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93
4. EXPERIMENTAL INVESTIGATIONS
4.1. ETHNOBOTANICAL SURVEY
An ethno-medicinal survey was conducted to find the traditional medicinal
plants and their uses by the tribe of Tripura. Ethnomedicinal information on folk
medicinal plants was collected from local peoples (generally more than 50 years old)
through interviewing them. Survey was conducted different hilly and remote villages
of West and South Tripura District of Tripura state. Semi-structured interview, group
discussion, and field observation were used to obtain the information on medicinal
plants. Information on local names of the plants, the parts used, method of preparation
and traditional medicinal uses was collected during the survey. Each area was visited
different seasons to determine the authenticity of information collected during field
work. Proper identification of plants were carried out with the help of forest guards,
traditional medical practitioner (locally known as auchai, kabiraj or vaidyas) and
knowledgeable or scientific persons.
4.2. SELECTION OF PLANTS
A number of ethanobotanical survey and few books published on medicinal
plants of Tripura were reviewed systemically to gather the information about the folk
medicinal plants of Tripura and their traditional uses. It was observed that more than
200 medicinal plants are used by the people of Tripura. The plants and/or plant
materials were selected on the basis of literature survey and information obtained
from local people. In first phase 12 plants were selected based on the folk medicinal
information, which is listed on Table 4.1.
94
Table 4.1: List of preliminary selected plants
Scientific Name Family Local Name Traditional Uses
Clerodendrum
viscosum
Verbenaceae bait gach,
chayakkhui
bufang, ghetu
fever, skin infection,
inflammation, laxative, cough,
blood dysentery, anthelmintic
Leea asiatica Leeaceae banchalita against guinea worms, in
wounds, bone fracture,
gastrointestinal diseases
Meyna spinosa Rubiaceae monkata skin disease, peptic ulcer,
diabetes, hepatic disorder
Microcos
paniculata
Tiliaceae pitchla, asar
bufang
bone fracture, cold, typhoid
fever
Phyllanthus
fraternus
Euphorbiaceae bhuiamla,
samuk gach
menstruation cycle disorders,
diabetes, dysentery
Paederia foetida Rubiaceae gandha
bhadali,
dukupui
rheumatic pain, amoebiasis,
tooth ach, stomach pain, as
blood purifier,
Cuscuta reflexa Cuscutaceae sarnalata cough, viral infection, itchy
skin
Galinsoga
parviflora
Asteraceae gangaful,
garingburani
sam
fever, liver problem, wound,
inflammation, insect
bite, toothache
Ipomea aquatica Convolvulaceae kalmi skin disease, dysentery
Meriandra
benghalensis
Labiatae bangal sage high blood pressure, diabetes,
tonsillitis, skin disease
Marsilea minuta Marsileaceae susni sak respiratory troubles, sleeping
disorders, diabetes
Oxalis
corniculata
Oxalidaceae amlati,
amchukai
dysentery, stomach disorders,
rheumatism, toothache
95
Scientific literatures on those plants were extensively reviewed. Reported
activities were matched with traditional knowledge. Initially in the view of relation
between oxidative stress and diseases, it was selected to screen antioxidant
potentiality of the plant first. It was observed that for some of the plants, antioxidant
and other activities were already been reported. Taking this as consideration 5 plants
(Meyna spinosa, Leea asiatica, Marsilea minuta, Meriandra benghalensis, Galinsoga
parviflora) were selected.
In next stage/phase information about freely available plants in Tripura among
the primarily selected plants were collected. It was observed that two indigenous
medicinal plants viz. Meyna spinosa and Leea asiatica has lot of traditional
importance, easily available in the state, widely used by the tribes of Tripura and
preliminary investigation of the leaves showed good DPPH radical scavenging effect.
But these plants were not scientifically investigated for their medicinal uses despite of
vast utility in folk medicine. Therefore I have concentrated on these two plants viz.
Meyna spinosa and Leea asiatica to bring their folk medicinal importance to a
scientific approach.
4.3. PLANT COLLECTION AND AUTHENTICATION
Fresh mature whole leaves of Meyna spinosa Roxb. were collected in October
2010 from Khowai subdivision of Tripura. While the leaves of Leea asiatica (L.)
Ridsdale were collected from Agartala subdivision of Tripura in April 2011. Both the
plants were identified by its vernacular name and later validated by Dr. B.K. Datta,
Department of Botany, Tripura University, Tripura, India. A voucher specimen of
herbarium of plants (TU/BOT/HEB/SS23072011a and TU/BOT/HEB/SS23072011c)
was deposited at the Plant Taxonomy & Biodiversity Laboratory, Department of
Botany, Tripura University.
96
4.4. DRUGS AND CHEMICALS
Chemicals, reagents, biochemical diagnostic kit, drugs were purchased from
reputed companies. List of the important chemicals and name their manufacturing
agencies were tabulated in table 4.2.
Table 4.2: List of chemicals and name of their manufacturing agencies
Company Chemicals
Sigma Aldrich
(Bangalore,
India)
2, 2-diphenyl-picrylhydrazyl (DPPH), Alloxan, Sodium
nitroprusside (SNP), Phenazine methosulfate (PMS), Nitro blue
tetrazolium (NBT), Naphthyl ethylene diamine dihydrochloride
SD Fine Ltd.
Mumbai
Hydrogen peroxide (H2O2), 2-deoxy-ribose, Trichloro acetic acid
(TCA), Thiobarbituric acid (TBA), Methanol, Ethyl acetate,
Petroleum ether, Ethanol, Chloroform, Quercetin, Aluminium
chloride, Sodium nitrite (NaNO2), Sodium hydroxide (NaOH),
Folin-Ciocalteu reagent, Tris-HCl buffer, Phosphoric acid.
Sisco Research
Laboratories
Pvt. Ltd.,
Mumbai
Linoleic acid, Ammonium thiocyanate, Ascorbic acid, α-
tocopherol, Butylated hydroxyanisole (BHA), Epinephrine,
Ascorbic acid, Gallic acid, Sulphanilamide, Nicotinamide adenine
dinucleotide-reduced (NADH).
Loba Chemie
Pvt Ltd.,
Mumbai
Ferric chloride (FeCl3), Ferrous chloride (FeCl2), Zinc chloride,
Chlorosulfonic acid, Lead acetate, Phenazone,
Ethelenediaminetetraacetic acid (EDTA), Potassium acetate,
Aluminium nitrate.
Agapee
Diagnostic Ltd.,
Kerala
Diagnostic kit for - Serum glutamic oxaloacetate transaminase
(SGOT), Serum glutamate pyruvate transaminase (SGPT),
Alkaline phosphatase (ALP), Bilirubin, Cholesterol, Triglycerides,
Blood urea nitrogen (BUN), Creatinine, Uric acid, Total protein,
Albumin, Glucose, High density lipids (HDL), Low density lipids
(LDL), and α–amylase.
All other chemicals used in the study were obtained commercially and were of
analytical grade.
97
4.5. INSTRUMENT USED
Following major instruments were used for the present study,
Double beam UV-Vis Spectrophotometer (Elico SL 164).
Digital pH Meter (Elico LI 127).
Research Centrifuge (Remi R-24).
Semi Auto Analyser (Mispa neo).
Tissue Homogenizer (Remi RQ-127A/D).
Ultrasonic Baths (Edutek Instrumentation, Ambala).
Vacuum Pump (Advanced Technocracy Inc., Ambala).
Melting point Apparatus (Advanced Technocracy Inc., Ambala).
Digital Balance (Contech CA-123)
4.6. PREPARATION OF EXTRACTS
Leaves of both the plants were cleaned to remove unwanted materials. The
leaves were dried in shade under room temperature, powdered mechanically and
sieved through No. 22 mesh sieve. The finely powdered leaves were kept separately
in an airtight container until the time of use. The leaf powder (1 kg) was extracted
with methanol (5 L) using Soxhlet apparatus. The procedure is repeated by taking
fresh leaves powder and extracted with ethyl acetate and petroleum ether separately.
Each filtrates were then concentrated under reduced pressure and evaporated
at 40ºC to obtain crude methanol extract of M. spinosa (MEMS), ethyl acetate extract
of M. spinosa (EEMS), petroleum ether extract of M. spinosa (PEMS), methanol
extract of L. asiatica (MELA), ethyl acetate extract of L. asiatica (EELA), and
petroleum ether extract of L. asiatica (PELA) leaves. All extracts were stored at 4ºC
till the time of use.
98
4.7. PHYSICOCHEMICAL PROPERTIES OF EXTRACT
Physiochemical evaluation of extracts was carried out by determining yield,
colour, pH, density and specific gravity of crude extract.
4.7.1. Colour and Yield Determination
Colour of the extracts was observed in naked eye. The physical properties of
extracts were noted, including percentage yield.
4.7.2. Determination of Specific Gravity
An empty pycnometer was taken and the weight (Wp) of the same was
recorded. Sample (10g) was placed in the pycnometer and the weight (Wps) was
recorded. Pycnometer was filled half or ¾ with distilled water and soaked for10
minute. Partial vacuum was applied to the content for 10 minute to remove entrapped
air. The pycnometer was filled with distilled water up to the mark; the exterior surface
of the pycnometer was cleaned with clean and dry cloth and the weight (Wb) was
determined. Then the pycnometer was cleaned fully by removing the content and
filled with distilled water, and the weight (Wa) was measured again. Specific gravity
was determined using the following formula [194],
Specific Gravity (Gs) = W0
W0 - (Wa - Wb)
W0 = weight of the sample (Wps - Wp)
Wa = Weight of pycnometer filled with water
Wb = Weight pycnometer filled with water and sample
99
Figure 4.1: Extraction of plant materials through Soxhlet apparatus
100
4.7.3. Determination of pH
The individual extract solution was filtered by using Whatman filter paper and
pH was recorded by using Elico digital pH meter [195].
4.8. PRELIMINARY PHYTOCHEMICAL INVESTIGATION
Qualitative phytochemical investigation of extracts was carried out to analyse
phytochemicals present in the MEMS, EEMS, PEMS, MELA, EELA and PELA.
Preliminary phytochemical screening will give the idea about the chemical
constituents present in the respective extracts.
Phytochemical test were carried out to find the presence of alkaloids,
carbohydrates, glycosides, protein, amino acids, tannins, flavonoids, triterpenoids and
steroids in the extracts. The screening was done as explained in the standard
literatures [196, 197].
Table 4.3: Procedure for preliminary phytochemical investigation
Test procedure Observation for
positive test
A. Detection of Alkaloids: A small quantity of solvent free extract was stirred
with little quantity of dilute hydrochloric acid (HCl) and filtered, which was used
to test to find the presence of alkaloids. The screening test for alkaloids were
performed using the following tests,
Mayer’s test: Few millilitres of filtrate were mixed with 2
drops Mayer’s reagent (potassium mercuric solution) along
the sides of tube.
White or creamy
precipitate.
101
Wagner’s test: Few millilitres of filtrate were mixed with
few drops of Wagner’s reagent (iodine-potassium iodide
solution) along sides of tube.
Reddish brown or
orange precipitate.
Hager’s test: Few millilitres of filtrate were mixed with
2.0 ml of Hager’s reagent (saturated solution of picric
acid).
Prominent yellow or
crystalline yellow
precipitate.
Dragendroff’s test: Few millilitres of filtrate were mixed
with 2.0 ml of Dragendroff’s reagent (potassium bismuth
iodide solution).
Prominent reddish
brown or orange red
precipitate.
B. Detection of Carbohydrates: A small quantity of the extract was dissolved in
few millilitre of distilled water and filtered. The filtrate was subjected to the
following tests,
Molish’s test: Two drops of Molish reagent (alcoholic
solution of α-naphthol) was added to 2.0 ml of filtrate.
The mixture was shaken well and 1.0 ml concentrated
sulphuric acid was added slowly along the sides of the
tube, cool the tube in ice water and allowed to stand.
A violet colour ring
appears at the junction of
layers.
Fehling’s test: One millilitre of filtrate was mixed with
1.0 ml each of Fehling’s solutions A and B. The solution
was heated on water bath.
Brick red precipitate
indicates presence of
reducing sugar.
Barfoed’s test: One millilitre filtrate was added with 1.0
ml Barfoed’s reagent in a test tube and heated on water
bath for 2 minute.
Red precipitate indicates
presence of
monosaccharides.
Benedict’s test: Equal volume of filtrate and Benedict
reagent was taken in a test tube and heated on water bath
for 2 minute.
A characteristic colored
precipitate indicates
presence of reducing
sugar.
102
C. Detection of Protein and Amino Acid: Small quantity of sample was
dissolved in few millilitre of distilled water and filtered through Whatman no.1
filter paper and the filtrate was subjected to tests for proteins and amino acids as
follows,
Millon’s test: Two millilitre of filtrate was mixed with few
drops of Millon’s reagent. (Test for amino acid)
White precipitate
indicates
Ninhydrin test: Two drops of ninhydrin solution was
added to 2.0 ml of filtrate and boiled.
Purple/blue/violet
colour for amino acid.
Biuret test: Two millilitre of filtrate was added to 2.0 ml
Biuret reagent.
Violet colour indicates
presence of protein
Xanthoprotein test: Five millilitre of test solution were
mixed with 1.0 ml concentrated HNO3 solution and boiled.
On cooling 40% NaOH solution was added.
Orange colour indicates
presence of protein.
D. Detection of Glycosides (General test for glycosides)
Test A: 200 mg of drug was extracted with 5.0 ml of dilute
H2SO4 by warming on a water bath. The solution was
filtered and extract was neutralised with 5% NaOH
solution. Fehling's solution A and B (each 0.1 ml) was
mixed until it becomes alkaline and heated on a water bath
for 2 minute. The quantity of red precipitate formed during
the test was measured.
Test B: About 200 mg of drug extracted with 5.0 ml of
water by warming on a water bath. Equal amount of water
was added as used for NaOH in the Test A. Fehling's
solution A and B (each 0.1 ml) was mixed until it becomes
alkaline and heat on water bath for 2 minutes. Quantity of
precipitate formed in Test B compared with that of forms
in Test A.
If the precipitate formed
in Test A is more than
in Test B then glycoside
may be present (Test A
represent free reducing
sugar plus those related
on acid hydrolysis of
any glycoside in the
crude drug, whereas
Test B represents the
amount of free reducing
sugar in the crude drug).
103
E. Detection of Specific Glycosides: Tests for specific glycosides like
anthraquinone, cardiac glycosides, coumarin, saponin glycosides was also
performed using the following methods,
Borntrager's test: Test sample was boiled with 1.0 ml of
sulphuric acid in a test tube for 5 minute. The solution was
filtered while hot, cool the filtrate and shake with equal
volume of chloroform. Lower layer of chloroform was
separated and shaken with half of its volume of dilute
ammonia.
A rose pink to red
colour will be produce
in the ammoniacal layer
if anthraquinone
glycosides present.
Keller-killiani test: The sample was extracted with
chloroform, and evaporates to dryness. Glacial acetic acid
(0.4 ml) containing trace amount of ferric chloride was
added. Concentrated H2SO4 (0.5 ml) was added carefully
by the side of the test tube.
Acetic acid layer shows
blue colour indicates
presence of cardiac
glycosides.
Raymond’s test: The test solution was treated with hot
methanolic alkali.
Violet colour indicates
presence of cardiac
glycosides.
A small amount of sample in test tube was taken and
covers the test tube with a filter paper moistened, with
dilute NaOH solution. The test tube was placed on water
bath for several minutes. The paper was removed and
expose to UV light.
Paper shows green
fluorescence indicates
presence of coumarin
glycosides.
Haemolysis test: Two millilitre NaCl solution (1.8%) was
taken to the two test tubes. To one of these 2.0 ml of
distilled water and to other 2.0 ml of sample solution was
added. The concentration of NaCl in each tube now is
isotonic to blood. Blood was obtained by pricking the
thumb at the base of the nail, and 5 drops of blood was
added to each and mixed gently.
Haemolysis observed
under the microscope in
the tube containing the
sample, but no
haemolysis in control
indicates presence of
saponin glycosides
104
Froth formation test: Small amount of extract was
shaken in a test tube with a little quantity of water.
The foam produced will
persists for 10 minute
indicates presence of
saponin glycosides.
F. Detection of Steroids and Triterpenoids: The test for flavonoids was
carried out by the following methods,
Libermann-Burchard’s test: The sample was treated
with few drops of acetic anhydride, heated to boiling, and
cooled. One millilitre of concentrated H2SO4 was added
along the side of test tube.
Brown ring at the junction
and upper layer turns to
green indicated presence
of presence of steroids.
Brown ring at the junction
and upper layer turns to
deep red indicates
presence of triterpenoids
Salkowski Test: The sample was treated with few drops
of concentrated H2SO4.
Red colour at lower layer
indicated presence of
presence of steroids.
Yellow colour at lower
layer indicates presence
of triterpenoids
Tschugajeu test: To the chloroform solution of sample
excess of acetyl chloride and a pinch of zinc chloride
were added. Test tube was kept aside for reaction and
then warm on water bath
Erosin red colour
produced indicates
presence of triterpenoids
Brieskorn and Brinar test: To the chloroform solution
of test sample a few drops of chrolosulfonic acid in
glacial acetic acid (7:3) was added.
Red colour in few
minutes indicates
presence of triterpenoids
105
G. Detection of Tannins: The presence of tannin in crude extract was
determined by the following methods,
Ferric chloride test: A small quantity extract was boiled
in few millilitre of distilled water in a test tube and then
filtered. A few drops of 0.1% ferric chloride were added.
Brownish green or a
blue-black colour.
Phenazone test: To 5.0 ml of aqueous extracts sodium
acid phosphate (0.5 g) was added. The mixture was
warmed and filtered. Phenazone solution (2%) was added
in the filtrate.
Bulky precipitate which
often coloured
Gelatin test: A small quantity of sample was dissolved in
distilled water, and 2.0 ml of 1% gelatine solution
containing 10% NaCl was added.
White precipitate
H. Detection of Flavonoids: The presence of flavonoids was determined by the
following methods,
Lead acetate test: To the alcoholic solution of the extract/,
few drops of 10% lead acetate solution was mixed
Yellow precipitate.
Alkaline reagent test: To the few millilitre of test
solution, few drops of 10% NH4OH was added
Intense yellow colour
formed which turns to
colourless on addition
of few drops of dilute
acid.
Zinc Hydrochloride test: To the alcoholic test solution of
the extract a pinch of zinc dust and few drops of
concentrated HCl was mixed.
Red/Megneta colour
developed after few
minute.
Ferric chloride test: To the alcoholic solution of extract,
few drops of ferric chloride were added.
Green colour indicated
presence of flavonoid in
the sample.
106
4.9. EXPERIMENTAL ANIMALS
Healthy Albino mice (20-30 g) and Wistar rats (250-250 g) were used for the
study. In acute toxicity study, nephroprotective activity mice were used, and rats were
used for ex vivo antioxidant, antidiabetic, hepatoprotective activity. Animals were
maintained under standard laboratory conditions at a temperature of 23±2ºC, with 12
h light-dark cycle, and relative humidity (5010%) for 1 week. All animals had free
access to pallet chow (Nutrivet Life Science, Pune) and water ad libitum until the day
before experiment, when only water was made available to them.
Indian adult earthworms (Pheretima posthuma) were used for anthelmintic
activity. The species Pheretima posthuma were supplied and authenticated by the
Yamuna Vermi Compost, Kurnool, Andhra Pradesh. The earthworms were collected
from moist soil and to remove all fecal matter washed with normal saline.
Earthworms with 3-5 cm length and 0.1-0.2 cm width were used for in vitro
anthelmintic activity.
All animal procedures have been approved by Institutional Ethical Committee
of Creative Educational Society’s College of Pharmacy, Kurnool, Andhra Pradesh
(1305/ac/09/CPCSEA) in accordance with animal experimentation and care.
4.10. DETERMINATION OF ANTIOXIDANT COMPOUNDS
4.10.1. Total Phenolic Content Determination
The amount of total phenolics in the crude extracts was determined using
Folin-Ciocalteu reagent following the colorimetric method [92]. The ethanol solution
107
of each extract (0.5 ml, l.0 mg/ml) were added into the test tubes containing 2.5 ml of
10% v/v Folin-Ciocalteu reagent and 2.0 ml of 2% w/v sodium carbonate. The tubes
were shaken thoroughly and incubated at 45°C for 15 minute with intermittent
shaking. Absorbance was observed at 765 nm using ELICO SL 164 UV–Vis
spectrophotometer. Gallic acid (0 to 800 µg/ml) was used as standard to get a
calibration curve, and results were expressed as gallic acid equivalents in milligram
per gram (mg GAE/g) of dried extract. The estimation was carried out in triplicate.
4.10.2. Total Flavonoid Content Determination
Total flavonoid content of extracts was estimated by aluminium nitrate-
potassium acetate reagent method. Briefly, 0.5 ml of sample solution (1.0 mg/ml) was
mixed with 0.1 ml of 10% aluminium nitrate, 0.1 ml 1 M potassium acetate and 4.3
ml of 80% ethanol. The mixture was mixed thoroughly and allowed to stand for 40
minute at room temperature. The absorbance of the supernatant was measured at 415
nm [94]. The estimation was carried out in triplicate and calculations were based on a
calibration curve obtained with quercetin (0 to 16 µg/ml). The results were expressed
as quercetin equivalents in milligram per gram (mg QE/g) of dried extract.
4.11. IN VITRO ANTIOXIDANT ACTIVITY
4.11.1. DPPH Radical Scavenging Assay
The hydrogen atom or electron-donation ability of the corresponding sample
was measured from the bleaching of a purple-coloured ethanol solution of DPPH. The
DPPH radical is a stable radical with a maximum absorption at 517 mm that can
108
readily undergo reduction by an antioxidant. Free radical scavenging capacity of
extracts/fractions was measured in terms of hydrogen-donating or radical scavenging
ability, using the stable DPPH radical [98].
Briefly, 1.0 ml of 0.1 mM DPPH methanolic solution was mixed with 3.0 ml
of sample solution of different concentrations. The reaction mixture was incubated in
room temperature in the dark for 30 min and the absorbance was recorded at 517 nm
[98]. The assay was carried out in triplicate for each sample. One millilitre methanol
with 3.0 ml extracts solution was used as a blank, DPPH solution (1.0 ml, 0.1 mM)
with ethanol (3.0 ml) was served as negative control. The radical scavenging activity
of ascorbic acid was also determined, which served as positive control. The decrease
in absorbance on addition of test samples was used to calculate the antiradical
activity, as expressed by the inhibition percentage (I %) of DPPH radical, following
the equation (1)
I % = [(Ac-As)/Ac] x 100
Where, Ac and As are the absorbance of the control and of the test/standard
sample respectively. From a plot of concentration against I%, a linear regression
analysis was performed to determine the IC50 (extract concentration resulting in a
50% inhibition) value for each sample.
4.11.2. Superoxide Anion Radical Scavenging Activity
The scavenging activity of the sample towards superoxide anion radicals was
measured by the method of Nagulendran et al. [93]. Superoxide anions were formed
in a non-enzymatic PMS-NADH system through the reaction of PMS, NADH, and
oxygen, which was assayed by the reduction of NBT. Superoxide anion reduces the
109
yellow dye NBT2+
to produce the blue formazan which was determined
spectrophotometrically at 560 nm.
Briefly, 3.0 ml of Tris-HCl buffer (100 mM, pH 7.4) containing 0.75 ml of
NBT (300 μM) solution, 0.75 ml of NADH (936 μM) solution were mixed with 0.3
ml of extract at different concentration. The reaction was started by mixing 0.75 ml
PMS (120 μM) to the mixture. After 5 minute of incubation at room temperature, the
absorbance of mixture was recorded at 560 nm. BHA was used as standard, all the
tests were carried out in triplicate. Scavenging activity was calculated according to the
equation 1.
4.11.3. Hydroxyl Radical Scavenging Activity
The hydroxyl radical scavenging activity of sample was assayed by using the
2-deoxyribose oxidation method. The 2-deoxyribose is oxidized by hydroxyl radical
that is generated by the Fenton reaction and degraded to malondialdehyde [100].
Therefore decomposing effect of sample on hydroxyl radicals was estimated by the
assay of malondialdehyde chromogen formation due to 2-deoxy 2-ribose degradation.
Briefly, 0.2 ml KH2PO4–KOH (100 mM), 0.2 ml deoxyribose (15 mM), 0.2 ml
FeCl3 (500 mM), 0.1 ml EDTA (1 mM), 0.1 ml ascorbic acid (1 mM), and 0.1 ml
H2O2 (10 mM) were mixed with 0.1 ml sample. The mixture was incubated at 37ºC
for 1 h. After incubation time, 1.0 ml of TBA (1% w/v) was added to mixture
followed by the addition of 1.0 ml of TCA (2.8% w/v). The resultant mixture was
heated on a water bath at 80ºC for 20 minute, so that pink coloured developed. The
absorbance of the solution was measured at 532 nm [106]. Quercetin was used as the
positive control. The scavenging activity of extracts or standard (I %) was calculated
using the equation (1).
110
4.11.4. Nitric Oxide Radical Scavenging Activity
Nitric oxide impulsively produced from an aqueous solution of sodium
nitroprusside at physiological pH, and interacts with oxygen to produce nitrite ions,
which may be quantified by the Griess Illosvoy reaction. The nitrite ions thus
produced react with Griess reagent that leads to a chromophore formation. The
concentration of this chromophore is proportional to that of the generated nitrite ions.
Antioxidant compounds compete with oxygen leading to reduced production of nitric
oxide [92, 98].
Four millilitres of sample solution at different concentrations were mixed with
1.0 ml of 25 mM sodium nitroprusside solution in a test tube, and incubated for 2 h at
37ºC. An aliquot (2.0 ml) of the incubated solution was taken and mixed with 1.2 ml
Griess reagent (1% sulfanilamide in 5% H3PO4 and 0.1% naphthyl ethylene diamine
dihydrochloride). The chromophore formed by diazotization of the nitrite with
sulfanilamide and subsequent coupling with naphthylethylenediamine
dihydrochloride. The absorbance was measured immediately at 570 nm [95]. Control
experiment was also carried out in similar manner, using distilled water in the place of
sample solution. The experiment was performed in triplicate, ascorbic acid was used
as positive control and I% was calculated using the equation (1).
4.11.5. Metal Chelating Ability
The capacity of antioxidants to form insoluble metal complexes with ferrous
ion or to generate steric hindrance which avert the interaction between metal and lipid
can be investigated using the iron chelating ability assay method. Ferrozine can make
111
red colored complexes with ferrous ions, which was interrupted by chelating agents.
Thus, the activity of antioxidants is determined by monitoring the decrease in
absorbance of the red Fe2+
-ferrozine complex as antioxidants compete with ferrozine
in chelating ferrous ion [93, 101].
Extracts at different concentration (0.4 ml) was mixed with 2 mM FeCl2
solution (0.05 ml). The reaction was started by the mixing of 0.2 ml of ferrozine (5
mM), and total volume of the solution was adjusted to 4.0 ml using ethanol. The
mixture was shaken vigorously and left standing for 10 minute at room temperature.
The absorbance of the solution was measured spectrophotometrically at 562 nm [104].
α-tocopherol was used as standard and percentage of inhibition of ferrozine–Fe2+
complex formation (I%) was calculated by using equation (1).
4.11.6. Reducing Power Ability
The potassium ferricyanide reduction method was used to evaluate the ability
of phenolic compounds to quench radicals through electron donation. Antioxidant
capacity of the sample was monitored by measuring the absorbance of Perl’s Prussian
blue complex at 700 nm, the absorbance was increases as antioxidants reduce the
ferric ion/ferricyanide complex to the ferrous form [101].
Briefly, different concentration of sample solutions (1.0 ml) were mixed with
0.2 M phosphate buffer (2.5 ml, pH 6.6) and 1% (w/v) K3Fe(CN)6 (2.5 ml). The
resulting mixture was incubated at 50ºC for 20 minute, and then 10% TCA (2.5 ml)
was added. The solution was centrifuged at 12000 rpm for 10 minute, and 2.5 ml of
supernatant solution was pipette out to mix with 2.5 ml of distilled water, 0.5 ml of
112
0.1% (w/v) FeCl3 solution. The absorbance of solution was determined at 700 nm
using ELICO SL 164 UV–Vis spectrophotometer [105]. The reducing power ability
of ascorbic acid (25-400 µg/ml) was also determined. The test was run in triplicate
and averaged.
4.11.7. Hydrogen peroxide Scavenging Activity
The hydrogen peroxide-scavenging ability of extracts was determined
according to the method of used by Bozin et al. [96]. Briefly, a solution of 40 mM
H2O2 and crude extracts/standard in different concentrations were prepared in
phosphate buffer (pH 7.4). An aliquot (3.4 ml) sample solution was added to 0.6 ml
of H2O2 solution. The absorbance of resulting solutions was taken at 230 nm. Gallic
acid was used as positive control. The percentage of H2O2 scavenging (I%) of
examined extracts was calculated by equation (1).
4.11.8. Ferric Thiocyanate Method (FTC)
The FTC method was employed to determine the peroxide level during the
initial stage of lipid oxidation. Linoleic acid oxidation cause the generation of
peroxides, which react with Fe2+
to form Fe3+
. The ferric ion form a red colored
complex with SCN– and this complex has a maximum absorbance at 500 nm [103].
Sample (200 µg) in 4.0 ml ethanol was mixed with 2.5% linoleic acid in
ethanol (4.0 ml), phosphate buffer (8.0 ml, 0.05 M, pH 7.0) and distilled water (4.0
ml). The mixture was placed in screw cap tube, mixed vigorously and incubated in the
113
dark conditions at 40ºC. One millilitre solution was drawn from the incubated reaction
mixture and mixed with 9.7 ml ethanol (75%) and 0.1 ml ammonium thiocyanate
(30%). Then, 0.1 ml 20 mM ferrous chloride in 3.5% HCl was added to the reaction
mixture, and exactly 3 minute later the absorbance of the red colour solution was
measured at 500 nm. The procedure was repeated every 24 h until the absorbance of
the control reached a maximum [103]. The control and standard were prepared as the
sample. Linoleic acid mixture without the addition of sample was used as the control;
α-tocopherol at the same concentration was served as the positive control.
4.12. EX VIVO ANTIOXIDANT ACTIVITY
4.12.1. Lipid Peroxidation Assay
A healthy Wister rat was fasted for 16 h, and sacrificed by decapitation. The
liver was dissected out carefully and washed with normal saline. The 5.0% w/v liver
homogenate was prepared in phosphate buffer saline. One millilitre liver homogenate
was mixed with 100 μl of sample solution and incubated for 2 h at 37°C. After
incubation time, 1.0 ml of 15% w/v TCA and 1.0 ml of 0.67% w/v TBA were added
to the mixture. This solution was heated on boiling water bath for 15 minute and
cooled. The final volume of the solution was made up to 5.0 ml with deionized water
and then centrifuged for 10 minute at 2800 rpm. The supernatant solution was
removed carefully and absorbance supernatant was recorded at 532 nm [99].
Control was prepared without extracts. Rutin was used as positive control. The
inhibition of lipid peroxidation was calculated according to equation (1), where,
114
Asample is the absorbance of an extract/standard in presence of liver homogenate and
Acontrol is the absorbance of the solution containing all reagents except the
test/standard sample.
4.12.2. Oxidative Haemolysis Assay
The assay method of Su et al. (2009) was performed with slight modifications
as described by Coulibaly et al. (2011) [97, 102]. Blood sample was collected from rat
eyepit under mild anaesthesia and centrifuged at 1500 × g for 10 minute at 4ºC. The
erythrocytes were isolated from the plasma and buffy coat, and suspended in 10.0 ml
phosphate buffer saline (PBS, 10 mM, pH 7.4). The erythrocyte in PBS was
centrifuged again at 1500 × g for 5 minute to remove buffy coats. The process was
repeated three times; the supernatant and buffy coats of white cells were carefully
removed after each wash. Washed erythrocytes made to 0.5% erythrocyte suspension
in PBS (10 mM, pH 7.4) for the assay.
Briefly, the reaction mixture was consisted of 0.5 ml of erythrocyte
suspension, 0.5 ml extract/standard drug solution at different concentration and 0.05
ml of 100 mM H2O2. Mixture was kept for incubation at 37ºC for 60 minute, and after
incubation period 4.2 ml of distilled water was added. The solution was centrifuged
for 10 minute at 1000 rpm, and absorbance of the supernatant was measured at 415
nm.
Control contains supernatant without extract. Ascorbic acid was used as
positive control. The protective effect of extract was calculated as inhibition
percentage of erythrocyte haemolysis (I%) using the following equation (1).
115
4.13. CHROMATOGRAPHIC FRACTIONATION OF EXTRACT
In vitro and ex vivo antioxidant study revealed that methanol extract of both
plant possess superior antioxidant activity. Therefore, most effective solvent extract
viz. methanol extract of leaves of M. spinosa and methanol extract of leaves of L.
asiatica was chromatographed in silica gel column (borosilicate column, 40 mm ×
600 mm). Silica gel (60-80 mesh) served as stationary phase. About 120 g of silica gel
was mixed with 200 ml of petroleum ether, and poured slowly to pack the column to
get a hight of 20 cm. The procedure was carefully performed to avoid deposition of
any air bubble. A quantity of 80 g of extract was dissolved in little quantity of
methanol and digested with 120 g of silica gel and dried under a stream of cold air,
then spread over the column packing and eluted successively with petroleum ether,
ethyl acetate and methanol. A volume of 2.5 l petroleum ether was eluted first
followed by 2 l of ethyl acetate and 2 l of methanol to obtain petroleum ether fraction
(PFMS), ethyl acetate fraction (EFMS), methanol fraction (MFMS) of methanol
extract of Meyna spinosa, and petroleum ether fraction (PFLA), ethyl acetate fraction
(EFLA), methanol fraction (MFLA) of methanol extract of Leea asiatica.
4.14. THIN LAYER CHROMATOGRAPHY (TLC) STUDY
Thin layer chromatography (TLC) was performed on pre-coated aluminium
sheets with silica gel 60 (20 × 20 cm, layer thickness 0.2 mm) procured from SD
Fine-Chen Limited, Mumbai. The plates were cut in smaller pieces to get 5 × 10 cm
size of each smaller plate.
116
Figure 4.2: Diagram of column chromatography
117
The 2-5 μl of the extract/fraction filtered solution (usually 1%) was spotted
using a fine bore capillary tube. The spot was placed (1 cm above the base) at
equidistance from each other. Chromatogram were developed using different solvent
systems in a room temperature (28°C and at an angle of 75°). The solvent system was
allowed to run to a distance of 8 cm from the application point of the extract/fraction
in the plates. The time required for the completion of run varied from 20-30 minute,
and then the plates were removed from the chamber and allowed to dry in air. These
plates were placed in iodine chamber and the presence of the spots was identified
[197]. Retardation factor (Rf) is the ratio of distance travelled by the solute (extract/
fraction) and the distance travelled by the respective solvent front. Rf values were
calculated using the following formula,
Distance traveled by the solute
Distance traveled by the solvent Rf value =
4.15. ACUTE TOXICITY STUDY
The acute toxicity for samples (potent extract and its fractions) was
determined in Albino mice. The animals were fasted overnight prior to the
experiment. Fixed dose [Organisation for Economic Co-operation and Development
(OECD) Guideline no. 423, Annexure 2d] method of Committee for the Purpose of
Control and Supervision of Experiments on Animals (CPCSEA) was adopted for
toxicity studies. The tested extracts suspended in 0.5% w/v sodium carboxy methyl
cellulose (CMC) and were administrated orally (1 ml/100 g) in 3 animals. The
presence or absence of any signs of toxicity or mortality was monitored at 2000
mg/kg in the all cases. Common side effects such as, mild diarrhea, lose of weight and
depression of treated groups of animals were recorded within the 7 days of
observation [198].
118
4.16. HEPATOPROTECTIVE AND IN VIVO ANTIOXIDANT
ACTIVITY
Hepatoprotective and in vivo antioxidant effect of MEMS, MELA and
fractions of MEMS, MELA were evaluated against paracetamol induced
hepatotoxicity [111, 112].
4.16.1. Paracetamol Induced Hepatotoxicity
Experiment was performed two stages. In first stage hepatoprotective activity
of extract was performed and in second stage effect of fractions on paracetamol
induced hepatotoxicity was evaluated.
In first stage, animals were divided into seven groups each containing six,
Group I served as control, Group II served as negative control, Group III treated with
standard drug silymarin, Group IV-V treated with MEMS (150 and 300 mg/kg, b.w.),
Group VI-VII treated with MELA (150 and 300 mg/kg, b.w.).
To determine hepatoprotective effect of fractions animals are divided into 15
groups (n=6) in following manner,
Group I (Control) : Vehicle (water, 1 ml/kg)
Group II (Negative control) : Vehicle (water, 1 ml/kg)
Group III (standard) : Silymarin (25mg/kg)
Group IV-IX : MFMS, EFMS, PEMS at 75 and 150 mg/kg
Group X-XV : MFLA, EFLA, PELA at 75 and 150 mg/kg
119
Animals of the test groups were given the respective drug treatment one daily
for consecutive three days. All the extracts/fractions, silymarin, and paracetamol were
administered orally by suspending in 0.5% sodium carboxy methyl cellulose (CMC).
Animals of all groups except normal control group received paracetamol (3 gm/kg) as
single dose on 3rd
day, thirty minutes after the administration of test samples or
vehicle respectively. After 48 h of paracetamol oral administration, the blood was
collected by direct cardiac puncture under light ether anaesthesia and serum was
separated by centrifugation of blood at 4000 × g, which was used for the biochemical
estimations.
4.16.2. Biochemical Determinations
Biochemical parameters like SGOT, SGPT, ALP, total bilirubin, direct
bilirubin, TC, TG were determined by using commercial biochemical kit obtained
from Agapee Diagnistic Ltd., Kerala
4.16.2.1. SGOT determination
Quantitative determination of SGOT in serum was performed according to the
manufacturer’s procedure (SGOT Kit Batch no: 4501, Manufacturing date: 9/11,
Expiry date: 11/13), which is based on the following reactions,
Aspartate + alpha Ketoglutarate Oxaloacetate + L-Glutamate
Aspartate aminotransferase
Oxaloacetate + NADH + H+
Malatedehydrogenase
L - Malate + NAD+
120
Laboratory procedure: Serum sample (0.1 ml) was mixed with 1.0 ml of working
reagent and incubated for 1 minute at 37°C. The absorption of the resulting was taken
at semi auto analyser. Following parameter were feed in auto analyser before the
determination of absorbance, mode of reaction – kinetic, slope of reaction –
decreasing, blank – distilled water, wavelength – 340 nm, factor – 1745 , linearity –
1000 U/l, delay time – 60 sec, no. of readings – 3, interval – 20 sec. Results was
expressed as U/l.
4.16.2.2. SGPT determination
Quantitative determination of SGPT in serum was performed according to the
procedure described by the manufacturer (SGPT Kit Batch no: 4063, Manufacturing
date: 9/11, Expiry date: 11/13), which is based on the following reactions,
Alanine + alpha Ketoglutarate Pyruvate + L-Glutamate
Aspartate aminotransferase
Pyruvate + NADH + H+
Lactatedehydrogenase
L - Lactate + NAD+
Laboratory procedure: Serum sample (0.1 ml) was mixed with 1.0 ml of working
reagent and incubated for 1 minute at 37°C. The absorption of the resulting was taken
at semi auto analyser. Following parameter were feed in auto analyser before the
determination of absorbance, mode of reaction – kinetic, slope of reaction –
decreasing, blank – distilled water, wavelength – 340 nm, factor – 1745 , linearity –
1000 U/l, delay time – 60 sec, no. of readings – 3, interval – 20 sec. Results was
expressed as U/l.
121
4.16.2.3. ALP determination
The level of ALP in serum was determined according to the procedure
described by the manufacturer (ALP Kit Batch no: 8721, Manufacturing date: 8/11,
Expiry date: 8/13), which is based on the following reactions,
p-Nitrophenylphosphate + H2
O
Alkaline
phosphatasep-Nitrophenol +
Inorganic phosphate
Laboratory procedure: Working reagent (1.0 ml) and test serum (0.02 ml) was mixed
and incubated again 1 minute at 37°C. The absorption of the resulting was taken at
semi auto analyser. Following parameter were feed in auto analyser before the
determination of absorbance, mode of reaction – kinetic, slope of reaction –
increasing, blank – distilled water, wavelength – 405 nm, factor – 2750 , linearity –
700 U/l, delay time – 60 sec, no. of readings – 3, interval – 60 sec. Results was
expressed as U/l.
4.16.2.4. Bilirubin determination
Total and direct bilirubin in serum was performed according to the
procedure described by the manufacturer (Bilirubin Kit Batch no: 670, Manufacturing
date: 06/11, Expiry date: 06/13). The assay is based on the principle that, salfanilic
acid (present in direct bilirubin reagent) reacts with sodium nitrate to produce in the
presence of diazotied sulfanilic acid to form azobilirubin. In the absence of dimethyl
sulfoxide only direct bilirubin reacts to give azobilirubin.
122
Laboratory procedure: Test and blank solution were prepared in the following
manner,
Reagents/ sample Total bilirubin Direct bilirubin
Sample blank Test Sample blank Test
Total bilirubin reagent 1.0 ml 1.0 ml - -
Direct bilirubin reagent - - 1.0 ml 1.0 ml
Serum 0.05 ml 0.05 ml 0.05 ml 0.05 ml
All samples were mixed well and incubated at room temperature for 5 minute.
The absorption of the resulting was taken at semi auto analyser. Following parameter
were feed in auto analyser before the determination of absorbance, mode of reaction –
end point, slope of reaction – increasing, wavelength – 546 nm, linearity – 20 mg/dl,
factor for total bilirubin – 20.5, factor for total bilirubin – 16.5. Result was expressed
as mg/dl.
4.16.2.5. Total cholesterol determination
Total cholesterol (TC) was estimated by CHOD-PAP method as per
manufacturer procedure (Total cholesterol Kit Batch no: CH-0612, Manufacturing
date: 1/12, Expiry date: 1/13). In this assay, cholesterol esters are dissociated into
cholesterol and fatty acids in presence of cholesterol esterase. In the subsequent
enzymatic oxidation by cholesterol oxidase, H2O2 and cholestenone was formed. In
presence of peroxidase, H2O2 reacts with p-aminoantipyrine and phenol to form a red
quinoeimine dye.
123
Chloesterol ester + H2OCholesterol esterase
Cholesterol + Fatty acids
Cholesterol + O2Cholesterol oxidase
4 Cholesten-3-one + H2O2
2H2O2 + Phenol + 4 Aminoantipyrine Red quinone + 4 H2OPeroxidase
Procedure: One millilitre of working reagent mixed with 0.01 ml of serum sample or
standard and mixed well. Solution was and incubated for 37°C for 5 minute. The
absorbance of sample and standard was measured against the blank (working reagent).
The absorption of the resulting was taken at semi auto analyser. Following parameter
were feed in auto analyser before the determination of absorbance, mode of reaction –
end point, slope of reaction – increasing, wavelength – 505 nm, linearity – 600 mg/dl,
standard concentration – 200 mg/dl. Result was expressed as mg/dl.
4.16.2.6. Total triglycerides determination
Total triglyceride (TG) in serum was estimated by GPO-PAP method using
the manufacturer procedure (Triglyceride Kit Batch no: 650, Manufacturing date: 5/11,
Expiry date: 5/13). The assay is based on following principle,
Triglycerides + H2O
Lipoprotein lipaseGlycerol + Fatty acid
Glycerol + ATP Glycerol kinase
Glycerol-3-phosphate + ADP
Glycerol-3-phosphate + O2
Glycerol-3
Dihydroxyacetone phosphate + H2O
2
H2O
2 + 4 Aminoantipyrine + p-chlorophenol
PeroxidaseRed quinoneimine
-phosphate oxidase
124
Procedure: One millilitre of working reagent was mixed with 0.01 ml of serum
sample or standard and mixed well. Solution was incubated for 37°C for 5 minute.
The absorbance of sample and standard was measured against the blank (working
reagent). The absorption of the resulting was taken at semi auto analyser. Following
parameter were feed in auto analyser before the determination of absorbance, mode of
reaction – end point, slope of reaction – increasing, wavelength – 505 nm, linearity –
1000 mg/dl, standard concentration – 200 mg/dl. Result was expressed as mg/dl.
4.16.3. In vivo Antioxidant Activity
In vivo antioxidant activity was evaluated by determining the level of SOD,
CAT, GPx, GSH in liver tissue, and blood plasma GSH level in drug treated and
hepatotoxic animals.
4.16.3.1. Preparation of liver homogenate
The liver was dissected out from animals and was perfused with cooled 0.15
M KCl. The 10% homogenate in 0.15 M KCl-10 mM potassium phosphate buffer, pH
7.4 was centrifuged at 3000 rpm for 10 minute [199].
4.16.3.2. Estimation of superoxide dismutase
SOD activity was determined by the inhibition of autocatalyzed adrenochrome
formation at 480 nm in the presence of tissue homogenate. The reaction mixture
contained 150 µl of homogenate, 1.8 ml of 30 mM carbonate buffer (pH, 10.2), and
0.7 ml of distilled water and 400 µl of epinephrine (45 mM). Auto oxidation of
125
epinephrine to adrenochrome was performed in a control tube without the
homogenate [200]. The enzymatic activity was calculated as nM of epinephrine
protected from oxidation/min/mg protein using a molar extinction coefficient of 4.02
× 103 M-1
cm-1
.
4.16.3.3. Estimation of catalase
The catalysis of H2O2 to H2O in an incubation mixture adjusted to pH 7.0 was
recorded at 254 nm. The reaction mixture contained 1.9 ml of 50 mM potassium
phosphate buffer pH 7.0 and 0.1 ml of supernatant and the reaction was started by the
addition of freshly prepared 0.1 ml of 30 mM H2O2. The rate of H2O2 decomposition
was measured spectrophotometrically at 240 nm [201]. One unit of catalase activity
was defined as the amount of enzymes causing the decomposition of µM H2O2/mg
protein/min at pH, 7.0 at 25ºC using a molar extinction coefficient of 43.6 M-1
cm-1
.
4.16.3.4. Estimation of glutathione peroxidase
The reduction of GSSG is coupled to the oxidation of NADPH through
glutathione reductase. The reaction mixture contained 100 µl tissue homogenate
solution and 800 µl 100 mM/l potassium phosphate buffer (pH 7.4), containing 1
mM/l EDTA, 1 mM/l sodium azide, 0.2 mM/l NADPH, 1 U/ml glutathione reductase
and 1 mM/l GSH. After 5 minute the reaction was started by the addition of 100 µl
2.6 mM H2O2 and the absorbance change at 340 nm in 3 min was recorded at 37ºC
[202]. Activity of GPx was determined using the molar extinction coefficient of
NADPH 6220 M−1
cm−1
and expressed as µM NADPH oxidized/min/mg protein at
37ºC.
126
4.16.3.5. Estimation of blood GSH
Blood glutathione was estimated by the method of Khynriam and Prasad
(2001). Briefly, fresh blood (0.1 ml) was mixed with 0.9 ml of water and 1.5 ml of
precipitating solution (1.67 g glacial metaphosphoric acid, 0.2 g Sodium EDTA, 30.0
g NaCl in 100 ml water) were added immediately, mixed well and incubated at room
temperature for 5 minute. After incubation, the mixture was incubated centrifuged at
3000 × g at 4°C for 15 minute. Clear supernatant (0.5 ml) was taken out and mixed
with 2.0 ml of 0.3 mol/l phosphate solution and 0.25 ml 0.2% DTNB in 1% sodium
citrate solution were added and mixed thoroughly. Blank solutions contain 1.0 ml 0.3
mol/l phosphate solution, 1.0 ml water, 0.5 ml precipitating solution and 0.25 ml
DTNB solution. Absorbance of both blank and sample reaction mixtures were taken
against water at 412 nm. The gluthathione concentration was calculated by following
formula taking extinction coefficient 13.6 and molecular weight of glutathione 307
[203].
Concentration of GSH (mg GSH/ 100 ml blood) = A x 2.75 x 2.75 x 307 x 100
13.6 x 0.1 x 0.5 x 1000
= A x 341.42
[Absorbance (A) = Absorbance of sample - Absorbance of blank]
4.16.3.6. Estimation of GSH in liver tissue
Briefly, 125 μl of 25% TCA was mixed with 0.5 ml of tissue homogenate in a
test tube to precipitate the protein. The test tubes were cooled in ice for 5 minute and
0.6 ml of 5% TCA again mixed with the previous mixture and centrifuged at 1500
rpm for 10 minute. After centrifugation 0.3 ml of supernatant was collected and
mixed with 0.7 ml of phosphate buffer (0.2 M, ph 8). Freshly prepared 2.0 ml DTNB
127
solution (0.6 mM in 0.2 M sodium phosphate buffer, pH 8) was mixed with above
mixture. The absorbance of mixture was measured after 10 minute 412 nm [204]. The
GSH content was calculated using a standard curve varying from 5-100 nm in 5%
TCA for assay, and results were expressed as nmol/mg protein.
4.17. NEPHROPROTECTIVE ACTIVITY
Nephroprotective effect of MEMS, MELA and fractions of MEMS (i.e.
MFMS, EFMS, PFMS), MELA (i.e. MFLA, EFLA, PFLA were evaluated against
cisplatin induced nephrotoxicity [126].
4.17.1. Experimental Design
Experiment was performed two stages. In first stage nephroprotective activity
of extract was performed and in second stage effect of fractions on cisplatin induced
nephrotoxicity was evaluated.
In first stage, animals were divided into seven groups each containing six,
Group I served as control, Group II served as negative control, Group III treated
with standard drug rutin, Group IV-V treated with MEMS (150 and 300 mg/kg,
b.w.), Group VI-VII treated with MELA (150 and 300 mg/kg, b.w.).
All the test drugs were administered for 6 days once daily. On fourth day
nephrotoxicity was induced in animals of group II-VII by a single i.p. injection of
cisplatin (20 mg/kg). Animals were monitored carefully and sacrificed at 72 h after
nephrotoxicity induced.
128
To determine nephroprotective effect of fractions animals were divided into 15
groups (n=6) in following manner,
Group I (Control) : Vehicle (Normal saline, 0.5 ml/animal/day)
Group II (Negative control) : Vehicle (Normal saline, 0.5 ml/animal/day)
Group III (standard) : Rutin (20 mg/kg)
Group IV-IX : MFMS, EFMS, PEMS at 75 and 150 mg/kg
Group X-XV : MFLA, EFLA, PELA at 75 and 150 mg/kg
All the test drugs, standard and vehicle were administered for six consecutive days
once daily by oral route. On fourth day nephrotoxicity was induced in animals of
group II-XVI by a single i.p. injection of cisplatin (20 mg/kg). Animals were
monitored carefully and sacrificed at 72 h after nephrotoxicity induced.
4.17.2. Sampling and Biochemical Analyses
The mice were sacrificed 72 h following cisplatin administration. Blood
samples were collected and centrifuged at 5000 rpm for 10 minute to obtain clear sera
which were used for the estimation of blood urea nitrogen (BUN), serum creatinine,
uric acid, total protein, and albumin.
All these estimation was carried out by using commercial biochemical kit
obtained from Agapee Diagnistic Ltd., Kerala. Kidney tissue was taken to determine
level of tissue MDA.
129
4.17.2.1. Estimation of blood urea nitrogen (BUN)
Quantitative determination of BUN in serum was determined by following
manufacturer procedure (Blood urea nitrogen Kit Batch no: 489, Manufacturing date:
1/12, Expiry date: 1/13). Standard sample was prepared by mixing of 1.0 ml of
working reagent with 0.01 ml of standard given with commercial kit. Sample solution
was prepared by mixing 1.0 ml of working reagent with 0.01 ml of serum sample. The
solutions were mixed well and optical density (T1) was determined after 30 sec at 340
nm. Second reading (T2) was taken exactly 60 sec after first reading. Distilled water
used as blank. The concentration of BUN was calculated using the following formula,
BUN concentration (mg/dl) =(T
1 - T
2) sample
(T1 - T
2) standard
X 23.4
4.17.2.2. Estimation of serum creatinine
Modified Jaffe’s method was used to determine the amount of creatinine in
serum as per procedure described by manufacturer (Serum creatinine Kit Batch no:
769, Manufacturing date: 1/12, Expiry date: 1/13). In this assay, creatinine reacts with
picric acid to form creatinine alkaline picrate, a coloured compound. The change in
absorbance is proportional to creatinine concentration.
Standard sample was prepared by mixing of 1.0 ml of working reagent with
0.1 ml of standard given with commercial kit. Sample solution was prepared by
mixing 1.0 ml of working reagent with 0.1 ml of serum sample. The solutions were
mixed well and optical density (T1) was determined after 60 sec. Second reading (T2)
was taken exactly 60 sec after first reading at 492 nm. Distilled water used as blank.
130
The concentration of creatinine was calculated using the following formula,
Creatinine concentration (mg/dl) =(T
1 - T
2) sample
(T1 - T
2) standard
X 2
4.17.2.3. Estimation of serum uric acid
Serum uric acid level was determined using the uricase methodology as
described by manufacturer (Uric acid Kit Batch no: 871, Manufacturing date: 6/11,
Expiry date: 6/13). The assay is based on the following reaction,
Uric acid + O2
+ 2H2O
UricaseAllantoine + CO
2 + H
2O
2H2
O2 + 4 -AAP + EHSPT
PeroxidaseRed quinone
EHSPT = N-ethyl N-(2-hydroxy-3-sulfopropyl) n-toluidine
4 -AAP = Amino-4-antipyrine
Standard sample was prepared by mixing of 1.0 ml of working reagent with
0.025 ml of standard given with commercial kit. Sample solution was prepared by
mixing 1.0 ml of working reagent with 0.025 ml of serum sample. The solutions are
mixed and incubated at 37°C for 5 minute. the absorption was taken at 546 nm.
Working reagent was used as blank. The concentration of uric acid was calculated
using the following formula,
Uric acid concentration (mg/dl) =Absorbtion of sample
Absorption of standardX 8
131
4.17.2.4. Estimation of serum total protein
Direct biuret method was employed to determine total protein concentration as
per manufacturer procedure (Total protein Kit Batch no: 298, Manufacturing date:
2/12, Expiry date: 2/12). In this colorimetric assay, protein in serum sample forms a
blue coloured complex when treated with cupric ions in alkaline solution (working
reagent). The intensity of the blue colour is proportional to the protein concentration.
Standard sample was prepared by mixing of 1.0 ml of working reagent with
0.02 ml of standard given with commercial kit. Sample solution was prepared by
mixing 1.0 ml of working reagent with 0.02 ml of serum sample. The solutions were
mixed and incubated at 37°C for 10 min. The absorption was measured at 546 nm
against reagent blank. The concentration of uric acid was calculated using the
following formula,
Total protein concentration (mg/dl) =Absorbtion of sample
Absorption of standardX 6
4.17.2.5. Estimation of serum albumin
Bromocresol green method was employed to determine albumin concentration
as per manufacturer procedure (Albumin Kit Batch no: 888, Manufacturing date:
10/11, Expiry date: 10/13). In this colorimetric assay, albumin from serum sample
reacts with a dye bromocresol-green (working reagent) to produce a colour change
that is proportional to the protein concentration.
Standard sample was prepared by mixing of 1.0 ml of working reagent with
0.01 ml of standard given with commercial kit. Sample solution was prepared by
132
mixing 1.0 ml of working reagent with 0.01 ml of serum sample. The solutions were
mixed and incubated for 1 minute. The absorption was measured at 630 nm against
reagent blank. The concentration of uric acid was calculated using the following
formula,
Albumin concentration (mg/dl) =Absorbtion of sample
Absorption of standardX 3
4.17.2.6. Estimation of MDA
Lipid peroxidation results oxidative degradation of polyunsaturated fatty acids
to MDA, which is usually determined by the chromogenic thiobarbituric acid reaction
and expressed as total TBARS. Animals were sacrificed by cervical dislocation; the
kidneys were isolated quickly and washed with saline, kidney was dried using filter
paper and weighed. A 10% (w/v) kidney tissue homogenate was prepared in
phosphate buffer (pH 7.4) using a homogenizer. Unbroken cells, cell debris and nuclei
were sedimented by centrifugation at 2000 × g force for 10 minute, and the
supernatant was pipetted into plastic tubes. This kidney homogenate thus obtained
was used for evaluate lipid peroxides (MDA production) in the kidney.
Briefly, 2.5 ml of 10% (w/v) TCA solution was mixed with 0.5 ml tissue
homogenate, and placed on a boiling water bath for 15 minute and then cooled in tap
water. The mixture was centrifuged at 1000 × g force for 10 minute. After
centrifugation 2.0 ml of the supernatant was added to 1.0 ml of 0.67% (w/v) TBA
solution in a test tube and the tube was placed again in a boiling water bath for 15
minute. The solution was then cooled in tap water and its absorbance of solution was
taken at 532 nm. The concentration of MDA was calculated by using absorbance
coefficient = 1.56×105 cm
−1M
−1) and the results were expressed as nM/min/mg tissue
protein [133].
133
4.18. ANTI-DIABETIC ACTIVITY
The effect of fractions of methanol extract of Meyna spinosa leaves was
investigated. MEMS showed better antioxidant activity therefore, MEMS and its three
fractions of methanol extracts viz, PFMS, EFMS and MFMS were selected for
antidiabetic activity.
4.18.1. Induction of Type 2 Diabetes
Type 2 diabetes was induced experimentally induced by intraperitoneal
injection of alloxan after administration of high fat diet for 10 days as described by
Shu et al. (2009) [140]. Healthy rats were treated orally with high fat diet (fat 20%,
cholesterol 5%, glucose 5%, fructose 5%, glutamine 5% and methylthiouracil 1%)
continuously for 10 days. On 11th
day alloxan was prepared in a concentration of 100
mg/ml in physiological saline solution and injected into the caudal vein of overnight
fasted rats, at a dose of 120 mg/kg body weight. Fifteen minute later 0.4 U insulin
was injected intraperitoneally; 2.5 and 5.0 hour later, 25% glucose (10 ml/kg)
administered orally to decrease animal mortality rate resulted from hypoglycemia and
hyperglycemia in rats with injection of alloxan. Twenty four hour after first alloxan
injection, freshly prepared alloxan solution (100 mg/kg body weight) was injected
again into the caudal vein of overnight fasted rats. Insulin (0.4 U) was injected (i.p.)
15 minute later to rats, then 25% glucose (10 ml/kg) administered orally 2.5 and 5.0
hour later respectively. The blood glucose was tested and animals with fasting blood
glucose more than 200 mg/dl were used for the experiments.
134
4.18.2. Antidiabetic Activity
The experiment was carried out in two stages, at first stage antidiabetic effect
of extracts were evaluated. Animals were divided into 6 groups (n=6).
Group I (normal control) : received vehicle (0.5% sodium CMC solution)
Group II (diabetic control) : received vehicle (0.5% sodium CMC solution)
Group III (standard drug) : diabetic rat treated with glibenclamide (5 mg/kg)
Group IV-VI : treated with MEMS at 100, 200, 300 mg/kg b.w. respectively.
Treatment was continued for 21 days body weight and serum glucose level
were estimated at 0, 7, 14, and 21 of treatment.
In second stage antidiabetic effect of fractions were evaluated. Healthy and
diabetic animals were grouped into nine groups of six rats each in following manner,
Groups I: Normal control received vehicle (0.5% sodium CMC solution)
Group II: Diabetic control received vehicle (0.5% sodium CMC solution)
Group III: Diabetic rat treated with 5 mg/kg/day glibenclamide
Group IV & V: Diabetic rat treated with the MFMS (75 and 150 mg/kg/day)
Group VI & VII: Diabetic rat treated with the EFMS (75 and 150 mg/kg/day)
Group VIII & IX: Diabetic rat treated with the PFMS (75 and 150 mg/kg/day)
135
The volume of administration was 5 ml/kg, and the treatment was lasted for 21
days. The animals were carefully monitored every day and weighed and serum
glucose level was estimated every week. Initial and final serum lipid profile, α–
amylase level were also evaluated in this stage.
4.18.3. Estimation of Biochemical Parameters
The blood samples were withdrawn through the retro orbital plexus from
overnight fasted rats under light ether anaesthesia using a glass capillary. Blood
samples were allowed to clot and serum separated by centrifugation at 3500 rpm for
10 minute, which was used to estimate different biochemical parameters.
Serum glucose level was determined in weekly basis. Initial (0 day) and final
(after 21 day treatment) serum lipid profile like total cholesterol (TC), triglyceride
(TG), high density lipids (HDL), low density lipids (LDL), very low density lipids
(VLDL), and α–amylase level in serum were estimated using a commercially
available standard kit in a semi-autoanalyzer. All diagnostic kits were stored at cool
place (2-8°C) till the time of use.
4.18.3.1. Serum glucose estimation
Serum glucose level was estimated using the manufacturer procedure using
GOP-PAP methodology (Glucose Kit Batch no: 9741, Manufacturing date: 3/12,
Expiry date: 3/13). The principle of this enzymatic colorimetric determination of
glucose was based on the following reaction,
136
Glucose + O2 + H2OGlucose oxidase
Gluconic acid + H2O2
2H2O2 + Phenol + 4-Aminoantipyrine Quinonimine + 4 H 2OPeroxidase
(Red Colour)
Procedure: To the sample tube, 0.01 ml of serum and 1.0 ml of working reagent were
added. In another tube, 0.01 ml of standard and 1.0 ml of working reagent were
mixed. The working reagent alone served as the blank. All solutions were mixed
properly and incubated for 10 minute at 37°C. The absorbance of the sample and
standard against the blank was measured at 505 nm. The concentration of glucose was
determined by the following formula,
Glucose concentration (mg/dl) = Absorbance of sample
Absorbance of standardX 100
4.18.3.2. Estimation of total cholesterol and triglycerides
Serum level of TC and TG in normal, diabetic, standard and fractions treated
animals were determined as per the commercial assay kit procedure, which is
described in ‘hepatoprotective activity’ section (section, 4.14.2.5 and 4.14.2.6).
4.18.3.3. Estimation of high density lipids (HDL)
Determination of HDL cholesterol was performed by manufacturer procedure
(HDL Kit Batch no: 822, Manufacturing date: 9/11, Expiry date: 11/12). In this assay
anti human β-lipoprotein antibody present in reagent 1 bind to lipoproteins like LDL,
VLDL, and chylomicrons. The antigen-antibody complexes formed block enzyme
reaction after addition of reagent 2. Cholesterol esterase and cholesterol oxidase of
regent 2 react only with HDL-C. Enzyme reaction with HDL-C yields a blue color
137
complex upon oxidative condensation with F-DAOS [N-ethyl-N-(2-hydroxy-3-
sulfopropyl)-3,5-dimethoxy-4-fluroaniline, sodium salt] and 4-aminoantipyrine in
presence of peroxidase. HDL-C concentration can be calculated when compared with
absorbance of the HDL-C calibrator. Blank, sample and HDL calibrator were
prepared by following way,
Reagents/Samples Blank Calibrator Sample
Reagent 1 0.45 ml 0.45 ml 0.45 ml
HDL-C direct Calibrator - 0.005 ml -
Sample - - 0.005 ml
Mix the samples and incubate for 5 minute at 37°C
Reagent 2 0.15 ml 0.15 ml 0.15 ml
After addition of R2 all samples were mixed well and incubated for 5 min at
37°C. The absorbance of calibrator and sample was measured against reagent blank.
And quantity of HDL-C was determined by following formula,
HDL-C concentration (mg/dl) =Absorbance of sample
Absorbance of standardx Calibrator concentration
4.18.3.4. Estimation of low density lipids (LDL)
Determination of LDL cholesterol was performed by manufacturer
procedure (LDL Kit Batch no: 866, Manufacturing date: 5/11, Expiry date: 5/13). In
this assay serum was mixed with reagent 1 (R1). An amphoteric surfactant present in
138
R1 protects LDL from enzyme reaction. Cholesterol esterase and cholesterol oxidase
react with non-LDL cholesterol and decomposed it to water by catalase. Reagent 2
(R2) enables conversion of LDL cholesterol to H2O2, which upon oxidative
condensation with HDAOS and 4-aminoantipyrine produces a blue colour complex,
which can be calculated when compared with the absorbance of LDL cholesterol
calibrator. Blank, sample and LDL cholesterol calibrator were prepared by following
way,
Reagents/Samples Blank Calibrator Sample
Reagent 1 0.45 ml 0.45 ml 0.45 ml
LDL-C direct Calibrator - 0.005 ml -
Sample - - 0.005 ml
Mix the samples and incubate for 5 minute at 37°C
Reagent 2 0.15 ml 0.15 ml 0.15 ml
After addition of R2 all samples were mixed well and incubated for 5 minute
at 37°C. The absorbance of calibrator and sample was measured against reagent
blank. And quantity of LDL-C was determined by following formula,
LDL-C concentration (mg/dl) =Absorbance of sample
Absorbance of standardx Calibrator concentration
4.18.3.5. Estimation of very low density lipids (VLDL)
VLDL level was calculated by the following formula [205]
VLDL (mg/dl) =Triglycerides
5
139
4.18.3.6. Estimation of α-amylase
Quantitative estimation of α-amylase level in serum was carried by using
CNPG3 methodology using as described by manufacturer.
The principal of α-amylase estimation was based on following formula,
5 CNPG3
Amylase3 CNP + 2 CNPG2 + 3 Maltotriose + 2 Glucose
CNPG3 = 2-Chloro-4-Nitrophenyl-alpha-Maltotrioside
CNP = 2-Choloro-4nitrophenol
CNP-G2 = 2-Chloro-4-nitrophenyl-a-maltoside
Procedure: Briefly, 0.025 ml of serum was mixed with 1.0 ml of working reagent.
The reaction mixture was incubated at 37°C for 1 minute. Change in the absorbance
per minute (▲ OD/min) was measured during 3 minute using semi Autoanalyzer.
Following parameter were feed in auto analyser before the determination of
absorbance, mode of reaction – kinetic, slope of reaction – increasing, wavelength –
405 nm, linearity – 2000 U/l, factor – 3178, temperature – 37°C, blank – distilled
water, no. of readings – 3, interval – 60 sec. The α-amylase activity was expressed in
U/l.
4.18.4. Normoglycaemic Plus Glucose-Hyperglycaemic Model (NG-OGTT)
To find the effect of fractions on normal as well as glucose loaded animal a
combined normoglycaemic plus glucose-hyperglycaemic model (NG-OGTT) was
used in this present study.
140
NG-OGTT in rats was performed according to the method used by Orhan et al.
(2005). This combined method was used in order to avoid use of excess animals.
Healthy rats are divided into eight groups each containing 6 animals.
After overnight fasting blood samples were collected from all the animals to
determine glucose level, and drugs were given immediately according to following
manner,
Groups I : received vehicle and served as control
Group II : served as standard and received glibenclimide (5 mg/kg)
Group III : treated with MFMS at a dose of 75 mg/kg, p.o.
Group IV: treated with MFMS at a dose of 150 mg/kg, p.o.
Group V: treated with EFMS at a dose of 75 mg/kg, p.o.
Group VI: treated with EFMS at a dose of 150 mg/kg, p.o.
Group VII: treated with PFMS at a dose of 75 mg/kg, p.o.
Group VIII: treated with PFMS at a dose of 150 mg/kg, p.o.
Blood glucose level was determined after 30 minute, 1 h, 2 h after drug
administration to assess the effect of the fraction on normoglycaemic animals.
After last measurement (at 2 h), 2 g/kg glucose was orally administered to each
animal and the blood was withdrawn in the following time pattern: 2.5, 3, 4 and 6 h to
access the effects of fractions on glucose-hyperglycaemic rats.
141
4.19. ANTHELMINTIC ACTIVITY
Anatomical and physiological characteristic of Indian earth worm resemblance
with the intestinal round worm parasite of human being, therefore Pheretima
posthuma have taken in this study to assess anthelmintic activity of fractions of
MELA.
Indian earth worms are divided into twelve groups each containing six
earthworms approximately of equal size in following manner,
Group I : Control (3% Tween 80 in normal saline)
Group II - IV : MFLA (10, 20 and 50 mg/ml)
Group V - VII : EFLA (10, 20 and 50 mg/ml)
Group VIII - X : PFLA (10, 20 and 50 mg/ml)
Group XI-XII : Reference standard piperazine citrate (10 and 20 mg/ml)
Fifty millilitres of respective drug solutions were taken in a petri dishes and
the earthworms were released in to the solution. Earth worms were monitored
carefully and observations were made for the time taken to paralyze and death of
individual worms. Time taken to till paralysis was recorded when no movement could
be observed except when the worms were shaken vigorously. Times taken for death of
worms were noted after ascertaining that the worms lost their motility completely
with fading of their body colour. To confirm, the death worms were shaken
vigorously or dipped in warm water at 50°C but no movement was observed [151].
142
Figure 4.3: Study of anthelmintic
activity
A: Control
B: Standard
C: MFLA (50 mg/ml)
D: EFLA (50 mg/ml)
E: FFLA (50 mg/ml)
143
4.20. ISOLATION OF CHEMICAL CONSTITUENTS
Present pharmacological investigation showed that methanol fraction of
MEMS and ethyl acetate fraction of MELA showed superior activity then other
fractions (detail observation given in result section). Therefore most effective
fractions viz. MFMS, EFLA were further fractionated to isolate chemical constituents.
MFMS and EFLA was fractionated by using column chromatography by
taking borosilicate column with a dimension of 40 mm × 600 mm. Silica gel (60-120
sieve) was used as stationary phase.
To fractionate MFMS chloroform was used as solvent, while benzene was
used to fractionate EFLA. Silica gel (400 g) was stirred with 500 ml of respective
solvent to make slurry and the column was packed at height of 30 cm and a diameter
of 4 cm carefully to avoid the deposition of air bubbles in between compact filling.
Solvent level was kept few centimetres above of the silica gel layer. About 20 g of
MFMS and EFLA was dissolved in minimum quantity of methanol and ethyl acetate
respectively, and then mixed with 30 g of silica gel. The mixture was air dried and
spreaded slowly on the top of the column, which was covered with silica gel slurry (in
respective solvent) followed by with a piece of cotton wool. Chloroform and benzene
were used as solvent system to elude the components from MFMS and EFLA
respectively.
A total of 11 fractions were eluted form MFMS (named as MM-1 to MM-11)
and 7 fractions (named as LA-1 to LA-7) eluted form EFLA. All the eluants were
dried under reduced pressure.
144
4.21. ANTIOXIDANT ACTIVITY AND TLC OF SELECTED
SUBFRACTIONS
Antioxidant activities of all sub-fractions obtained from MFMS and EFLA (MS-1
to MS-11 & LA-1 to LA-7) were screened by taking 2 fixed concentrations of
fractions (20 and 40 μg/ml) using DPPH● and NO
● scavenging activity method.
Procedures to perform the same were discussed in section 4.11. Based on the
antioxidant study 3 ‘more potent’ sub-fractions from L. asiatica and 4 ‘more potent’
sub-fractions from M. spinosa were selected for further analysis.
Selected sub-fractions were subjected for TLC using the same procedure as
discussed earlier, benzene was used as solvent (mobile phase). TLC was performed to
check purity of compound. One fraction of L. asiatica and 3 fractions of M. spinosa
were selected for spectral analysis as these compounds showed single spot in TLC.
4.22. SPECTRAL ANALYSIS
The compounds (one fraction form L. asiatica and one fractions of M. spinosa)
were subjected to IR spectroscopy in KBr (PwrkinElmer Spectrum Version 10.03.06),
1H and C13 NMR (Bruker DRX-300) and mass spectroscopy (ESI-HRMS, Agilent
6520 Q-Tof).
4.23. STATISTICAL ANALYSIS
The data were subjected to analysis of variance (ANOVA) and expressed as
mean ± SEM (n=3 for in vitro and ex vivo model; n = 6 for in vivo tests). Statistical
analysis was carried out by analysis of variance followed by Tukey tests. A level of p
< 0.05 was used as the criterion for statistical significance. For in vitro and ex vivo
antioxidant activity IC50 value was the concentration which cause 50% scavenging
effect and determined graphically.
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