Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
111
4. EXPERIMENTAL INVESTIGATIONS
4.1. SELECTION OF PLANTS
A number of ethnobotanical survey and books describing the ethno-medicinal
plants of North East India were reviewed systemically to gather the information about
the folk medicinal knowledge of tribes of the region and to find potential medicinal
plants used by them. It was observed that more than thousands of plants are used by
more than 450 ethnic groups live in this region. The ethnomedicinal information
obtained from local people was the primary basis for the selection of medicinal plants.
In first phase 15 plants were selected based on the folk medicinal information, which
were distributed throughout the entire region commonly used by ethnic peoples. List
of those plants with their traditional and reported pharmacological effect given in
Table 4.1.
Table 4.1: List of preliminary selected plants
Scientific
name
Family Traditional uses Investigated
pharmacological effect
Dillenia
indica
Dilleniaceae Gastrointestinal pain,
diarrhoea, respiratory
diseases, cough, fever,
dysmenorrhoea,
infection
Antioxidant effect,
maltase inhibitory
effect, anti-amylase,
anti-glucosidase,
antihistamine release,
anti-leukemic activity
Litsea
glutinosa
Lauraceae Treat muscular, non-
bleeding injuries,
gonorrhea,
constipation,
diarrhoea,
rheumatism, wound,
antispasmodic
Antibacterial,
antioxidant, anti-
inflammatory, wound
healing, anthelmintic,
free radical scavenging,
aphrodisiac activity
112
Oxalis
corniculata
Oxalidaceae Treat dysentery,
stomach disorders,
rheumatism,
toothache, cold, fever,
skin diseases,
diarrhoea,
dysmenorrhoea
Anti-inflammatory,
anxiolytic,
anticonvulsant,
antifungal, antiulcer,
antinociceptive,
anticancer, antidiabetic,
hepatoprotective,
hypolipedemic,
abortificient,
antimicrobial and
wound healing
properties
Phyllanthus
acidus
Phyllanthaceae Astringent, cathartic,
antihypertensive,
emetic, purgative,
laxative; to treat
respiratory disorders,
cough, bronchitis,
liver disorders,
psoriasis, diabetes,
pain, fever
Antidiabetic,
antimicrobial, cytotoxic
and selective
antioxidant, treatment of
cystic fibrosis,
hepatoprotective against
CCl4 induced
hepatotoxicity
Smilax
zeylanica
Smilacaceae Treat hepatitis, urinary
disease, skin disease,
veneral diseases, skin
disorders, sores,
swellings, rheumatism
and pain
Antidiabetic, pesticidal,
anthelmintic, analgesic,
antibacterial,
antipyretic,
anticonvulsant,
antiepileptic,
hepatoprotective,
antioxidant effect
Marsilea
minuta
Marsileaceae Treatment of cough,
bronchitis, insomnia,
headache, mental
problems,
hypertension,
gastrointestinal
disorders, renal
diseases
Hypocholesterolemic,
anxiolytic, antifertility,
anti stress,
antidepressant,
hepatoprotective against
CCl4 induced
hepatotoxicity, few in
vitro antioxidant study
113
Aphanamixis
polystachya
Meliaceae Treat stomach pain,
skin disease, tumours,
cancer, spleen
diseases, rheumatism,
liver disease
Hepatoprotective,
antimicrobial, antiviral
and
antibacterial activity,
radioprotective,
antioxidant, antipyretic,
antiulcer, hematinic,
diuretic
Asparagus
racemosus
Liliaceae Treatment of
diarrheoa, fever,
measles, stomach pain,
ulcerative disorders,
menstruation problem
and to increase milk
secretion during
lactation
Antiulcer, galactogogue,
immunomodulating,
antineoplastic,
hepatoprotective,
antioxidant, cardio
protection
Coccinia
grandis
Cucurbitaceae Treatment of diabetes,
scabies, skin infection,
jaundice, bronchitis,
skin eruptions, burns,
insect bites, fever,
indigestion, nausea,
eye infections, allergy,
syphilis, gonorrhoea
Antidiabetic,
antioxidant,
antinociceptive and
anti-inflammatory
activity, antipyretic,
hypolipedemic, anti-TB,
anthelmintic, antitussive
effect
Ipomea
aquatic
Convolvulaceae Treatment of nervous
and general debility,
piles, worm infections,
leucoderma, leprosy,
jaundice, liver
disorders, high blood
pressure, skin disease,
dysentery; used as
purgatives, diuretics
Nootropic,
antihyperglycemic,
antiulcer antimicrobial,
cytotoxic, diuretic,
antioxidant,
antiepileptic effect
114
Mimosa
pudica
Mimosaceae Treatment of leprosy,
burning sensation,
fever, alopecia, tumor,
diarrhoea, dysentery,
insomnia, and various
urogenital infections
Wound healing activity,
regeneration of sciatic
nerve, antidepressant,
anticonvulsant,
hyperglycemic, diuretic,
antifertility,
hepatoprotective,
antioxidant,
antimicrobial effect,
aphrodisiac property,
Paederia
foetida
Rubiaceae Treatment of
amoebiasis, tooth
ache, stomach pain,
asthma, bowel
complaints, diarrhea,
diabetes, seminal
weakness, and as
antiarthritic,
diaphoretic,
antispasmodic,
expectorant
Anti-diarrhoeal,
antioxidant,
hepatoprotective,
antitussive,
anthelmintic,
antispasmodic
Typhonium
trilobatum
Araceae Treatment of bowl
disease, constipation,
body pain,
rheumatism, traumatic
injury, tuberculosis,
bronchitis, vomiting,
cough, asthma,
headache, ulcer
Antimicrobial,
nematocidal, larvicidal
activity, analgesic, anti-
inflammatory and anti
diarrheal effect
Alternanther
a
philoxeroides
Amaranthaceae Treatment of stomach
pain, dysentery,
stomach disorders,
wound, ulcer
Analgesic,
antiinflammatory,
anthelmintic,
antioxidant, antipyretic,
antibacterial,
hepatoprotective,
antiulcer, diuretic effect
115
Centella
asiatica
Apiaceae Treatment of stomach
and liver disorder,
asthma, leprosy
Wound healing,
sedative and anxiolytic,
epatoprotective,
antioxidant,
antiepileptic, antiulcer,
antileprotic
cardioprotective,
antidepressant,
antinociceptive, anti-
inflammatory,
neuroprotective effect
Scientific literatures on these plants were extensively reviewed. Reported
activities were observed to be matched with traditional knowledge. In the view of
relation between oxidative stress and diseases, it was planned to screen antioxidant
potential of those plant first. It was observed that for some of the plants antioxidant,
hepatoprotective and other activities were already been reported. Taking this as a
consideration 4 plants viz. Marsilea minuta (whole plant), Oxalis corniculata (leaf),
Phyllanthus acidus (leaf), Dillenia indica (leaf) were selected for primary study.
In next stage a preliminary antioxidant study was conducted using DPPH and
nitric oxide radical scavenging assay method. This study was very initial study using
only two concentrations, to find more effective plant among these four. All methanol
extracts were taken to perform activity. It was observed that all the extract showed
antioxidant activity, but two plants viz, Marsilea minuta and Phyllanthus acidus
showed better effect as compared with other two. It was also observed that having a
lot of traditional medicinal importance of these plants was not scientifically
investigated despite of vast utility as folk medicine. Therefore I have concentrated on
these two plants (Marsilea minuta and Phyllanthus acidus) to focus their folk
medicinal importance by a scientific approach.
116
4.2. PLANT COLLECTION AND AUTHENTICATION
Whole plant of Marsilea minuta L. was collected during the month of
March/April 2011 and the leaves of Phyllanthus acidus were collected in February
2011 from Tripura, India. The time of collection purposefully taken based on the
suggestion of botanis/phytochemist. The plant was identified vernacular name, later
validated and authenticated by Dr. BK Datta, Department of Botany, Tripura
University, Tripura, India. Voucher specimens (TU/BOT/HEB/SS23072011b and
TU/BOT/HEB/RC25092011b) were deposited at the herbarium of Plant Taxonomy &
Biodiversity Laboratory, Tripura University.
4.3. DRUGS AND CHEMICALS
Chemicals, reagents, biochemical diagnostic kit, drugs were purchased from
reputed companies. List of the important chemicals and name of their manufacturing
agencies were tabulated in Table 4.2.
Table 4.2: List of chemicals and name their manufacturing agencies
Company Chemicals
Sigma Aldrich
(Bangalore,
India)
DPPH, Sodium nitroprusside (SNP), Phenazine methosulfate
(PMS), Nitro blue tetrazolium (NBT), Naphthyl ethylene
diamine dihydrochloride
SD Fine Ltd.
Mumbai
H2O2, 2-deoxy-ribose, Trichloro acetic acid (TCA),
Thiobarbituric acid (TBA), Methanol, Ethyl acetate, Petroleum
ether, Ethanol, Chloroform, Quercetin, Aluminium chloride
(AlCl3), Sodium nitrite (NaNO2), Sodium hydroxide (NaOH),
Folin-Ciocalteu reagent, Tris-HCl buffer, Phosphoric acid,
Carragennan, Formalin
117
Sisco Research
Laboratories
Pvt. Ltd.,
Mumbai
Linoleic acid, Ammonium thiocyanate, Ascorbic acid, α-
tocopherol, Folin–Ciocalteau reagent, BHA, Epinephrine,
Ascorbic acid, Gallic acid, Sulphanilamide, NADH
Loba Chemie
Pvt Ltd.,
Mumbai
Ferric chloride (FeCl3), Ferrous chloride (FeCl2), Zinc chloride,
Chlorosulfonic acid, Lead acetate, Phenazone, Ethelene diamine
tetra acetic 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, and Albumin.
All other chemicals used in the study were obtained commercially and were of
analytical grade.
4.4. INSTRUMENTS USED
Following major instruments were used for the study,
Double beam UV-Vis Spectrophotometer (Elico SL 164).
Plethysmometer (AARSON Scientific Works, Haryana)
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)
118
4.5. PREPARATION OF EXTRACTS
Collected whole plant/leaves were washed in running water to remove
unwanted materials, and dried under shade. The air dried materials were powdered
mechanically by using a grinder and sieved through sieve 22 mesh to obtain fine
powder and stored in air tight container. Dried powdered plant material was extracted
using Soxhlet apparatus with several solvents like methanol, ethyl acetate, petroleum
ether separately. About 2 kg of Marsilea minuta and 1kg of Phyllanthus acidus leaves
were extracted by using 10 L and 5 L of each solvent respectively. The extracts were
concentrated under reduced pressure by using rotary vaccume evaporator to obtain
methanol extracts of Marsilea minuta (MEM) and Phyllanthus acidus (MEP), ethyl
acetate extract of Marsilea minuta (EEM) and Phyllanthus acidus (EEP), petroleum
ether extract Marsilea minuta (PEM) and Phyllanthus acidus (PEP) which were used
for further study.
Figure 4.1: Extraction of plant materials
119
4.6. PHYSICOCHEMICAL PROPERTIES OF EXTRACT
Physiochemical properties of the extracts were evaluated. The yield, colour,
pH, density and specific gravity of crude extracts were determined by following
standard procedure.
4.6.1. Colour and Yield Determination
Colour of the extracts was observed in bare eye. The physical properties i.e.
colour, percentage yield of the extracts were also observed and reported.
4.6.2. Determination of pH
The individual extract solution was filtered by using Whatman filter paper and
the pH was recorded by using Elico digital pH meter [199].
4.6.3. Determination of Specific Gravity
The weight of an empty pycnometer (Wp) was recorded. About 10 g of sample
was placed in the pycnometer and weight (Wps) was taken. Half or ¾ of pycnometer
was filled with distilled water and soaked for10 min. Partial vacuum was applied to
remove entrapped air. And then pycnometer was filled with distilled water up to the
mark. The exterior surface of the pycnometer was cleaned properly and the weight
(Wb) was determined. After removing the content, the pycnometer was cleaned, filled
with distilled water, and the weight (Wa) was measured again. Specific gravity was
determined using the following formula [200],
120
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
4.7. PRELIMINARY PHYTOCHEMICAL INVESTIGATION
The presence of Alkaloids, carbohydrates, glycosides, protein, amino acids,
tannins, flavonoids, triterpenoids and steroids were carried out by quantitative
phytochemical investigation using the standard chemical methods [201, 202].
4.7.1. Detection of Alkaloids
Small quantity of dried extract/fractions was stirred with dilute hydrochoric
acid and filtered. Filtrate was tested for alkaloids with various reagents.
Mayer’s test: Reddish brown or orange precipitate obtains when few millilitres of
filtrate were mixed with 2 drops of Mayers reagent (potassium mercuric solution)
along with the side of the test tube.
Wagner’s test: Few drops of Wagner’s reagent (iodine-potassium iodide solution)
were mixed with few millilitres of filtrate along sides of the tube to obtain precipitate.
Hager’s test: Prominant yellow or crystalline yellow precipitate was the positive test
when few millilitre of filtrate mixed with 2.0 mL of Hager’s reagent (saturated
solution of picric acid).
121
Dragendroff’s test: Prominent reddish brown or orange red precipitate can be
observed, when few millilitres of filtrate were mixed with 2.0 mL of Dragendroff’s
reagent (potassium bismuth iodide solution).
4.7.2. Detection of Carbohydrates
A solution was prepared by taking a small quantity of the extract/fraction in
few mL of distilled water, filtered and was subjected for the following tests.
Molish’s test: Around 2 mL filtrate was taken in a test tube, two drops of Molish
reagent (alcoholic solution of α-naphthol) was added. Solution was mixed well and
1.0 mL concentrated sulphuric acid added slowly along the sides of the tube. Cooled
the tube in ice water and allowed to stand. Appearance of violet colour ring at the
junction of layers was the conformation for carbohydrate.
Fehling’s test: The test used to identify reducing sugar, and was performed by taking
few millilitre of filtrate mixing with 1.0 mL each of Fehling’s solutions A and B. The
solution was heated on water bath. Formation of brick red precipitate was the positive
test.
Benedict’s test: Few mL of filtrate and few mL of Benedict reagent was taken in a
test tube and heated on water bath for 2 min. reducing sugar confirmed by formation
of colored precipitate.
Barfoed’s test: One mL Barfoed’s reagent with few milliliter of filtrate was added in
a test tube and heated on water bath for 2 min. Monosaccharides confirmed when red
precipitate forms.
122
4.7.3. Detection of Protein and Amino Acid
A solution was prepared by taking small quantity of sample in few mL of
distilled water and filtered through Whatman filter paper no 1. The filtrate was
subjected for the following tests.
Millon’s test: Formation of white precipitate, when the filtrate was mixed with few
drops of Millon’s reagent.
Ninhydrin test: Two drops of ninhydrin solution was added to 2.0 mL of filtrate and
boiled in a water bath. Purple/blue/violet colour could be observed for positive test.
Biuret test: Formation of violet colour was the conformation, when equal volume of
filtrate and Biuret reagent mixed together.
Xanthoprotein test: A solution was prepared by taking 5 mL of filtrate with 1mL
concentrated HNO3 and boiled in water bath, 40% NaOH was added after cooling.
Orange colour indicates the presence of protein.
4.7.4. Detection of Glycosides
4.7.4.1. General test for glycosides
Test A: About 200 mg of drug and 5.0 mL of dilute sulphuric acid warmed on a water
bath. The filtrate was neutralised with 5% NaOH solution and mixed with 0.1 mL of
Fehling's solution A and B until it becomes alkaline (tested with pH paper) and heated
on a water bath for 2 min. The quantity of red precipitate formed during the test was
measured.
Test B: Warmed about 200 mg of drug extract with 5.0 mL of water on a water bath.
Equal amount of water as used for sodium hydroxide in the Test A was added.
Fehling's solution A and B was added untill the solution became alkaline (tested with
123
pH paper) and the mixture heated on water bath for 2 minutes. Quantity of red
precipitate formed was noted and compared with Test A.
Presence of glycoside is confirm if the precipitate formed in Test A is more
than in Test B (Test A correspond to free reducing sugar plus those associated on acid
hydrolysis of any glycoside in the crude drug, whereas Test B stands for the amount
of free reducing sugar present in the crude drug).
4.7.4.2. Tests for specific glycosides
Tests for specific glycosides also performed using the following methods,
Borntrager's test: Crude extract was boiled with 1.0 mL of sulphuric acid in a test
tube for 5 min and filtered while hot. Cooled the filtrate and shaked with equal
volume of chloroform. Lower layer of chloroform was separated and mixed with half
of its volume of dilute ammonia and shaked well. Anthraquinone glycoside confirms
if a rose pink to red colour produces in the ammoniacal layer.
Keller-killiani test: Extracted the crude extract with chloroform, and evaporated to
dryness. Add 0.4 mL glacial acetic acid containing trace amount of ferric chloride. A
volume of 0.5 mL concentrated H2SO4 was added carefully by the side of the test
tube. Blue colour in acetic acid layer indicates the presence of cardiac glycosides.
Raymond’s test: If a violet colour develops when the test solution treated with hot
methanolic alkali, confirms cardiac glycosides.
A small amount of sample in test tube was taken and heated for several minutes on a
water bath by covering the test tube with a filter paper moistened, with dilute sodium
hydroxide solution. On exposure to UV light the paper shows green fluorescence
indicates presence of coumarin glycosides.
124
Froth formation test: Small amount of extract/fraction was shaken in a test tube
with a little quantity of water. If it results in formation of foam persists for 10 min
indicates presence of saponin glycosides.
Haemolysis test: A volume of 2 mL 1.8% NaCl solution was taken to the two test
tubes, made the solution isotonic to blood after adding one of these test tube 2.0 mL
of distilled water and to other 2.0 mL of sample solution. Each of these test tubes was
gently mixed with 5 drops of blood which was obtained by pricking the thumb at the
base of the nail. Haemolysis observed under the microscope in the tube containing the
sample, but no haemolysis in control indicates presence of saponin glycosides.
4.7.5. Detection of Tannins
Tannin in crude extract or fraction was determined by the following methods,
Ferric chloride test: A small quantity extract was boiled in few mL of distilled water
in a test tube and then filtered. Few drops of 0.1% ferric chloride was added to the
filtrate. A brownish green or a blue-black colour develops after represents the
presence of tannin.
Phenazone test: In a test tube 5 mL of aqueous extract was mixed with 0.5 g of
sodium acid phosphate, warmed the mixture and filtered. A solution of 2% phenazone
was added to the filtrate. Formation of bulky precipitate (which often coloured)
represents the positive test for tannin.
Gelatin test: Small quantity of sample dissolved in distilled water and mixed with a
solution of 1% gelatin (2.0 mL) containing 10% NaCl. Formation white precipitate
indicates the presence of tannin.
125
4.7.6. Detection of Flavonoids
Flavonoids in crude extract or fraction was determined by the following
methods,
Lead acetate test: Alcoholic solution of the extract/fraction mixed with few drops of
10 % lead acetate solution. Yellow precipitate indicates the presence of flavonoid.
Alkaline reagent test: Few mL of test solution, was mixed with few drops of 10%
NH4OH. Intense yellow colour develops and turns to colourless on addition of few
drops of dilute acid confirms the presence of flavonoid.
Zinc Hydrochloride test: Red colour/Megneta colour cis conformation when
alcoholic test solution of the extract/fraction mixed with a pinch of zinc dust and few
drops of concentrated HCl.
Ferric chloride test: To the alcoholic solution of extract, few drops of ferric chloride
were added. If it gives green colour, flavonoid is confirmed.
4.7.7. Detection of Steroids and Triterpenoids
Flavonoids in crude extract or fraction was determined by the following
methods,
Libermann-Burchard’s test: The sample and few drops of acetic anhydride, heated
to boil, and cooled. Along with the side of the test tube, 1 mL of concentrated H2SO4
was added. If it results in formations of brown ring at the junction and upper layer
turns to green indicates presence of steroids and brown ring at the junction and upper
layer turns to deep red indicates presence of triterpenoids.
126
Salkowski Test: The sample was mixed with few drops of concentrated sulphuric
acid. Red colour at lower layer indicates presence of steroids where as yellow colour
at lower layer indicates the presence of triterpenoids.
Tschugajeu test: Chloroform solution of sample mixed with excess of acetyl chloride
and a pinch of zinc chloride in a test tube and was kept aside for few minute. It was
warmed on a water bath. Formation of erosin red colour indicates the presence of
triterpenoids.
Brieskorn and Brinar test: Chloroform solution of test sample was added to a
mixture of chrolosulfonic acid in glacial acetic acid (7:3). Development of red colour
in few minutes indicates presence of triterpenoids.
4.8. EXPERIMENTAL ANIMALS
Healthy Wistar rats (150–200 g) and albino mice (20–30 g) between 2 and 3
months of age were used. Animals were housed under standard environmental
conditions (24±1°C) with 12 h light – 12 h dark cycles. The animals had free access to
water and food. The animal studies were carried out in CES College of pharmacy,
Kurnool, Andhra Pradesh. All animal procedure have been approved by institutional
animal ethical committee of CES college of Pharmacy (Reg. no:
1305/ac/09/CPCSEA) in accordance with animal experimentation and care.
4.9. DETERMINATION OF ANTIOXIDANT COMPOUNDS
4.9.1. Total Phenolic Content Determination
The total phenolic content in the crude extracts was determined by using
Folin-Ciocalteu reagent by colorimetric method [76]. A solution of 0.5 mL ethanol
solution of extract/fraction (l.0 mg/mL) was mixed in a test tube with 2.5 mL of 10%
127
v/v Folin-Ciocalteu reagent and 2.0 mL of 2% w/v sodium carbonate. The tubes were
shaken thoroughly and incubated for 15 min at 45°C with intermittent shaking.
Absorbance was taken at 765 nm using ELICO SL 164 UV–Vis spectrophotometer. A
calibration curve was plotted by taking gallic acid as standard (0 to 800 µg/mL), 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.
4.9.2. Total Flavonoid Content Determination
Aluminium nitrate-potassium acetate reagent method was used to determine
total flavonoid content of extracts. A volume of 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 was mixed thoroughly and allowed to stand for 40
min at room temperature. Absorbance of the supernatant solution was measured at
415 nm [86]. A calibration curve was plotted by taking quercetin (0 to 16 µg/mL) as
standard. The estimation was carried out in triplicate. The results were expressed as
quercetin equivalents in milligram per gram (mg QE/g) of dried extract.
4.10. IN VITRO ANTIOXIDANT ACTIVITY OF EXTRACTS
4.10.1. DPPH Radical Scavenging Assay
Ability of the sample to donate hydrogen atom or electron was measured from
the bleaching of a purple-coloured methanol solution of DPPH. Antioxidants can
readily reduce DPPH a stable radical with an absorption maximum at 517 mm. Free
radical scavenging capacity of extracts/fractions was measured using the stable DPPH
radical [87].
128
Methanolic solution of DPPH (1.0 mL, 0.1 mM) was mixed with 3.0 mL of
sample solution of different concentrations. The reaction mixture was incubated in
dark at room temperature for 30 min and the absorbance was recorded at 517 nm [87].
The assay was carried out in triplicate for each sample. Methanol (1 mL) with 3.0 mL
extracts/fraction solution was used as a blank and DPPH solution (1.0 mL, 0.1 mM)
with methanol (2.5 mL) served as negative control. The radical scavenging activity of
ascorbic acid was also determined as positive control. The decrease in colour intensity
on addition of test samples was used to calculate the antiradical activity, measured by
taking absorbance and compared with negative control. The activity was 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.
4.10.2. Superoxide Anion Radical Scavenging Activity
The scavenging activity of the sample towards O2•−
was measured by the
method described by Nagulendran et al. [77]. Superoxide anions can be formed in a
non-enzymatic phenazine methosulfate-nicotinamide adenine dinucleotide (PMS-
NADH) system. PMS, NADH, and oxygen reacts to give O2•−
which was assayed by
the reduction of nitroblue tetrazolium (NBT).
Briefly, different concentration of 0.3 mL of extract was mixed with 3.0 mL of Tris-
HCl buffer (100 mM, pH 7.4) containing 0.75 mL of NBT (300 μM) solution, 0.75
129
mL of NADH (936 μM) solution. The reaction was started by mixing 0.75 mL of
PMS (120 μM) to the mixture and after 5 min of incubation at room temperature, the
absorbance of mixture was recorded at 560 nm. For comparison, BHA was used as
standard; all the tests were carried out in triplicate. The super oxide anion scavenging
activity was calculated by the equation 1.
4.10.3. Hydroxyl Radical Scavenging Activity
Hydroxyl radical was generated by oxidising 2-deoxyribose (Fenton reaction)
and degraded to malondialdehyde [85]. Therefore decomposing effect of sample on
hydroxyl radicals was estimated by the assay of formed malondialdehyde chromogen
formation.
Briefly, 0.2 mL of 100mM KH2PO4–KOH, 0.2 mL of 15mM deoxyribose, 0.2 mL
of 500mM FeCl3, 0.1 mL of 1mM ethylene diamine tetra acetic acid (EDTA), 0.1 mL
of 1mM ascorbic acid, and 0.1 mL of 10mM H2O2 were mixed with 0.1 mL sample
with different concentration. The mixture was incubated for 1 h at 37ºC. After
incubation, 1.0 mL of 1% w/v TBA and followed by 1.0 mL of 2.8% w/v TCA was
added to the mixture. The resultant mixture was heated for 20 min at 80ºC on a water
bath results in development of pink colour which was measured at 532nm [89].
Quercetin was used as the positive control. The scavenging activity (I %) was
calculated using the equation (1).
4.10.4. Nitric Oxide Radical Scavenging Activity
Aqueous solution of sodium nitroprusside at physiological pH produces nitric
oxide, which interact with oxygen to produce nitrite ions, which was quantified by the
130
Griess Illosvoy reaction. The nitrite ions thus produced react with Griess reagent that
leads to formation of a chromophore which can be determined
spectrophotometrically. The concentration of chromophore is proportional to that of
the generated nitrite ions. Antioxidant compounds present in the sample compete with
oxygen leading to reduced production of nitric oxide [76, 87].
An aliquot of extract solution (4 mL) at different concentrations were mixed
with 1.0 mL of 25 mM sodium nitroprusside (SNP) solution in a test tube, and
incubated for 2 h at 37ºC. Incubated solution (2 mL) was mixed with 1.2 mL Griess
reagent (1% sulfanilamide in 5% H3PO4 and 0.1% naphthylethylenediamine
dihydrochloride) which results in diazotization of the nitrite with sulfanilamide and
subsequent coupling with naphthylethylenediamine dihydrochloride to form a
chromophore. The absorbance of chromophore was measured immediately at 570 nm
[78]. Control experiment was also carried out in similar manner taking same volume
of distilled water in the place of sample solution. The experiment was performed in
triplicate, ascorbic acid was used as positive control and percentage scavenging
activity was calculated using the equation (1).
4.10.5. Hydrogen Peroxide Scavenging Activity
A solution of 40 mM H2O2 and crude extracts/standard in different
concentration were prepared in phosphate buffer (pH 7.4). An aliquot (3.4 mL)
sample solution was added to 0.6 mL of H2O2 solution and the absorbance of resulting
solutions was measured at 230 nm. Gallic acid was used as standard. The percentage
of H2O2 scavenging (I %) of extracts was calculated by equation (1) [79].
131
4.10.6. Reducing Power Ability
The ability of phenolic compounds to quench radicals by electron donation
was determined by potassium ferricyanide reduction method. The absorbance of
Perl’s Prussian blue complex was at 700nm. Increase in absorbance may be due to
reduction of ferric ion/ferricyanide complex by antioxidants to ferrous form [88].
Briefly, 1 mL of sample solutions with different concentration were mixed
with 0.2 M phosphate buffer (2.5 mL, pH 6.6) and 1% (w/v) K3Fe(CN)6 (2.5 mL) and
the mixture was incubated at 50ºC for 20 min, and then mixed with 2.5mL, 10%
TCA. The solution was centrifuged at 12000 rpm for 10 min. A volume of 2.5 mL
supernatant solution was mixed with 2.5 mL of distilled water, 0.5 mL of 0.1% (w/v)
FeCl3 solution. The solution was mixed properly and the absorbance of solution was
measured at 700 nm using ELICO SL 164 UV–Vis spectrophotometer [80]. Ascorbic
acid (25-400 µg/mL) was used as standard. The test was run in triplicate and average
was calculated.
4.10.7. Metal Chelating Ability
Ferrozine with ferrous ion makes red colored complex, which was interrupted
by chelating agents. Thus, the activity of antioxidants was determined by monitoring
the decrease in absorbance of the red Fe2+
/ferrozine complex as antioxidants present
in the test sample compete with ferrozine to chelate ferrous ion [77, 88].
A volume of 0.4 mL different concentration extracts was mixed with 0.05 mL,
2 mM FeCl2 solution. The reaction was started by the mixing 0.2 mL, 5mM ferrozine.
132
Total volume of the final solution was adjusted to 4.0 mL using ethanol. The mixture
was mixed by shaking vigorously and incubated for 10 min at room temperature. The
absorbance of the solution was measured at 562nm spectrophotometrically [81]. The
procedure was also performed using α- tocopherol as standard and percentage of
inhibition of ferrozine–Fe2+
complex formation (I%) was calculated by using equation
(1).
4.10.8. Ferric thiocyanate method (FTC)
Oxidation of Linoleic acid causes the generation of peroxides, which react
with Fe2+
to form Fe3+
. The ferric ion forms red colored complex with SCN– and this
complex has a maximum absorbance at 500 nm. The method was employed to
determine the peroxide level during the initial stage of lipid oxidation [15].
A quantity of 200 µg of sample in 4.0 mL ethanol was mixed with 4 mL,
linoleic acid (2.5 % in ethanol), 8.0 mL phosphate buffer (0.05 M, pH 7.0) and 4 mL
distilled water. The mixture was mixed vigorously in a screw cap tube and incubated
in dark at 40ºC. One millilitre of above solution was mixed with 9.7 mL ethanol
(75%) and 0.1 mL ammonium thiocyanate (30%) followed by 0.1 mL 20 mM ferrous
chloride in 3.5% hydrochloric acid and exactly 3 min later the absorbance of the red
colour solution was measured at 500 nm. The sample was withdrawn after every 24hr
and the procedure was repeated until the absorbance of the control reached a
maximum [15]. Linoleic acid mixture without the addition of sample was used as the
control and α-tocopherol was used as the positive control at the same concentration.
133
4.11. EX VIVO ANTIOXIDNT ACTIVITY OF EXTRACTS
4.11.1. Lipid Peroxidation Assay
A healthy Wister rat was sacrificed by decapitation after fasten for 16 h. The
liver was dissected out carefully and washed with normal saline and 5.0 %w/v liver
homogenate was prepared in phosphate buffer saline. From the above mixture 1 mL
was mixed with 100 μL of sample solution and incubated for 2 h at 37°C. A volume
of 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 min and kept aside
until it comes to room temperature. Deionised water was used to make the final
volume 5.0 mL. The mixture was then centrifuged for 10 min at 2800 rpm. The
supernatant solution was removed carefully and absorbance was recorded at 532 nm
[82]. Control was prepared by following the same procedure without extracts. Rutin
served as positive control. The inhibition of lipid peroxidation was calculated using
the equation (1), where, Asample was the absorbance of an extract/standard in presence
of liver homogenate and Acontrol was the absorbance of the solution containing all
reagents except the test/standard sample.
4.11.2. Oxidative Haemolysis Assay
Oxidative haemolysis assay method was followed as described by Su et al.
(2009) with slight modification as represent by Coulibaly et al. [83]. Blood sample
was collected from rat eyepit under mild anaesthesia and followed by centrifuged at
1500 × g for 10 min at 4ºC. The erythrocytes were separated from the plasma and
buffy coat. The erythrocytes were suspended in 10 mL phosphate buffer saline (PBS,
134
10 mM, pH 7.4). The erythrocyte in PBS centrifuged again at 1500 × g for 5 min to
take out buffy coats. The process was repeated three times and washed erythrocytes
made to 0.5% erythrocyte suspension in PBS (10 mM, pH 7.4) for the assay.
Prepared 0.5 mL of erythrocyte suspension, 0.5 mL extract/standard drug
solution at different concentration and 0.05 mL of 100 mM H2O2 was mixed together
and kept for incubation at 37ºC for 60 min. After incubation period 4.2 mL of distilled
water was added and the solution was centrifuged for 10 min at 1000 rpm. The
absorbance of the supernatant was measured at 415 nm. Control was prepared by
taking supernatant without extract and 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).
4.12. ACUTE TOXICITY STUDY
Albino mice maintained under standard condition used for the acute toxicity
study for the samples (potent extract and its fractions). The overnight fasted animals
were taken and fixed dose (OCED Guideline no. 423, Annexure 2d) procedure of
CPCSEA was adopted for toxicity studies. Solvent free dried extract or fraction was
orally administered in acute toxicity and in screening of other biological activity to
experimental animal. In acute toxicity study, the test material suspended in 0.5% w/v
sodium carboxy methyl cellulose (CMC) and was orally administrated (1 mL/100 g)
in 3 animals. The mortality was observed at a dose of 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 observed for 7 days.
135
4.13. ANIMAL GROUPING
The experiments were performed in two stages. At first stage activity of crude
extracts were evaluated, and in second phase the activity of fractions of more potent
extract were determined. For each experiment animals were divided into eight groups
in following manner.
Group I : Normal control (treated with only vehicle)
Group II : Negative control (diseased group)
Group III : Standard
Group IV : Lower dose of methanol extract or methanol fraction
Group V : Higher dose of methanol extract or methanol fraction
Group VI : Lower dose of ethyl acetate extract or ethyl acetate fraction
Group VII : Higher dose of ethyl acetate extract or ethyl acetate fraction
Group VIII : Lower dose of Pet ether extract or pet ether fraction
Group VIII : Higher dose of Pet ether extract or pet ether fraction
Lower dose of extracts indicates 250 mg/kg b.w. higher dose indicates 500
mg/kg b.w. For fractions lower dose indicates 75 mg/kg b.w. and higher dose
indicates 150 mg/kg b.w. Hepatoprotective and nephroprotective activity of both the
plants were evaluated therefore Group I, II. III were common for both the plants.
136
4.14. ANALGESIC AND ANTI-INFLAMMATORY ACTIVITY OF
P. ACIDUS
4.14.1. Nociceptive Tests
4.14.1.1. Writhing reflex induced by acetic acid in mice
The extracts/fraction, indomethacin, and vehicle were administered orally.
Indomethacin at a dose of 5mg/kg was used as standard. After 1 h each mouse was
tested with acetic acid (0.6%, v/v, 10 mL/kg), and the intensity of nociceptive
behaviour was quantified by counting the total number of writhes over a period of 25
min [102]. The percentage analgesic activity was calculated by using the following
equation:
Percentage analgesic activity = [(Nc − Nt)/ Nc] × 100%
Where Nc was the average number of stretches of the control group, and Nt was the
average number of stretches of the test drug group.
4.14.1.2. Tail immersion test
In hot water (temperature was maintained at 55±0.5°C) extreme 3 cm of the
Albino mouse tail immerged in that water. Within a time period each mouse was
reacted by withdrawing the tail, and the reaction time was recorded with a stopwatch.
The drugs were given orally to the respective groups as described above. The
experiment was repeated at 0, 0.5, 1, 2, 3, 5 h after administration of extracts/fraction
and standard drug [104]. Morphine was used as standard at a dose of 10mg/kg.
137
4.14.1.3. Formalin-induced licking response in mice
One hour after oral administration of vehicle, extract/fraction and diclofenac
sodium (10 mg/kg), 25 μL of 1% formalin in saline was injected subcutaneously in
subplantar area of right hind paw. The mice were instantly placed in a cleaned jar.
The time spent on licking the injected paw was recorded. The first period was
recorded at 0-5 min considered as early phase response and the second period was
recorded at 10-35 min considered as late phase response [114].
4.14.2. Anti-inflammatory Activity
4.14.2.1. Carrageenan-induced paw oedema in rats
A solution of 1%, 0.1 mL carrageenan was injected subcutaneously into the
plantar surface of left hind paw to induced paw inflammation in rats after respective
drug treatment to each group. Indomethacin (5mg/kg) used as standard. The volume
of the rat paws was measured with a plethismometer before and after 1, 2, 3, 4, and 5
h [109].
14.4.2.2. Granuloma formation induced by cotton pellet in rats
A 30 mg sterilized cotton pellet was put subcutaneously into the interscapular
area of anaesthetized rats using 25 mg/kg of pentobarbitone sodium under sterilized
condition. The extract/fraction solution, indomethacin (5 mg/kg, p.o.), and vehicle
water were administered 5 consecutive days once in a day. On the 5th
day, animals
were killed via cervical dislocation after 1h of drug treatment and the cotton pellets
with granuloma tissue around them were dissected out carefully. The weights of these
pellets (wet and dry) were measured and the anti-proliferative effects of
extracts/fraction and indomethacin were determined by comparing with control group
[103].
138
14.4.2.3. Membrane stabilizing activity
The membrane stabilizing activity was performed by the method used by
Shinde et al. [101].
Preparation of erythrocyte suspension
Under anaesthesia whole blood was collected in a heparinized tube from mice.
Blood sample was washed with 0.9% saline for three times. Isotonic buffer solution
(pH 7.4) was used to make 40% (v/v) erythrocyte suspension. Buffer solution was
prepared by following formula, NaH2PO4. 2H2O – 0.26 g, NaHPO4 – 1.15 g, NaCl – 9
g (10 mM sodium phosphate buffer) in 1 L distilled water.
Heat induced haemolysis
Isotonic buffer containing different concentration of extract/fraction were
taken into 2 duplicate sets of centrifuge tubes. Vehicle was prepared by adding same
amount of water to another centrifuge tube. To each tube 30 µL erythrocyte
suspension was added and mixed gently by inversion. A pair of tubes was kept in
incubator for 20 min at 54ºC, another pair of test tube kept in an ice bath at 0–5ºC.
After incubation, reaction mixture was centrifuged at 1300 × g for 3 min. The
absorbance (A) was read at 540 nm using UV-Vis spectrophotometer. Acetyl salicylic
acid used as standard.
Percent (%) activity = [1 – (A2-A1)]/ [A3-A1] × 100
A1 = test sample unheated; A2 = test sample heated; A3 = control sample heated
139
Hypotonic solution induced haemolysis
The isotonic buffer solution was prepared by taking 154 mM NaCl in 10 mM
sodium phosphate buffer (pH 7.4). The test was performed in duplicate pair of test
tubes. Hypotonic solution containing different concentration of extract/fraction was
taken in test tube and mixed with 30 µL of stock erythrocyte suspension, while
control sample was prepared with drug free solution. The test tubes were incubated
for 10 min in room temperature, and then centrifuged at 1300 × g for 3 min. The
absorbance (A) was measured at 540 nm. Acetyl salicylic acid used as standard.
Percent (%) activity = [1 – (A2-A1)]/ [A3-A1] × 100
A1 = test sample in isotonic solution; A2 = test sample in hypotonic solution; A3 =
control sample in hypotonic solution.
4.15. EXPECTORANT AND ANTITUSSIVE ACTIVITY OF M.
MINUTA
4.15.1. Antitussive Activity
4.15.1.1. Ammonium liquor induced cough
Briefly, extract/fraction, vehicle, standard was orally administered 1h prior to
each mouse, was placed in a glass chamber exposed to 0.3 mL 25% NH4OH produced
by a nebulizer for 45 s. Animals were monitored during ammonia exposure and the
cough frequency and latent period of cough were recorded for 6 min. Codeine
phosphate (30mg/kg) used as standard [99].
140
4.15.1.2. Sulphur dioxide (SO2) induced cough
Mice were treated with extract/fraction, standard, vehicle continuously for 5
days. NaHSO3 solution was prepared in water (500 mg/kg, 2.0 mL) was placed at the
base of special deigned desiccator and 0.2 mL of sulphuric acid was added using a
pipette; results in formation of sulphur dioxide gas. After 15 seconds, each mouse was
placed in desiccator and exposed to SO2 for 45 s. The mice were then removed and
placed in a clear glass chamber for counting of bouts of cough for next five minutes.
Codeine phosphate (30mg/kg) used as standard [92].
4.15.2. Expectorant Activity
After 1 week of acclimatization, mice were divided into different groups as
described earlier and 0.5%, 1.0 g/kg ammonium chloride was used as positive control.
The treatment was last for 5 days; after 30 min of last administration mice were
injected with 5% phenolsulfonphthalein and after 40 min animals were dissected to
take out trachea and bronchia parts. NaHCO3 solution (5 %) was used to flush inside
of these parts three times with 0.5 mL for each time, which were collected and
centrifuged to measured the absorbance of supernatant at 546 nm using UV–Vis
spectrophotometer [93].
4.16. CHROMATOGRAPHIC SEPARATION OF EXTRACTS
Most potent bioactive extract from each plant was identified by in vitro and ex
vivo antioxidant activity, analgesic and antiinflammatory activity, expectorant and
antitussive activity. Identified potent extract (methanol extract of both plants) was
fractionated using column chromatography technique by taking borosilicate column
(40 mm × 600 mm). Silica gel (60-80 mesh) served as stationary phase. About 120 g
141
of silica gel was mixed with 200 mL of petroleum ether, and poured slowly to pack
the column up to a hight of 20 cm. The procedure was carefully performed to avoid
deposition of any air bubble during packing. A quantity of 30 g of extract was
digested with 30 g of silica gel and dried under a stream of cold air, then spread over
the column packing. Fractions were eluted successively with petroleum ether, ethyl
acetate and methanol. A volume of 2.5 L of each solvent was used to elute
successively with petroleum ether followed by EtOAc and MeOH to obtain petroleum
ether fraction (PFM), ethyl acetate fraction (EFM), methanol fraction (MFM) of
methanol extract of Marsilea minuta, and petroleum ether fraction (PFP), ethyl
acetate fraction (EFP), methanol fraction (MFP) of methanol extract of Phyllanthus
acidus. All the eluents were dried by using rotary vaccume evaporator.
4.17. DETERMINATION OF RETARDATION (Rf) VALUE
TLC was carried out using pre-coated aluminium sheets with silica gel 60 (20
× 20 cm, layer thickness 0.2 mm) (SD Fine-Chen Limited, Mumbai). The plates were
cut into smaller pieces (5 × 10 cm size). The 1% extract/fraction filtered solution (2-5
μl) was spotted using a fine bore capillary tube 1 cm above the base at same distance
from each other. Chromatograms were developed in a room temperature (28°C) using
different solvent systems at an angle of 75°. The solvent system was allowed to run to
a distance of 9 cm from the base of the plates. The time required for the completion
chromatogram varied from 20-30 min, and then the plates were taken out from the
chamber and allowed to dry in air. The plates were placed in iodine chamber to
identify the presence of the spots [202]. Rf (Retardation factor) values were calculated
using the following formula,
Distance traveled by the solute
Distance traveled by the solvent R
f value =
142
4.18. IN VITRO AND EX VIVO ANTIOXIDANT ACTIVITY OF
FRACTIONS
DPPH radical scavenging activity, nitric oxide radical scavenging activity, total
antioxidant activity, lipid peroxidation activity was performed for the fractions using
the same method discussed earlier.
4.19. HEPATOPROTECTIVE AND IN VIVO ANTIOXIDANT
ACTIVITY OF FRACTIONS
Hepatoprotective and in vivo antioxidant effect of MFP, EFP, PFP, MFM,
EFM, and PFM were evaluated against paracetamol induced hepatotoxicity as
described by Singh and Handa (1995) and Manokaran et al. (2008) [128, 129].
4.19.1. Paracetamol Induced Hepatotoxicity
Fractions of different concentrations, paracetamol, and silymarin were
administered orally and the dose was prepared by suspending in 0.5% sodium CMC.
Respective drug treatment was continued once in a day for consecutive three days. A
single dose paracetamol on 3rd
day at a dose of 3g/kg was administered orally to the
animals of all groups except normal control, after 30 min the test sample in vehicle
was administered. Blood sample was collected by direct cardiac puncture under light
ether anaesthesia after 48 h of paracetamol oral administration, and serum was
separated for the biochemical estimations.
143
4.19.2. Biochemical Determinations
Biochemical parameters related to hepatotoxicity like SGOT, SGPT, ALP,
total bilirubin, direct bilirubin, total cholesterol, total triglycerides were assayed by
using commercial diagnostic kits.
4.19.2.1. SGOT determination
Quantitative determination of SGOT in serum was performed by using Agapee
Diagnostic kit (Batch no: 4501, Manufacturing date: 9/11, Expiry date: 11/13) and the
principle mechanism of estimation is based on the following reactions,
Aspartate + Alpha Ketoglutarate Oxaloacetate + L-Glutamate
Aspartate aminotransferase
Oxaloacetate + NADH + H+
Malatedehydrogenase
L - Malate + NAD+
Laboratory procedure: the estimation was performed by Mispa semi auto analyser
and the following parameter was imputed in the system before analysing the samples.
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.
Sample was prepared by mixing 0.1 mL of serum sample with 1.0 mL of working
reagent, incubated for 1 min at 37°C and the sample was analysed. Results were
expressed as U/L.
144
4.19.2.2. SGPT determination
Quantitative determination of SGPT in serum was performed by using Agapee
Diagnostic kit (Batch no: 4063, Manufacturing date: 9/11, Expiry date: 11/13) and the
principle mechanism of estimation is based on the following reactions,
Alanine + Alpha Ketoglutarate Pyruvate + L-Glutamate
Aspartate aminotransferase
Pyruvate + NADH + H+
Lactatedehydrogenase
L - Lactate + NAD+
Laboratory procedure: the estimation was performed by Mispa semi auto analyser
and the following parameter was imputed in the system before analysing the samples.
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.
Sample was prepared by mixing 0.1 mL of serum sample with 1.0 mL of working
reagent, incubated for 1 min at 37°C and the sample was analysed. Results were
expressed as U/L.
4.19.2.3. ALP determination
Quantitative determination of ALP in serum was performed PNPP kinetic
method by using Agapee Diagnostic kit (Batch no: 4781, Manufacturing date: 8/11,
Expiry date: 8/13) and the principle mechanism of estimation is based on the
following reactions,
p-Nitrophenylphosphate + H2O
Alkalinephosphatase
p-Nitrophenol + Phosphate
145
Laboratory procedure: the estimation was performed by Mispa semi auto analyser
and the following parameter was imputed in the system before analysing the samples.
Mode of reaction – kinetic, slope of reaction – increasing, blank – distilled water,
wavelength – 405 nm, factor – 2754 , linearity – 1000 U/L, delay time – 60 sec, no. of
readings – 4, interval – 60 sec.
Sample was prepared by mixing 0.1 mL of serum sample with 1.0 mL of working
reagent (mixed after incubating 37°C for 1 min), incubated for 1 min at 37°C and the
sample was analysed. Results were expressed as U/L.
4.19.2.4. Bilirubin determination
Quantitative determination of total and direct bilirubin in serum was
performed by using Agapee Diagnostic kit (Batch no: 670, Manufacturing date: 6/11,
Expiry date: 6/13) and the principle mechanism of estimation is based on the reaction
between sulfanilic acid (present in direct bilirubin reacgent) with sodium nitrate in
presence of diazotied sulfanilic acid to form azobilirubin. Direct bilirubin only reacts
in absence of methyl sulfoxide to give azobilirubin.
Laboratory procedure: The test and blank solution was prepared in the following
manner,
Reagents/ sample Total bilirubin Direct bilirubin
Sample blank Test Sample blank Test
Total bilirubin reagent 1000 mL 1000 mL - -
Direct bilirubin reagent - - 1000 mL 1000 mL
Serum 50 mL 50 mL 50 mL 50 mL
146
All solutions were mixed well, incubated for 5 min at room temperature and the
absorption of the resulting solutions was taken using 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.
Results were expressed as mg/dL.
4.19.2.5. Total cholesterol determination
Quantitative determination of total cholesterol in serum was performed
CHOD-PAP method by using Agapee Diagnostic kit (Batch no: CH-0612,
Manufacturing date: 1/11, Expiry date: 1/13) and the mechanism of estimation is
based on the principle that, cholesterol esters are dissociated into cholesterol and fatty
acids in presence of cholesterol esterase. Subsequently cholestenone forms by
enzymatic oxidation with cholesterol oxidase, hydrogen peroxide. Red quinoeimine
dye forms when hydrogen peroxide reacts with p-aminoantipyrine and phenol in
presence of peroxidase.
Chloesterol ester + H2OCholesterol esterase
Cholesterol + Fatty acids
Cholesterol + O2Cholesterol oxidase
4 Cholesten-3-one + H2O2
2H2O2 + Phenol + 4 Aminoantipyrine Red quinone + 4 H2OPeroxidase
Procedure: Working reagent (1 mL) mixed with 0.01 mL of serum sample or standard
and mixed well. Solution was incubated for 5min at 37°C. The absorbance of sample
147
and standard was measured by taking working reagent as blank. 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.
4.19.2.6. Total triglycerides determination
Quantitative determination of triglyceride in serum was performed CHOD-
PAP method by using Agapee Diagnostic kit (Batch no: 650, Manufacturing date: 5/11,
Expiry date: 5/13), Hyderabad and the mechanism of estimation is based on the
following principle,
Triglycerides Lipoprotein lipase
Glycerol + Free fatty acids
Glycerol + ATP
Glycerol kinaseGlycerol 3 phosphate + ADP
Glycerol 3 phosphate + O2 Glycerol 3 PO
Dihydroxyacetone phosphate + H2O2
H2O2 + 4 Aminoantipyrine + PhenolPeroxidase
Red quinoneimine dye + H2O
Procedure: Working reagent (1 mL) was mixed with 0.01 mL of serum sample or
standard. Solution was and incubated for 37°C for 10 min and the absorbance of
sample and standard was measured using working reagent as blank in semi auto
analyser. Following parameter were feed in auto analyser before the determination of
absorbance.
148
Mode of reaction – end point, slope of reaction – increasing, wavelength – 546 nm,
linearity – 1000 mg/dL, standard concentration – 200 mg/dL. Result was expressed
as mg/dL.
4.19.3. In vivo Antioxidant Activity
The level of SOD, CAT, GPx, GSH in liver tissue, and blood plasma GSH
level in drug treated and hepatotoxic animals was evaluated to determine in vitro
antioxidant activity.
4.19.3.1. Preparation of liver homogenate
Animal was anesthetised and the liver was dissected out and was perfused
with cooled 0.15 M KCl. The 10% homogenate was prepared in 0.15 M KCl-10 mM
potassium phosphate buffer, pH 7.4 and was centrifuged at 3000 rpm for 10 min
[203].
4.19.3.2. Estimation of superoxide dismutase
Inhibition of autocatalyzed adrenochrome formation in the presence of tissue
homogenate was determined at 480 nm to estimate SOD activity. The reaction
mixture was prepared by taking 150 µl of homogenate, 1.8 mL of 30 mM carbonate
buffer (pH, 10.2), 0.7 mL of distilled water and 400 µl of epinephrine (45 mM).
Control was prepared by performing auto oxidation of epinephrine to adrenochrome
without the homogenate [204]. The enzymatic activity was represented as nmol of
epinephrine protected from oxidation/min/mg protein using a molar extinction
coefficient of 4.02 × 103 M-1
cm-1
.
149
4.19.3.3. Estimation of catalase
The catalysis activity was determined by the conversion of H2O2 to H2O in an
incubation mixture adjusted at pH 7.0 and was recorded at 254 nm. The reaction
mixture was prepared by mixing 1.9 mL of 50 mM potassium phosphate buffer pH
7.0 and 0.1 mL supernatant of tissue homogenate and the reaction was started with the
addition of 0.1 mL of 30 mM H2O2. Absorbance was taken at 240 nm [205].One unit
of catalase activity was represents the amount of enzymes causing the breakdown of
µM H2O2/mg protein/min at pH, 7.0 at 25ºC using a molar extinction coefficient of
43.6 M-1
cm-1
.
4.19.3.4. Estimation of glutathione peroxidase
The reduction of oxidised glutathione occurs simultaneously with oxidation of
NADPH catalysed by of glutathione reductase. The reaction mixture was prepared by
taking 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. The reaction was started after 5 min
with the addition of 100 µl 2.6 mM H2O2 and the absorbance change was measured at
340 nm in 3 min was recorded at 37ºC [206]. Activity of GPx was determined using
the molar extinction coefficient of NADPH 6220 M−1
cm−1
and represented as µM
NADPH oxidized /min /mg protein at 37ºC
150
4.19.3.5. Estimation of GSH in liver tissue
To precipitate protein 0.125 mL of 25% TCA was mixed with 0.5 mL of tissue
homogenate in a test tube and cooled in ice for 5 min. Again 0.6 mL of 5% TCA was
mixed with the previous mixture and centrifuged at 1500 rpm for 10 min. After
centrifugation 0.3 mL of supernatant was transferred in another test tube and mixed
with 0.7 mL of phosphate buffer (0.2 M, ph 8), freshly prepared 2.0 mL DTNB [5'-5'
Dithiobis (2- nitro benzoic acid)] solution (0.6 mM in 0.2 M sodium phosphate buffer,
pH 8). The absorbance of mixture was measured after 10 min at 412 nm [207]. The
GSH was calculated using a standard curve plotted by taking different concentration
of GSH (5-100 nM) in 5% TCA, and results were expressed as nmol/mg protein.
4.19.3.6. Estimation of blood GSH
Blood glutathione was estimated by mixing fresh blood (0.1 mL) 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 g NaCl in 100 mL water). The solutions mixed well and
incubated at room temperature for 5 min and followed by centrifuged at 3000 × g at
4°C for 15 min. Clear supernatant (0.5 mL) was taken out and mixed thoroughly with
2.0 mL of 0.3 mol/L phosphate solution, 0.25 mL 0.2% DTNB in 1% sodium citrate
solution. A solution was prepared by taking 1.0 mL 0.3 mol/L phosphate solution, 1.0
mL water, 0.5 mL precipitating solution and 0.25 mL DTNB solution which served as
blank. Absorbance of both blank and sample reaction mixtures were taken against
water at 412 nm [208]. The gluthathione concentration was calculated by following
formula taking extinction coefficient 13.6 and molecular weight of glutathione 307.
151
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) = Asample - Ablank
4.20. NEPHROPROTECTIVE ACTIVITY OF FRACTIONS
Nephroprotective effect of MFP, EFP, PFP, MFM, EFM, PFM were evaluated
against cisplatin induced nephrotoxicity [131].
Fractions, standard drug and vehicle were administered orally for six
consecutive days once daily. On fourth day cisplatin (20 mg/kg) injected
intraperitoneally to induce nephrotoxicity to all animals expect normal control group.
All animals were sacrificed 72 h after nephrotoxicity induced.
4.20.1. Sampling and Biochemical Analyses
Cisplatin was administered in mice and were sacrificed after 72 h. Blood
samples were collected and centrifuged at 5000 rpm for 10 min to obtain clear sera
which were used for the estimation of different biochemical parameters like BUN,
serum creatinine, uric acid, total protein, and albumin. Commercial biochemical kit
was used to estimate all biochemical parameters obtained from Agapee Diagnistic
Ltd., Kerala. Kidney tissue was used to determine level of tissue MDA.
152
4.20.1.1. Estimation of BUN
Quantitative determination of BUN in serum was determined by following
manufacturer procedure (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 are 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) =(T1 - T2) sample
(T1 - T2) sandardX 23.4
4.20.1.2. Estimation of serum creatinine
Serum creatinine was determined by Modified Jaffe’s method as per procedure
described by manufacturer (Batch no: 769, Manufacturing date: 1/12, Expiry date:
1/13). Creatinine reacts with picric acid to form creatinine alkaline picrate, a coloured
compound. Observed change in absorbance is proportional to creatinine
concentration.
Briefly, standard and sample solution was prepared by mixing of 1.0 mL of
working reagent with 0.1 mL of standard given with commercial kit and 0.1 mL of
serum sample respectively. The solutions were mixed well and optical density (T1)
was determined after 60 sec and second reading (T2) was taken immediately after 60
sec after first reading at 492 nm using distilled water as blank. The concentration of
creatinine was calculated using the following formula,
153
Creatinine concentration (mg/dL) =(T1 - T2) sample
(T1 - T2) sandardX 2
4.20.1.3. Estimation of serum uric acid
Uricase methodology was used to determine serum uric acid level as described
by manufacturer. The principle of the assay is based on the following reaction,
Uric acid + O2 + 2H2O Uricase
Allantoine + CO2 + H2O
2H2O2 + 4 -AAP + EHSPTPeroxidase
Red quinone
EHSPT = N-ethyl N-(2-hydroxy-3-sulfopropyl) n-toluidine
4 -AAP = Amino-4-antipyrine
Standard and sample solution was prepared by mixing of 1.0 mL of working
reagent with 0.025 mL of standard given with commercial kit and 0.025 mL of serum
sample respectively. The solutions are mixed and incubated for 5min at 37°C and the
absorption was taken at 546 nm using working reagent as blank. The concentration of
uric acid was calculated using the following formula,
Uric acid concentration (mg/dL) =Absorbtion of sample
Absorption of satndardX 8
4.20.1.4. Estimation of serum total protein
Protein concentration was determined by direct biuret method as per
manufacturer procedure (Batch no: 298, Manufacturing date: 2/12, Expiry date: 2/13).
Briefly, cupric ion present in working reagent forms blue colour complex with protein
in serum sample. The intensity of the blue colour determined by calorimetric assay is
proportional to the protein concentration.
154
Standard and sample solution was prepared by mixing of 1.0 mL of working
reagent with 0.02 mL of standard given with commercial kit and 0.02 mL of serum
sample respectively. The solutions are mixed and incubated for 10min at 37°C. The
absorption was measured at 546 nm taking reagent blank. The concentration of uric
acid was calculated using the following formula,
Total protein concentration (mg/dL) =Absorbtion of sample
Absorption of satndardX 6
4.20.1.5. Estimation of serum albumin
Albumin concentration was determined using bromocresol green method as
per manufacturer procedure (Batch no: 888, Manufacturing date: 10/11, Expiry date:
10/13). Briefly, albumin from serum sample reacts with a dye bromocresol-green
(working reagent) cause colour change which is proportional to protein concentration.
Standard and sample solution was prepared by mixing of 1.0 mL of working
reagent with 0.01 mL of standard given with commercial kit and 0.01 mL of serum
sample respectively. The solutions were mixed and incubated for 1 min and the
absorption was measured at 630 nm using reagent blank. The concentration of uric
acid was calculated using the following formula,
Albumin concentration (mg/dL) =Absorbtion of sample
Absorption of satndardX 3
4.20.1.6. Estimation of MDA
Lipid peroxidation responsible for oxidative degradation of polyunsaturated
fatty acids to MDA, and this formation of MDA is usually estimated by the
155
chromogenic thiobarbituric acid reaction and expressed as total TBARS. After
physical euthanasia the kidneys were isolated quickly form each animal and washed
with saline. Kidney was dried using filter paper, weighed, and homogenised using
phosphate buffer (pH 7.4) 10% (w/v). Unbroken cells, cell debris and nuclei were
sedimented by centrifugation for 10 min at 2000 × g, and the supernatant was taken
out which was used for evaluate lipid peroxides (MDA production) in the kidney.
A volume of 0.5 mL tissue homogenate was mixed with 2.5 mL of 10% (w/v)
trichloro acetic acid in a tube. Tubes were placed in a boiling water bath for 15 min
and then cooled in tap water followed by centrifugation at 1000 × g for 10 min. After
centrifugation 2.0 mL of the supernatant was mixed to 1.0 mL of 0.67% (w/v) TBA
solution in a test tube and placed again in a boiling water bath for 15 min. The
solution was then cooled in tap water and its optical density was measured at 532 nm.
The concentration of MDA was calculated by using absorbance coefficient 1.56×105
cm−1
M−1
) and the results were represented as nM/min/mg tissue protein [144].
4.21. ANTI TUBERCULAR ACTIVITY OF FRACTIONS
The anti-TB activity of PFM, EFM, MFM, MFP, EFP, PFP were assessed
against M. tuberculosis using MABA as described by Lourenco et al. (21). MABA is
a non-toxic method, uses a thermally stable reagent and shows outstanding correlation
with propotional and BACTEC radiometric assay. Briefly, sterile 96 wells plate were
taken and sterile deionzed water (200 µl) was added to all outer perimeter wells of
sterile wells plate to reduce the evaporation of medium in the test wells throughout
156
incubation. Middlebrook 7H9 broth (100 µl) was mixed in all wells plate and serial
dilution of test sample was made directly on plate. The final fraction concentrations
tested were 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50, and 100µg/mL. Each plate was
enclosed and sealed with parafilm and incubated at 37ºC for five days. Exactly 25 µl
of freshly prepared mixture of Almar Blue reagent was mixed after incubation period
and 10% tween 80 (1:1) was mixed to the plate and then incubated for 24 hrs. A blue
color in the well represents no bacterial growth, while pink colour interpreted as
bacterial growth. The MIC was determined as minimal sample concentration which
prevented the colour change from blue to pink.
4.22. ISOLATION AND IDENTIFICATION OF CHEMICAL
CONSTITUENTS
Pharmacological investigations reveal that methanol fraction from M. minuta
and P. acidus leaves methanol extract showed superior activity. To investigate and to
identify the possible active ingredients/chemical constituent, both the fractions were
further fractionated by using preparative column chromatography.
MFM and MFP were fractionated by using column chromatography by taking
borosilicate column with a dimension of 40 mm × 600 mm. Silica gel (60-120 sieve)
used as stationary phase. Chloroform was used as mobile phase. A weight of 400 g of
silica gel was stirred with 500 mL of chloroform to make slurry and the column was
packed at height of 30 cm and a diameter of 4 cm carefully to form a compact
packing, special care has been taken to avoid the deposition of air bubbles. Solvent
level was maintained few centimeters above of the silica gel layer. About 20 g of
157
fraction was mixed with minimum volume of methanol and mixed with 30 g of silica
gel. The mixture was air dried under stream of cold air and spreaded slowly on the top
of the column. The top layer (sample-silica gel mixture) was covered with silica gel
slurry (in chloroform) followed by a piece of cotton wool.
A total of 13 fractions were eluted form MFM (named as MM-1 to MM-13)
and 6 fractions (named as PA-1 to PA-6) eluted form MFP. All the eluants were dried
under reduced pressure.
Figure 4.2: Fractionation of extract
158
4.23. ANTIOXIDANT ACTIVITY AND SELECTION OF POTENT
SUB-FRACTION
Fractions PA 1-6 and MM 1-13 were screened for antioxidant activity by
taking 2 fixed concentrations ((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 P. acidus
and 3 ‘more potent’ sub-fractions from M. minuta were selected for further analysis
(details are given in result section).
4.24. TLC AND SPECTRAL ANALYSIS
Selected sub-fractions were subjected for TLC using the same procedure and same
solvent system as discussed earlier.
One fraction from P. acidus and one fraction from M. minuta were selected for
spectral analysis as they showed single spot in TLC. Spectra study like IR (in KBr),
1HNMR,
13CNMR were performed to find the possible structure of constituents.
Melting point was determined for isolated compounds by open capillary method
and was uncorrected.
4.25. 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.