TABLE OF CONTENTS

1. INTRODUCTION ..2

1.1 DRUG DEVELOPMENT AND CELL BIOLOGY ..3

1.2 TYPES OF MARKERS ..4
1.3 STRUCTURAL AND FUNCTIONAL PROPERTIES OF CELL ..4
MEMBRANE

2. METHODS OF ISOLATION AND PURIFICATION OF ..5

MARKERS

2.1 AFFINITY CHROMATOGRAPHY ..5

2.2 ISOELECTRIC FOCUSING ..6
2.3 IMMUNOAFFINITY CHROMATOGRAPHY ..6
2.4 IMMUNO PRECIPITATION ..7
2.5 SELECTIVE PRECIPITATION ..7
2.6 GEL EXCLUSION CHROMATOGRAPHY ..7

3. BOTANICALS FOR HEPATOPROTECTION ..8

3.1 SILYMARIN ..8

3.2 METHOD OF ISOLATION ..9
3.3 MECHANISM OF ACTION ..11

4. APPROACHES TO THE CHRACTERIZATION OF ..12

MEMBRANE PROTEINS

4.1 PHOTOAFFINITY LABELLING ..12

4.2 AMINO ACID SEQUENCING AND PEPTIDE MAPPING ..12
4.3 MONOCLONAL ANTOBODIES ..12
4.4 CONSTRUCTION OF CDNA AND GENOMICS LIBRARIES ..12
4.5 SOUTHERN AND NORTHERN BLOTTING ANALYSIS ..13
4.6 CELL TRANSFECTION ..13
5. ANAYLITICAL PROFILE FOR SILYMARIN ..14
6. CONCLUSION ..22
7. APPENDIX ..28
8. REFERENCE ..29

1
1.INTRODUCTION
A biomarker, or biological marker, is in general a substance used as an
indicator of a biological state. It is a characteristic that is objectively measured and
evaluated as an indicator of normal biological processes, pathogenic processes, or
pharmacologic responses to a therapeutic intervention. It is used in many scientific
fields. Molecular marker may be define as a DNA sequence used for chromosome
mapping as it can be located at a specific site in chromosome . A molecular marker may
be either (1) anonymous or (2) defined. Anonymous marker is a cloned random DNA
fragment whose function or specific feature is not known. But a defined marker may
contain a gene or some other specific features eg. restriction site for rare cutting
restriction enzymes, etc. some common markers are as follow :
1) Restriction fragment length polymorphism (RFLP).
2) Random amplified polymorphic DNA (RAPD).
3) Variable number of tandem repeat (VNTR).
1) Restriction fragment length polymorphism (RFLP) :-
Restriction fragment length polymorphism denotes that a single
restriction enzyme produces fragments of different length from the same stretch of
genomic DNA of different strains of a species or from different related species . The
pattern of RFLPs generated will depend mainly on the following: (1) differences in the
DNAs of selected strains/species, (2) the restriction enzyme used and (3) the DNA probe
employed for southern hybridization.
RFLPs may have the following applications:
(1) Identification and isolation of any gene known to be linked with an RFLP locus,
(2) Fingerprinting of strains for their unequivocal identification,
(3) Linkage mapping of quantitative trait loci,
(4) Identification of the most important loci affecting a quantitative trait etc.

2) Random amplified polymorphic DNA (RAPD):-

RAPDs are obtained by using a PCR (polymerase chain reaction) equipment or a
thermal cycler . The procedure, in simple terms, for obtaining RAPDs is as follows.
1. The genomic DNA of a selected strain /variety/species is isolated in a high molecular
weight condition.
2. To this DNA is added an excess of a selected oligonucleotide that serves as the
primer.
3. This mixture is subjected to repeated cycles of DNA denaturation-renaturation-DNA
replication in PCR equipment.
4. After several cycles of amplification, the DNA is subjected to gel electrophoresis. The
amplified DNA will form a distinct band, which is usually detected by ethidium

2
 bromide staining and visible fluorescence under UV light.

3) Variable Number of Tandem Repeat (Vntr):-

VNTRs are dispersed throughout the genome and are made up of a variable
number of end-to-end duplications of identical or almost identical sequences of 2-80bp
each. Polymorphism in VNTRs is usually associated with the number of repeats, i.e.
different individuals show different numbers of repeat units at a given locus or position
in a chromosome; these variations constitute the alleles of VNTR loci. The VNTRs are
generally classified into two groups: (1) minisatellite and (2) microsatellite DNAs; these
together constitute the hypervariable region or DNA.
1.1 BIOMARKER: DRUG DEVELOPMENT AND CELL
BIOLOGY:-
Biomarkers are characteristic biological properties that can be detected
and measured in parts of the body like the blood or tissue. They may indicate either
normal or diseased processes in the body. Biomarkers can be specific cells, molecules,
or genes, gene products, enzymes, or hormones. Complex organ functions or general
characteristic changes in biological structures can also serve as biomarkers. Although
the term biomarker is relatively new, biomarkers have been used in pre-clinical research
and clinical diagnosis for a considerable time.
In medicine, a biomarker can be a substance that is introduced into an
organism as a means to examine organ function or other aspects of health. For example,
rubidium chloride is used as a radioactive isotope to evaluate perfusion of heart muscle.
It can also be a substance whose detection indicates a particular disease state, for
example, the presence of an antibody may indicate an infection. More specifically, a
biomarker indicates a change in expression or state of a protein that correlates with the
risk or progression of a disease, or with the susceptibility of the disease to a given
treatment
In cell biology, a biomarker is a molecule that allows for the detection and isolation of a
particular cell type (for example, the protein Oct-4 is used as a biomarker to identify
embryonic stem cells).

3
1.2 TYPES OF BIOMARKER:-
Biomarkers In Medicine
Biomarkers validated by genetic and molecular biology methods can be classified into
three types.
• Type 0 - Natural history markers
• Type 1 - Drug activity markers
• Type 2 - Surrogate markers
Disease-Related Biomarkers And Drug-Related Biomarkers
It is necessary to distinguish between disease-related and drug-related biomarkers.
Disease-related biomarkers give an indication of whether there is a threat of disease (risk
indicator or predictive biomarkers), if a disease already exists (diagnostic biomarker), or
how such a disease may develop in an individual case (prognostic biomarker). In
contrast, drug-related biomarkers indicate whether a drug will be effective in a specific
patient and how the patient’s body will process it.

Biomarkers In Drug Development

Once a proposed biomarker has validated, it can be used to diagnose disease risk,
presence of disease in an individual, or to tailor treatments for the disease in an individual
(choices of drug treatment or administration regimes). In evaluating potential drug
therapies, a biomarker may be used as a surrogate for a natural endpoint such as survival
or irreversible morbidity. If a treatment alters the biomarker, which has a direct
connection to improved health, the biomarker serves as a surrogate endpoint for
evaluating clinical benefit.

1.3 STRUCTURAL & FUNCTIONAL PROPERTIES OF CELL

MEMBRANE PROTEIN: -
The plasma membrane is a fundamental structural component of cells. The phospholipids
bilayer is a basic structure of biological membrane. The amount of protein & lipid
material varies depending on the specific function of the cell membrane.
Plasma protein membrane functions as a semi permeable barrier to different
nutrients, ions & molecules. Primarily the protein constituents of the membrane are
responsible for the transport of the molecules across the lipid bilayer of the
membrane. These are 2 types of protein associated with cell membrane. Those
loosely bound. Known as peripheral membrane proteins & those embedded within
the membrane referred to as integral membrane protein.
Eukaryotic & prokaryotic cells posses’ proteins, including reception, that
are exposed to the external side of the membrane & monitored the cell
surroundings. The Trans membrane protein extended through the bilayer.

4
2. METHOD FOR ISOLATION & PURIFICATION OF
MEMBRANE PROTEIN

2.1 AFFINITY CHROMATOGRAPHY

Affinity chromatography relies upon the specific binding capabilities of
the membrane proteins as the means of purification this types of adsorption
chromatography involve the use of a bed material with biological affinity for the
component to be purified.

An example of a purification protocol for isolation of mammalian β-

adrenergic receptor is as follow:-

STEPS INVOLVE IN PURIFICATION OF MAMMALIAN β -ADRENERGIC RECEPTORS

5
2.2 ISOELECTRIC FOCUSING
. It has often been the method of choice when separating amphoteric
proteins. The influence of an electric field on these proteins causes the migration
& focusing of a protein at a specific point which corresponds to the isoelectric
point of the protein
2.3 IMMUNOAFFINITT CHROMATOGRAPHY
Immunoaffinity chromatography is a means of membrane protein
purification that focus on the functional properties of a protein. Immunoaffinity
chromatography involves the use of specific “Ab” directed against a given
protein.

6
 Steps involved in the immuno affinity purification of the turkey erythrocyte β-
adrenergic receptors using a ligand binding site specific monoclonal Antibody.

2.4 IMMUNOPRECIPITATION
The precipitation method is dependant on the formation of an immune
complex between the protein “Ag” & its specific “Ab”. The advantage in using
“Ab” for the isolation & purification of membrane proteins are the high
specificity & the high degree of sensitivity of the method to precipitate specific
membrane protein found in low concentration in the cell membrane.
2.5 SELECTIVE PRECIPITATION
Selective precipitation by polyethylene glycol or by ammonium sulphate has been
used to precipitate free proteins or protein hormone complexes from a mixture of
binding proteins & ligands.
2.6 GEL EXCLUSION CHROMATOGRAPHY
It has been employed as a means of purifying a mixture of membrane proteins
based on their structural properties of molecular weight & size. A column is packed
with inert polymer or polysaccharide beads of varying pore size. The lower
molecular weight proteins penetrate the beads & are retained longer on the column
than the higher molecular weight proteins.

7
3. BOTANICALS FOR HEPATOPROTECTION

3.1 SILYMARIN

PHARMACOGNOSTIC STUDY

SYNONYMS
Marian Thistle, Our Lady’s Thistle, Milk Thistle, The Wild Artichoke

BIOLOGICAL SOURCE
It is reported in the ripe seeds of milk thistle silybum marianum gaerth belonging
to family asteraceae.

MACROSCOPIC STUDY

Height 5-10 ft
Leaves • oblong to lanceolate
• milk white veins
Flowers Reddish purple flower with spiny apex
flower heads 4 to 5cm long and wide

DESCRIPTION
Milk Thistle contains three potent liver protective flavonoids: silybin, silydianin, and
silychristin, known collectively as silymarin. Numerous clinical trials have shown that
silymarin and milk thistle extract can protect the liver. Silymarin counteracts the toxic
effects of a wide variety of poisons, including alcohol, carbon tetrachloride,
acetaminophen overdose, and the Deathcap mushroom.

8
 3.2 METHOD FOR ISOLATION OF SILYMARIN FROM
SYLIBUM MARIANUM SEEDS

The method of isolation of silymarin includes grinding the seeds, extraction of oil via a
hot defatting process and extraction of defatted seeds with a medium polarity solvent.
After filtration, the extract is evaporated to dryness and the residue is azeotropically
dried. The dried extract is then further defatted in hot ether wherein, after the chilling,
filtration and drying, a concentrate of silymarin in the form of a homogeneous yellow-
orange crystalline substance is obtained having a high melting point (ca. 140-
165.degrees. C.). Yield of silymarin is 2.0-2.5% according to the content of total
silymarin of 86-97% and approx. 20% of the oil calculated by a crude seed.
In comparison with the previously described methods, the process described in the instant
invention does not use the toxic solvents, dichloromethane, methanol or acetonitrile. The
described method applies acetone as the least toxic and by far the cheapest medium
polarity solvent. The method is developed for the use of minimal (optimum) amounts of
organic solvents with a regeneration rate of approx. 90-95%. All chemicals and
technological details are stated in the detailed description of the method. The method
comprises the following steps:

1. Grinding the seeds of Slybum marianum

2. Extracting the seed powder as a means for defatting
3. Filtering the defatted seed powder
4. Extraction of the defatted seed powder with acetone
5. Filtration of the extracted seed powder
6. Evaporation of the acetone extract filtrate
7. Azeotropic drying of the extract with toluene
8. Secondary defatting of the extract with diisopropyl ether
9. Filtration of the resulting silymarin crystals
10. Drying of the silymarin crystals under vacuum
Crude seeds of Sylbum marianum are powdered using a jet mill with rotating blades with
the application of screens up to 40 meshes. After this, defatting of the resultant seed
powder is conducted by extraction with a solvent suitable for defatting, such as a
hydrocarbon. In one preferred practice of this invention, n-hexane is used as the
hydrocarbon solvent. In another preferred practice of this invention, petroleum ether is
used. A suspension of the seed powder in the applied ratio of seed powder : solvent
(m/V) can normally be stirred with a mechanical stirrer.
The filtration is conducted in via vacuum filtration. The filtrate has intense yellow color
and contains the oil of the seeds in n-hexane. The filtered powder is then heated under
vacuum at 70° C. for 2-3 hours to provide complete removal of extraction solvent traces
According to this invention, defatted seeds do not need to be further dried in order to rid

9
them of the traces of absorbed n-hexane but rather are extracted with a medium polarity
solvent, such as acetone, immediately after vacuuming.

Extraction with acetone is conducted at a temperature of between 18° C. and 56° C.

Optimum extraction time is approx. from 24 to 72 hours, depending on the temperature at
which the extraction is conducted. Filtration follows the extraction.
In another version of the invention, defatting is conducted in common percolator at room
temperature during at least 48 hours. After defatting and removal of traces of n-hexane,
percolation with acetone for isolation of silymarin is continued.
Azeotropic distillation follows, by which water and toluene is removed from the residue
after evaporation of the acetone filtrate After evaporation of acetone in the filtrate, a
mixture of toluene and water is collected. The toluene and water mixture is easily
separated in an extractor and 80% of the used toluene is regenerated. A vacuum system is
used to provide drying at a temperature as low as possible.
For purification, i.e. removing the residual oil, according to the invention ethers are
applied as a means of extraction. According to the invention, ethers with 4 to 8 carbon
atoms such as tetrahydrofuran, or diisopropyl ether, or diethyl ether are appropriate for
secondary defatting.

It has been found that diisopropyl ether acts as the most efficient solvent for defatting the
extracts wherein the oil is completely dissolved while silymarin constituents remain
almost completely suspended. One part of the evaporated residue can stay attached to the
walls of the extraction flask and it is not removed spontaneously during heating at the
reflux temperature of the solvent. Therefore, mechanical stirring is needed. On an
industrial scale, a reactor with mechanical mixer whose shape follows the geometry of
the extraction flask is used. The purpose of it is to avoid the silymarin getting glued to the
walls during azeotropic drying with toluene. Once formed, the suspension of silymarin
constituents is nicely defatted and easily filtered in vacuo after the cooling. Further
cooling of the suspension does not substantially affect the level of product purity, since
the solubility of silymarin constituents in diisopropyl ether is extremely low. The next
example severs only as an illustration of the invention and is not meant to be used for
defining the range and content of the invention.

10
3.3 MECHANISMS OF ACTION
The hepatoprotection provided by silymarin appears to rest on four properties:
• Activity against lipid peroxidation as a result of free radical scavenging and the
ability to increase the cellular content of GSH;
• Ability to regulate membrane permeability and to increase membrane stability in
the presence of xenobiotic damage;
• Capacity to regulate nuclear expression by means of a steroid-like effect; and
• Inhibition of the transformation of stellate hepatocytes into myofibroblasts, which
are responsible for the deposition of collagen fibres leading to cirrhosis.

Mechanism of action of silymarin as proposed by Valenzuela and Garrido.

Silymarin and silibinin inhibit the absorption of toxins, such as phalloidin or -amanitin,
preventing them from binding to the cell surface and inhibiting membrane transport
systems. Furthermore, silymarin and silibinin, by interacting with the lipid component of
cell membranes, can influence their chemical and physical properties. Studies in
erythrocytes, mast cells, leucocytes, macrophages and hepatocytes have shown that
silymarin renders cell membranes more resistant to lesions.

11
4. APPROCHES TO THE CHARACTERIZATION OF
MEMBRANE RECEPTORS:-
4.1 PHOTOAFFINITY LABELING
Photoaffinity labeling is a useful method for investigating the chemical
structure & mode of action of a protein.
This methodology involves covalently & specifically linking a radioligand
to the reception protein successfully utilized in adrenergic system by a number of
groups to label ligand binding subunits of β & α & dopamine receptors.

4.2 Amino Acid Sequencing & Peptide Mapping: -

Peptide mapping has proved to bean extremely useful tool in the structural
characterization of the relationship between the receptior domainded their
involvement is cell function if the receptor could be specifically radiolabeled or
photolabeled in its native environment, more information related to structural
changes due to ligand binding or protein intraction with cellular components
could be obtained.

4.3 Monoclonal Antibodies:

Monoclonal “Ab” have been used as probes of receptor structure &
function on cell protein of low concentration & stability. Monoclonal “Ab” can be
directed against many parts of a receptor & the site of attachment of the “Ab” can
be determined precisely.
4.4 Construction of cDNA & Genomic libraries :
Recombinant DNA technology has evolved as an excellent method for
obtaining the protein sequence encoding a particular receptor. The isolation &
selection of a specific receptor gene is made easier if it display a phenotypic
marker.
The optimal cDNA library contains at least one cDNA clone for each m-
RNA sequence in the cell & expresserthe desired m-RNA sequence at high levels.

12
4.5 Southern & Northern Blot Analysis
A useful technique for analyzing the DNA of positive clones from
genomic libraries is southern blot analysis. Northern blot analysis is used to
analyze RNA sequences from positive clones from c-DNA libraries.
4.6 Cell Transfection
Cell transfection can be accomplished in 3 different ways: - calcium
phosphate transfection, diethylamino ethyl (DEAE) Dextran transfection &
electroporation. The first 2 methods involve attachment of the DNA to the cell
surface under specific chemical environmental conditions.
Electroporation uses electric currents to open pores in the cell allowing for
DNA diffusion in to the cell this procedure can be performed on virtually any type
of cell.

13
5) ANALYTICAL PROFILE OF SILYMARIN

IDENTIFICATION OF MILK THISTLE

1. PURPOSE OF METHOD

The method for identification of Milk Thistle seeds (fruits) by HPTLC fingerprint is
suitable to identify a given sample of plant material as Milk Thistle (Silybum
marianum) based on its flavonolignane fingerprint.

The method may be used to identify an extract or finished product as derived from
Milk Thistle, provided that the material was made from a single herb and is intended
to contain the constituent profile seen in Milk Thistle.

2. MATERIALS

Wear lab coat, protective goggles and gloves at all times when handling chemicals.

2.1 Chemicals and solvents

Methanol, acetone, formic acid, dichloromethane, chloroform, diphenylborinic acid

aminoethylester, polyethylene glycol (Macrogol) 400, ethyl acetate.

2.2 Samples and reference materials (optional)

Botanically authenticated and freshly dried Milk Thistle seeds (fruits), and silybin
(=silibinin), silydianin, silychristin, and taxifolin (available from ChromaDex).

2.3 Plates

Glass plates HPTLC Si 60 F254, 10x10 or 20x10 cm, Merck (Darmstadt, Germany),
or others if equivalence was shown.

2.4 Lab ware and instruments

* Analytical mill or mortar,

* Ultrasonic bath,

* Centrifuge with centrifuge tubes, or suitable set-up for filtration with beakers or
small flasks (10 or 20 mL)

* Analytical balance,

14
 * graduated pipettes (1, 5, and 10 mL),

* graduated cylinder (50 mL),

* Glass bottles (with tightly closing lid, 100 mL and 200 mL),

* TLC Twin Trough Chamber or Flat Bottom Chamber 20x10 cm, alternatively
automatic developing chamber,

* Sample application device using the spray-on technique (such as Linomat, ATS
[CAMAG] or AS 30 [Desaga]),

* Chromatogram immersion device [CAMAG],

* Plate heater or oven,

* Documentation system consisting of an illumination device for UV 254 nm, UV

366 nm, and white light and a video or digital camera,

* suitable TLC software,

* thermometer and hygrometer

* device for humidity control of plates

* lab coat, protective goggles and gloves.

3. DESCRIPTION OF METHOD

3.1 Preparation of test solutions

3.1.1 Raw materials

Mill each sample to a fine powder. Weigh 1 g each of powder in individual centrifuge
tubes or flasks. Add 10 mL of methanol each and mix well. Close the lid or cap and heat
at 700C for 5 min. Alternatively the samples can also be heated under reflux for 5 min.
Centrifuge or filter the solutions and use the supernatants / filtrates as test solutions.

3.1.2 Dry extracts and dry finished products

Weigh an amount of each extract powder or finished product equivalent to 1 g of raw

material in individual centrifuge tubes or flasks. Add 10 mL of methanol each and mix
well. Close the lid or cap and heat at 70[degrees]C for 5 min. Alternatively the samples
can also be heated under reflux for 5 min. Centrifuge or filter the solutions and use the
supernatants / filtrates as test solutions.

15
3.1.3 Liquid extracts and liquid finished products

Dilute the liquid samples with the same solvent (as on the label) to obtain a solution with
the same concentration as that of a test solution from raw material as described under
3.1.1.

3.2 Preparation of reference solutions (optional)

3.2.1 Botanical reference solution As 3.1.1

3.2.2 Chemical reference solutions

Weigh 1 mg of silybin (= silibinin) in a flask. Add 10 mL of methanol. Individually

dissolve silydianin, silychristin, and taxifolin in the same way.

3.3 Preparation of derivatizing reagents

Natural Products reagent (NP): 1 g of diphenylborinic acid aminoethylester is dissolved

in 200 mL of ethyl acetate.

Macrogol reagent (PEG): 10 g of polyethylene glycol (macrogol) 400 are dissolved in

200 mL of dichloromethane.

3.4 Stationary phase

10x10 cm (or 20x10 cm) glass plates HPTLC silica gel 60 F254 (Merck).

3.5 Sample application

Apply 10 [micro]L of test solution, 10 [micro]L of botanical reference solution, and 10

[micro]L of each chemical reference solution each as 8 mm band, at least 2 mm apart, 8
mm from the lower edge and at least 15 mm from left and right edges of the plate.

3.6 Temperature and humidity

Record temperature and humidity in the laboratory. If the relative humidity exceeds
50%RH, condition the plate to about 30%RH using a suitable device.

3.7 Chromatography

3.7.1 Developing solvent

Place 75 mL of chloroform, 16.5 mL of acetone, and 8.5 mL of formic acid in a bottle,

close lid tightly and mix content by shaking. Larger or smaller amounts of solvent can be
prepared once a day.

16
3.7.2 Chamber

Line one side of a 10x10 cm Twin Trough Chamber with filter paper. Pour 10 mL of
developing solvent over the paper, and tilt the chamber to equilibrate solvent level in both
troughs, close the lid. Allow the chamber to saturate for 20 min. If using a 20x10 cm
chamber, use 20 mL of developing solvent. If using a Flat Bottom Chamber, use enough
solvent to cover the bottom with a 5 mm level. If using an automatic chamber, refer to the
manufacturer's instructions.

3.7.3 Development

Measure and mark on the plate the developing distance of 70 mm from lower edge of
plate (62 mm from application position). Open the saturated chamber and introduce the
plate with the layer facing the inside, close the chamber and wait for the solvent to reach
the mark. Remove the plate from the chamber. 3.7.4 Drying

GENERAL METHODOLOGY FOR HPTLC

I. PURPOSE

This SOP provides general guidance for analysis by HPTLC.

II. APPLICATION

This SOP applies to all work regarding HPTLC analysis unless the
resulting chromatograms or the applied method require adjustments.

III. PROCEDURE

GENERAL

1. Prior to using an instrument, log-in the corresponding instrument log according to

GDL 50.000.01 Use of CAMAG equipment and software.

2. Observe safety and housekeeping rules according to GDL 60.000.01 Housekeeping

rules and schedule and SOP 60.000.02 Chemical Hygiene Plan.

3. For use of instrument refer to GDL 50.000.01 Use of CAMAG equipment and
software.

4. Record any performed work in appropriate worksheets as specified in GDL 50.002.01

Record keeping. Include temperature and humidity at least once a day in any active
worksheets.

17
A) Plate material Handle plates with extreme caution to avoid any damage to the layer.
Store plates in the original package with the lid closed. Remove plate from storage
only immediately prior to use. Plates are generally handled only at the upper edge to
avoid contamination. Unless otherwise necessary Merck HPTLC plates silica gel 60 F
254 in the format 10x10 cm or 20x10 cm are used. For most work plates are used
without pre-treatment unless chromatography produces impurity fronts due to
contamination of the plate. For reproducibility studies and quantitative analyses plates
are pre-washed as follows.

2. Develop plate with 20 mL methanol per trough in a 20 x 10 cm Twin Trough Chamber

(TTC) to the upper edge. Up to two 20 x 10 cm or four 10 x 10 cm plates can be
developed back to back in each trough of the TTC.

3. Remove the plate and dry for 20 minutes in a clean drying oven at 120[degrees]C.

4. Equilibrate plate with lab atmosphere (temperature, relative humidity) in a suitable

container providing protection from dust and fumes.

B) SAMPLE APPLICATION

Unless otherwise necessary apply samples using the spray-on technique with ATS 4 or
Linomat 5. In case of spot application by contact use the Nanomat or the ATS 4 and
dissolve the sample in the solvent of lowest suitable solvent strength.

C) PREPARATION AND STORAGE OF DEVELOPING SOLVENTS

Developing solvents consisting of more than 1 component are prepared by measuring the
required volume (respectively weight) of each component separately and transferring
them into a solvent bottle of appropriate size. The bottle is closed with a lid and shaken to
ensure proper mixing of the content.

Volumes smaller than 1 mL are measured with a suitable micropipette. Volumes up to 20

mL are measured with a graduated volumetric pipette of suitable size. Volumes larger
than 20 mL are measured with a graduated cylinder of appropriate size.

To minimize volume errors developing solvents are prepared in a volume that is

sufficient for one working day.

D) DEVELOPMENT

Plates are developed in a saturated Twin Trough Chamber according to the following
procedure:

1. Prepare the appropriate volume (10 mL for 10x10 cm, 20 mL for 20x10 cm TTC) of
developing solvent.

18
2. Open chamber and place a correctly sized (10x10 cm; 20x10 cm) piece of filter paper
in the rear trough.

3. Pour solvent into chamber so that the filter paper is thoroughly wetted and adheres to
rear wall of TTC.

4. Tilt chamber to the side (about 45[degrees]) so that the solvent volume in both troughs
equalizes.

5. Set chamber on bench, replace the lid and let chamber equilibrate for 20 minutes.

6. Mark the desired developing distance (70 mm from lower edge of plate) with a pencil
on the right edge of the plate.

7. Slide off the lid to the side.

8. Insert the plate into the front trough. The layer should face the filter paper and the back
of the plate is resting against front wall of TTC.

9. Replace lid.

10. Develop plate to the mark.

11. Open lid, remove plate.

12. Dry the plate (vertically in direction of chromatography) 5 minutes in a stream of cold
air.

13. After each development remaining mobile phase and filter paper are discarded. Prior
to being prepared for the next run the chamber is dried and, if necessary, also cleaned.
Alternatively the ADC 2 can be used for development. Unless otherwise specified the
standard method S1 is applied.

E) DERIVATIZATION

Transfer of reagent for derivatization of samples on a HPTLC plate may be

accomplished by spraying or dipping. Dipping is the preferred method and should be
used whenever possible. Spraying is done in the TLC spray cabinet. If derivatization
includes heating the plate heater is used. Always refer to the HPTLC method for
details of the derivatization procedure.

DIPPING (CHROMATOGRAM IMMERSION DEVICE)

1. Charge tank of immersion device with 200 mL of reagent.

2. Place plate in holder of immersion device, set parameters according to method
and press start.

19
 3. Let excess reagent drip off plate, wipe off back of the plate with paper towel.
Remove plate from plate holder.
4. Dry plate with cold air (vertically in direction of chromatography).

Spraying (Glass sprayer)

1. Charge the bottle of the sprayer with up to 50 mL of reagent.

2. Place plate in spray cabinet against a filter paper or a paper towel.
3. Spray plate with horizontal and vertical motion until it is homogeneously covered
with reagent.
4. Dry plate with cold air.

HEATING (PLATE HEATER)

1. Turn on plate heater and select temperature.

2. Wait until the temperature is stable.

3. Place plate onto plate heater.

4. After the time specified by the method remove hot plate from heater.

F) Documentation of plates (DigiStore)

Each developed plate is documented with an electronic documentation system under UV

254 nm, UV 366 nm and white light.

If a type of light does not produce usable information that fact must be documented. If a
plate is derivatized images are taken prior and after derivatization.

G) QUANTITATIVE EVALUATION

Generally quantitative evaluation is performed with the TLC Scanner 3 using winCATS
software. The analysis files are labeled to reflect the plate ID and any additional
descriptive information if multiple evaluation under different conditions is performed.

H) DOCUMENTATION OF WORK

20
SYSTEMIC MEDICINE AND SYLIBUM MARIANUM

21
6. CONCLUSION

1. PROTECTIVE EFFECTS IN MODELS OF OXIDATIVE STRESS

Oxidative stress is defined as structural and/or functional injury produced in tissues by
the uncontrolled formation of pro-oxidant free radicals. Oxidative stress usually develops
when the pro-oxidant action of an inducer exceeds the anti-oxidant capacity of the cell
defense system, altering its homeostatic capacity. Numerous substances induce oxidative
stress, including carbon tetrachloride, TBH, ethanol, paracetamol (acetaminophen) and
phenylhydrazine. It has been shown in rats that silibinin protects neonatal hepatocytes
from cell damage produced by erythromycin, amitriptyline, nortriptyline and TBH

2 ACTIVITY AGAINST LIPID PEROXIDATION

Lipid peroxidation is the result of an interaction between free radicals of diverse origin
and unsaturated fatty acids in lipids. Lipid peroxidation involves a broad spectrum of
alterations, and the consequent degeneration of cell membranes may contribute towards
the development of other disorders of lipoprotein metabolism, both in the liver and in
peripheral tissues.

3 EFFECTS ON LIVER LIPIDS

The influence of silymarin on cellular permeability is closely associated with qualitative
and quantitative alterations of membrane lipids (both cholesterol and phospholipids).This
suggests that silymarin may also act on other lipid compartments in the liver; this may
influence lipoprotein secretion and uptake. It has been shown that silymarin and silibinin
reduce the synthesis and turnover of phospholipids in the liver of rats.

4. EFFECTS ON PLASMA LIPIDS AND LIPOPROTEINS

The administration of silymarin reduces plasma levels of cholesterol and low-density

lipoprotein (LDL) cholesterol in hyperlipidaemic rats, whereas silibinin does not
reduce plasma levels of cholesterol in normal rats; however, it does reduce
phospholipid levels, especially those transported in LDL.Data obtained in
experimental models of hepatic injury have shown that silymarin is able to normalize
the increase in plasma lipids observed after administration of carbon tetrachloride and
to antagonize the reduction in serum free fatty acids induced by thioacetamide. In the
experimental model of hepatic injury produced by thioacetamide, silymarin did not
appear to be able to normalize the reduction in triglycerides in serum. In the
experimental model of hepatic injury produced by paracetamol in rats, it was evident
that silymarin improves LDL binding to hepatocytes, an important factor for the
reduction of LDL in plasma.

22
5. STIMULATION OF LIVER REGENERATION

One of the mechanisms that can explain the capacity of silymarin to stimulate liver
tissue regeneration is the increase in protein synthesis in the injured liver. In in vivo
and in vitro experiments performed in the liver of rats from which part of the organ
had been removed, silibinin produced a significant increase in the formation of
ribosomes and in DNA synthesis, as well as an increase in protein synthesis.
Interestingly, the increase in protein synthesis was induced by silibinin only in injured
livers, not in healthy controls. The mechanism whereby silibinin stimulates protein
synthesis in the liver has not been defined; it may be the physiological regulation of
RNA polymerase I at specific binding sites, which thus stimulates the formation of
ribosomes. In rats with experimental hepatitis caused by galactosamine, treatment with
intraperitoneal silymarin 140 mg/kg for 4 days completely abolished the inhibitory
effect of galactosamine on the biosynthesis of liver proteins and glycoproteins.

These data support the results of previous experiments in a similar model of acute
hepatitis in the rat, in which silymarin protected hepatic structures, liver glucose stores
and enzyme activity in vivo from injury produced by galactosamine.

The capacity of silymarin to stimulate protein synthesis has also been studied in
neoplastic cell lines, in which no increase in protein synthesis, ribosome formation or
DNA synthesis has been found after treatment with silymarin.

6. ANTI-INFLAMMATORY AND ANTICARCINOGENIC

PROPERTIES

A significant anti-inflammatory effect of silymarin has been described in liver tissue.

Studies have shown that silymarin exerts a number of effects, including inhibition of
neutrophil migration, inhibition of Kupffer cells, marked inhibition of leukotriene
synthesis and formation of prostaglandins.

The protection afforded by silymarin against carcinogenic agents has been studied in
various experimental animal models. A series of experiments have been performed in
nude mice with nonmelanoma skin cancer produced by UVB radiation, studying its
initiation, promotion and complete carcinogenesis. In all the stages studied, silymarin
applied onto the skin at different doses appeared to reduce significantly the incidence,
multiplicity and volume of tumours per animal. Furthermore, in a short-term
experiment (using the same experimental model), the application of silymarin
significantly reduced apoptosis, skin oedema, depletion of catalase activity and
induction of cyclo-oxygenase and ornithine decarboxylase activity. This effect
provides protection against photocarcinogenesis. Similar results were also obtained in
the model of skin carcinogenesis produced by chemical carcinogenic agents in
carcinogenesis-sensitive (SENCAR) mice.

23
 The molecular bases of the anti-inflammatory and anticarcinogenic effects of silymarin
are not yet known; they might be related to the inhibition of the transcription factor
NF- B, which regulates the expression of various genes involved in the inflammatory
process, in cytoprotection and carcinogenesis. It has also been hypothesised that
silymarin may act by modulating the activation of regulating substances of the cellular
cycle and of mitogen-activated protein kinase.

7. INHIBITION OF CYTOCHROME P450

Silymarin can inhibit the hepatic cytochrome P450 (CYP) detoxification system
(phase I metabolism). It has been shown recently in mice that silibinin is able to
inhibit numerous hepatic CYP enzyme activities,[ whereas other researchers have not
detected any effect of silymarin on the CYP system.
This effect could explain some of the hepatoprotective properties of silymarin,
especially against the intoxication due to A phalloides. The Amanita toxin becomes
lethal for hepatocytes only after having been activated by the CYP system. Inhibition
of toxin bioactivation may contribute to the limitation of its toxic effects.
Additionally, silymarin, together with other antioxidant substances, could contribute
towards protection against free radicals generated by enzymes of the CYP system.

24
OTHER HEPATOPROTECTIVE DRUGS
FUMARIA OFFICINALIS LINN. (Fumitory)

Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Ranunculales
Family: Papaveraceae
Genus: Fumaria

ANDROGRAPHIS PANICULTATA

Kingdom: Plantae
(unranked): Angiosperms
(unranked): Eudicots
(unranked): Asterids
Order: Lamiales
Family: Acanthaceae
Genus: Andrographis
Species: A. paniculata

BOERHAVIA DIFFUSA LINN.(PUNARNAVA)

Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Caryophyllales
Family: Nyctaginaceae
Genus: Boerhavia
Species: B. diffusa

25
CICHORIUM INTYBUS LINN. (CHIKORY)

Kingdom: Plantae
(unranked): Angiosperms
(unranked): Eudicots
(unranked): Asterids
Order: Asterales
Family: Asteraceae
Tribe: Cichorieae
Genus: Cichorium
Species: C. intybus

PHYLLANTHUS NIRURI

Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Malpighiales
Family: Phyllanthaceae
Genus: Phyllanthus
Species: P. niruri

PICRORHIZA KURROA

Kingdom: Plantae - Plants

Genus: Picrorhiza
Specific epithet: kurroa - Royle
Botanical name: - Picrorhiza kurroa Royle

RHEUM OFFICINALE

Kingdom Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Caryophyllales
Family: Polygonaceae
Genus: Rheum
Species: R. officinale

26
RICINUS COMMUNIS

Kingdom: Plantae
Phylum: Magnoliophyta
Class: Magnoliopsida
Order: Malpighiales
Family: Euphorbiaceae
Subfamily: Acalyphoideae
Tribe: Acalypheae
Subtribe: Ricininae
Genus: Ricinus
Species: R. communis

SOLANUM NIGRUM

Kingdom: Plantae
(unranked): Angiosperms
(unranked): Eudicots
(unranked): Asterids
Order: Solanales
Family: Solanaceae
Genus: Solanum`
Species: S. nigrum

TEPHROSIA PURPUREA

Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Fabales
Family: Fabaceae
Tribe: Millettieae
Genus: Tephrosia
Species: T. purpurea

TINOSPORA CORDIFOLIA

Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Ranunculales
Family: Menispermaceae
Genus: Tinospora
Species: T. cordifolia

27
 7. APPENDIX
LIST OF ABBREVIATIONS

RFLP Restriction Fragment Length Polymorphism

RAPD Random Amplified Polymorphic DNA
VNTR Variable Number Of Tandem Repeat
PCR Polymerase Chain Reaction
DTAE Dextran Transfecsn And Electroporasn
HPTLC High Pressure Thin Layer Chromatography
NP Natural Product
TTC Twin Through Chamber
LDL Low Density Lipoproteins
TLC Thin Layer Chromatography
PEG Polyethylene Glycol

28
8 REFERENCE:
1. Khare Cp; “Indian Medicinal Plants An Illustrated Dictionary”; CP
khare Edition; Springer Science + Business Media, LLC., Spring Street,
New York, NY, USA; 2007; 49, 274, 482, 485, 554, 551, 613, 650, 662.
2. ^ Rose, Francis (1981). The Wild Flower Key. Frederick Warne &
Co. pp. 388-389. ISBN 0-7232-2419-6.(morphology/ description)

29