Plant Extracts for in Vitro in Vivoscreening and Their Characterization

8/10/2019 Plant Extracts for in Vitro in Vivoscreening and Their Characterization

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Giulio LUPIDI

University of Camerino

Dept. Biology M.C.A.

Plant Extracts for in vitro/ in vivo

screening and their characterization


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Evolution against challenges

Structural diversity

Apparently unlimited quantity

Potency

Natural Products

Advantages of Natural Products

Disadvantages of Natural Products

Synthesis

Isolation

Identification


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Medicinal Plants Researches

Drugs and

inhibitors

In vitro

studies

Animalmodels

Human

studiesMedicinal

plants in

vivo effects

In vitro

studies

Animal

models

Active

components


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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


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products, endpoints & methodologies

Bioprospecting for active molecules: new leads for

conventional drug development

Phytomedicines: standardised herbal extracts.

Traditional medicine:prepared according to traditional

formulations


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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 ..

.


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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.


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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


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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


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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


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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


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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


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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


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Water extract

Ethanol tandem

extract

Ethanol extract

concentrate by liophylization

evaporation

We can obtain a powder from different extracts


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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.


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THERAPEUTIC ACTION OF NATURAL

PRODUCTS

- ACTIVE EXTRACTS

- COMBINED ACTIVE EXTRACTS

- ACTIVE COMPOUNDS


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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.


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Separation

Isolation

Identification

Extraction

Crude Extracts

Extract Fractions

Pure Compounds

Medicinal Plant

Active

Lead Compound

Determination of

Biological Activity


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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


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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)


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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


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Dosage Effects

Site

ofAction

Plasma

Concen.

Pharmacokinetic

Toxicokinetics

Pharmacodynamics

Toxicodynamics


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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


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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


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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.


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-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


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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.


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DRUGS TO BE DISCOVERED

FOR EXAMPLE: IF PRIORITY GIVEN

TO:

-ANTITUMORS,-ANTICARCINOGENIC,

-IMMUNOMODULATORY,

-ANTIVIRAL AGENTS


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Curiosity

COSMECEUTICAL ANDCOSMECEUTICAL ANDPHYTOPHARMACEUTICAL PRODUCTSPHYTOPHARMACEUTICAL PRODUCTS

FROM MEDICINAL PLANTSFROM MEDICINAL PLANTS


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-NEXT STEPS FOR THERAPY DEVELOPMENT

-VALIDATION OF TRADITIONAL MEDICINE

-QUALITY CONTROLS

-CONSERVATION and SUSTAINABLE USE of

MEDICINAL PLANTS


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Thanks for your attention

Documents

Plant Extracts for in Vitro in Vivoscreening and Their Characterization