Upload
firdoushyder
View
217
Download
0
Embed Size (px)
Citation preview
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
1/34
Giulio LUPIDI
University of Camerino
Dept. Biology M.C.A.
Plant Extracts for in vitro/ in vivo
screening and their characterization
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
2/34
Evolution against challenges
Structural diversity
Apparently unlimited quantity
Potency
Natural Products
Advantages of Natural Products
Disadvantages of Natural Products
Synthesis
Isolation
Identification
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
3/34
Medicinal Plants Researches
Drugs and
inhibitors
In vitro
studies
Animalmodels
Human
studiesMedicinal
plants in
vivo effects
In vitro
studies
Animal
models
Active
components
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
4/34
The limitations of traditional medicine
There is little clinical data on safety and efficacy
Content of active compounds in plants is variable
There is no consensus on what plants, preparations
and dosages to use
These are all remediable, through research
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
5/34
products, endpoints & methodologies
Bioprospecting for active molecules: new leads for
conventional drug development
Phytomedicines: standardised herbal extracts.
Traditional medicine:prepared according to traditional
formulations
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
6/34
The African continent abounds of plants that
reportedly have anti-malarial activitywhich could be further studied and exploited
THE PLANT
-Evidence of efficacy of a plant used by traditional medical
practitioners to treat a number of diseases including malaria.
-Name of plant.
-Local name ..
.
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
7/34
WHAT IS KNOWN ON PLANT
Scientific studies on preparations available, indicating the effects
of plant.
They also include preliminary results on:
-Anti-plasmodial, i.e., works against the malaria parasite
Plasmodium falciparum outside the body.
-Anti-microbial, i.e., works against small or micro organisms
(germs, bacteria);-Anti-hyperglycaemic, i.e., against diabetes.
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
8/34
The competitive advantage of herbal antimalarials Affordable
Available
Sustainable
Reach the parts that modern drugs dont reach
Ways of using plants against malaria
Insect repellents
Vector Control
Prophylaxis
Treatment
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
9/34
Natural Products
Typically process of drug discovery from natural sources
Pre-knowledge helpful (cultural folklore,
traditional use.)
Collect source (plant, leaves,root .) Screen extracts (organic + aqueous
extractions)
Chromatographic separation Screen individual components
Structural identification
Independent synthesis and re-bioassay
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
10/34
Screening of extracts for in vitro and in vivo
activity
Question:
Which is the most active extract from selected
plants?
Answer:
Extracts can be obtained, applying the
technique used by most of the traditional
healers
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
11/34
Powdered plant
Decoction
in water
ethanol extract
Centrifugation
Centrifugation
Water extract
PrecipitateTandem extraction
with ethanol
Centrifugation
Ethanol tandem
extractEthanol extract
Optional solvents:acetone, alcohols, chloroform, glyceroldimethylsuphoxide, dioxane, others
Parameters:
1-Temperature
2-Time
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
12/34
Powdered plantDecoction
in waterCentrifugation
Centrifugation
Water extract
(1)
Precipitate Decoctionin water
Centrifugation
Water extract
(2)
Water extract
(3)
PrecipitateDecoction
in water
Precipitate
Parameters:
1-Temperature
2-Time
Multiple extractions
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
13/34
With different extracts it is possible to perform in
vitro and in vivo studies
Questions:
-We want to study the dependence of antimalaria
activity as function of extract concentration .
-We want to know the toxicity of extracts in animal
models or their cytotoxicity on human cells (es.
fibroblasts (HeLa))
A cytotoxicity/antiplasmodial index can be
calculated,
Problem: the extracts are diluted
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
14/34
Water extract
Ethanol tandem
extract
Ethanol extract
concentrate by liophylization
evaporation
We can obtain a powder from different extracts
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
15/34
Problem: Standardised or quantified extracts
The method must be simple, rapid, solution-based so a
spectrophotometer could be used for quantification, amendable tothe simultaneous analysis of multiple samples, and able to
incorporate a commercially available standard or established
extinction coefficient.
Colorimetric methods used for other plant secondary metabolites
(e.g., flavonoids (flavanones , anthocyanins, simplephenolics, and carotenoids )
-Total polyphenol contents, estimated by FolinCiocalteu method
-Characterization and measurement of anthocyanins by UV-visible
spectroscopy-Carotenoids (analytical methods).
-Total limonoids compounds estimated with colorimetric method
-Chromatographycs methods (HPLC) to estimate the concentration
of leader components.
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
16/34
THERAPEUTIC ACTION OF NATURAL
PRODUCTS
- ACTIVE EXTRACTS
- COMBINED ACTIVE EXTRACTS
- ACTIVE COMPOUNDS
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
17/34
ADVANTAGES AND DISADVANTAGES
In-Vitro Bioassays
-Rapid, less selective, but diagnostic, less expensive,
-suitable for minute amounts of materials,
-suitable for screening large number of
samples in Drug Discovery programs.
In-Vivo Bioassays
More selective, expensive, mostly Time Consuming,Animal model, require large amounts of material,
hardly could guide the fractionation of extracts or mixture of compounds.
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
18/34
Separation
Isolation
Identification
Extraction
Crude Extracts
Extract Fractions
Pure Compounds
Medicinal Plant
Active
Lead Compound
Determination of
Biological Activity
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
19/34
BIOACTIVITY APPROACH:
-Extracts are screened for a specific medicinal activity (an in-vitro
bioassay).
-Only BioActive extracts are
fractionated.
-Active fractions are further fractionatedto isolate the active compound(s) in
pure form.
-Characterization of structure.
METHODS OF DRUG DISCOVERY
FROM NATURAL PRODUCTS
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
20/34
Studies the influence of all the factors affecting the ability of
a drug to reach the systemic blood circulation and to
perform its pharmacological activity
The drugs action is performed through three different phases:
11. The BIOPHARMACEUTICAL phase
It is related to the drug release from the pharmaceutical dosage
form, the drug solubilization and the drug absorption throughspecific cellular compartments in order to reach the systemic blood
circulation.
22. The PHARMACOKINETIC phase
It is related to the distribution, metabolism, removal of the drug.
Every step influences the drug half-life.
33. The PHARMACODYNAMIC phase
It is related to the pharmacological activity of the drug (interaction
with specific molecules)
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
21/34
Pharmacokinetics
Or: Does the drug actually reach its site of action?
This is governed by three factors:
1. Absorption: Uptake of the drug from thecompartment of application into the blood
2. Distribution: Transport / equilibration between the
blood and the rest of the organism
3. Elimination: Filtration and secretion in the kidneys;
chemical modification in the liver
Determine the timeneeded to reach the
target
Determines the time
available
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
22/34
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
23/34
Dosage Effects
Site
ofAction
Plasma
Concen.
Pharmacokinetic
Toxicokinetics
Pharmacodynamics
Toxicodynamics
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
24/34
Pharmacokinetics
Onset: Time to reachminimum effective plasma
concentration
Therapeutic window:
Range of plasmaconcentrations where the
drug is effective but not
toxic
Duration: Time within the
therapeutic windowPlasmaconcentration
Time
Minimum effective concentration
Maximum safe concentration
Amount adsorbed
Clearance
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
25/34
The analyses are generally conducted in a two-stepprocess in which the total concentration of a given family
of metabolites is first estimated in equivalents of the
predominate metabolite through a colorimetric assay
followed by, if needed, a more complete analysis byHPLC or LC-MS to identify individual metabolites and
establish their relative concentrations
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
26/34
PHARMACEUTICALTECHNOLOGY
It is the science that studies how to chose an
appropriatepharmaceutical dosage form*** toadminister and delivery a drug or plant extracts at
appropriate rate and site of action.
Whats a pharmaceutical dosage form?Tablets, capsules, injectables, any systems that
enables drug or extracts administration.
The BIOPHARMACEUTICAL phase.
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
27/34
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
28/34
-INJECTABLE DOSAGE FORMS
-PREPARATION OF STERILE PRODUCTS
-FREEZE DRYING (LYOPHILIZATION)
STORAGE ( Advantages Disadvantage)
-THE ORAL ROUTE (Dosage forms forthe oral route administration :Tablets, Capsules,
Scirops, Solutions, Suspensions) (Fillers,diluents, bulking agents)
-DRUG DELIVERY (from molecular
packaging to targeting)
PHARMACEUTICAL TECHNOLOGY
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
29/34
The PHARMACODYNAMIC phaseIt is related to the pharmacological activity of the
drug (interaction with specific molecules)
Interaction with isolated Biochemical systems
(specific enzymes,factors, receptors..) or cells.
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
30/34
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
31/34
DRUGS TO BE DISCOVERED
FOR EXAMPLE: IF PRIORITY GIVEN
TO:
-ANTITUMORS,-ANTICARCINOGENIC,
-IMMUNOMODULATORY,
-ANTIVIRAL AGENTS
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
32/34
Curiosity
COSMECEUTICAL ANDCOSMECEUTICAL ANDPHYTOPHARMACEUTICAL PRODUCTSPHYTOPHARMACEUTICAL PRODUCTS
FROM MEDICINAL PLANTSFROM MEDICINAL PLANTS
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
33/34
-NEXT STEPS FOR THERAPY DEVELOPMENT
-VALIDATION OF TRADITIONAL MEDICINE
-QUALITY CONTROLS
-CONSERVATION and SUSTAINABLE USE of
MEDICINAL PLANTS
8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization
34/34
Thanks for your attention