5. materials and methods -...

Materials and Methods

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5. MATERIALS AND METHODS

5.1. Collection and authentication of plant materials

The plants Phyllanthuspolyphyllus&Prosopis cinerariawere collected in the

month of November 2009 from the Kolli hills and around in Salem District,

Tamilnadu, India. The plant material was taxonomically identified by the Botanical

Survey of India, Coimbatore, Tamil Nadu.

Preparation of extracts

The leaves of Phyllanthuspolyphyllus&Prosopis cineraria were dried under

shade and then powdered with a mechanical grinder. The powder was passed

through sieve No 40 and stored in an airtight container for further use.

Chemicals

Petroleum Ether (60-80o C), Methanol (95% v/v)

Extraction procedure

Petroleum Ether

The coarse powder was extracted with 1-1.5 liters of Pet. Ether (60-80o C) by

continuous hot percolation using Soxhlet apparatus. After the completion of

extraction, it was filtered and the solvent was removed by distillation under reduced

pressure.

Methanol extract

The marc left after Pet. ether extraction was dried and extracted with 1-1.5

liters of methanol (70-80oC) by continuous hot percolation using Soxhlet apparatus.


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After the completion of extraction, it was filtered and the solvent was removed by

distillation under reduced pressure. The extract was stored in desiccator. The

extractive values are presented in table 3.

Table 3.\Data showing the Extractive Values of Leaves of Phyllanthuspolyphyllus &

Prosopis cineraria.

Plant Name Part used Method ofExtraction

Yield in percentage(w/w)

PetroleumEther Methanol

Phyllanthuspolyphyllus Leaves Continuous hot percolation 4.5 5.2

Prosopis cineraria Leaves Continuous hot percolation 3.5 4.2

5.2. PRELIMINARY PHYTOCHEMICAL STUDIES [117-120]

Test for Carbohydrates and Glycosides

A small quantity of the extracts were dissolved separately in 4 ml of distilled

water and filtered. The filtrate was subjected to Molisch’s test to detect the presence

of Carbohydrates.

Molisch's Test

Filtrate was treated with 2-3 drops of 1% alcoholic - napthol solution and 2

ml of Con sulfuric acidwas added along the sides of the test tube. Appearance of

brown ring at the junction of two liquids shows the presence of carbohydrates.

Another portion of the extract was hydrolysed with hydrochloric acid for few

hours on a water bath and the hydrolysate was subjected to Legal’s and Borntrager’s

test to detect the presence of different glycosides.


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Legal’s Test

To the hydrolysate 1 ml of pyridine and few drops of sodium nitroprusside

solutions were added and then it was made alkaline with sodium hydroxide solution.

Appearance of pink to red colour shows the presence of glycosides.

Borntrager’s Test

Hydrolysate was treated with chloroform and then the chloroform layer was

separated. To this equal quantity of dilute ammonia solution was added. Ammonia

layer acquires pink color, showing the presence of glycosides.

Test for Alkaloids

A small portion of the solvent free pet ether, alcohol extracts were stirred

separately with few drops of diluted hydrochloric acid and filtered. The filtrate was

tested with various reagents for the presence of alkaloids.

Mayer’s reagent - Cream precipitate

Dragendroff’s reagent - Orange brown precipitate

Hager’s reagent - Yellow precipitate

Wagner’s reagent - Reddish brown precipitate

Test for Phytosterol

The extracts were refluxed with solution of alcoholic potassium hydroxide

till complete saponification has taken place. The mixture was diluted and extracted

with ether. The ether layer was evaporated and the residue was tested for the

presence of Phytosterol.


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Liebermann Burchard Test

The residue was dissolved in few drops of dilutedaceticacid, 3 ml of acetic

anhydride was added followed by few drops of concentratedsulphuric acid.

Appearance of bluish green colour shows the presence of Phytosterol.

Tests for Fixed Oils

Spot Test

Small quantities of various extracts were separately pressed between two

filter papers. Appearance of oil stain on the paper indicates the presence of fixed oil.

Few drops of 0.5N alcoholic potassium hydroxide were added to a small quantity of

various extracts along with a drop of phenolphthalein. The mixture was heated on a

water bath for 1-2 hours. Formation of soap or partial neutralization of alkali

indicates the presence of fixed oils and fats.

Test for Gums and Mucilages

Small quantities of the extracts were added separately to 25 ml of absolute

alcohol with constant stirring and filtered. The precipitate was dried in air and

examined for its swelling properties for the presence of carbohydrates.

Test for Saponins

The extracts were diluted with 20 ml of distilled water and it was agitated in

a graduated cylinder for 15 minutes. The formation of 1 cm layer of foam shows the

presence of Saponins.

Test for Proteins and Free Amino Acids

Small quantities of the extracts were dissolved in few ml of water and treated

with following reagents. a. Million’s reagent-Appearance of red color shows the


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presence of protein and free amino acid. b. Ninhydrin reagent- Appearance of

purple colourshowsthe presence of proteins and free amino acids. c. Biuret test-

Equal volumes of 5% sodium Hydroxide solution & 1% copper sulphate solution

was added. Appearance of pink or purple color Shows the presence of proteins and

free amino acids.

Test for Phenolic Compounds and Tannins

Small quantities of the extracts were taken separately in water and tested for

the presence of phenolic compounds and tannins were carried out with the following

reagents.

1. Dil Ferric chloride solution (5%) – Violet color.

2. 1% solution of gelatin containing 10% sodium chloride-White precipitate

3. 10% lead acetate solution-White precipitate

Test for Flavonoids

1. with Aqueous Sodium Hydroxide Solutions

Blue to violet colour (anthocyanins) yellow colour (flavones), yellow to

orange (flavonones)

2. with Sulphuric Acid

Yellow orange colour (anthocyanins) yellow to orange colour (flavones)

orange to crimson (flavonones)

3. Shinoda’s Test

Small quantities of the extracts were dissolved in alcohol, to them piece of

magnesium followed by Con: hydro chloric acid drop wise added and heated.


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Appearance of magenta color shows the presence of flavonoids. The results of

phytochemical test are presented in table 4 & 5.

5.3. INVITRO CYTOTOXICITY STUDIES

Tissue culture has been used to screen many anticancer drugs, as there is

clear correlation between the in vitro and in vivo activities of potential

chemotherapeutic agents. There is scientific justification for cytotoxicity testing in

tissue culture, since animal models are in many ways inadequate for predicting the

effects of chemicals on humans since there are many metabolic differences between

species. Cytotoxicity studies involve the analysis of morphological damage or

inhibition of zone of outgrowth induced by the chemicals tested (Table 6).

Assay for Proliferation Studies

5.3.1. Trypan blue dye exclusion assay [121]

Principle

Trypan blue is a vital stain used to selectively colour dead tissues or cells

blue. It is a diazo dye. Trypan blue is recommended in dye Exclusion procedures for

viable cell counting based on the principle that live (viable) cells actively pump out

the dye by efflux mechanism where as dead (non-viable) cells do not. So in this

assay white transparent cells are viable cells and blue cells taking up the dye are

dead cells.

Cell line used: HepG2 - Human Liver Cancer cells

Procedure:

1. Cell suspension was prepared in a medium.


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2. Transferred 700 l of a cell suspension to 24 well plate and incubate 24 hrs

in 5% CO2. After incubation add 300 l of drug with varying concentration

of MPP & MPC (62.5-1000 g/ml) and incubate 48 h.

3. 100 l of cell suspension was taken and added 100 l of 0.4% Trypan Blue

Solution to anEppendorf tube and mixed thoroughly. Allowed to stand for 5

to 15 minutes.

4. With the cover-slip in place, using a Pasteur pipette transferred a small

amount of trypan blue-cell suspension mixture to both chambers of a

hemocytometer. Carefully touched the edge of the cover-slip with the pipette

tip and allowed each chamber to fill by capillary action.

5. Starting with chamber 1 of the hemocytometer, counted all the cells in the 1

mm center square and four 1 mm corner squares. Non-viable cells stainedand

appeared in blue color. Viable and non-viable cells were counted separately.

Calculation

Number of non viable cells (Stained)% Inhibition = ------------------------------------------------- X 100

Total Cells (Stained and Unstained)

5.3.2. MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide)

method

Materials for MTT assay

RPMI-1640 media (Himedia, Mumbai, India)

Fetal bovine serum (Gibco’s, USA)

Penicillin-G (Himedia, Mumbai, India)


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Streptomycin (Himedia, Mumbai, India)

Amphotericin-B

Phosphate buffered saline (PBS) (Himedia, Mumbai, India)

Trypsin (Trypsin-EDTA [1x] in HBSS, Gibco’s UK)

Ethylenediamine tetra-acetic acid (EDTA) (Himedia, Mumbai, India)

Trypan blue (Himedia, Mumbai, India)

Dimethyl sulphoxide (DMSO) (Merck India Ltd, Mumbai, India)

SDS lysis buffer (Himedia, Mumbai, India)

MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) (Himedia,

Mumbai, India)

Additional equipments required

CO2 incubator (WTC Binder, Germany)

Laminar air flow cabin (Klenzaids, Chennai, India)

Refrigerated centrifuge (Biofuge Fresco, by Heraeus, Germany)

ELISA-reader (for MTP) (Anthos 2010, Germany)

Deep freezer (Polar Angelantoni Industries, Italy)

Ultrasonic bath (Transonic [460/H], by Elma®, Germany)

Vaccum pump (Zenith [model: PDF-2-2.5], Mumbai, India)

Pipettes (Eppendorf, Hamburg, Germany)

Culture plates (Felcon, Germany)

Centrifuge tubes (Felcon, Germany)


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Eppendorf tubes (Tarsons, Mumbai, India)

Aerosol resistant tips (Tarsons, Mumbai, India)

Flat-bottomed 96-well MTP, tissue culture grade (Tarsons, Mumbai, India)

Procedure

Preparation of antibiotic solutions

Penicillin-G

1 0.61 g (one vial) of penicillin-G was weighed and dissolved in 1 ml of sterile

phosphate buffered saline (PBS).

2 Contents were stirred for 5 minutes.

3 The contents were sterilized with syringe by passing through 0.22 micron

filter aliquot of 5 ml reactions in 15 ml storage vials.

4 Stored at –200 C until use.

Streptomycin sulfate

1. 10 g (one vial) of streptomycin sulfate was weighed and dissolved in 10ml of

sterile PBS.

Amphotericin-B

250 mg of Amphotericin-B was weighed and dissolved in 10 ml of sterile

PBS.

Preparation of Dulbecco’s PBS

1. 9.6 g (one vial) of D-PBS was suspended in 800 ml triple distilled water and

mixed until dissolved.


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2. It was then autoclaved at 1210 C (15 lbl) for 15 minutes.

3. After cooling to room temperature aseptically, sterile 100ml CaCl2 (1mg/ml)

solution and 100 ml MgCl2 (1mg/ml) solution were added and mixed.

Preparation of Lysis buffer

15 g of sodium chloride was weighed and dissolved in 1:1 mixture of

dimethylformamide and distilled water. The final pH was adjusted to 3 with ortho-

phosphoric acid.

Preparation of RPMI-1640 Medium

1 10.39 g (one vial) of RPMI-1640 was weighed in a sterile conical flask.

2 The contents were dissolved in 900 ml of Milli-Q (triple distilled) water by

stirring and the antibiotic solutions were added (100 U/ml Penicillin-G

Sodium, 50 g/ml of streptomycin and 2 g/ml of Amphotericin-B)

3 Once all constituents of the medium were completely added, the pH was

adjusted to 7.2 to 7.5 with 0.1 N HCl.

4 The volume was made up to one liter with triple distilled water.

5 Contents were transferred to the Millipore filtration kettle (Duran,

Germany) supported by 0.22 microns membrane filter, kept in laminar flow

hood that was connected to the outlet by negative pressure pump and

filtered.

6 Pressure was adjusted so that the flow rate of medium from the filtration

unit is 10-12 min/L i.e., 100 ml/min.


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7 5 ml of the sample is taken in T25 tissue culture flask; kept for 24 hours in

CO2 incubator for sterility check.

8 100 ml of sterile Fetal Bovine Serum was added to 900 ml medium.

9 Reconstituted medium was checked for sterility by transferring 5ml of

medium into a T25 tissue culture flask and incubated for 24 hours in CO2

incubator.

Trypsinization

1 The media was aspirated from each flask, being sure to change pipettes

between cell lines to prevent cross contamination.

2 Monolayer was rinsed with 5-10 ml PBS to remove traces of serum and the

rinsing solution was aspirated.

3 1 ml of 0.25 % trypsin-EDTA (Himedia) was added to each flask and

spread evenly over cell monolayer and depending on cell type, the flask was

either placed in hood or in incubator for 2-5 minutes.

4 The flask was gently ‘tapped’ for dislodging the cells.

5 Then the cells were resuspended in 8 ml of the medium containing serum to

stop the action of the trypsin. Gentle pippetting was carried out up and

down for breaking up the clumps.

6 Cell suspension was transferred to a properly labelled 15 ml centrifuge tube.

7 The tubes were centrifuged at 1000 rpm for 5 minutes.

8 The pellet was resuspended in 5-10 ml of medium depending upon the size

of the pellet or cell number.


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MTT assay [122]

Principle

MTT[(3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide] measures

the metabolic activity of the viable cells. The assay is non-radioactive and can be

performed entirely in a microtiterplate (MTP). It is suitable for measuring cell

proliferation, cell viability or cytotoxicity. The reaction between MTT and

‘mitochondrial dehydrogenase’ produces water-insoluble formazan salt. This

method involves culturing the cells in a 96-well microtiterplate, and then incubating

them with MTT solution for approximately 2 hours. During incubation period,

viable cells convert MTT to a water-insoluble formazan dye. The formazan dye in

the MTP is solubilized and quantified with an ELISA plate reader. The absorbance

directly correlates with the cell number. This is applicable for adherent cells cultured

in MTP.

Materials

RPMI-1640, (Himedia, Mumbai India)

TRYPSIN-0.25% ( Gibcos USA)

FBS(Fetal bovine serum) ( Gibcos USA)

MTT 4mg/ml (Himedia)

DMSO (Emerck India)

Lysis buffer (15%SLSin 1:1DMFand water)

Composition of RPMI; 9.54 g/lit, 10%FBS, 2000mg sodium bicarbonate, 250 l each

of penicillin (60mg/ml), streptomycin (100mg/ml), Amphotericin (200mg/ml)

Cell lines used: HepG2 - Human Liver Cancer cells


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Method

0.1ml of the cell suspension (containing 1x105cells) and 0.1ml of the MPP

& MPC (31.25-500 g/ml) in DMSO such that the final concentration of DMSO in

media is less than 1%) were added to the 96 well plates and kept in carbondioxide

incubator with 5% CO2, at 370 C for 72 hours. Blank contains only cell suspension

and control wells contain 1% DMSO and cell suspension

After 72 hours, 20 l of MTT was added and kept in corbondioxide incubator

for 2 hours followed by 80 l of lysis buffer (15%SLSin 1:1 DMF and water). The

plate was covered with aluminum foil to protect it from light. Then the 96 well

plates are kept in rotary shaker for 8 hours

After 8 hours, the 96 well plates were processed on ELISA reader for

absorption at 562nm. The readings were averaged and viability of the test samples

were MPP & MPC with DMSO control (Table 7)

The percentage growth inhibition was calculated using the following formula

Mean OD of Individual Test Group% Growth Inhibition = 100 - ------------------------------------------- X 100

Mean OD of Control Group

5.3.3. Apoptosis

Apoptosis, or programmed cell death, is a highly conserved, tightly

controlled cell suicide process that is regulated by many different intracellular and

extracellular events to ablate neoplastic cells in normal physiological functions.

Apoptosis is controlled by two potential pathways, the mitochondrial pathway and

the death receptor pathway. The mitochondrial pathway is characterized by the loss

of mitochondrial transmembrane potential and release of cytochrome c [123]. The

death receptor pathway is mediated by serial activation of Fas [a cell surface death


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receptor of the tumor necrosis factor (TNF) family of cytokines]. Apoptosis as an

intrinsic suicide serves to remove excess, damaged or infected cells in metazoans.

It's known that apoptosis is characterized by a variety of morphological and

biochemical events, including phosphatidylserine (PS) externalization, chromatin

condensation, genomic DNA fragmentation, and plasma-membrane blebbing

[124,125].

It occurs under physiological conditions (eg, tissue and organ development,

tissue maintenance),anddysregulated apoptosis has been associated with

autoimmune disease and cancer (126,127). Toxic stimuli capable of inducing

apoptosis in susceptible cells include irradiation, DNA-damaging drugs, and

activation of the Fas antigen (126-128). The importance of understanding apoptosis

is underscored further by the fact that the “therapeutic index” of different

antineoplastic therapies may correlate with the differential capacity of tumor and

normal cells to undergo apoptosis (129). “Necrosis” is a form of cell death that

differs from apoptosis (130). Such death usually results from overwhelming damage

to cells, leading to their death without the involvement of a genetically encoded

suicide program (131).

Hoechst staining and photomicroscopy

To analyse the morphological apoptotic changes, 1 X 105 cells seeded in 96-

well plates (370C, 5%CO2), when logarithmic growth phase of cells was reached,

the extractsMPP&MPCwith final concentration of 500 µg/ml or 0.1% DMSO

(negative control) was added, respectively. After 48 h the cells were washed in

phosphate-buffered saline (PBS) and stained for 10 min at room temperature in PBS

containing 40% paraformaldehyde and 10 mg/ml Hoechst 33258. Human Liver

Cancer cells (HepG2) cells for Hoechst staining were grown on sterilized cover slips

and processed as described (132), with modifications. Briefly, after washing one


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time with PBS, cells were fixed with 3.7% formaldehyde in PBS for 10 min, washed

one time with PBS, stained with 0.4 mg/ml Hoechst (Molecular Probes, Eugene,

OR) in PBS for 15 min, washed two times with PBS, and then one time with water.

Cover slips were then air-dried and mounted with Slow Fade (Molecular Probes)

mounting media. Morphological evaluations of nuclear condensation and

fragmentation were performed immediately after staining by means of fluorescent

microscope (Olympus, Japan) at 550 nm of emission.

5.4. DNA BINDING ASSAY

Deoxyribonucleic acid plays an important role in the life process because it

carries heritage information and instructs the biological synthesis of proteins and

enzymes through the process of replication and transcription of genetic information

in living cells. Studies on the binding mechanism of some small molecules with

DNA have been identified as one of the key topics during the past few decades

[133,134]. Moreover it is of great help to understand the structural properties of

DNA, the mutation of genes, the origin of some diseases, the action mechanism of

some antitumour and antivirus drugs and, therefore, to design new and more

efficient DNA targeted drugs to deal with genetic diseases. Anticancer drugs interact

with DNA in many different ways. These include intercalation, non-covalent groove

binding, covalent binding/cross-linking, DNA cleaving and nucleoside-analog

incorporation [135]. As a result of complex formation occurring between DNA and

drug, the thermo dynamic stability and the functional properties of DNA change

[136]. Understanding how complexation affects both the structural and mechanical

properties of DNA is an important step towards elucidating the functional

mechanism of binding agents and may also.

Numerous biological experiments have demonstrated that DNA is the

primary intracellular target of anticancer drugs due to the interaction between small


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molecules and DNA, which cause DNA damage in cancer cells, blocking the

division of cancer cells and resulting in cell death.[137-139] Of these studies, the

interaction of plant extract with DNA has gained much attention. This is due to their

possible application as new therapeutic agents and their phytochemical properties

which make them potential probes of DNA structure and conformation.[140,141]

In order to develop new antitumor drugs which specifically target DNA, it is

necessary to understand the different binding modes a complex is capable of

undertaking. Basically, plant extract interact with the double helix DNA in either a

non-covalent or a covalent way. The former way includes three binding modes:

intercalation, groove binding and external static electronic effects. Among these

interactions, intercalation is one of the most important DNA binding modes as it

invariably leads to cellular degradation. It was reported that the intercalating ability

increases with the planarity of ligands.[142,143].Additionally, the coordination

geometry and ligand donor atom type also play key roles in determining the binding

extent of complexes to DNA [144].

An understanding of the modes of binding of MPP and MPC extracts to

DNA is required to illustrate the principles governing the DNA recognition by such

functional molecules, that is, the factors that decide the affinity and specificity of the

complexes for DNA base sequence. Cationic complexes have been found to both

intercalate into DNA and bind non-covalently in a surface-bound groove-bound

fashion [145]. To assess the mode of DNA binding have been employing several

spectroscopic, electrochemical and other techniques.

Spectrophotometric methods

A compound, when being united to the DNA, modifies its phantom of

absorption, since it undergoes modifications in his electronic structure. In the

absorption phantom displacements in the maximums with respect to binding take


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place free. This one can be by displacement to greater bathochromic effect,

wavelengths, along with a diminution of the molar extinction coefficient. These

spectral change sallow knowing aspects like the binding constant of the complex

[146]. In case of substances, which are non-absorbing, this technique cannot be

employed.

Electrochemical approach

One of the practical applications of electrochemistry is the determination of

electrode redox processes. Due to the existing resemblance between electrochemical

and biological reactions it can be assumed that the oxidation mechanisms taking

place at the electrode and in the body share similar principles[147,148].

Electrochemical investigations of nucleic acid binding molecules–DNA interactions

can provide a useful complement to the spectroscopic methods, e.g.

spectroscopically inactive species, and yield information about the mechanism of

intercalation and the conformation of anticancer drug–DNA adduct [149]. In recent

years, there has been a growing interest in the electrochemical investigation of

interaction between anticancer drugs and DNA. The recent developments of DNA

biosensors have attracted substantial research efforts directed toward clinical

diagnostics as well as forensic and biomedical applications. Electrochemical DNA

biosensors enable us to evaluate and predict anticancer drugs–DNA interaction.

Biosensors are small devices, which utilize biological reactions for detecting

target analytes [150]. A typical biosensor construct has three features a recognition

element, a signal transducing structure and an amplification/processing element.

Various transduction mechanisms such as electrochemical, optical, thermal and

piezoelectric have been employed [151].

There are two types of biosensors depending on the nature of recognition

element. Bio affinity devices rely on the selective binding of the target analyte to a


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surface confined ligand partner (e.g. antibody and oligonucleotide). In contrast, in

bio catalytic devices, an immobilized enzyme is used for recognizing the target

substrate. For example, sensor strips with immobilized glucose oxidase have been

widely used for personal monitoring of diabetes [152].Electrochemical DNA

biosensors comprise a nucleic acid recognition layer, which is immobilized over an

electrochemical transducer. The role of the nucleic acid recognition layer is to detect

the changes occurred in the DNA structure during interaction with DNA-binding

molecules or to selectively detect a specific sequence of DNA. The signal transducer

must determine the change that has occurred at the recognition layer due to the

binding molecules or due to the hybridization; converting this into an electronic

signal which then be relayed to the end user [153].Observing the electrochemical

signal related to DNA–DNA interactions or DNA–drug interactions can provide

evidence for the interaction mechanism, the nature of the complex formed, binding

constant, binding site size and the role of free radicals generated during interaction

in the drug action.

These investigations form a theoretic guide for the design of new anticancer

drugs and chemical treatments of tumor and virus. They are also very valuable for

probing the mechanism of the interaction between anticancer drugs and DNA and

establishing convenient methods to effectively choose specific anticancer drug.

Experimental methodology

Chemicals

All reagents and chemicals were procured from Merck, Mumbai, India. Solvents

used for electrochemical and spectroscopic studies were purified by standard

procedures [154]. DNA was purchased from Bangalore Genei (India). Agarose

(molecular biology grade), ethidium bromide (EB) were obtained from


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Sigma,St.Louis (USA). Tris (hydroxymethyl)amino methane-HCl (Tris–HCl) buffer

solution was prepared using deionized, sonicated triply distilled water.

Experiments

All the experiments involving the interaction of MPP and MPC with Calf

thymus (CT) DNA were carried out in Tris–HCl buffer (50 mMTris–HCl, pH 7.2)

containing 5% ethanol at room temperature. A stock solution of CT DNA was

prepared by dissolving the CT DNA in the Tris-HCl buffer. Solutions of CT DNA

in the above buffer gave a ratio of UV absorbance at 260 and 280 nm, A260/A280 of

1.87, indicating that the CT DNA was sufficiently free from protein [155]. The CT

DNA concentration per nucleotide was determined by absorption spectroscopy at

260 nm using the molar absorption coefficient 260 (6600 M-1 cm-1) [156].

DNA binding experiments

5.4.1. Absorption spectroscopic studies

Electronic absorption spectra were measured on a Shimadzu UV-1601

spectrophotometer in 5 mMTris-HCl buffer (pH 7.1) containing 50 mMNaCl at

room temperature. MPP & MPC were dissolved in absolute ethanol at a

concentration of 5 × 10-3 M. Working solutions were prepared by dilution of the PP

in the absolute ethanol in 5mM Tris-HCl buffer to concentration of 50 M.

Absorption titration experiments were performed by maintaining the extract

concentration as constant at 50 µM while varying the concentration of the CT DNA

within 0 to 400 µM. While measuring the absorption spectra, equal quantity of CT

DNA was added to both the extract solution and the reference solution to eliminate

the absorbance of CT DNA itself. From the absorption data, the intrinsic binding

constant Kb was determined from the following equation (1):

[DNA]/( a f) = [DNA]/( b f) + [Kb b f)]–1 ------------------ (1)


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where a, f, b correspond to Aobsd/[extract], the extinction coefficient for the

free extract, and the extinction coefficient for the extract in the fully bound

form,respectively. A plot of [DNA]/( a f) versus [DNA], where [DNA] is the

concentration of CT DNA in base pairs, gives Kb as the ratio of slope to intercept.

5.4.2 Viscosity measurements

Viscosity experiments were carried on an Ostwald viscometer, immersed in a

thermostated water-bath maintained at a constant temperature at 30.0 ± 0.1°C. DNA

samples of approximately 0.5 mM were prepared by sonicating in order minimize

complexities arising from DNA flexibility [157]. Flow time was measured with a

digital stopwatch three times for each sample and an average flow time was

calculated. Data were presented as ( 0)1/3 versus the concentration of the MPP &

MPC, where is the viscosity of DNA solution in the presence of complex, and 0 is

the viscosity of DNA solution in the absence of complex. Viscosity values were

calculated after correcting the flow time of buffer alone (t0), = (t – t0)/ t0 [158].

5.4.3 Electrochemical methods

Cyclic voltammetric study was performed on a CHI 620C electrochemical

analyzer with three electrode system of glassy carbon (GC) as the working electrode,

a platinum wire as auxiliary electrode and Ag/AgCl as the reference electrode. All

the voltammetric experiments were carried out in single-compartment cells of

volume 5-15 mL. Solutions were deoxygenated by purging with N2 prior to

measurements. Increasing amounts of CT DNA were added directly in to the cell

containing the MPP &MPCsolution (5 X 10-3 M, 5 mMTris-HCl/50 mMNaCl

buffer, pH 7.1). The concentration ranged from 0 to 400 M for CT DNA. The

solution in the cuvette was thoroughly mixed before each scan. All the experiments

were carried out at room temperature.


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5.5 ACUTE AND SUB ACUTE TOXICITY STUDY

Principles and Methods

Life of modern man has been greatly improved by the development of

chemicals in various ways. Like good additives, curing diseases, house ware etc.,

has been said “Without chemicals life wouldn’t be same. At the same times we must

be cautions of over expanded use of chemicals, about which we know so little.

Following decades of reckless and unconscious handling of chemicals which

have resulted in several disasters incidence of pollution. Man has recognized the

need for better control of the present use and the future development of chemical

which should chemically tested and retested before reaching the consumer before

making a judgment, the toxicologist needs the knowledge of the chemical primary

and cumulative toxicity and its mutagenic, teratogenic and carcinogenic potential

which can be obtained from animal studies.

There are four factors contributed to the argument for change. The first is

logistical. New toxicology’s approaches must be established to provide minimum

acceptable databases for these materials. Second existing toxicity testing

methodology using whole animal is expensive and time consuming and cheaper can

significantly reduce economic border. Third, the development of a creative testing

methods which encompass the three R’s (Refinement, reduction, and replacement)

and there incorporation into accepted testing practices. Finally dramatic advances in

biotechnology both on the area of cell, tissue, and organ culture as well as bio-

analytical technique.

Toxicity is defined as any harmful effect of a chemical or a drug on a target

organism. Acute and sub chronic toxicities have been defined by various experts.

The organization for economic corporation and development (OECD) panel of

experts [159] defines acute toxicity as “The adverse effect occurring within a short


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time of (oral) administration of a single dose of a substance or multiple doses given

within 24 hrs. And sub chronic toxicity as “ the advance effects occurring as result

of the repeated daily (oral) dosing of a chemical to experimental annual for part (not

exceeding 10 %) of the life span. Although opinions different on the length of

exposure. Exposure in a sub chronic study generally from 1-3 months. The National

Academy of science (NAS) defines sub chronic exposure from a few days to 6

months [160]. When these times are translated terms of human exposure, acute

toxicity represents life threatening crises of accidental catastrophes and over doses,

suicidal attempts chronic toxicity, on the other hand, represents daily ingestion or

additive or agricultural chemical residues food.

Objectives of Toxicity Testing:

The purpose of toxicity testing is to provide adequate database to make

decisions concerning the toxicology properties of chemical and commercial

products. In some situations, the purpose is to decide whether a material will be safe.

Under the conditions of expected use in other situations, the objective is to establish

the safe limits in condition of use. This process is called hazard evaluation and

would contribute to the introduction of new industrial chemicals and household

products.

Toxicological information which is used in the decision – making process

can be derived from various sources:

1. Historical database

2. Structure – Activating relationship

3. In vivo toxicological testing data from whole animal testing

4. In vivo Toxicological testing data from clinical trials, human volunteers

studies and / or epidemiological studies, and


56

5. In vitro toxicological testing data.

It is mainly focused mainly on the contribution in vitro toxicity testing can

make in the toxicological decision. With respect to the type of testing, In vitro

toxicity test can be classified in to three categories.

(1) Screening test

(2) Adjunct test, and

(3) Replacement test.

Screening test provides preliminary data, for definitive testing, whether in

vivo or vitro. Adjunct test provide information which directly taking in to

consideration in to wakening or regulatory decisions. Finally sufficient information

to make on the basic of another existing test.

The second scheme to categorise the test based on the level of toxicological

information.

(1) Toxicity potential

(2) Toxicity potency

(3) Hazard

Toxicity potential are designed to give a qualitative evaluation (often yes /

No response).The next higher level of test are those which can directly measure

relative potency of test materials and thus rank materials as to their relative toxicity.

Final test, which tests hazard i.e., the probability of given toxicological response in

an organism as a function of exposure to provide the highest level of toxicological

information.


57

The 28 Day Sub acute Oral Toxicity Study:

It gives the valuable information on the cumulative toxicity of a substance,

target organs, physiological changes and metabolism of a compound at low dose for

a prolonged exposure. A wide variety of adverse effect can be detected. The result

from such studies can provide information’s which will aid in selecting dose level.

For chronic, reproductive and carcinogenicity studies.

The long term safety level of a compound can be predicted from acute or

shorter than sub chronic studies [161, 162]. Acutely non-toxic compounds may be

toxic from prolong exposure, even at low dose level, due to cumulation, changes in

enzyme level and disruption of physiological and biochemical homeostasis.

Oral and inhalation sub chronic studies are generally carried out in three

months. In shorter live animals (Rodents) and one year in longer lived animals

(Dogs and Monkeys). Sub chronic plural studies are usually performed in one month

or tests. The common routes of administration employed in sub chronic toxicities

studies are oral, dermal and respiratory.

Oral administration of a test substance can be carried out by gavage in

capsules or in diet or drinking water 3-4 dose levels of the substance should be

employed. At the end of the dosing period and before, daily observations such as

periodic physical examination, monitoring of body weight and food consumption

and analysis of hematology, biochemistry and urinary parameters have been

analysed. When the animals have been sacrificed at the end of the study, organ

weights are recorded and histopathalogic evaluation is performed.

In Life Observation and Measurements:

Detailed observation of each animal should be done at least once and

preferably twice daily by the duration of the study. If the recovery groups are


58

included in the study, these animals should be observed for period of time after the

termination of treatment.

Daily, observation should include close scrutiny for changes in fur texture,

skin, eyes, mucous membrane orifices, and clinical sciences of respiratory,

circulatory, and autonomic and central nervous system and behavioral changes.

Special attention should be given to examine any palatable mass for tumor

incidence. All signs should be recorded, and if details are found during the study.

They should be necrotised within a short period of time (16 hr of death) and the

tissue placed in 10 % neutral buffered formalin or another appropriate fixative,.

Haematology:

Haematology analysis should include haematocrit and hemoglobin

concentration, red blood cell count, white blood cell count, morphology of the red

blood cells, a measure of clotting ability such as prothrombin and thromboplastin

time or platelet count. These should be determined prior to treatment, once during

the study and at the terminal sacrifices. However one must be cognizant of the fact

that the retro orbital bleeding can interfere with ophthalmological examination.

Biochemical Measurement:

Biochemical analysis from the blood sample includes electrolyte balance

carbohydrate and protein metabolism and organ functional tests. It is extremely

important in the establishment for target organ toxicity. To collect the blood sample

animals or starved overnight depending upon the type of animals. However 12 to 16

hr fast is adequate for most animals. For animal such as mice with a high

metabolism rate, 3-4 hrs starving may be sufficient.

Good clinical chemistry analysis will include most, if not all sub chronic

study fasting glucose, serum GOT (AST) and GPT (ALT), alkaline phosphatase,


59

gamma glutamyltranspeptidase, blood urea nitrogen (BUN) total protein, albumin,

globulin, total bilirubin, blood creatine, cholesterol acid / base balance, lipids,

Cholinesterase (Plasmas, red blood cell, and / or brain and other biochemical

parameter which facilitate the definition of adverse effects.

Pathological Examination

All tissues from the high dose and control animals and any tissues with

lesions in other groups should be further examined microscopically. If it is having

microscopic lesions, the tissues should be examined from animals in lower dose

groups. Because it is generally expensive and time consuming.

Analysis and Interpretation of Data

With early acute to computers most daily data are stored and analysed

automatically all quantitative and qualitative data, including histopathological

finding, should be processed with appropriate statistics.

Food consumption data along with body weight data may include change in

appetite or change in the efficiency of food utilization by the body, Elevated level of

serum plasma enzymes which have a specific tissues origin are of great value it

identifying target organs. For example out of normal range and statistically

significantly increase in serum glutamic pyruvate tranaminase (SGPT) may suggest

hepatotoxicity. The level of serum alkaline phosphatase is elevated in certain

orleologic disease (e.g. estrogenic sarcoma, rickets) or biliary disorder such as

obstructive jaundice and cirrhosis. Elevated acid phosphatase activity has been

related to carcinoma of the prostate.

Any significant deviation may indicate malfunction of these regulatory

mechanisms, which may be related to the chemical exposure. Acid / base balance is

regulated by renal and respiratory systems, and a significant deviation may indicate


60

respiratory or renal problems. Renal damage may be substantiated by an increase in

BUN level, the appearance of stones, calculi protein or epithelial cells in the wire.

Any changes in the morphology of blood cells may indicate blood dyscrasia.

EXPERIMENTAL METHODS OF TOXICITY TESTING

5.5.1. Acute Oral Toxicity Studies

Animals

Adult female Wistar rats (125-150 g) were used for acute toxicity. These

animals were kept in polypropylene cages under identical animal house condition

and provided with standard pellet and water ad libitum. Each cage contained 3 rats

of the same sex with a bedding of husk, and 12-hour light/dark cycles were

provided. Environmental conditions were maintained at a temperature of 22°C ± 2°C

and a relative humidity of 60% ± 10%.

Toxicity studies conducted as per internationally accepted protocol drawn

under OECD No 420 guidelines. Test drugs used weremethanolic extracts of P.

polyphyllus (MPP)andP.cineraria(MPC). The overnight fasted rats were divided

into 6 groups, each group consisting of 3 animals. The MPP& MPCextracts were

given separately in various doses (50, 500, 1000, 2000 mg/kg) by oral route. After

administration of the extracts, the animals were observed continuously for the first

two hours and 24 hrs to detect changes in the behavioural responses and also for

tremors, convulsion, salivation, diarrhoea, lethargy, sleep and coma and monitored

for any mortality.

5.5.2.SUB ACUTE TOXICITY STUDIES

Experimental design

Seven groups of rats were used in sub-acute toxicity study of MPP &

MPCandeach group consists of 6 rats. The groups and treatment were designed as

follows


61

Group 1 - Control treated with saline (2 ml/kg, p.o.)

Group 2 - MPP (200 mg/kg, p.o.)



Group 5 - MPC (200 mg/kg, p.o.)



Seven groups of rats received MPP& MPC extracts at dose of 200, 500 and

1000 mg/kg (low dose, intermediate dose and high dose) orally for 28 days. The

group which served as control received equivalent quantity of normal saline orally.

Animals were observed for signs and symptoms, behaviour alteration, food and

water intake and body weight changes. All animals were observed twice daily for

mortality during the 28 day period of study. The weight of each rat was recorded on

day 0 and at weekly intervals throughout the course of the study. The group mean

body weights were calculated.

At the end of the 28-day period the animals were fasted overnight. The

following morning, each animal was heparinised and blood samples were collected

from the orbital sinus. The blood sample was collected after 24 h of the last doses of

alcoholic extracts.

Haematological analyses were performed in total blood was collected in

heparinised funnels and tubes with EDTA The relative blood indices like white

blood cell count (WBC), red blood cell count (RBC), haemoglobin (Hb), erythrocyte

sedimentation rate (ESR), platelets, clotting time and packed cellular volume (PCV)

were determined using routine method [163]. In addition, Biochemical analyses

were performed in serum obtained after centrifugation of total blood without

anticoagulants, at 2400 rpm for 15 min. The analysis of blood glucose[164],


62

Cholesterol[165], Serum aspartate amino transferase (AST)[166],Alanine amino

transferase(ALT)[167], Alkaline phosphatase, Total bilirubin[168], Urea and

BUN[169], Total Protein & Albumin[170]and creatinine[171] were estimated in

serum. Urine samples were also collected at the end of the study period and

analyzed for specific gravity, pH, glucose, proteins, ketones, and occult blood. At

the end of 28 days, experiment animals were autopsied and vital organs viz. Liver,

kidney, spleen, lung, heart and brain were removed, weighed and subjected to gross

pathological examination. [172,173]

Since liver and kidney are organs of metabolism and excretion, potentially

toxic agents are likely to affect them. So, portions of these organs were fixed in

buffered 10% formalin and 5 m thick paraffin sections were made and stained with

haematoxylin and eosin [174] for microscopic examination. The results on sub

chronic toxicity studies are presented in Tables 11 to 16 and Fig. No 14.

Statistical analysis

The values were expressed as mean ± SEM. Statistical analysis was

performed by one way analysis of variance (ANOVA) followed by Tukey multiple

comparison tests. P values < 0.05 were considered as significant.

5.6 CHEMOPREVENTIVE ACTION AGAINST DEN INDUCED LIVERCARCINOMA

Animals

Healthy Male Wistar albino rats (6-8 weeks old) were used throughout the

study. The animals were purchased from King Institute of Preventive Medicine,

Chennai-600 034 and Animal House Facility, Ratnam Institute of Pharmacy,

Nellore, India, and maintained in a controlled environmental condition of

temperature (23 2 C) and relative humidity (50-70%) on alternatively 12 hr

light/dark cycles. All animals were fed standard pellet diet (Gold Mohor rat feed,


63

M/s. Hindustan Lever Ltd., Mumbai) and water ad libitum. This research work on

Wistar albino Male rat was sanctioned and approved by the Institutional animal ethical

committee (IAEC. No. 02/2010).

Sources of Chemicals

N-NitrosoDethylamine [DEN], bovine serum albumin, 2,4,6-Trinitro benzene

sulfonate, reduced glutathione, ribonuclease were obtained from Sigma Chemical

Company, St. Louis, MO, USA.

Ascorbic acid, adenosine triphosphate, 1-amino 2-naphthol 4-sulphonic acid

(ANSA) and glutathione were obtained from Sisco Research Laboratories, Mumbai,

India. 1-choloro-2, 4-dinitrobenzene (CDNB) and 5,5’-dithionitrobenzoic acid

(DTNB) were obtained from S.D. Fine Chemicals, Mumbai, India. All other

chemicals used were of analytical grade obtained from Sisco Research Laboratories

Pvt. Ltd., Mumbai, India and Glaxo Laboratories, CDH division, Mumbai, India.

Experimental design

The rats were divided into 6 groups, each group consisting of six animals.

Liver tumor was induced in group 2, 3, 4, 5 and 6 with single intraperitoneal

injection of DEN at a dose of 200 mg/kg body weight in saline. Two weeks after

DEN administration, the carcinogenic effect was promoted by 0.05% phenobarbitol,

which was supplemented to the experimental animals through drinking water for up

to 20successive weeks [175].

Group I - Control animals treated with distilled water orally for 20 weeks

Group II - N-NitrosoDiethylamine (DEN) (200 mg/kg b. wt, by i.p) treated

at single dose.


64

Group III - DEN (as in group II) and MPP (200 mg/kg b.wt, dissolved in

0.3% cmc) simultaneously for 20 weeks from the first dose of

DEN (as in Group II)

Group IV - MPP (400 mg/kg b.wt, dissolved in 0.3% cmc) (as in Group III)

simultaneously for 20 weeks from the first dose of DEN (as in

Group II)

Group V - DEN (as in group II) and MPC (200 mg/kg b.wt, dissolved in

0.3% cmc) simultaneously for 20 weeks from the first dose of

DEN (as in Group II)

Group VI - MPC (400 mg/kg b.wt, dissolved in 0.3% cmc) (as in Group III)

simultaneously for 20 weeks from the first dose of DEN (as in

Group II)

After the experimental period of 20 weeks, the animals were

sacrificed and the parameters of interest were assayed.

Collection of blood and organs

All the experimental animals were killed by cervical decapitation after the

experimental period. The blood was collected for the separation of serum to

determine blood parameters. The liver was removed, weighed, morphological

changes and tumour incidence were observed. A portion of liver tissues were

homogenized with motor driven Teflon coated homogeniser in ice-cold 0.1M Tris-

HCl buffer pH 7.4 to obtain 10% homogenate.

Assay of liver marker enzymes

Alkaline phosphatase (Ortho phosphoric acid – monoester phospho hydrolase)

Alkaline phosphatase was assayed by the method of Kind (1971).[176]


65

Reagents

1. Carbonate-bicarbonate buffer: 0.1 M pH 10.0

2. Disodium phenyl phosphate: 0.1 M

3. MgCl2: 0.1M

4. Sodium Carbonate: 15%

5. Folin’s phenol reagent

6. Standard: 100 mg of phenol was dissolved in 100 ml of distilled water.

Procedure

The incubation mixture containing 1.5 ml of buffer, 1 ml of substrate and 0.1

ml of MgCl2 were preincubated at 37oC for 10 minutes. Then 0.1 ml of homogenate

was added and incubated at 37oC for 15 minutes. The reaction was arrested by the

addition of 1.0 ml of folin’s phenol reagent. Control without enzyme was also

incubated and the homogenate was added after the addition of folin’s phenol

reagent. Then 1.0 ml of sodium carbonate was added. After 10 minutes the blue

colour developed was read at 640nm using a photochem colorimeter. The enzyme

activity was expressed as µmoles of phenol liberated /min/mg protein under

incubation conditions.

Aspartate amino transferase (L-Aspartate: 2-oxoglutarate amino transferase)

Aspartate amino transferase was estimated by the method of King

(1965).[177]

Reagents

1. Phosphate buffer: 0.1M pH 7.5.

2. Substrate: 1.33gm of aspartic acid and 15 mg of 2- oxoglutarate were

dissolved in 20.5 ml of 1N NaOH and made up to 100ml with buffer.


66

3. 2,4-Dinitropheneyl hydrazine (DNPH): 0.02% 0f DNPH in 1N HCl

4. NaOH: 0.4N

5. Standard: 11mg of sodium pyruvate was dissolved in 100 ml of phosphate

buffer. This contains 1M of pyruvate/ml.

Procedure

One ml of substrate was incubated at 37oC for 10 minutes. Then 0.2ml of

enzyme was added and the mixture was incubated at 37oC for 1 hour. To the control

tubes, the enzyme was added after the reaction and it was arrested by the addition of

1.0 ml of DNPH reagent. The tubes were kept at room temperature for 30 minutes.

Then 5ml of NaOH was added. A set of pyruvate solution standard were also treated

in a similar manner. The colour developed was read at 540nm using a photochem

colorimeter.

The enzyme activity was expressed as µmoles of pyruvate liberated /min/mg

protein under incubation conditions.

Alanine amino transferase (L – Alanine: 2- oxoglutarate amino transferase)

Alanine amino transferase was assayed by the method of King (1965).[177]

Reagents

1. Phosphate buffer: 0.1M pH 7.5.

2. Substrate: 1.78 gm of DL- alanine and 30 mg of 2- oxoglutarate were

dissolved in 20ml of buffer 0.5ml of 1N sodium hydroxide was added and

made up to 100ml with distilled water.

3. DNPH: 0.02% 0f DNPH in 1N HCl


67

4. NaOH: 0.4N

5. Standard: 11mg of sodium pyruvate was dissolved in 100 ml of phosphate

buffer. This contains 1M of pyruvate/ml.

Procedure

1.0 ml of substrate was incubated at 37oC for 10 minutes. Then 0.2ml of

enzyme solution was added. The tubes were incubated at 37oC for 30 minutes. To

the control tubes, enzyme was added after arresting the reaction with 1.0ml of

DNPH reagent. The tubes were kept at room temperature for 20 minutes. Then

5.0ml of 4N NaOH was added and the colour developed was read at 540nm using a

photochemcolorimeter.The enzyme activity was expressed as µmoles of pyruvate

liberated /min/mg protein under incubation conditions.

Assay of -Glutamyltranspeptidase

The activity of -glutamyltranspeptidase was estimated according to the method

of Orlowski and Meister (1965). [178]

Reagents

1. 0.1 M Tris-HCl buffer, pH 8.5

2. Substrate (30.37 mg of L- -glutamyl-p-nitroanilide was dissolved in 10 ml of

water by heating at 50-60 C)

3. Glycyl glycine (13.2 mg/10 ml)

4. p-Nitroaniline


68

Procedure

The incubation mixture contained 0.5 ml of substrate, 1 ml of Tris-HCl buffer

2.2 ml of glycyl glycine, 0.2 ml of homogenate and the total volume was made up to 4

ml with water. After incubation for 30 min at 37 C, the samples were heated at 100 C

for 5 min and centrifuged. The amount of p-nitroaniline in the supernatant was

measured at 410 nm.

The activity of -glutamyltransferase was expressed as mole of p-nitroaniline

formed/min/mg protein.

Estimation of Protein

Protein was estimated by the method of Lowry et al. (1951).[179]

Reagents

1. Alkaline copper reagent

Solution A: 2% sodium carbonate in 0.1 N NaOH

Solution B: 0.5% copper sulphate in 1% sodium potassium tartarate 50 ml of

solution A was mixed with 1 ml of solution B just before use.

2. Folin's phenol reagent (commercial reagent, 1:2 dilution)

3. Bovine serum albumin (BSA).

Procedure

To 0.1 ml of suitably diluted plasma/homogenate/hemolysate, 0.9 ml of water

and 4.5 ml of alkaline copper reagent were added and kept at room temperature for10

min. Then 0.5 ml of Folin’s reagent was added and the colour developed was read after

20 min at 640 nm. The level of protein was expressed as mg/g tissue or mg/dl plasma.


69

ENZYMIC AND NONENZYMIC ANTIOXIDANTS

Assay of superoxide dismutase (SOD)

The activity of superoxide dismutase was determined by the method of

Marklund and Marklund (1974).[180]

Reagents

1. 0.4 mMpyrogallol in air-saturated 0.1 M Tris-cacodylic acid buffer, pH 8.2

containing 2 mMdiethylenetriaminepenta acetic acid (DETPA).

Procedure

The assay mixture contained 1 ml of pyrogallol-Tris-DETPA, 0.2 ml of

homogenate and 0.8 ml of water. The rate of pyrogallol autoxidation is taken from

the increase in absorbance at 420 nm. The activity of SOD was expressed as

units/min/mg protein. One unit of the enzyme is defined as the amount of enzyme,

which inhibits the rate of pyrogallol autoxidation by 50%.

Assay of catalase

The activity of catalase was assayed by the method of Sinha (1972).[181]

Reagents

1. Dichromate/acetic acid reagent (5% solution of potassium dichromate in

acetic acid (1:3))

2. 0.01 M Phosphate buffer, pH 7.0

3. 0.2 M Hydrogen peroxide (H2O2)


70

Procedure

The assay mixture contained 4 ml of H2O2, 5 ml of phosphate buffer and 1 ml

of homogenate. One ml portions of the reaction mixture was withdrawn and blown into

2 ml of dichromate/acetic acid reagent at 1 min intervals. Then the mixture was heated

for 10 min in a boiling water bath. After cooling, the OD was measured at 570 nm.

The activity of catalase was expressed as moles of H2O2 consumed/min/mg

protein.

Assay of glutathione peroxidase (GPx)

The activity of GPx was assayed by the method of Rotrucket al. (1973).[182]

Reagents


2. 0.8 mM EDTA

3. 10 mM Sodium azide

4. 3 mM reduced glutathione

5. 2.5 mM H2O2

6. 10% TCA

7. 0.3 M Disodium hydrogen phosphate

8. DTNB solution (40 mg of DTNB in 100 ml of 1% sodium citrate)

9. Reduced glutathione

Procedure

The reaction mixture consisted of 0.2 ml each of EDTA, sodium azide, H2O2,

0.4 ml of phosphate buffer, 0.1 ml homogenate and was incubated at 37 C at different

time intervals. The reaction was arrested by the addition of 0.5 ml of TCA and the


71

tubes were centrifuged at 2000 rpm. To 0.5 ml of supernatant, 4 ml of disodium

hydrogen phosphate and 0.5 ml DTNB were added and the colour developed was read

at 420 nm immediately.

The activity of GPx was expressed as moles of glutathione oxidized/min/mg

protein.

Assay of glutathione reductase (GR)

The activity of GR was measured by the method of Staalet al. (1969).[183]

Reagents


2. 0.25 M EDTA

3. 12.5 mM oxidized glutathione

4. 3 mM NADPH

Procedure

The reaction mixture containing 1 ml of phosphate buffer, 0.5 ml of EDTA, 0.5

ml of oxidized glutathione and 0.2 ml of NADPH was made up to 3 ml with water.

After the addition of 0.1 ml of homogenate the change in optical density at 340 nm was

monitored for 2 min at 30 sec intervals. The activity of GR was expressed as nmoles of

NADPH oxidized/min/mg protein.

Estimation of reduced glutathione (GSH)

The level of reduced glutathione was measured by the method of Moron et al.

(1979).[184]


72

Reagents

1. 10% TCA

2. 0.6 mM 5, 5 -dithiobis-2-nitrobenzoic acid (DTNB) in 0.2 M sodium phosphate


Procedure

One ml of homogenate was precipitated with 1 ml of TCA and the precipitate

was removed by centrifugation. To 0.5 ml of supernatant, 2 ml of DTNB was added

and the total volume was made up to 3 ml with phosphate buffer. The absorbance was

read at 412 nm. The level of glutathione was expressed as g/mg protein.

Estimation of ascorbic acid

The level of ascorbic acid was estimated by the method of Omayeet al.

(1979).[185]

Reagents

1. 5% TCA

2. DTC reagent (3 g of 2,4-dinitrophenyl hydrazine, 0.4 g of thiourea and 0.05

g of copper sulphate were dissolved in 100 ml of 9 N sulphuric acid)

3. 65% sulphuric acid

4. Ascorbic acid

Procedure

To 0.5 ml of homogenate, 0.5 ml of water and 1 ml of TCA were added, mixed

thoroughly and centrifuged. To 1 ml of the supernatant, 0.2 ml of DTC reagent was

added and incubated at 37 C for 3 hrs. Then 1.5 ml of sulphuric acid was added, mixed


73

well and the solutions were allowed to stand at room temperature for another 30 min.

The colour developed was read at 520 nm. The level of ascorbic acid was expressed

as g/mg protein.

Estimation of vitamin E

The level of vitamin E was estimated by the method of Desai (1984).[186]

Reagents

1. Ethanol

2. Petroleum ether

3. 0.2% 4,6-diphenyl-1,10-phenanthroline in ethanol

4. 0.001 M Ferric chloride in ethanol

5. 0.001 M Ortho-phosphoric acid in ethanol

6. -Tocopherol acetate

Procedure

To 1 ml of homogenate, 1 ml of ethanol was added and thoroughly mixed.

Then 3 ml of petroleum ether was added, shaken rapidly and centrifuged. 2 ml of

supernatant was taken and evaporated to dryness. To this 0.2 ml of

bathophenanthroline was added. The assay mixture was protected from light and 0.2 ml

of ferric chloride was added followed by 0.2 ml of Ortho-phosphoric acid. The total

volume was made up to 3 ml with ethanol. The colour developed was read at 530

nm.The level of vitamin E was expressed as g/mg protein.

5 -Nucleotidase

5 -Nucleotidase was assayed by the method of Lulyet al. (1972).[187]


74

Reagents

1. Tris-HCl buffer 184 mM, pH 7.5: 2.23 g Tris was dissolved in 100 ml of

demonized water and pH was adjusted to 7.5 with HCl.

2. Magnesium sulphate 50 mM: 616.2 mg of MgSO4 was dissolved in 50 ml of

demonized water.

3. Potassium chloride 650 mM: 1.211 g of KCl was dissolved in 25 ml of

demonized water.

4. EDTA 1 mM: 37.23 mg of EDTA was dissolved in 100 ml of demonized

water.

5. TCA 10%.

6. Substrate 40 mM: 5 -Adenosine monophosphate was prepared by dissolving

69.4 mg in 5.0 ml of demonized water.

Procedure

The reaction mixture contained 1.0 ml of Tris-HCl buffer and 0.1 ml each of

magnesium sulphate, potassium chloride, EDTA, substrate and water. The reaction was

initiated by the addition of 0.2 ml plasma or tissue homogenate and incubated at 37 C

for 15 minutes. The reaction was arrested by the addition of 2.0 ml of 10% TCA and

centrifuged. The phosphorus liberated in the supernatant was estimated by the method

of Fiske and Subbaroa (1925). The enzyme activity was expressed as nmoles of

inorganic phosphorus liberated/min/mg protein.

Assay of lactate dehydrogenase

The activity of lactate dehydrogenase was assayed by the method of King

(1965). [188]


75

Reagents

1. 0.1 M Glycine buffer, pH 7.4

2. Buffered substrate (4 g of lithium lactate was dissolved in 75 ml of 0.1N

NaOH and was made up to 200 ml with glycine buffer)

3. NAD+ (5 mg/ml)

4. 0.02% DNPH in 1 N HCl

5. 0.4 N NaOH

6. Pyruvate

Procedure

To 1 ml of buffered substrate, 0.1 ml of serum, 0.2 ml of water and

0.2 ml of NAD+ were added and incubated at 37 C for 15 min. Then 1 ml of DNPH

was added and again incubated for another 15 min at 37 C. Then 0.5 ml of NaOH was

added and the colour developed was read at 420 nm. The activity of lactate

dehydrogenase was expressed as mole of pyruvate liberated/min/mg protein.

AssayofAryl hydrocarbonhydroxylase(AHH)

The AHHassaywas modifiedfromthe methodofMildredetal. (1981).[189]

Reagents

1. 62.5mMTris-HClbuffer

2. 3.75mMMgCl2

3. Bovineserumalbumin

4. 10 mMNADPH

5. 2mMBenzo(a)pyreneinmethanol

6. 1NNaOH


76

Procedure

TheassayusingB(a) pwasperformedinareactionmixturecontaininga total

volume of 1ml which included Tris-HCl buffer, MgCl2: bovineserum albumin

(1:25mg/ml); 0.1 ml supernatant suspension and 50µl of NADPH in Tris-HCl buffer,

with 50 ml of B(a) pinmethanol (0.54mg/ml) added to initiate the reaction. The

reaction was incubated for 10 min in a covered, shaking water bathat 37°C and wast

erminated by adding 1.0 ml acetone and 3.25 ml hexane. The mixture was shakenvig

orously in the dark to extract the B(a) panditsmetabolites. 1 ml sample of the organic

phase was extracted with 3.0 ml of 1N NaOH and the AHH were examined in

spectrophotometer. The enzyme activity was expressed as phenolicmetabolite

formed/min /mgprotein.

Estimation of Adenosine triphosphatase

Na+, K+-ATPase (Adenosine triphosphatase)

Na+, K+-ATPase was estimated by the method of Bonting (1970).[190]

Reagents

1. Tris-HCl buffer 184 mM, pH 7.5.

2. MgSO4, 50 mM

3. KCl, 50 mM

4. NaCl, 600 mM

5. EDTA, 1 mM

6. ATP, 40 mM

7. TCA, 10%

8. Ammonium molybdate, 2.5%

9. ANSA.


77

Procedure

The incubation mixture contained 1 ml of Tris-HCl buffer, 0.2 ml each of

magnesium sulphate, potassium chloride, sodium chloride, EDTA, ATP and the

homogenate. The mixture was incubated at 37 C for 15 minutes. The reaction was

arrested by the addition of 1 ml of 10% TCA, mixed well and centrifuged. The

phosphorus content of the supernatant was estimated as described in phosphorus

estimation.The enzyme activity was expressed as moles of inorganic phosphorus

liberated/min/mg protein.

Ca2+ATPase

The activity of Ca2+ATPase was assayed according to the method of Hjerten

and Pan (1983).[191]

Reagents

1. Tris-HCl buffer 125 mM, pH 8.0

2. CaCl2, 50 mM

3. ATP, 10 mM

4. TCA, 10%.

Procedure

The incubation mixture contained 0.1 ml each of Tris-HCl buffer, calcium

chloride, ATP and enzyme preparation. After incubation at 37 C for 15 minutes, the

reaction was arrested by the addition of 1 ml of TCA. The amount of phosphorous

liberated was estimated as described in phosphorus estimation. The enzyme activity

was expressed as moles of inorganic phosphorous liberated/min/mg protein.


78

Mg2+ATPase

The activity of Mg2+ ATPase was assayed by the method of Ohinishi

et al. (1962).[192]

Reagents

1. Tris-HCl buffer 375 mM, pH 7.6

2. MgCl2, 25 mM

3. ATP, 10 mM

4. TCA, 10%.

Procedure

The incubation mixture contained 0.1 ml each of Tris-HCl buffer, magnesium

chloride, ATP, and enzyme preparation. The reaction mixture was incubated at 37 C

for 15 minutes. The reaction was arrested by the addition of 1 ml TCA. The liberated

phosphorous was estimated as described in phosphorous estimation. The enzyme

activity was expressed as moles of inorganic phosphorous liberated/min/mg.

Estimation of Glycoproteins

Acid hydrolysis of tissues for estimation of hexose and hexosamine

To the weighed amount of defatted tissues, 2 ml of 4 N HCl was added and the

mixture was refluxed at 100 C for 4 hrs in a test tube with suitable marble lids. The

hydrolysate was then neutralised with NaOH. Aliquots of the neutralised samples were

taken for the analysis.

Estimation of hexose

Hexose level was estimated by the method of Niebes (1972).[193]


79

Reagents

1. Orcinol - Sulphuric acid reagent

Solution A : 60 ml of concentrated sulphuric acid was mixed with 40 ml of

distilled water.

Solution B: 1.6 g of orcinol (recrystallized from benzene) was dissolved in

100 ml of distilled water. 7.4 ml of solution A was mixed with 1 ml of solution

B before use.

2. Standard: 5 mg of each of galactose and mannose were dissolved in 100

ml of distilled water (100 g/ml).

Procedure

0.5 ml of the neutralized solution was made up to 1 ml with distilled water and

added 8.5 ml of ice-cold orcinol reagent. The mixture was heated at 80 C for 15

minutes, cooled and left in the dark for 25 minutes for colour development. Then the

absorbance was read at 540 nm in a Photochem colorimeter. Standard solutions

containing 25-100 g of hexose were treated in a similar manner. The hexose content

was expressed as mg/g wet tissue.

Hexosamine

Hexosamine content was estimated by the method of Wagner (1974).[194]

Reagent

1. Acetyl acetone reagent

Solution A: Trisodium phosphate 0.1 M

Solution B: Potassium tetra borate 0.5 N


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2. 5 ml of acetyl acetone was added to mixture of solution A and solution

B in the ratio of 98:2 (v/v)

3. Ehrlich's reagent

320 mg of p-dimethyl aminobenzaldehyde was dissolved in 21 ml of

isopropanol and 3 ml of concentrated HCl

4. Standard: Galactosamine was prepared in the concentration range of

100 g/ml in water.

Procedure

0.5 ml of the neutralized sample was made up to 1 ml with distilled water.

Standard galactosamine (in the range of 10-40 g) was also made up to 1 ml with

distilled water. 0.6 ml of acetyl acetone reagent was added to all the tubes and heated in

a boiling water bath for 30 minutes. After cooling, 2 ml of Ehrlich's reagent was added

and the contents were shaken well. The pink colour developed was measured at 540

nm against the reagent blank. Hexosamine content was expressed as mg/g wet tissue.

Sialic acid

The weighed amounts of defatted tissues were hydrolysed with 1.0 ml of 0.1 N

sulphuric acid at 80 C for 60 minutes to release sialic acid bound to the proteins. The

solution was neutralised with NaOH. Sialic acid level was determined by the method of

Warren (1957).[195]

Reagents

1. Periodic acid, 0.25 M.

2. 4% Sodium metaarsenite


81

3. Thiobarbituric acid

4. Acidified butanol

5. Standard sialic acid: 10 mg of N-acetyl neuraminic acid was dissolved in

100 ml of distilled water.

Procedure

0.5 ml of the neutralised sample was taken along with the standards

(in the range of 10-40 g). Blank contained 0.5 ml of 0.1 N sulphuricacid. 0.25 ml of

periodate was added to all tubes at 37 C. After 30 minutes, 0.25 ml of arsenite solution

was added to inhibit the reaction. Contents were mixed well and 2 ml of thiobarbituric

acid was added and the tubes were heated in a boiling water bath for 6 minutes. After

cooling, the pink colour developed was extracted into 5 ml of acidified butanol phase

and was measured at 540 nm against a reagent blank. Thesialic acid content was

expressed as mg/g wet tissue.

ISOLATION OF MITOCHONDRIA AND MICROSOMES

The mitochondria were isolated by the method of Johnson and Lardy (1967)

[196] and microsomes by Omuraet al. (1964). [197]

Reagents

1. 0.05 M Tris-HCl buffer, pH 7.4 containing 0.25 M sucrose

2. 0.05 M Tris-HCl buffer, pH 7.4 containing 0.15 M potassium chloride

A 10% (w/v) homogenate was prepared in 0.05 M Tris-HCl buffer, pH 7.4

containing 0.25 M sucrose and centrifuged at 600 x g for 10 min. The supernatant

fraction was decanted and centrifuged at 15,000 x g for 5 min. The resultant

mitochondrial pellet was then washed and resuspendedin the same buffer. The post


82

mitochondrial fraction was further centrifuged at 105,000xgfor 60 min. The

microsomal pellet was suspended in 0.05 M Tris-HCl buffer, pH 7.5 containing 0.15 M

KCl.The purity of mitochondrial and microsomal fractions were assessed by measuring

the activities of succinate dehydrogenase and glucose-6-phosphate dehydrogenase

respectively.

ESTIMATION OF PHASE-I ENZYMES

Estimation of Cytochrome P450

Cytochrome P450 was estimated by the method of James et al (2008).[198]

Reagents


2. Sodium dithionate

3. Carbon monoxide

Procedure

Microsomes suspended in phosphate buffer (4 mg/ml) were reduced by a few

milligrams of solid sodium dithionate. Then 1 ml of water saturated with carbon

monoxide was added. The absorbances of the samples were scanned at 400-500

nm.The level of cytochrome P450 was expressed as nmole/mg protein based on molar

extinction coefficient.

Estimation of Cytochrome b5

The amount of cytochrome b5 was measured by the method of James et al

(2008).[198]


83

Reagents


2. 0.4 mM NADH

Procedure

To the microsomal suspension, containing 4 mg of protein/ml in phosphate

buffer, 1 ml of NADH was added. The absorbance spectrum between 400-500 nm was

read against the blank containing microsomal suspension alone. The level of

cytochrome b5 was calculated using the molar extinction coefficient of 185 mM/cm

between 424-409 nm and was expressed as nmoles/mg protein.

Assay of NADPH-cytochrome P450reductase

The activity of NADPH-cytochrome P450reductase was assayed by the method

of James et al (2008).[198]

Reagents


2. 0.045 mM NADPH

3. 1 mM Potassium cyanide

4. 0.05 mM Cytochrome c

Procedure

The assay mixture containing 2.5 ml of buffer, 0.2 ml of potassium cyanide,

0.1 ml of cytochrome c was mixed gently. After 3 min, 0.1 ml of NADPH was added

and the change in optical density was recorded at 30 seconds intervals for 3 min at

550 nm.The activity of NADPH-cytochrome P450reductase was expressed as nmoles of

cytochrome C reduced/min/mg protein.


84

PHASE-II ENZYMES

UDP-GlucuronylTransferase

The UDP-Glucuronyltransferase was estimated by the method by Reitman and

Frankel(1957).[199]

Reagents

1. Tris-HCl buffer 1 M, pH 7.4: 12.11 g of Tris was dissolved in 100 ml of

water and pH was adjusted to 7.4 with HCl.

2. Triton X-100 (0.25%)

3. MgCl2 50 mM: 47.5 mg of MgCl2 was dissolved in 10 ml of water.

4. p-Nitrophenol 5 mM: 0.84 mg of p-nitrophenol was dissolved in 1 ml of

water.

5. UDP-Glucuronic acid 30 mM: 19.38 mg of UDP-glucuronic acid was

dissolved in 1 ml of water.

6. TCA 5%.

7. NaOH 2 M.

Procedure

The incubation mixture containing 0.5 ml buffer, 0.2 ml Triton X-100, 0.05 ml

MgCl2, 0.05 ml p-nitrophenol, 0.18 ml water and 0.1 ml enzyme was incubated at 37 C

for 2 minutes. Then 0.1 ml of UDP-glucuronic acid was added. Then 0.1 ml aliquot of

this mixture was arrested at 0, 10 and 15 minutes with TCA and centrifuged. To one ml

of the supernatant, 0.25 ml of NaOH was added and read at 450 nm using a

photochemcolorimeter.The enzyme activity was expressed as nmoles/min/mg protein.


85

Glutathione S-transferase (EC 2.5.1.18)

Glutathione S-transferase was assayed by the method of Habiget al.

(1974).[200]

Reagents


2. 30 mM 1 chloro-2, 4-dinitrobenzene (CDNB)

3. 30 mM GSH

Procedure

To 1.0 ml of phosphate buffer, 0.1 ml of CDNB, 1.7 ml of water and 0.1 ml of

enzyme source was added. After 5 minutes of incubation at 37 C, 0.1 ml of GSH was

added and the change in optical density was measured immediately for 3 minutes. A

complete assay mixture without enzyme was used a control. Optical density was

measured at 340 nm in a Shimadzu spectrophotometer.Activity of glutathione S-

transferase was expressed as moles of CDNB of conjugate formed/minute/mg

protein.

Estimation of Macromolecular Damages

Assay of lipid peroxidation (LPO)

The level of lipid peroxides was assayed by the method of Ohkawaet al.

(1979).[201]

Reagents

1. 8.1% Sodium dodecyl sulphate (SDS)

2. 20% Acetic acid, pH 3.5 adjusted with NaOH


86

3. 0.8% Thiobarbituric acid (TBA)

4. n-Butanol/pyridine mixture (15:1 v/v)

5. 1, 1, 3, 3-tetraethoxypropane

Procedure

To 0.2 ml of homogenate, 0.2 ml of SDS, 1.5 ml of acetic acid and 1.5 ml of

TBA were added. The mixture was made up to 4 ml with water and then heated in an

oil bath at 95 C for 60 min using glass ball as a condenser. After cooling, 1 ml of water

and 5 ml of n-butanol/pyridine mixture were added and shaken vigorously. After

centrifugation at 4000 rpm for 10 min, the absorbance of organic layer was measured at

532 nm. The level of lipid peroxides was expressed as nmoles of MDA formed/mg

protein.

Extraction of nucleic acids

The nucleic acids were extracted by the method of Schneider (1957).[202]

Known amount of tissues were homogenised in 5.0 ml of ice-cold distilled water using

Potter-Elvehjemhomogeniser with a Teflon pestle. 5.0 ml of 5% TCA was added to the

homogenate and this was kept in ice for 30 minutes to allow complete precipitation of

proteins and nucleic acids. The mixture was centrifuged and the precipitate obtained

was washed thrice with ice cold 10% TCA. Then it was treated with 95% ethanol to

remove lipids. The final precipitate was heated at 90 C for 15 minutes with occasional

shaking, which facilitated the quantitative separation of nucleic acids from protein. The

supernatant after centrifugation was used for the estimation of DNA and RNA.

Deoxy ribonucleic acid (DNA)

DNA was estimated by the method of Burton (1956).[203]


87

Reagents

1. Diphenylamine reagent: 1.5 g diphenylamine was dissolved in 100 ml of

redistilled acetic acid and 1.5 ml of concentrated H2SO4 was added. The

reagent was stored at 4 C in dark. Before use, 0.1 ml of aqueous acetaldehyde

(0.16%) was mixed with every 20 ml of the reagent.

2. Stock standard: Highly polymerised calf-thymus DNA was dissolved in

5 mMNaOH to give a concentration of 0.4 mg/ml.

3. Working standard: This was prepared by mixing 2.0 ml of the stock solution

with an equal volume of 1 N perchloric acid and was heated at 70 C for 15

minutes.

Procedure

A known volume of the nucleic acid extract was made up to 3.0 ml with 1 N

perchloric acid. This was mixed with 2.0 ml of diphenylamine reagent. A reagent blank

and standards were also run out concurrently. This mixture was kept in a boiling water

bath for 10 minutes and the blue colourdeveloped was read at 640 nm in a

photochemcolorimeter.The amount of DNA was expressed as mg/g wet tissue.

Ribonucleic acid (RNA)

RNA was estimated by the method of Rawalet al. (1977).[204]

Reagents

1. Orcinol-ferric chloride reagent: One gram of orcinol was dissolved in 100 ml

of concentrated HCl containing 0.5 g of ferric chloride. This reagent was

prepared freshly.


88

2. Standard: Standard was prepared by dissolving 2.0 mg of yeast RNA in 100

ml of 5% TCA.

Procedure

Aliquots of nucleic acid extracts were made up to 2.0 ml with 5% TCA. To this

3.0 ml of orcinol-ferric chloride reagent was added and mixed well. The tubes were

heated in a boiling water bath for 20 minutes. Reagent blank and standards were also

treated in the same way. The tubes were cooled and the colour developed was

measured at 640 nm using a photochemcolorimeter.The RNA level was expressed as

mg/g wet tissue.

Statistical analysis

Statistical analysis was done by using Graph Pad INSTAT-V3 Version

computer software programme. Values are mean ± SEM for six animals in each

group and the significance of difference between mean values were determined by

one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison

test.

5.7. Isolation and characterization of the active principle of P. Polyphyllus

5.7.1. Isolation of compound I

The concentrate of the methanol extract of P. polyphyllus was fractionated

using benzene (3 300 ml), chloroform (3 300 ml) and ethyl acetate (4 300 ml).

The chloroform fraction on concentration yielded a brownish solid, which was non

homogenous in TLC and hence was further subjected to separation and purification

on column chromatography.[205,206]


89

Column chromatographic analysis

The residue obtained from the chloroform fraction (15g) of P. polyphyllus

was chromatographed in silica gel column (60-120 mesh, 300 gm, 100 5 cm) using

gradient elution with the solvents of increasing polarity. Fractions of 100 ml were

collected each time and the homogeneity was examined on TLC with suitable

solvents. The details of the fractionations and their characteristics are given in Table

27.

Fractions 1-5, 6-13, 14-31, 32-53,54-77, 78-82 and 83-95 each of which on

concentration yielded residues with varying intensities of green colour. These were

tested individually by TLC and further purification was not carried out because of

paucity of the samples. Fractions 83-95 on concentration yielded a pure brownish

homogeneous solid and were designated as compound I.

FT-IR Spectral data of compound I [207]

The FTIR spectrum of compound I was recorded using JASCO- FTIR 5300

spectrometer (Plate 1) using CDCl3 as the solvent and complete assignment of band

regions are shown in table 28.

1H-NMR Spectral data of compound I[208, 209]

The 1H-NMR spectrum of compound I was recorded using AMX 400 (400

MHz) spectrometer (Plate 2) using CDCl3 as the solvent and complete assignment of

protons are shown in table 29.

13C-NMR spectral data of compound I[208, 209]

13C-NMR spectrum of compound I was recorded using AMX 400 (100

MHz) spectrometer (Plate 3)using CDCl3 as the solvent and the complete

assignment of carbon are given in table 30.


90

LC-MS study of compound I [210]

LC-MS 2010 spectrum (Shimadzu) of compoundI(Plate 4) was taken and it

gave various fragments at m/z: (M) + 638

5. 7. 2. IsolationandCharacterisationofthe Active Principle fro ProsopisCineraria

Isolation of compound II

The concentrate of the methanol extract of ProsopisCinerariawas

fractionated using benzene (3 300 ml), diethyl ether (3 300 ml) and ethyl

acetate (4 300 ml). The ethyl acetate fraction on concentration yielded a yellow

solid, which was non homogenous in TLC and hence was further subjected to

separation and purification on column chromatography. [205,206]

Column chromatographic analysis

The residue obtained from the ethyl acetate fraction (15g) of

ProsopisCinerariawas chromatographed in silica gel column (60-120 mesh, 300

gm, 100 5 cm) using gradient elution with the solvents of increasing polarity.

Fractions of 100 ml were collected each time and the homogenity was examined on

TLC with suitable solvents. The details of the fractionations and their

characteristics are given in table 31.

Fractions 1-5, 6-10, 11-48, 49-64 and 65-76 each of which on concentration

yielded residues with varying intensities of yellow colour. These were tested

individually by TLC and further purification was not carried out because of paucity

of the samples. Fractions 77-90 on concentration yielded a pure yellowish

homogeneous solid and were designated as compound II. It gave dark green

colouration with neutral ferric chloride and violet colouration with


91

Molish’sreagent.TheRf values of compound II in various solvent systems are given

in table32.

Acid hydrolysis of compound II [212]

In order to find out the nature of the glycoside, compound II was subjected to

acid hydrolysis. To a solution of the glycoside (10 mg) in hot methanol (10 ml), an

equal volume of H2 SO4 (7%) was added and the mixture was gently refluxed at

100 C for 2 hours. The excess of alcohol was distilled off invacuoand the resulting

aqueous solution was partitioned with ether to separate the ether soluble aglycone

and the aqueous sugar.

Identification of the sugar [213]

The aqueous layer was treated with BaCO3 to remove excess sulphuric acid

and the barium sulphate formed was filtered off using Whatman No. 42 filter paper

and the filtrate (sugar portion) was concentrated.The concentrate was analyzed by

Paper chromatography (PC) with various authentic sugar samples on a Whatman

No.1 filter paper strip and identified using Aniline hydrogen phthalate spray reagent

(prepared by dissolving 9.2 ml of aniline and 16 g of phthalic acid in 490 ml of n-

butanol, 490 ml of ether and 20 ml of water). The various solvent systems used for

PC and Rf values of the identified sugar.in these solvent systems are presented in

table 33.

UV spectral characteristics of compound II [214]

Basic flavonoid structure of compound II and position of attachment of

hydroxyl group and other substituents were conveniently studied by recording UV

spectrophotometer (Shimadzu 1601) in MeOH as well as in various Shift reagents

shown in table 34..


92

1H-NMR Spectral data of compound II [208, 209]

The 1H-NMR spectrum of compound II was recorded using AMX 400 (400

MHz) spectrometer (Plate 1) using DMSO-d6 as the solvent and complete

assignment of protons are shown in table 35.

13C-NMR spectral data of compound II [208, 209]

13C-NMR spectrum of compound II was recorded using AMX 400 (100

MHz) spectrometer (Plate 2)using DMSO-d6 as the solvent and the complete

assignment of carbon are given in table 36.

EI-MS study of compound II [215]

EI-MS spectrum (Fenniganmat 8230, 70 eV) of compoundII (Plate 3) was

taken and it gave various fragments at m/z: 330, 213 and 118.

5.8. Cytotoxicity studies of Compounds I & II

The cytotoxicity studies of compound I & II on HepG2 - Human Liver

Cancer cells exactly in the same way as per described MTT assay method and

Hoechst staining and photomicroscopy [Apoptosis] in the Chapter 5.3.2. and

5.3.3.; Page No ----------.

5. materials and methods -...

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