39
107 Materials and Methods
107
Materials and Methods
108
Chapter 3
MATERIALS AND METHODS
. .
3.1. Materials
3.1.1. Collection of Plant Material
The leaves of V. leucoxylon of two different seasons namely summer and winter of
2007 were collected together with bark and seeds from kalryan hills of the Eastern
Ghats of South India. They were duly processed and mounted in standard
Herbarium sheets. A voucher specimen was deposited in the Rapinat Herbarium
(RHT) of St.Joesph‟s College, Tiruchirapalli, South India and the voucher specimen
number is RHT 56156.
3.1.2. Plant Powder Preparation
The disease free plant parts (leaf, stem and seeds) were spread out and dried in the
laboratory at room temperature for 5-8 days or until they were easily broken by
hand. Once completely dried, plant parts were grounded to a fine powder using an
electronic blender. Plants were stored in a closed container at room temperature
until required.
3.1.3. Purification of Solvents
The solvents light petroleum b.p.40-60ºC (petroleum ether), ethanol, ethyl acetate
and dichloromethane were used for the isolation of co-active principles from plant
materials which were purified before use by the method reported in the literature1.
109
3.1.3.1. Purification of Light petroleum (Petroleum ether b.p.40-60°C)
Petroleum ether was shaken two or three times with 10% of the volume of
concentrated sulphuric acid; vigorous shaking was then continued with successive
portions of a concentrated solution of potassium permanganate in 10% sulphuric
acid until the colour of the potassium permanganate remained unchanged. The
solvent was then thoroughly washed with water, dried over anhydrous calcium
chloride and distilled2.
3.1.3.2. Purification of Ethanol3,4
Ethyl alcohol was purified by drying with anhydrous potassium carbonate. It was
filtered and distilled at 780C.
3.1.3.3. Purification of Dichloromethane5
Perinn‟s technique was adopted with some modifications. Reagent grade
dichloromethane with concentrated sulphuric acid was shaken until the acid layer
remained colourless. It was washed with water, saturated aqueous sodium
carbonate, and then with water again. It was predried with calcium chloride. Then it
was refluxed for two hours over phosphorous pentoxide and distilled onto fresh
phosphorous pentoxide under inert atmosphere, discarding the first 10% and the last
20% of the solvent. The dichloromethane was stored over phosphorous pentoxide
and distilled as needed.
3.1.3.4. Purification of Ethylacetate3,4
Ethylacetate was shaken with anhydrous potassium carbonate, filtered and distilled.
Pure ethylacetate was distilled and collected at 770C.
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3.1.4. Preparation of Solvent Extracts
Fifty grams of the dried and powdered plant materials (leaves and stems) were
soaked separately with 300 ml of each of the solvents viz. ethanol, methanol,
chloroform, hexane, benzene, dichloromethane, petroleum ether and acetone, in a
soxhlet apparatus for 48 hrs at 310 C until complete extraction of the materials. At
the end of 48 hrs, each extract was filtered through Whatman No.1 filter paper and
filtrates were concentrated at room temperature in order to reduce the volume. The
sample was concentrated using rotary evaporator and freeze dried to powdered
form. The paste like extracts were stored in pre-weighed screw scapped bottles and
the yield of extracts were weighed. These screw scrapped bottles were kept in
refrigerator at 40C. Each extract was individually reconstituted using minimal
amounts of the extracting solvent prior to use.
3.2. Methods
3.2.1. Separation of Plant Phytoconstituents
Among the various methods of separating plant constituents, the chromatographic
technique is one of the most commonly used methods of general application.
Chromatography represents a group of methods for separating molecular mixture
that depend on the differential affinities of the solute between the immiscible phase,
the stationary phase and mobile phase .The stationary phase may be a porous or
finely divided solid or liquid, that has been coated as thin layer on a inert support
material, the mobile phase may be a pure liquid or a mixture of solutions or may be
a gas or mixture of gases.
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The separation and purification of plant phytoconstituents is mainly carried out
using one or other or a combination of four chromatographic technique ,viz, paper
chromatography (PC), thin layer chromatography(TC), column chromatography,
Gas liquid chromatography (GLC) and High pressure liquid chromatography
(HPLC). The choice of technique depends largely on the solubility properties and
volatilities of the compounds to be separated. TLC is the method of choice for
separating all liquid soluble components, the lipids, steroids, carotenoids, quinines
and chlorophylls. TLC and HPLC technique now–a-days are important analytical
tools for the separation and determination of natural products. They have the
following advantages:
Simple to operate, economical and rapid.
Always available for use (pre-coated TLC plates are commercially available).
Method of detection is easy.
The whole system is flexible since neutral, basic, acidic or purely aqueous
eluents can be employed.
TLC and column chromatography can be exploited in the investigation and
cultivation of medicinal plants. It is possible to run many samples of extracts from
different chemical races simultaneously with authentic standard and high
performance individuals that can be recognized, selected and bred. In this study, the
separation of preparation plant phytoconstituents was made by preparative TLC
technique.
112
3.3. Preliminary Phytochemical Analysis
Chemical tests were also conducted on the aqueous extract of each plant sample and
also of the powdered form of the plant samples by using standard methods of
Harborne and Edeoga6,7
.
Plants have an almost limitless ability to synthesise not only chemical substances
but also secondary metabolies. It is said that less than 12000 have been isolated, a
number which is less than 10 of the total8. In many cases, these substances serve as
plant defence mechanism against microorganisms, insects and herbivores. Many
compounds are responsible for plant flavour and some of them are used by humans
as season food9. Several species have been screened for antimicrobial properties. A
systematic phytochemical study therefore involves the complete screening of
primary as well as secondary metabolites derived as a result of plant metabolism.
3.3.1. Preliminary Identification
The plant extract may contain along with derived compounds, some other
substances viz. chlorophyll or other pigments, inorganic and organic acids, resins,
fatty substances etc. Depending upon the type of impurities present, the purification
varies largely, separation of constituents by partitioning between two immisible
solvents in which the compound dissolves preferentially or precipitation of either
the desired product or the impurity by a certain reagents are quinine widely used.
However the most convenient modern technique for purification is chromatography,
which was done by the precipitation of impurities by HCl.
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3.3.2. Qualitative Phytochemical Screening10
The different Qualitative chemical tests can be performed for establishing a profile
of given extract for its chemical composition. The following tests may be performed
on extracts to detect various phyto constituents present in them.
3.3.2.1. Detection of Alkaloids
Solvent free extract 50 mg was stirred with few ml of dil. HCl and filtered. The
filtrate was tested carefully with various alkaloidal reagents as follows.
a) Mayer’s Test
To a few ml of filtrate, one or two drops of Mayer‟s reagent were added by the side
of the test tube. A white creamy precipitate indicated the test as positive.
b) Mayer’s Reagent
Mercuric chloride (1.358g) was dissolved in 60 ml of water and KI (5.0 g) dissolved
in 10 ml of water. The two solutions were mixed and made upto 100 ml with water.
c) Wagner’s Test
To a few ml of filtrate, few drops of Wagner‟s reagent was added by the side of the
test tube. A reddish brown precipitate confirmed the test as positive.
d) Wagner’s Reagent
Iodine (1.27 g) and KI (2 g) were dissolved in 5 ml of water and made upto 100 ml
with distilled water.
114
e) Hager’s Test
To a few ml of filtrate 1 or 2 ml of Hager‟s reagent (Saturated aqueous Solution of
picric acid) was added. A prominent yellow precipitate indicated the test as
positive.
f) Dragendorff’s Test
To a few ml of Filtrate 1 or 2 ml of Dragen droff‟s reagent was added. A prominent
yellow precipitate indicated the test as positive.
g) Dragen Droffs Reagent
Bismuth carbonate (5.2 g) and sodium iodide (4 g) were boiled for a few minutes
with 50 ml Glacial acetic acid. After 12 hrs the precipitated sodium acetate crystals
were filtered off using a sintered glass funnel. Clear, red-brown filtrate, 40ml was
mixed with 60 ml ethyl acetate and 1 ml water and stored in amber-coloured bottle.
3.3.2.2. Detection of Carbohydrates and Glycosides
The extract (100mg) was dissolved in 5 ml water and filtered. The filtrate was
subjected to the following tests:
a) Molish’s Test
To 2 ml of filtrate two drops of alcoholic solution of α- napthol was added, the
mixture was shaken well and 1 ml of con H2SO4 was added slowly along the sides
of the test tube and allowed to stand. A violet ring indicated the presence of
carbohydrates.
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b) Fehling’s Test
1 ml of filtrate was boiled on water bath. To this, 1 ml of Fehling solutions A and B
were added. A red precipitate indicated the presence of sugar.
Fehlings‟s solution A: CuSO4(34.66 g) was dissolved in distilled water and made
upto 500 ml using distilled water.
Fehling‟s solution B: (Potassium sodium tartarate (173 g) and NaOH(50g) )was
dissolved in water and made upto 500 ml.
c) Barfoed’s Test
To 1 ml of filtrate, 1 ml of Barfoed‟s reagent was added and heated on a boiling
water bath for 2 min. Red Precipitate indicated the presence of sugar Barfoed‟s
Reagent. Copper acetate 30.5 g was dissolved in 1.8 ml of glacial acetic acid.
d) Benedict’s Test
To 0.5 ml of filtrate, 1 ml of Bendict‟s reagent was added. The mixture was heated
on a boiling water bath for 2 mins. A characteristic coloured precipitate indicated
the presence of sugar.
e) Benedict’s Reagent
Sodium citrate (173 g) and Na2CO3 (100g) were dissolved in 800 ml of distilled
water and boiled to make it clear. CuSO4 (17.3 g) dissolved in 100 ml distilled
water was added to it.
3.3.2.3. Detection of Glycosides
50 mg of extract was hydrolysed with concentrated HCl for 2 hours on a water bath,
filtered and the hydrolysate was subjected to the following tests:
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a) Borntrage’s Test
To 2 ml of filtered hydrolysate, 3 ml of chloroform is added and shaken, chloroform
layer was separated and 10% ammonia solution was added to it. Pink colour
indicated the presence of glycosides.
b) Legal’s Test
50 mg of the extract was dissolved in pyridine. Sodium nitro prusside solution was
added and made alkaline using 10% NaOH. Presence of glycosides was indicated
by pink colour.
3.3.2.4. Detection of Saponins
The extract (50 mg) was diluted with distilled water and made upto 20 ml. The
suspension was shaken in a graduated cylinder for 15 min. A layer of foam
indicated the presence of saponins.
3.3.2.5. Detection of Proteins and Amino Acids
The extract (100 mg) was dissolved in 10 ml of distilled water and filtered through
whatmann no: 1 filter paper and filtrate was subjected to tests for proteins and
amino acids.
a) Millon’s Test
To 2 ml filtrate, few drops of millon‟s reagent were added. A white precipitate
indicated the presence of proteins.
b) Millon’s Reagent
Mercury (1 g) was dissolved in 9 ml of fuming HNO3 when the reaction was
completed equal volume of distilled water was added.
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c) Biuret Test
An aliquot of 2 ml of filtrate was heated with 1 drop of 2 % CuSO4 solution. To this
1 ml of ethanol (95%) was added, followed by excess of KOH Pellets. Pink colour
in the ethanolic layers indicated the presence of proteins.
d) Ninhydrin Test
2 drops of Ninhydrin solution (10 mg of Ninhydrin in 200 ml of acetone) was added
to 2 ml of aqueous filtrate. A characteristics purple colour indicated the presence of
amino acids.
3.3.2.6. Detection of Phytosterols
a) Libermann – Burchard’s Test
The extract (50 mg) was dissolved in 2 ml acetic anhydride. To this one or two
drops of concentrated H2SO4 were added slowly along the sides of the test tube. An
array of colour change showed the presence of phytosterols.
3.3.2.7. Detection of Fixed Oils and Fats
a) Spot Test
A small quantity of extract was pressed between two filter papers. Oil stain on the
paper indicated the presence of fixed oil.
b) Saponification Test
A few drops of 0.5N alcoholic KOH solution were added to a small quantity of
extract along with a drop of phenolphthalein. The mixture was heated on water bath
for 2 hours. Formation of soap or partial neutralization of alkali indicated the
presence of fixed oils and fats.
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3.3.2.8. Detection of Phenolic Compounds and Tannins
a) Ferric Chloride Test
The extract (50 mg) was dissolved in 5 ml of distilled water. To this few drops of
neutral 5% ferric chloride solution was added. A dark green colour indicated the
presence of phenolic compounds.
b) Lead Acetate Test
The extract (50 mg) was dissolved in distilled water and to this 3 ml of 10% lead
acetate solution was added. A bulky white precipitate indicates the presence of
phenolic compounds.
c) Gelatin Test
The extract (50 mg) was dissolved in 50 ml of distilled water 2 ml of 1% solution of
gelation containing 10% sodium chloride was added to it. White precipitate
indicated the presence of phenolic compounds.
d) Alkaline Reagent Test
An aqueous solution of the extract was heated with 10% NH4OH solution. Yellow
fluorescence indicated the presence of flavonoids.
e) Magnesium and Hydrochloric Test
The extract (50 mg) was dissolved in 5 ml of alcohol and few fragments of
magnesium ribbon and concentrated HCl were added (dropioire). If any pink to
crimson developed, presence of flavanol glycoside was inferred.
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3.3.2.9. Detection of Gum and Mucilages
The extract (100 mg) was dissolved in 10ml of distilled water and to this 25 ml of
absolute alcohol was added with constant strirring. White or cloudy precipitate
indicated the presence of gums mucilages.
3.3.2.10. Detection of Volatile Oil
In volatile oil estimation apparatus, 50 g of powdered material (crude drug) was
taken and subjected to hydro distillation. The distillate was collected in graduated
tube of the assembly wherein the aqueous portion automatically separated out from
the volatile oil.
3.3.2.11. Test for Steroids
10 ml of all extract of the test plant was evaporated to a dry mass and the mass
dissolved in 0.5 ml of chloroform. Acetic anhydride (0.5 ml) and 2 ml of
concentrated sulphuric acid were added. A blue or green colour or a mixture of
these two shades shows the presence of steroidal compounds.
3.3.3. Quantitative Phytochemical Analysis
3.3.3.1. Total Phenols Estimation
The total phenols of all extracts were measured at 765 nm by Folin Coicalteu
reagent11
. The dilute Methanolic extract (0.5 ml of 1:10 g ml-1
), Gallic acid
(Standard phenolics compound) was mixed in the Folin Coicalteu reagent (5 ml,
1:10 diluted with dis. H2O) and aqueous Sodium carbonate (4 ml, 1M). The mixture
was allowed to stand for 15 min. and the total phenols were determined by
spectrophotometer at 765 nm. The standard curve was prepared using 0, 50,100,
120
150,200,250 mg/ml-1
solutions of Gallic acid in methanol: water (50:50V/V). Total
phenol values were expressed in terms of Gallic acid equivalent (mg/ml-1
of dry
mass), which was a common reference compound.
3.3.3.2. Total Flavonoids Estimation
Aluminum chloride colorimetric technique was used for Flavonoids estimation12
.
Each extract (0.5 ml of 1:10 g/ml-1
) in methanol was separately mixed with 1.5 ml
of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1M potassium acetate and
2.8 ml of distilled H2O. It was left at room temperature for 30 min., after which the
absorbance of the reaction mixture was measured at 415 nm with a double beam
UV visible spectrophotometer, SHEMADZU (Japan). The calibration curve was
plotted by preparing the quercetin solutions at concentrations 12.5 to 100 g/ml-1
in
methanol.
3.3.3.3. Preparation of Fat Free Sample
2 g of the sample was defatted with 100 ml of diethyl ether using a Soxhlet
apparatus for 2 hours.
3.3.3.4. Saponin Determination13
The samples were ground and 20 g of each were put into a conical flask and 100 cm
of 20% aqueous ethanol were added to each sample. The samples were heated over
a hot water bath for 4 hours with a continuous stirring at about 55°C. The mixture
was filtered and the residue re-extracted with another 200 ml 20% ethanol. The
combined extracts were reduced to 40 ml over water bath at about 90 C. The
concentrate was transferred into a 250 ml separatory funnel and 20 ml of diethyl
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ether was added and shaken vigorously. The aqueous layer was recovered while the
ether layer was discarded. The purification process was repeated. 20 ml of n-butanol
was added. The combined n-butanol extracts were washed twice with 10 ml of 5%
aqueous sodium chloride. The remaining solution was heated in a water bath. After
evaporation, the samples were dried in the oven and weighted.
3.3.3.5. Flavonoid Determination
Flavonoid determination was made using bohm and Kocipai Abyazan method14
.
10g of the plant sample was extracted repeatedly with 100 ml of 80% aqueous
methanol at room temperature. The whole solution was filtered through whatmann -
filter paper No: 42 (125mm). The filtrate was later transferred into a crucible and
evaporated into dryness over a water bath and weighted to a constant weight.
3.3.3.6. Alkaloid Determination7, 15
2.5 g of the powder was extracted using 100 ml of 20% acetic acid in ethanol. The
solution was covered for almost 4 hours. The filtrate was concentrated to 25 ml.
Concentrated ammonium hydroxide was added stepwise to attain precipitation. The
whole solution was kept as such so that precipitate will settle. The Collected
precipitate was washed with dilute ammonium hydroxide and finally filtered.
Filtrate was discarded and pellet obtained was dried and weighed.
3.4. Hydrodistillation Method of Essential Oil
Essential oil content in leaves Vitex shows seasonal variation. The air shade dried
leaves were cut into small pieces and hydro-distilled for 3 hours in a Clevenger
apparatus. The distillate was extracted with diethyl ether. The ethereal layer was
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dried over anhydrous sodium sulphate and ether was distilled off on a gently heated
water bath. The yield of oil was measured for every month.
The above method was modified for other analysis for HPLC and GC-MS. shade
dired leaves were pulverized and extracted successively with petroleum ether +
ethyl acetate + CH3OH + C2H5OH + H2O in a Soxhlet extractor for 18 hours. The
solvents were evaporated under vacuum and percentage yield of each extract was
calculated. Each of the extract was stored in a sealed glass bottle in a refrigerator
until analysis.
3.5. UV-Visible Spectroscopy
Ultraviolet-visible spectroscopy is useful as an analytical technique to identify some
functional groups in molecules, thus aiding in structural elucidation in
biomolecules.
3.5.1. Principle
Absorption of ultraviolet or visible light electromagnetic radiation causes electrons
to move from lower energy levels to higher energy levels. Because the spectrum of
an atom or molecule depends on its electron energy levels, UV-visible absorption
spectra are useful for identifying unknown substances.
3.5.2. Procedure
The Plant samples used in solutions (Petroleum ether, Dichloromethane and
ethanol) were placed in a small silica cell. A hydrogen or deuterium lamp for the
ultraviolet region and a halogen lamp for the visible region were used. In this way
radiation across the whole range was scanned by the spectrometer. A reference cell
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containing only solvent was used. Light was passed simultaneously through the
sample cell and reference cell. The spectrometer compared the light passing through
the sample with that passing through the reference cell. The transmitted radiation
was detected and the spectrometer recorded the absorption spectrum by scanning
the wave length of the light passing through the cells.
UV study was done at Archbishop Casimir Instumentation Centre (ACIC),
St.Joseph‟s College, Tiruchirappalli.
3.6. Infrared (IR) Spectroscopy
Infrared spectroscopy is a well developed technique to identify chemical
compounds.
3.6.1. Principle
Infrared spectroscopy is based on the study of reflected, absorbed or transmitted
radiant energy in region of electromagnetic spectrum ranging from wavelength 0.8
to 500 nm. A more commonly used measurement is the frequency and is expressed
in wave number. The IR spectrum is usually divided into three regions namely near
IR (12500 to 4000 cm–1
) mid IR (4000 to 400 cm–1
) and far IR (400 to 20 cm–1
)
only the mid IR region is usually referred to as Infrared and is widely used in the
analysis of pharmaceuticals.
3.6.2. Procedure
The plant substance was measured in an automatic recording IR spectrophotometer
(Perkin Elemer - FT-IR Spectrophotometer) as a mull with nujol oil or in the solid
state, mixed with potassium bromide (KBr). In the later case, a thin disc was
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prepared under anhydrous conditions from a powder containing about 1 mg of
material and 10-100 mg potassium bromide, using a mould and press. The range of
measurement was from 4000 to 667 cm–1
(or 2.5 to 15 m) and the spectrum was
recorded within three minutes. IR spectroscopy is most frequently used in
phytochemical studies as a finger printing device for comparing a natural with a
synthetic sample, which is very important in the complete identification of many
types of plant constituents. It can also usefully contribute to structural elucidation,
when new compounds are encountered in plants.
3.7. Isolation and Identification of Compounds
3.7.1. Methods for Separation, Isolation and Identification of Compounds in
Leaves
Melting points were determined on Electric Sun-Vic melting point apparatus and
are uncorrected. The IR spectra (In KBr) were recorded on a 460 Shimadzu
spectrometer. The UV spectra were recorded on a Hitachi UV-3200
spectrophotometer (ëmax in nm). Silica gel (Fine G & H as well as 60-120 mesh)
were used in TLC and Column chromatography respectively. TLC plates and pre-
coated silica gel G-25-UV254 plates were used to check the purity and isolation of
the compounds. Visualization of the TLC plates was carried out under UV at 254
and 366 nm and by spraying with cerric sulphate reagent (with heating). The 1H and
13C-NMR, and other 2D spectra were recorded on Bruker spectrometers operating at
400MHz for 1H and 100MHz for
13C-NMR respectively ä values in ppm downfield
from TMS. EI-MS measurements were carried out on micro mass QUATTRO II
triple quadrupole mass spectrometer.
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3.7.2. Methods for Separation, Isolation and Identification of Compounds 1
and 2 in Bark
The air dried, coarsely powdered (2kg) of V. leucoxylon was sequentially extracted
with petroleum ether (60º-80ºC), acetone and methanol by the soxhlet apparatus
(5 times x 1 lit each). The fractions of each extract were mixed together and the
excess of solvent was evaporated under reduced pressure to get their mass. Out of
these extracts only acetone extract was considered for further examination. The
semisolid brownish mass (6 gm) obtained from acetone extract was dissolved in
small amount of acetone and a slurry was made with (6 gm) of silica gel. The slurry
was loaded on a column of silica gel (120 gm) and eluted with petroleum ether,
benzene, chloroform, ethyl acetate, methanol and their mixtures of different
proportions of increasing polarity. Several fractions were obtained which were
monitored by TLC and the fractions showing single spot on TLC were combined
together.
3.7.3. Methods for Separation, Isolation and Identification of Compounds 3
and 4 in Bark
The air dried and coarsely powdered bark (2kg) was sequentially extracted with
petroleum ether (600-80
0C), acetone and methanol by the soxhlet apparatus (5 times
x 1 lit each). The fractions of each extract were mixed together and the excess of
solvent was evaporated under reduced pressure. Out of these extracts only acetone
extract was considered for further examination. A column of silica gel was prepared
well stirred with petroleum ether. Then slurry of acetone extract (6gm) was made
and digested over this column. The column was eluted with different solvents like
petroleum ether, benzene, chloroform, ethyl acetate, methanol and their mixtures of
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increasing polarity. Form the eluent two compounds could be separated. One
compound (3) was obtained by eluting the column with benzene-methanol (1:1)
while other compound (4) was obtained by eluent chloroform-methanol (8:2). These
compounds were further purified by crystallization. The compounds were then
characterized by m.p., solubility and different spectral studies like IR and NMR
(1H and
13C).
3.8. Gas Chromatography - Mass Spectrum Study (GC-MS)
Gas Chromatography-Mass Spectrometry (Finnigan Matt GCO Mass Spectrometer)
is one of the hyphenated analytical techniques. Gas chromatography separates the
components of a mixture and mass spectrometry characterizes each of the
components individually. By combining the two techniques one can evaluate a
solution (both qualitatively and quantitatively) containing a number of chemicals
which are used extensively in the medical, pharmacological and law enforcement
fields.
3.8.1. Principle
GC-MS is a hyphenated experimental technique that incorporates two widely used
methods in tandem. The GC portion is the Gas chromatography used for separating
components in a mixture, and the MS portion is the mass spectrometry used in the
qualitative and quantitative analysis of each component that was separated by the
Gas Chromatography (GC).
127
3.8.2. Preparation of plant Extracts for GC-MS Analysis
Ethanol extract of leaf samples collected in summer was prepared using soxhlet
apparatus and the dried extract treated with 100 ml of ethyl acetate. Ethyl acetate
fraction was filtered and filtrate named as Sample A.
Ethanol extract of leaves collected in winter was prepared using soxhlet apparatus
and the dried extract treated with 100 ml of ethyl acetate. Ethyl acetate fraction was
filtered and filtrate named as Sample B.
Ethanol extract of bark was prepared using soxhlet apparatus and the dried extract
treated with 100 ml of ethyl acetate. Ethyl acetate fraction was filtered and filtrate
named as Sample C
Dichloromethane extract of bark was prepared using soxhlet apparatus and the dried
extract treated with 100 ml of ethyl acetate. Ethyl acetate fraction was filtered and
filtrate named as Sample D.
Petroleum ether extract of bark was prepared using soxhlet apparatus and the dried
extract treated with 100 ml of ethyl acetate. Ethyl acetate fraction was filtered and
filtrate named as Sample E.
Ethanol extract of seed of Vitex leucoxylon was prepared using soxhlet apparatus
and the dried extract treated with 100 ml of ethyl acetate. Ethyl acetate fraction was
filtered and filtrate named as Sample F.
3.8.3. Application of GC-MS Detector in Phytochemical Analysis
GC-MS plays a key role in the analysis of unknown components of plant origin.
GC-MS ionizes compounds and measures their mass numbers. Ionization is
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typically, the Chemical Ionization (C.I.) and Electron Ionization (E.I.). The E.I.
method provides good results for quantitative analysis as well of the compounds
and it is a highly selective method for interfering components. Gas chromatography
technique involves the separation of volatile components in a test sample using
suitable capillary column coated with polar and non-polar or intermediate polar,
chemicals.
Elite-1 column (100% Dimethyl poly siloxane) is a non-polar column used for
analysis of phytocomponents in medicinal plants and pesticide residues. Elite-5
column (5% phenyl and 95% methyl poly siloxane) is an intermediate column used
for the estimation of pesticide residues in soft drinks and food grains. Elite wax
(polyethylene glycol) is a polar column used in the estimation of fragrances in rice,
alcohol, flowers and fatty acid profile of edible oils. An inert gas such as hydrogen
or nitrogen or helium is used as a carrier gas.
The components of test sample was evaporated in the injection port of the GC
equipment and segregated in the column by adsorption and adsorption technique
with suitable temperature programme of the oven controlled by software. Different
components were eluted from the column based on the boiling point of the
individual components. The GC column was heated in the oven between 60 to
270˚C. The time at which each component eluted from the GC column was termed
as Retention Time (RT).
a) GC-MS Programme
Column: Elite-1 (100% Dimethyl poly siloxane), 30m×0.25mm ID ×1µm df.
Equipment: GC Clarus 500 Perkin Elmer.
129
Carrier Gas: Helium 1 ml/min.
Detector: Mass detector – Turbo mass gold – Perkin Elmer, Software –
Turbomass 5-1.
Sample injected: 1µl (one Micro litre) was injected with a Hamilton syringe
to the GC-MS manually.
Split: 10:1.
b) Oven Temperature Programme
110˚ – 2 min hold
Up to 280˚ at the rate of 5˚ / min – 9 min hold
Injector temp: 250˚C.
Total GC time: 45 min.
c) MS Programme
Library used: NIST ver.2.1.
Inlet line temperature: 200˚C
Source temperature: 200˚C
Electron energy: 70 ev
Mass scan: (m/z) 45-450
MS time: 46 min.
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3.8.4. Identification of Components
Interpretation of mass spectrum (GC-MS) was conducted using database of
National Institute Standard and Technology (NIST) having more than 62,000
patterns. The spectrum of the unknown component was compared with the spectrum
of known components stored in the NIST library. The retention time, molecular
weight, molecular formula, and composition percentage of the sample material was
recorded.
3.8.5. Compound and Its Identification, Nature and Activity
Compound‟s identification and nature and activity were done using database of
Dr.Duke's Phytochemical and Ethnobotanical Database. GC-MS study was carried
out at Crop Processing Research Institute, Tanjore.
3.9. Method of Extracting Corosolic Acid
The dried leaves of Vitex leucoxylon were powdered and then it was perculated
about 3-5 times with polar or non-polar solvents. The powder was boiled with the
percolated solvent and filtered to remove insoluble particles. The solvent under
vacuum was evaporated to get an active extract, and it contained corosolic acid
ranging between 0.5 and 2%. The extract was partitioned with other solvents to
enrich the extract to contain corosolic acid in the range of 2-10%. The pure
compound corosolic acid was isolated by column chromatography in the range of
90-100% purity by crystallizations using polar and non-polar solvents. The isolated
pure corosolic acid was confirmed by HPLC and also by its physical and spectral
data.
131
3.9.1. HPLC Instrumentation
The HPLC system, supplied by M/s Shimadzu comprising LC-10AT VP
pumps,SCL-10A VP auto injector and Phenomenex Luna C18, 5 μ, (250 X 4.6 mm)
column was used at ambient temperature. Isocratic elution was carried out with
acetonitrile : 0.1% (v/v) phosphoric acid in water (75 : 25, v/v) at a flow rate of
1ml/min, detection was at 210 nm using SPD – M10 AVP photodiode arraydetector.
Class VP software was used for integration and calibration. Evaluation was via peak
areas with linear regression.
3.10. Antimicrobial Study
3.10.1. Microbial samples
a) Test Bacteria
Seven bacterial species were collected from Microbial Type Culture Collection
(MTCC), from the Institute of Microbial Technology (IMT), Chandigarh in Punjab,
for the study. The Microbial strains used were Enterobacter aerogenes MTCC
2823; Escherichia coli MTCC 443, Klebsiella pneumoniae MTCC 190, Salmonella
paratyphi; Vibrio cholerae; 441, Staphylococcus aureus MTCC 87 and
Streptococcus faecalis.
b) Test Fungi
Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Candida albicans and
Black mold were also collected from Microbial Type Culture Collection, the
Institute of Microbial Technology, Chandigarh in Punjab, India.
132
3.10.2. Maintenance of Microorganisms
The test bacteria‟s were maintained in Nutrient Agar (Himedia Laboratories Pvt.
Ltd., Mumbai) and test fungi were maintained in Potato Dextrose Agar (PDA)
slants (Himedia Laboratories Pvt. Ltd., Mumbai). The microbial cultures were
subcultured and the cultured strains were allowed to grow one week for fungi and
two days for bacteria and they were stored at 5°C for further studies.
3.10.3. Methods of Antibacterial Screening
The antimicrobial screening of the aqueous and other organic solvent extracts of
different parts of Vitex leucoxylon was investigated through different methods. The
assays consisted of both antibacterial and antifungal evaluations.
3.10.4. Turbidity Standard for Inoculum Preparation
To standardize the inoculum density for a susceptibility test, a BaSO4 turbidity
standard, equivalent to a 0.5 McFarland standard or its optical equivalent (e.g., latex
particle suspension), should be used. A BaSO4 0.5 McFarland standards were
prepared as follows:
A 0.5-ml aliquot of 0.048 mol/L BaCl2 (1.175% w/v BaCl2, 2H2O) was
added to 99.5 ml of 0.18 mol/L H2SO4 (1% v/v) with constant stirring to
maintain a suspension.
The correct density of the turbidity standard should be verified by using a
spectrophotometer with a 1-cm light path and matched cuvette to determine
the absorbance. The absorbance at 625 nm should be 0.008 to 0.10 for the
0.5 McFarland standards.
133
The barium sulfate suspension should be transferred in 4 to 6 ml aliquots into
screw-cap tubes of the same size as those used in growing or diluting the
bacterial inoculum. These tubes should be tightly sealed and stored in the
dark at room temperature.
The barium sulfate turbidity standard should be vigorously agitated on a
mechanical vortex mixer before each use and inspected for a uniformly
turbid appearance. If large particles appear, the standard should be replaced.
Latex particle suspensions should be mixed by inverting gently, not on a
vortex mixer
The barium sulfate standards should be replaced or their densities verified
monthly.
3.11. Antibacterial Assays
3.11.1. Disc Diffusion Assay - Principle
Disc diffusion method is used for the rapid determination of the drug or a particular
substance on a specific bacterium. This method consists of impregnating small
circular disc of standard filter paper with given amount of a chosen concentration of
substance. The discs are placed on plates of culture medium that has been seeded
with a test bacterial inoculum. After incubation the diameter of the clear zone of
inhibition surrounding the deposit of substance is taken as a measure of the
inhibitory power of the particular substance against the particular test organism.
134
3.11.2. Disc Preparation
The filter paper discs of uniform size were impregnated with the compound (plant
extract) usually consisting of absorbent paper. It is most convenient to use Whatman
No.1 filter paper for preparing the discs. Dried discs of 6 mm diameter were
prepared from Whatman No.1 filter paper and sterilized in an autoclave. These
dried discs were used for the test.
3.11.3. Procedure
The freeze dried extract was reconstituted with DMSO to obtain a stock solution of,
100 mg/ml, 50 mg/ml, 25 mg/ml, and 12.5 mg/ml. Nutrient agar (Hi Media
Laboratories Pvt. Ltd. Mumbai) plates were swabbed using sterile cotton swabs
with the adjusted broth culture of the respective bacterial strains. Discs of 6 mm
were punched from Whatman No.1 filter paper. Up to 10 µl of each concentration
of the extract were respectively introduced in the discs using sterile automatic
pipettes. The discs were allowed to dry at room temperature for 2 hours and were
placed at equidistance in each of the plates using a sterile forceps. The plates were
incubated to 37°C for 24 hours. The control antibiotic Gentamycin (10 µg) for
gram-positive bacteria and Kanamycin (30 µg) for gram-negative bacteria
(Hi Media Laboratories Pvt. Ltd. Mumbai) were used. Diameters of the inhibition
zones were measured. The antibacterial activity was expressed as the mean zone of
inhibition diameters (mm) produced by the plant extract
3.11.4. Dilution Method for Antibacterial Testing to Calculate MIC16, 17
Niobium (NB) tubes were labelled with name, date of inoculation,
experiment number, and name of bacterium.
135
The microdilution was performed in 96 well microtitre plates with U-shaped
wells, label with name and date of inoculation, experiment number and name
of bacterium.
The stock solution was prepared with DMSO (400 mg/ml).
500 µl (250 mg) of the extract was added to 4.5 ml of NPBG (Nutrient Broth
containing 0.05% Phenol red and supplemented with 10% Glucose).
The extract was transferred to 200 µl/well and serially diluted (base 2
logrithmatic dilutions) with NPBG (i.e. two-fold) to obtain concentrations
ranges of (4 mg/ml – 0.008 mg/ml) in use the micropipetted to dispense 100
µl of test extract to the first two–fold dilution. The micropipette with the
same tip should be used to carry out a second two–fold dilution. The series of
two–fold dilutions should be continued until the 11th
well (Colum) of the
microwell plate. The quantity in the micropipette from this well should be
discarded.
24 hours culture of bacterial strains were stirred with 0.9% NaCl of achieve
0.5 McFarland (108 cells/ml for bacteria and 10
6 for fungi) diluted 1/100 to
achieve 106 and 10
4 cells/ml. for bacteria and fungi respectively and
inoculated in the 96 wells plates (100µl/well) each organism in a single row
from A to H (24 µl + 1174 µl).
The first well consisted of the negative control with standard inoculums and
the last well (12th
well Column) control of positive control with 15µg
(Gentamycin (15 µl of 1 mg/1 ml stock) for gram-positive bacteria and
Kanamycin (15 µl of 1 mg /1 ml stock) for gram-negative bacteria)
136
Microbial growth was determined by observing the change on growth and
yellow when there is growth).
The plates were sealed, placed in plastic bags and incubated at 37˚C (for
bacteria) for 24 hours in ambient atmosphere.
The MIC should be measured which is defined as the lowest concentration of
extract that exhibited growth by visual reading. It should be expressed in mg
or µg/ml.
The plates should be discarded by appropriate procedure.
The Minimum Inhibitory Concentration (MIC) was the lowest concentration of
extracts that completely inhibited the growth of microorganism in the microdilution
wells. It would be detected by unaided eye (i.e. red colour when there are no growth
negative wells).
3.12. Antifungal Assays
3.12.1. Disc Diffusion Method - Principle
The antimycotic activity of aqueous and other solvent extracts were evaluated by
disc diffusion method18
. This method would consist of impregnating small circular
discs of standard filter paper with given amount of a chosen concentration of
substance. The discs would be placed on plates of culture medium previously
spread with the fungal spores (inoculum). After incubation the degree of sensitivity
would be determined by measuring the inhibition zone produced by the diffusion of
the antibiotic substances from the disc into the surrounding medium.
137
3.12.2. Preparation of Disc
The disc–diffusion method would provide a simple and reliable test in routine
clinical Mycology in order to find out the effect of a particular substance on a
specific fungus. This method would consist of impregnating small circular discs of
standard filter paper with a given amount of a chosen concentration of substance.
The discs would be placed on plates of culture medium previously spread with
fungal inoculums to be tested. After incubation the degree of sensitivity would be
determined by measuring the inhibition zone produced by the diffusion of the
antibiotic substances from the discs into the surrounding medium.
3.12.3. Procedure
Test plates (petridishes) were prepared with potato dextrose Agar medium and
inoculated on the surface with a spore suspension of 106 CFU/ml. Sterile paper
discs of 6mm diameter impregnated with the extract at the concentration of
10mg/ml were placed over the test plates and were incubated at 25˚C for 48 hours.
The diameter of growth inhibition zone around each disc was measured against each
concentration after 48 hours.
3.12.4. Dilution Method for Antifungal Testing to Calculate MIC16, 17
PDB (Potato Dextrose Broth) tubes were labelled with name, date of
inoculation, experiment number, and name of fungus.
The microdilution was performed in 96-well micro liter plates with
U-shaped wells, label with name and date of inoculation, experiment number
and name of fungus.
138
The stock solution was prepared with DMSO (400 mg/ml).
500 µl (250 mg) of the extract was added to 4.5 ml of PDBPG (Potato
Dextrose Broth containing 0.05% Phenol red and supplemented with 10%
Glucose).
The extract was transferred to 200 µl/well and serially diluted (base 2
logrithmatic dilutions) with PDBPG (i.e. two-fold) to obtain concentrations
ranges (4 mg/ml – 0.004 mg/ml)of test extract to the first two - fold dilution.
The micropipette with the same tip should be used to carry out to the second
two-fold dilution. The series of two–fold dilutions would be continued until
the 11th
well (Colum) of the microwell plate. The quantity in the micropipette
from this well should be discarded.
24 hours culture of microbial strains were stirred with 0.9% NaCl of achieve
0.5 McFarland (108 cells / ml for bacteria and 10
6 for fungi) diluted 1/100 to
achieve 106 and 10
4 cells /ml. for bacteria and fungi respectively and
inoculated in the 96 wells plats (100 µl / well) each organism in a single row
from A to H (24 µl + 1174 µl).
The first well would consist of the negative control with standard inoculums
the last well (12th
well Colum) control of positive control with 15 µg
(Nystatin 15 µl of 1mg /1 ml stock).
Microbial growth was determined by observing the change on growth and
yellow when there is growth).
The plates were sealed, placed in plastic bags and incubated at 28˚C (fungi)
for 24 hours in ambient atmosphere.
139
The MIC which is defined as the lowest concentration of extract that
exhibited on growth by visual reading would me measured. It is expressed in
mg or µg/ml.
The plates were discarded by appropriate procedure.
The Minimum Inhibitory Concentration (MIC) was the lowest concentration of
extract that completely inhibited the growth of microorganism in the microdilution
wells, detected by unaided eye (i.e. red colour when there are no growth negative
wells).
3.13. Antioxidant Study
3.13.1. Chemicals
The chemicals used were as follows:
Nitro blue tetrazolium (NBT), glutathione (GSH), glutathione oxidized (GSSG),
nicotinamide adenine dinucleotide phosphate reduced (NADPH), and 5-5‟-dithiobis
(2-nitro benzoic acid) (DTNB). 2,2-Diphenyl-1-picryl hydrazyl (DPPH) and 2,2-azo
bis-3-ethylbenzthiazoline-6-sulfonicacid (ABTS), RPMI 1640, Fetal calf serum
(FCS), Phorbol-12-myristate-13-acetate (PMA) All other chemicals and reagents
used were of analytical grade.
The extraction was done as per standard pharmacopoeia. Leaves bark and seed
(700 g) of Vitex leucoxylon were separetely extracted with 450 ml ethyl alcohol.
The material was placed in a widemouth bottle and alcohol was added. The jar was
stoppered and sealed to prevent evaporation. It was placed in a dark room at room
temperature and shaken every day for 2 weeks. Thereupon the clear liquid was
140
decanted and the residue was pressed out through clean linen, which was added to
the decanted liquid. Volume was made up to 1 litre with alcohol. One hundred
milliliters of this tincture of Vitex leucoxylon parts were evaporated to dryness in a
shaker water bath at 42°C. The yield was found to be 1.1 g. One gram of the dried
extract was redissolved in known amount of distilled water and used for all
experiments.
3.13.2. Experimental Animals
Female Swiss albino mice (20–25 g) were obtained from the animal house of Amala
Cancer Research Centre. They were housed in well-ventilated cages and fed with
normal mouse chow (Sai Durga Feeds and Food, Bangalore, India) and water ad
libitum. All the animal experiments were done after approval from the institutional
animal ethical committee.
3.13.3. In Vitro Antioxidant Activity
3.13.3.1. Determination of DPPH Radical Scavenging Activity
In this method, a commercially available and stable free radical DPPH
(2,2-diphenyl-1-picryl hydrazyl) soluble in methanol was used. DPPH in its radical
form has an absorption peak at 515 nm, which disappeared on reduction by an
antioxidant compound22
. Different concentrations of the extract (22 to 550 mg)
were added to 1.5 ml of freshly prepared DPPH solution (0.25 g/l in methanol).
Absorbance was measured at 515 nm. 20 min., after the reaction was started. The
141
percentage inhibition of DPPH+
in the reaction medium was calculated by
comparing with the control.
3.13.3.2. Determination of Superoxide Radical Scavenging Activity
Superoxide radical scavenging activity was determined by the NBT reduction
method19
. The reaction mixture contained EDTA (6 mm), NaCN (3 mg), riboflavin
(2 mm), NBT (50 mm), various concentrations of the extract (0.22 to 2.2 mg), and
phosphate buffer (67 mm, ph 7.8) in a final volume of 3 ml. The tubes were
uniformly illuminated with an incandescent lamp for 15 minutes, and the optical
density was measured at 560 nm before and after illumination. The percentage
inhibition of superoxide generation was evaluated by comparing the absorbance
values of the control and experimental tubes.
3.13.3.3. Determination of Hydroxyl Radical Scavenging Activity
Hydroxyl radical scavenging activity was measured by studying the competition
between deoxyribose and test compound for hydroxyl radicals generated from the
Fe3+
/ascorbate/EDTA/H2O2 system. The hydroxyl radical attacks deoxyribose,
which results in thiobarbituric acid reacting substance (TBARS) formation20
. The
reaction mixture contained deoxyribose (2.8 mM), FeCl3 (0.1 mM), EDTA (0.1
mM), H2O2 (1mM), ascorbic acid (0.1 mM), KH2PO4-KOH buffer (20mM, pH 7.4),
and various concentrations of the extract (0.22 to 2.2 mg) in a final volume of 1ml.
The reaction mixture was incubated for 1 hr. at 37oC. Deoxyribose degradation was
measured as TBARS and percentage inhibition was calculated21
.
142
3.13.3.4. Determination for Inhibition of Lipid Peroxidation
Reaction mixture (0.5 ml) containing rat liver homogenate (0.1 ml, 25% w/v) in
Tris-HCl buffer (40 mm, ph 7.0), KCl (30 mm), ferrous ion (0.16 mm), and ascorbic
acid (0.06 mm) were incubated for 1 hr. at 370C in the presence (0.22 to 2.2 mg)
and absence of the extracts. The lipid peroxide formed was measured by TBARS
formation21
. Incubation mixtures (0.4 ml) were treated with sodium dodecyl sulfate
(SDS; 8.1%, 0.2 ml), thiobarbituric acid (TBA; 0.8%, 1.5 ml), and acetic acid (20%,
1.5 ml, pH 3.5). The total volume was then made up to 4 ml with distilled water and
kept in a water bath at 1000C for 1 hour. After cooling, 1 ml of distilled water and
5 ml mixture of n-butanol and pyridine (15:1 v/v) were added and vortexed. After
centrifugation, the absorbance of the organic layer was measured at 532 nm. The
percentage inhibition of lipid peroxidation was determined by comparing the results
of the test compound with those of the control which was not treated with the
extracts.
Antioxidant study was conducted availing the facility at Amala Cancer Research
Institute, Thrissure, Kerala.
143
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