434- Part III [Lec-3..

advertisement
434 PHG
Recent Approaches in
Medicinal Plants Analyses
Prof. Mohammed Abdulaziz Al-Yahya
1
BIOLOGICAL
SCREENING
2
TYPES OF ASSAY
Chemical Assays:
Spectrophotometry, Turbidimetry
Spectrofluorimetry, Gravimetry,
Chromatography etc.
 Bioassays :
In-vitro Assay
In-vivo Assay
Microbiological assays

3
BIOASSAY: Estimation of the conc. /
potency of a substance by measuring its
biological response in living systems.

Choosing the right bioassay or test system is crucial to the
success of a biological screening program.

The test should be simple, quick and relevant as there are
usually a large number of samples to be analyzed.

Human testing is not possible at such early stage, so the test
has to be done in vitro first. Because in vitro tests are cheaper,
easier to carry out, less controversial and can be automated
than in vivo one.

In vivo tests needed to check the drugs interaction with specific
4
target and to monitor their pharmacokinetics properties.
IN-VITRO ASSAY

They do not involve live animals. Instead, specific tissues,
cells, or enzymes are isolated and used.

Enzyme inhibitors can be tested on pure enzyme in
solution.

Receptor agonist and antagonists can be tested on isolated
tissues or cells.

Antibacterial drugs are tested in vitro by measuring how
effectively they inhibit or kill bacterial cells in culture
5
IN-VIVO ASSAY

In vivo tests on animals often involve inducing a clinical
condition in the animal to produce observable symptoms.

The animal is then treated to see whether the drug
alleviates the problem by eliminating the observable
symptoms. For example, the development of non-steroidal
inflammatory
drugs
was
carried
out
by
inducing
inflammation on test animals.
6

There are several problems associated with in vivo testing.
It is slow and it also causes animal suffering. There are also
many problems of pharmacokinetics and the result obtained
may be misleading. For example, penicillin methyl ester is
hydrolyzed in mice into active penicillin, while it is not
hydrolyzed in humans or rabbits. Also, thalidomide is
teratogenic in rabbits and humans while it is not in mice.
7
● After a new substance (expected to be a new drug)
has been developed (extracted, isolated).
-The next step is to subject it to a specified set of
procedures in experimental animals.
● The aim is to determine whether or not the compound
possesses the desired action.
8
Pharmacological screening:
Requirements:
1- Animals: have a large problem such as:
a- Cost
b- Feeding
c- Selection: e.g. : Dogs (heart/ vascular)
Rats (analgesia/ inflammation)
Rabbits ( CNS)
Monkey ( tumor, virus)
2- Tissues:
All types of muscle, heart, nerve structure.
3- Time:
It take from 2-3 months and also need duplication for
experiments.
4- Solubility: drug action depend on solubility so most probably
9
in animal used: oral, i.p (Intraperitoneal).
Phytopharmacological screening:
● Antimicrobial activity
Eugenia caryophyllus
(Eugenol)
Thymus vulgaris
(Thymol)
Lavendula officinalis
(Linalool)
● Antineoplastic activity
Catharanthus roseus
vinblastin)
Taxus brevifolia
(Taxol)
● Antimalarial:
Cinchona succirubra
Artemisia annua
(Quinine)
(Artemisinin)
(Vincrestine,
10
● Hypoglycemic:
Garlic (Allium sativum)
(Allicin)
● Cardiotonic
Digitalis purpurea
Strophanthus kombe
(Digoxin and Digitoxin)
(K-strophanthoside)
● Antiarrythemic
Cinchona succirubra
(Quinidine)
11
Type of pharmacological screening:
1-Blind screening
The aim is to see if a new compound has any useful
pharmacological activity.
2- Simple screening
The aim is to find a substance having a particular property.
e.g. A single test for the conc. of sugar in blood may be
used to screen compounds for hypoglycemic activity.
3- Programmed screening
In this type of screening the most important pharmacological
action of the new compound is fully investigated.
12
The organization of blind screening
Screening for pharmacological activities proceeds from general
observation on intact animals to the more specific techniques
using isolated organs.
It might include the following tests:
A- Preliminary observations on the behaviour of conscious
animals (neuropharmacological tests)
B- The cardiovascular test.
C- Isolated organ preparations especially the isolated
guinea pig ileum.
13
a) Neuropharmacological tests:
These tests were developed to determine the central
actions of drugs.
● It can detect: sedatives, hypnotics, tranquillizers,
pscychomotor stimulants, muscle relaxant,
analgesics, atropine-like drugs, antipyretics,
peripheral vasodilators.
● The animal used are: mice.
● The route of administration is important:
- The compound is given parenterally in order to avoid
the possibility of poor absorption from the intestinal tract.
● Different doses are used.
- For the performance of the test 3 animals are used per dose.
14
The signs observed in the primary test for pharmacological
activity are:
1- Consciousness or awareness.
2- Stereotype: is the frequental mechanical repetition of
a movement.
e.g. - Morphine: cause the mice to have circular
movement.
- Amphetamine: cause them to have anxiety
movement.
3- Motor activity and spontaneous activity, this measures
stimulation and depression of the autonomic nervous
system.
4-The pain response: this measures analgesia and sedation.
15
5- The touch response, may indicate the presence of
anesthetic activity.
6- Central excitation, is indicated by:
- Tremors.
- Convulsions.
- Restlessness and irritability.
7- The autonomic responses includes:
- Pupil size.
- Hypothermia
- Secretion
- Skin color
- Heart rate
- Respiratory rate
16
b) The cardiovascular tests:
These tests describe the action of the new
substance on blood pressure of the intact
anesthetized animals.
The following conclusions can be made:
1- If the test substance causes brief fall in b.p. (↓in b.p.)
therefore it may have:
- Muscarinic activity like ACH
- Direct vasodilator activity
- Cardiodepressant activity.
17
2- If the test substance causes a brief rise in blood
pressure
(↑ in b.p.) therefore it may have:
- Sympathomimetic activity or
- Direct vasopressor activity.
3- If the test substance causes a prolonged fall in b.p. (↓↓b.p.)
(more than 5 min), it may have: Ganglion blocking
4- If the test substance causes a prolonged rise in b.p. (↑↑b.p.)
(5minutes or more) it may have:
Monoamine oxidase inhibiting activity.
18
5- If the test substance causes a fall in b.p. after a delay
of 45 sec. it may have:
Histamine-releasing activity like morphine.
6- If after administration of the test substance,
the response to ACH is diminished.
- Parasympatholytic activity like atropine.
7- If after administration of the test substance,
the response to epinephrine injection is diminished
- Sympatholytic activity.
19
8- If the test substance causes the response to ACH to be
augmented above the control, the test substance may
have:
- Anticholinesterase activity like neostigmine.
9- If the test substance causes increase in response of
epinephrine above to the response obtained by giving
only epinephrine (i.e. without test substance). The test
substance may be monoamine oxidase inhibitor.
This will confirm the observation of prolonged rise in b.p.
produced by the test substance itself.
20
c) Isolated organ preparations especially the isolated
guinea pig ileum.
Isolated preparation have the following advantages:
1- Free from interference by central action (e.g. circulating
hormones).
2- Relatively small amounts of the material is required.
3- Drug effect is tested directly without the factor of
absorption, metabolism, etc.
4- Several preparation can be tested on a single animal.
21
Screening methods:
Ideal methods such as:
1- Antimicrobial
2- Anticancer
3- Antiviral
4- Hepatoprotective
5- Antioxidant
22
Antimicrobial Screening of Natural Products Using
Agar Dilution Method
- The Screening methods for the detection of antimicrobial
activity of natural products fall into three groups, including:
- Bioautographic
- Dilution methods (turbidimetric assay).
- Agar diffusion (plate assay)
The bioautographic is known as:
Qualitative technique since this method will only give
an idea of the presence or absence of substances with
antimicrobial activity.
23
Procedure:
1- The desired concentration of natural products is dissolved in
very small amount of DMSO (dimethyl sulfoxide).
2- The solution is added to 10 ml of the melted agar medium and
mixed will.
3- The agar is poured into the Petri dish often desired dimensions
and cooled.
4- The desired test organism are then applied a lines from the edges
to the center of the plate.
5- The plate is then incubated and growth of organisms is recorded.
24
Results:
1- In the above figure five organisms were used to screen natural
products A, B and C at 1mg/10 ml agar.
2- The used organisms are:
E: Escherichia coli
B: Bacillus subtillus
S: Staphylococcus aurous
C: Candida albicans
Ps: Pseudomonas aurogenosa
3- The results revealed that:
a. Natural products A and B are active against:
B: Bacillus Subtillus
S: Staphylococcus aurous
C: Candida albicans
b. Natural products C is active against:
B: Bacillus Subtillus
S: Staphylococcus aurous
c. The fourth plate is the control showing growth of all organisms
25
The Dilution methods (turbidimetric assay) and agar diffusion
(plate assay) are considered as:
Quantitative assays once they determine the minimal
inhibitory concentration (MIC).
 MIC is the lowest concentration of an antimicrobial that will
inhibit the visible growth of a microorganism after overnight
incubation.
 MIC are important in diagnostic laboratories to confirm
resistance of microorganisms to an antimicrobial agent and
also to monitor the activity of new antimicrobial agents.
26
Turbidimetric assay:
 The test organisms growing in test tubes that contain
different concentrations of the antibiotic.
 There is a direct relationship of the concentration of
antibiotic to the growth of the test organism, and by
measuring the growth of the organism which is indicated
by the turbidity of the content of the test tube, the
antibiotic titer can be determine.
 Clear tubes indicate a higher antibiotic conc. than turbid
tubes, and the lowest concentration of antibiotic that
completely prevents the appearance of turbidity is known
as MIC .
27
Plate assay:
 Filter paper discs are impregnated with solutions of
antibiotic of varying concentrations , allowed to dry,
placed on agar media seeded with an appropriate test
organism, and incubated.
 As the concentration of the antibiotic increase, its
diffusion through the agar medium increases; therefore,
the size of the clear zone of growth inhibition around
the filter paper disc is related to the concentration of
antibiotic.
28
Antioxidant Screening of Natural Products Using TLC
 Oxidation of a wide range of chemical compounds and the production
of free radicals (contains one or more unpaired electrons) at the
cellular level are believed to be involved in the cause of many cancers.
 Plant derived natural products such as flavonoids, terpenoids and
steroids have received considerable attention in recent years due to
their diverse pharmacological properties including antioxidant and
antitumor activity.
 Antioxidants are compounds that help to inhibit many oxidation
reactions caused by free radicals which will prevent or delay damage to
the cells and tissues.
29
Free radicals and cancer
Many forms of cancer are thought to be the result of reactions between
free radicals and DNA, resulting in mutations that can adversely affect
the cell cycle.
Antioxidants: classification
Non-Enzymatic antioxidant:



Alpha tocopherol (vitamin E)
Beta Carotene
Ascorbic acid (vitamin C)
Antioxidant enzymes:



Superoxide dismutase (SOD)
Glutathione peroxidase enzyme
The catalase enzyme
30
Natural antioxidants are presents
in fruits and vegetable
WHO recommendation:
eat 5 fruit or vegetable per a day
31
EXPERIMENT
Conditions:
* Plant extract rich in antioxidant are:
- Orange or lemon peel (dry and coarsely powder).
- Green Tea leaves (coarsely powder).
* Standard
antioxidant (References):
Gallic acid, Ellagic acid, Arbutin, Hydroquinone & Rutin
(at concentration = 1-2 mg/ml MeOH).
* Solvent
System:
-For orange peel, Lemon peel and green tea leaves is (for flavonoids):
Ethylacetate : Formic acid : H2O (8 : 1 : 1)
Or CHCl3 : Ethylacetate (6 : 4)
32
* Detection :
1. DPPH solution (20 mg/100 ml in MeOH), must be freshly
prepared. (DPPH= 1,1 Diphenyl-2-picrylhydrazyl)
2. Heat in Oven at 60 οC.
Procedure:
1. Spotting of the silica gel TLC plate.
2. Develop the plate.
3. Dry plates at open air for 15-20 seconds.
4. Dip the plate in DPPH which in Petri dish.
5. Dry plate in dark for 90 seconds at room temperature then for 30
seconds at 60 οC.
6. Free radical scavenging zone were identified immediately as
yellow area against violet purple background plate
33
Anticancer Screening of Natural Products :
 Cancer is a class of diseases in which a group of cells display
uncontrolled growth, invasion (intrusion on and destruction of
adjacent tissues) and sometimes metastasis (spread to other
locations in the body via lymph or blood).
 Causes:
- Tobacco smoke
- Infectious agents
- Radiation
- Chemical (Benzene)
- Diet (coloring agent, preservative)
- Pesticide
- Drug
34
 There are currently some well known structural classes of plant
derived anticancer agents in the market. These are:
1. The vinca alkaloids (vinblastine, vincristine): were isolated from
Catharanthus roseus and are used for the treatment of a wide
variety of cancers, including leukemia, bladder cancer and testicular
cancer.
2. The taxanes (paclitaxel and taxol): were isolated from Taxus brevifolia
and are used for the treatment of a wide variety of cancer including
Ovarian, Lung, Gastric, Cervical, Prostate & Colon cancer
3. The camptothecin derivatives (topotecan and irinotecan).
Uses: Ovarian cancers, Colorectal cancer.
35
Cytotoxicity assays
1- Brine shrimp lethality assay:
 Artemia
salina is commonly known as the brine shrimp.
 Larvae were hatched at 26 ºC in filtered seawater.
 A lamp was placed above the tank. After 24 hrs, hatched nauplii
were used for the assay.
 Plant extract with different concentration was added to brine solution.
 The experiment was run in five replicates of each concentration.
 The number of dead and survivor brine shrimps in each tube was
counted after 24 h.
 To ensure that mortality observed in the bioassay is related to
bioactive compounds, dead larvae in each treatment were compared
to the dead larvae in the control.
 The percentage of mortality was calculated as: percentage of survival
in the control-percentage of survival in the treatment
The brine shrimp assay is one of the best and rapid biological and
36
toxicological assays for all the labs.
Hepatoprotective Screening of Natural Products
 Cirrhosis is the formation of fibrous tissue in the liver from replacing dead
liver cells. The death of the liver cells can be caused by viral hepatitis,
alcoholism or contact with other liver-toxic chemicals.
 Diagnosis is done by checking levels of Serum glutamic oxaloacetic
transaminase and Serum glutamic pyruvic transaminase
Method:
1- Animals (rabbits/ rats)
- Control (saline) - Standard (Silymarin)
- Extracts or isolated compounds
2- Induction of cirrhosis in animals by using:
- CCl4 or CHCl3 (reversible)
- Galactosamine or α- amanitin (irreversible)
3- Administer extract and standard 24 hours measure enzymes
37
Although random compound screening in animal models
is still a useful approach to discover new drugs.
Disadvantages:
- It requires a large amount of compound.
- Its sensitivity is low.
- It is extremely laborious.
- Since the amount of active constituents present in natural
products is usually very small, it is impractical in most cases,
to supply sufficient quantities of pure natural compounds for
animal experimentation.
38
Problems with natural product screening
1. Isolation of an active component is problematic
because of very small amount of active constituent
along with large amount of background “rubbish”.
2.The mixtures are often very complex and contain
many large macromolecules. These
can often
“hide” biological activity.
3.Compound isolation and structure determination
difficult.
4. Structures often complex, therefore difficult
to
synthesize and identify the pharmacophore.
5.Plant materials are chemically and naturally variable.
39
6. Herbal medicines contain a large variety of different
compounds. Several of these may have biological activity,
but there is a significant risk of side effects and toxicity. The
active principle present in small amount, so herbals are
expected to be less active than pure compound.
7.Herbal medicines may be interacting with prescribed
medicines and there is no regulations or control of this
matter and their uses.
8. But it is an important lead to discover and design new drugs.
9.Selective analytical methods or reference
not be available commercially.
40
compounds may
10. The active principles are frequently unknown;
11. Standardization, stability and quality control are
feasible but not easy.
12. The availability and quality of raw materials are
frequently problematic.
13.Well-controlled
double-blind
clinical
and
toxicological studies to prove their efficacy and
safety are rare.
14. The occurrence of undesirable side effects seems
to be less frequent with herbal medicines, but wellcontrolled randomized clinical trials have revealed
that they also exist.
41
High-throughput technologies:
• High-throughput screening (HTS) is an approach to drug discovery
that has gained widespread popularity over the last three or four
years.
• HTS is the process of assaying a large number of potential effectors
of biological activity against targets.
• The methods of HTS are applied to the screening of stereo chemistry
of compounds, genomics, protein and peptide libraries.
• The goal of HTS is to accelerate drug discovery by screening large
libraries often composed of hundreds of thousands of compounds.
42
Advantage:
• HTS decreases the amount of testing compound required such
that only microgram quantities are needed. This is
advantageous for certain natural products that are difficult to
isolate and purify, and permits compounds that are difficult to
synthesize to be assayed.
• The number of compounds assayed has increased from
100000 per year to 100000 per day.
• HTS need:
Highly specialized and expensive screening lab to run an
HTS operation .
43
High-throughput screening (HTS)
 Using robotics, data processing and control software, liquid
handling devices, and sensitive detectors.
 HTS allows a researcher to quickly conduct millions of
biochemical, genetic or pharmacological tests.
 The results of these experiments provide starting points for
drug design and for understanding the interaction or role
of a particular biochemical process in biology.
44
45
Methodology and equipment
 HTS uses automation to run an assay, or screen, of a library
of candidate compounds against a target.
Note:
Automation: Typically, an integrated robot system consisting of
one or more robots transports assay-microplates
from station to station for sample and reagent
addition, mixing, incubation, and finally readout
or detection.
46
Assay plate preparation
 The key labware or testing vessel of HTS is the microtiter plate:
a small container, usually disposable and made of plastic, that
features a grid of small, open divots called wells.
 Modern microplates for HTS generally have either 384, 1536,
or 3456 wells. These are all multiples of 96, reflecting the original
96 well microplate with 8 x 129mm spaced wells.
 To prepare for an assay, the researcher fills each well of the plate
with some biological matter such as protein or some cells, or an
animal embryo.
 After some incubation time has passed to allow the biological
matter to absorb, bind to, or to react with the compounds in the
wells, measurements are taken across all the plate's wells, either
manually or by a machine.
47
 Manual measurements are often necessary when the researcher is
using microscopy to seek changes or defects in embryonic
development caused by the wells' compounds, looking for effects
that a computer could not easily determine by itself. Otherwise, a
specialized automated analysis machine can run a number of
experiments on the wells. In this case, the machine outputs the
result of each experiment as a grid of numeric values, with each
number mapping to the value obtained from a single well.
 A high-capacity analysis machine can measure dozens of plates in
the space of a few minutes like this, generating thousands of
experimental data points very quickly.
48
High-Throughput Screening
Up to 100,000 samples/24 hours
49
50
BIOLOGICALLY DIRECTED PHYTOCHEMICAL STUDY
The isolation of biologically active constituents
Target: Pharmacological Activity






Fractionation of the active extracts
Send fractions for biological screening
Isolate compounds from active fractions
Structural elucidation of isolated compounds
Send pure compounds for biological screening
Hopefully active molecule/s is/are found
51
Fractionation and isolation
The right chromatography techniques: CC,
TLC, VC, MPLC, HPLC; solvent systems
and stationary phase critical in hunting
down the bioactive compound/s.
52
Conventional Phytochemical Evaluation
1. To study cpd. which were most easily separated
(present in the largest quantities and crystallized
readily)
2. To study the specific group of interest
(e.g. alkaloids, terpenoids, phenols, etc.)
3. To elucidate the isolated compounds
4. To test for their biological activities
(this would depend on sufficient material being
available)
Countless medicinally useful cpd. Have been
missed in this type of approach.
53
The isolation of biologically
constituents
Systematic investigation
active
1. Every portion of the plant and every fraction
of the extract is biologically tested before
isolation or characterization
2. Any class of active cpd. may be isolated
3. May not be traditionally associated with
a particular plant family
54
Download