Experimental 61 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
49
Embed
5.1 Materials 5.1.1. List of Chemicals Chemicals Suppliershodhganga.inflibnet.ac.in/bitstream/10603/73011/13/13_chapter 5.pdf · List of Chemicals Chemicals Supplier ... Barfoed’s
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Experimental
61
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
62
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
63
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
64
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
65
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
66
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
Experimental
67
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.
Experimental
68
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.
Experimental
69
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
Experimental
70
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
71
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 --
Experimental
72
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.
Experimental
73
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
74
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
75
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
76
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
77
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