 1 1 1 1 1 1 1 1
Alcohol 1 1 1 1 1 1 1 1 1 1 1 1
Water q.s
to
100
q.s
to
100
q.s
to
100
q.s
to
100
q.s
to
100
q.s to
100
q.s
to
100
q.s
to
100
q.s
to
100
q.s
to
100
q.s
to
100
q.s
to
100
TEA to
adjust pH
7
q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s q.s
Experimental
93
Physical evaluation of the gels
The prepared gels were evaluated for physical parameters like homogeneity, clarity and
consistency.
From the physical evaluation, it was observed that gels comprising of carbopol 940(1%),
gave consistent gels. Gels with lower concentration of gelling agent (0.5%) were poor in
consistency. Gels with higher concentration of gelling agents (1.5%) were viscous and
does not spread easily on skin surface, hence unsuitable for application. Formulations
prepared with formula 2 were selected for further characterization.
5.11.2.3 Evaluation of topical cream and gel formulations.216,217,218,219
1. pH
5g of the cream/gel was weighed in 100ml beaker and 45ml of water was added. The gel
was completely dispersed in it. pH of suspension was measured using the pH meter. The
pH meter was previously standardized using pH 4, pH 7 and pH 9. The assay was done in
triplicates.
2. Spreadability
Spreadability (g/cm/sec) is expressed in terms of time taken in seconds by two slides to
slip off from the gel placed between them, under certain load. Spreadability of the
cream/gels was determined using a spreadability tester. The apparatus consists of two
glass plates of dimension 10cm x 20cm. One of the glass plate is fixed on the wooden
block, while the other plate is free to slide onto the former one. One end of the movable
plate is tied to the string, which passes over a pulley. The other end of the string is
attached to a pan meant for holding the weights. 1g of the cream/gel was placed between
two glass plates. A weight of 300g was allowed to rest on the upper plate for 5 min in
order to expel any entrapped air from between the plates. This led to the formation of
uniform film of cream/gel. The weights were removed and a weight of 100g was placed
in the pan, leading to a pull being exerted on the free glass plate. The time in seconds
required for the upper plate to travel a distance of 10cm across the length of the lower
Experimental
94
plate was recorded. This time was indicative of the relative spreadability of the
cream/gel.
3. Viscosity
Brookfield viscometer was used to determine the viscosity of the cream/gel. The cream or
gel was placed in the beaker. The spindle was lowered in it such that it was completely
immersed in the gel but not touching the bottom of the beaker. On rotating of the spindle
at the fixed speed of 5 rpm, the dial reading on the viscometer was noted. Direct
multiplication of the dial reading with the factors given, gave the viscosity of the
cream/gels in centipoises.
5. Extract content
Preparation of Standard Stock Solution
1mg of each extract was accurately weighed, transferred to 10ml volumetric flask and
dissolved in 10ml of methanol to give a standard solution of 100µg/ml. The volumetric
flask was placed in an ultrasonic bath to affect dissolution of extracts.
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 for RD-ME and ethanol
for MEB-ME. The absorbance of the resultant solution was scanned in UV range (200-
400 nm) for maximum absorbance after enabling blank correction for methanol/ethanol
in the above region.
Procedure for plotting calibration curve
The standard solutions were prepared by proper dilutions of the primary stock solution
with methanol to obtain working standards in the concentration range of 1-10µg/ml. The
absorbance was measured against a solvent blank and the calibration curve was plotted.
Similarly absorbance of sample solution was measured and the amount of RD-ME and
MEB-ME extracts were determined by referring to the calibration curve.
Experimental
95
Estimation of extracts in cream and gel formulations
For estimation of RD-ME extract in cream/gel, the RD-ME was extracted from 1gm of
each cream/gel formulation with 100ml methanol for 30 min and the resultant extract was
filtered through membrane filter. The concentration of RD-ME was estimated from the
regression equation of calibration curve.
For estimation of MEB-ME extract in cream/gel, the MEB-ME was extracted from 1gm
of each cream/gel formulation with 100ml ethanol for 30 min and the resultant extract
was filtered through whatmann filter. The concentration of MEB-ME was estimated from
the regression equation of calibration curve.
5.12 PRIMARY SKIN IRRITATION STUDIES220,221
5.12.1 Background
Dermal acute studies are designed to provide information on local effects, particularly
skin irritation and corrosion. The principle of the invitro skin model irritation assay is
based on the premise that irritant chemicals are able to penetrate the stratum corneum by
diffusion and are cytotoxic to the cells in the underlying layers.
5.12.2 Procedure
Primary skin irritation studies were carried out using Draize test. Animals were divided
into 11 groups with 5 animals each. Dorsal hair were depilated from the back of the rats
one day prior to commencement of study with the help of depilator (Veet cream) and area
of 4cm2
was marked.
Animals: Male Wistar rats of 2-3 month old weighing 180-250g
Groups:
Group 1-4: Cream formulations (F2C1-F2C4),
Group 5-8: Gel formulations (F2G1-F2G4)
Group 9: 0.8 % v/v aqueous solution of formalin (standard irritant).
Experimental
96
The control, cream, gel formulations (0.5g/rat) and formalin solution were applied daily
for seven days. The application sites were covered with cotton bandage and were
observed for any signs of edema and erythema and were graded according to the draize
scoring scale as given in table 5.6.
Table 5.6 Draize scoring scale for evaluation of skin reaction
A. Erythema formation Score
Very slight erythema (barely perceptible) 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) 4
B. Edema formation
Very slight edema (barely perceptible) 1
Well defined edema (edges of area well defined by definite raising) 2
Moderate to severe edema (area raised approximately 1 mm.) 3
Severe edema (raised more than 1 mm, extending beyond area of
exposure) 4
5.13 WOUND HEALING STUDIES OF TOPICAL CREAM AND GEL
FORMULATIONS IN FRESH WOUNDS
Wound healing activity of cream (F2C1- F2C4) and gel (F2G1- F2G4) formulations were
carried out on fresh wounds by 3 invivo models viz;
i. Excision wound model: The procedure followed is described in section 5.9.1.1
ii. Incision wound model: The procedure followed is described in section 5.9.1.2
iii. Dead space wound model: The procedure followed is described in section 5.9.1.3
Experimental
97
STATISTICAL ANALYSIS
All statistical analyses were made using the software InStat for windows. All results were
expressed as Mean ± SEM post Hoc Dunnet’s test was used to determine statistical
significance. The values were considered statistically significant when p≤0.001.
5.14 WOUND HEALING STUDIES OF TOPICAL CREAM AND GEL
FORMULATIONS IN EXPERIMENTALLY INDUCED DIABETES RATS.
5.14.1 Selection of formulations
Cream and gel formulations which exhibited good wound healing activity in normal
wounds were selected further for the evaluation of wound healing activity in diabetic rats.
The formulations selected for the study were F2C2, F2C3, F2G2 and F2G3.
5.14.2 Induction of Experimental diabetes222, 223
Diabetes was induced in overnight-fasted rats by a single intraperitoneal (i.p) injection of
alloxan monohydrate 120mg/kg. The rats were fed with 5% glucose water and ad libitum
basal diet during the next 24 hrs to avoid sudden hypoglycemia. On day 2, glucose water
was replaced with normal drinking water. Blood samples were withdrawn from the retro-
orbital plexus of animals at 72 hrs and on day 7 after an overnight fast. Fasting blood
glucose levels were estimated using glucose strips (Accu check active Glucometer, Roche
diagnostics, Germany). Hyperglycemia was confirmed by elevated blood glucose levels
determined at 72hrs after injection. Rats with fasting blood glucose levels above
200mg/dl were considered diabetic and selected for the study.
These animals were then used for the evaluation of wound healing activity by excision,
incision and dead space wound model
i. Excision wound model: The procedure followed is described in section 5.9.1.1
ii. Incision wound model: The procedure followed is described in section 5.9.1.2
Experimental
98
iii. Dead space wound model: The procedure followed is described in section 5.9.1.3
STATISTICAL ANALYSIS
All statistical analyses were made using the software InStat for windows. All results were
expressed as Mean ± SEM post Bonferroni test was used to determine statistical
significance. The values were considered statistically significant (p≤0.001) when
compared with negative and diabetic control group.
5.15 IN VITRO WOUND HEALING ACTIVITY OF OPTIMIZED
FORMULATION
5.15.1 Background
Key to wound healing processes are the proliferation, migration, and functioning of
fibroblasts and keratinocytes, thus they are the basis of in vitro studies. Hence, the
fibroblast in vitro model is integral to correlating the contractile events of wound healing.
In vitro assays are useful for examining the effect of agents on particular cell types.224
They are quick, relatively inexpensive, and can be used to screen a wide variety of
conditions or samples simultaneously but are incapable of replicating all the factors
involved in complex processes of wound healing. In vitro assays are useful in wound
healing research for determining the possible effectiveness of various treatments,
particularly antimicrobial and healing enhancing agents. Another noteworthy attribute of
in vitro testing is the ability to screen multiple agents or samples simultaneously.225
5.15.2 Selection of formulation/s:
Optimized formulation which exhibited potent wound healing activity in normal and
diabetic wounds, so was selected further for the evaluation of in vitro wound healing
activity. The formulation selected for the study was gel F2G2.
Experimental
99
5.15.3 Scratch Assay
Principle: Tissue wounds undergo a complex and ordered series of events to repair
tissue. These events may include infiltration of inflammatory immune cells as part of the
process to remove and destroy necrotic tissue, increased vascularization by angiogenic
factors, and increased cell proliferation and extracellular matrix deposition. Wound
healing assays have been carried out in tissue culture for many years to estimate the
migration and proliferation rates of different cells and culture conditions. These assays
generally involve first growing a confluent cell monolayer. A small area is then disrupted
and a group of cell destroyed or displaced by scratching a line through the layer. The
open gap is then inspected microscopically over time as the cells move in and fill the
damaged area. This “healing” can take from several hours to over a day depending on the
cell type, conditions, and the extent of the “wounded” region.226
Groups
Group 1- Control (PBS)
Group 2 – Formulation F2G2
Procedure227
Cells were cultured to confluence or near (>90%) confluence in 35 mm dishes.
Depending on the conditions, cell were rinsed with PBS and starved in low serum media
overnight. Base media with the test compound was filter sterilized and stored at 4oC
(warmed up before using). Using a sterile, disposable scratch loop, wound was scratched
through the cells. Any cellular debris was removed by washing with phosphate buffer
saline (PBS) and replaced with 1.5ml of media containing the test and without test
compound. Cell migration in the induced wound was observed for 72 hrs. The
observations of the test samples were compared with the PBS control on the basis of cell
migration rate.
Experimental
100
5.16 IN VITRO SKIN AGING ACTIVITY OF OPTIMIZED FORMULATION
5.16.1 Background
Aging of skin is accompanied by thinning of epidermis and concomitant loss of dermal
connective tissue. This dermal connective matrix is made up of glycosaminoglycans
(GAG’s) interwoven with fibrous matrix proteins like collagen, elastin, tyrosine,
hyaluronic acid and fibronectin forming a cross linked meshwork that gives ECM
strength and resilience.228
Collagen is the important structural component of skin that
represents 70% to 80% of the dry weight. Elastin accounts for only about 1-2% of the dry
weight of skin but is important for the maintenance of skin’s elasticity and resilience.
Hyaluronic acid is mucopolysaccharide that holds the water and keeps the body moist,
lubricated and smooth.229
Tyrosine is a non essential aminoacid that helps produce
melanin, the pigment responsible for hair and skin color. These GAG’s are important for
maintaining a healthy and younger skin. The connective tissue proteins are constantly
attacked by several enzymes like collagenases, elastases, tyrosinase, hyaluronidase and
matrix metalloproteinases, which leads to decrease in thickness of skin, dry and wrinkled
and consequently aged skin.230
Efficacy of any antiaging compound/substances can be
determined in vitro by measuring the amount of these enzymes inhibited by the test
substances. Hence by assessing anti-collagenase, anti-elastase, anti-hyaluronidase and
anti-tyrosinase activity; skin antiaging potential of test substances can be determined.
5.16.2 Selection of formulation/s
Optimized formulation/s which exhibited potent wound healing activity in normal and
diabetic wounds were selected further for the evaluation of in vitro skin aging activity.
The formulation selected for the study was gel F2G2.
Gel F2G2 was evaluated for in vitro skin aging activity by following assays:
1. Anti-Collagenase Activity
2. Anti-Elastase activity
3. Anti-Hyaluronidase activity and
Experimental
101
4. Anti-Tyrosinase activity
5.16.3 Anti-Hyaluronidase Activity
Principle231
The colorimetric assay (Reissig assay, Morgan-Elson assay) is based on the reaction of
the N-acetyl-D-glucosamine (GlucNAc) at the reducing ends of hyaluronan and its
fragments with p-dimethylaminobenzaldehyde resulting in a red coloured product. The
postulated main product of the degradation of hyaluronic acid by the bovine
hyaluronidase is a tetrasaccharide with N-acetyl-D-glucosamine at the reducing end (fig
5.3). The chromogens I and II are formed under alkaline conditions (100 °C, pH 9) of the
Morgan-Elson reaction. The chromogen III, which is formed by elimination of water
under acidic conditions (conc.HCl / glacial acetic acid) react in the final step with p-
dimethylaminobenzaldehyde (Ehrlich´s reagent) to give the unstable red – coloured
product, which can be photometrically measured at 586 nm.
Procedure232
50µl bovine hyaluronidase (7900 units/ml) was mixed with 50µl of various
concentrations of gel F2G2 dissolved in 5% DMSO. The mixture was incubated at 37oC
for 20 min. The control group was treated with 50µl of DMSO instead of the sample.
Hyaluronidase was activated by adding 50µl of 12.5mM calcium chloride in reaction
mixture and incubated at 37oC for 20mins. The Ca
+2activated hyaluronidase was
subjected to 250µl of sodium hyaluronate (1.2mg/ml) and then incubated in waterbath at
100oC, exactly for 3min. Reaction mixture was allowed to cool to room temperature.
1.5ml of p-Dimethyl amino benzaldehyde was added to the reaction mixture and it was
then incubated in water bath at 37oC for 20mins. The absorbance was measured at 585
nm. The assay was performed in triplicates.
Calculation
Abs of control - (Abs of test – abs of blank)
____________________________________________ X 100
% Anti-hyaluronidase =
Abs of control
Experimental
102
Figure. 5.3 Schematic representation of Reissig assay231
Experimental
103
5.16.4. Anti-collagenase activity
Principle233
Figure 5.4 Breakdown of collagen into collagen fragments233
The most basic method for determining enzyme activity is the ninhydrin (2,2-
dihydroxyindane-1,3-dione) assay which was originally developed by Mandl et al.
(1953). The ninhydrin assay detects the release of amino acids and peptides librated from
the breakdown of collagen (fig 5.4). Collagen is incubated with the enzyme and the
librated peptides are measured by colormetric ninhydrin methods to detect the
nonspecific protease activity after incubation for 5hrs at 37°C.
Procedure
25mg of bovine collagen was weighed into four test tubes. Additional two test tubes were
included to serve as blanks which contained no enzyme. 5ml of 0.05 M TES buffer was
added to the tubes and incubated at 37°C for 15 min. The reaction was started by adding
0.1ml of enzyme dilution to appropriate tubes. After 5hrs, collagenase reaction was
terminated by transferring 0.2ml of solution (leaving behind the collagen) to test tubes
containing 1ml of ninhydrin-citric acid mixture. The reaction mixture was heated for 20
min in a boiling water bath. After cooling, the mixture was diluted with 5ml of 50 % n-
propanol. After 15 min the absorbance was read at 600 nm. The assay was performed in
triplicates
Calculation
Abs of control - (Abs of test – abs of blank)
% Anti-collagenase = ____________________________________________ X 100
Abs of control
Experimental
104
5.16.5. Anti- Elastase activity235
Principle
Porcine pancreatic elastase enzyme (Sigma) is incubated with the inhibitor
(investigational sample) under study in the cuvette. The amount of enzyme left
uninhibited is detected by reacting it with substrate N-Succ-(Ala)-nitroanilide that gives
p-nitroaniline as the final product. This is read spectrophotometrically at 405 nm. Higher
the amount of product formed, lower is the inhibitory effect of the investigational sample
on the enzyme.
Procedure
The test sample was added to 200 mM Tris HCl buffer (pH 8). The elastase enzyme (10
μg/ml) was added and the reaction was initiated by adding substrate N-Succ-(Ala)-
nitroanilide. The test reaction was incubated for 30 min at 25°C and the absorbance was
read at 410 nm. Appropriate blanks were also run. The assay was performed in triplicate
Calculation:
Abs of control - (Abs of test – abs of blank)
____________________________________________ X 100
% Anti-elastase =
Abs of control
5.16.6. Anti-Tyrosinase Activity
Principle236
Tyrosinase plays a vital role in the melanin pathway. The anti-tyrosinase activity of the
test substance is determined by incubating the enzyme with the test and substrate. The
uninhibited enzyme will oxidize the substrate to DOPA-quinone, which can be detected
spectrophotometrically at 475 nm (fig 5.5). Higher the product formed, more intense is
the color and consequently, lesser is the inhibitory effect of test sample on the enzyme.
Experimental
105
Figure 5.5 Oxidation of tyrosine to DOPA quinone236
Procedure237
Each test sample was incubated with enzyme (tyrosinase) and substrate (tyrosine) in
phosphate buffer for 45 min at 37°C. The test tubes were placed in an ice bath to
terminate the reaction. The amount of DOPA-quinone released was determined by
measuring the absorbance at 475nm. Appropriate control and blanks were used in the
experiments. The assay was performed in triplicates.
Calculation:
Abs of control - (Abs of test – abs of blank)
____________________________________________ X 100
% Anti-htyrosinase =
Abs of control
5.17 STABILITY STUDIES217,218
The stability studies were carried out in all formulations at different temperature
conditions (4º ± 2 º C and 37 º± 2 º C) for 3 months. All evaluation parameters i.e pH,
viscosity, spreadability, and phase seperation were studied at different time intervals i.e 1
month, 2 month and 3 month.
Experimental
106
5.18 STANDARDIZATION OF THE FORMULATION
5.18.1 Background
Standardization is defined as adjusting the herbal drug preparation to a defined content of
a constituent or a group of substances with known therapeutic activity respectively by
adding excipients or by mixing herbal drug extracts.238
Standardization of herbal formulations is essential in order to assess of quality drugs,
based on the concentration of their active principles, physical, chemical, phytochemical
and invitro, invivo parameters.239
One of the major problems faced by the herbal industry
is the unavailability of rigid quality control profiles for herbal materials and their
formulations. In India, the department of AYUSH, Government of India, launched a
central scheme to develop a standard operating procedures for the manufacturing process
to develop pharmacopeial standards for ayurvedic preparations.240
The quality assessment of herbal formulations is of paramount importance in order to
justify their acceptability in modern system of medicine With the use of modern methods
of analysis such as UV and IR spectroscopy, GC, TLC, HPTLC, HPLC and more, it is
possible to set up certain standards and analyze a particular constituent i.e. “Marker
compound/s” from Ayurvedic preparations.241
5.18.2 ANALYTICAL METHOD DEVELOPMENT
High-pressure liquid chromatography (HPLC), is a technique in analytic chemistry used
to separate the components in a mixture, to identify each component, and to quantify each
component in a herbal drug. It relies on pumps to pass a pressurized liquid solvent
containing the sample mixture through a column filled with a solid adsorbent material.
Each component in the sample interacts slightly differently with the adsorbent material,
causing different flow rates for the different components and leading to the separation of
the components as they flow out of the column.242
Experimental
107
5.18.2.1 HPLC Method Development243
Instrumentation
The Younglin(S.K) Gradient HPLC system with a multiple-wavelength ultraviolet-
visible (UV-Vis) detector was used for HPLC studies.
Standard stock and working solutions
The reference standards were dissolved in mobile phase solution to obtain the required
stock and working solutions.
Betulinic Acid stock: 1mg/ml in mobile phase.
Betulinic Acid stock: 10-100µg/ml in mobile phase
Quercetin stock solution: 1mg/ml in mobile phase.
Quercetin working solution: 10-100µg/ml in mobile phase.
Kaempferol stock solution: 1mg/ml in mobile phase.
Kaempferol working solution: 10-100µg/ml in mobile phase.
Sample Preparation
MEB-ME and RD-ME extracts were dissolved in mobile phase solution to prepare stock
solutions (1mg/ml) of each. The stock solutions were subsequently diluted to
prepared different working solutions (10-100µg/ml). All aliquots were filtered through
Whatman’s syringe filters (NYL 0.45μm) before analysis.
Method Development
HPLC method was developed for marker compounds (betulinic acid quercetin and
kaempferol) by optimizing the HPLC parameters such as wavelength, mobile phase
composition and flow rate, and stationary phase. The fingerprint chromatograms were
recorded.
5.18.2.2 HPLC Method Validation
The HPLC method developed was validated for linearity, limit of detection and
quantification, accuracy, precision and robustness.
Experimental
108
i. Linearity
The linearity was established for marker compounds betulinic acid, quercetin and
kaempferol. The calibration curve was constructed by linear regression analysis of the
peak area against the respective concentration of the marker compound.
ii. Limits of detection (LOD) and limit of quantification (LOQ)
The LOD and LOQ were calculated based on standard deviation and slope of the y-
intercepts of regression lines. The following equations were used to determine LOD and
LOQ.
LOD= 3.3 σ/S and LOQ=10 σ/S where, σ is standard deviation of response and S is the
slope of calibration curve.
iii. Accuracy
The accuracy of the method was measured through the analyte recovery test in triplicate.
The method was employed by the addition of known quantities of marker compound with
the pre-analyzed extract sample followed by the re-analysis of the contents. The recovery
of the marker compound was expressed as %RSD from mean recovery of the each
theoretical concentration.
iv. Precision
Precision of the method was evaluated by analyzing different concentrations of marker
compound, three times on the same day for intra-day and on three successive days for
inter-day precision. The mean and % RSD were calculated for intra-day and inter-day
runs.
v. Robustness
Robustness study was carried out by analyzing the marker compound under critical
modifications of optimum conditions set for the developed method. The marker compound
was analyzed with the small changes in the mobile phase flow rate, detection wavelength
and pH to determine their effect on the RT and peak area response. The % RSD of
retention time and peak area response was calculated.
Experimental
109
5.18.3 QUANTIFICATION OF THE MARKER COMPOUNDS PRESENT IN THE
FORMULATION BY HPLC
The developed HPLC method was used to quantify the marker compounds present in
formulation.
Quantification was carried out considering the peak area response. Percentage w/w of
marker compound in the formulation was quantified using the formula-
Area of sample X Weight of standard X Dilution factor of sample X % Purity of standard
% w/w = ______________________________________________________________________________________________
Area of standard X Weight of sample X Dilution factor of standard