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Plant Extracts for in vitro/ in vivo

screening and their characterization

Giulio LUPIDI
University of Camerino
Dept. Biology M.C.A.

Natural Products
9 Advantages of Natural Products



Evolution against challenges
Structural diversity
‘Apparently’ unlimited quantity
Potency

9 Disadvantages of Natural Products


Synthesis
Isolation
Identification

Medicinal Plants Researches
Human
studies

Animal
models

In vitro
studies

Drugs and
inhibitors

Medicinal
plants in
vivo effects

Animal
models

In vitro
studies

Active
components

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. through research… . preparations and dosages to use ‹ These are all remediable.

‹ Traditional medicine: prepared according to traditional formulations . endpoints & methodologies ‹ Bioprospecting for active molecules: new leads for conventional drug development ‹ Phytomedicines: standardised herbal extracts.products.

.”. .The African continent abounds of plants that reportedly have anti-malarial activity which 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. -Local name “……. -Name of plant.

. works against small or micro organisms (germs. i.e... indicating the effects of plant. i. They also include preliminary results on: -Anti-plasmodial. -Anti-microbial. works against the malaria parasite Plasmodium falciparum outside the body. i.e. bacteria).WHAT IS KNOWN ON PLANT Scientific studies on preparations available. . against diabetes. -Anti-hyperglycaemic.e.

The competitive advantage of herbal antimalarials ‹ Affordable ‹ Available ‹ Sustainable ‹ Reach the parts that modern drugs don’t reach… Ways of using plants against malaria ‹ Insect repellents ‹ Vector Control ‹ Prophylaxis ‹ Treatment .

Natural Products 9 Typically process of drug discovery from natural sources • • • • • • • Pre-knowledge helpful (cultural folklore. leaves.) Screen extracts (organic + aqueous extractions) Chromatographic separation Screen individual components Structural identification Independent synthesis and re-bioassay . traditional use….root ….) Collect source (plant.

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 .

glycerol dimethylsuphoxide. others 2-Time . chloroform.Water extract Decoction in water Centrifugation Precipitate Tandem extraction with ethanol Powdered plant ethanol extract Centrifugation Centrifugation Ethanol extract Ethanol tandem extract Parameters: Optional solvents: 1-Temperature acetone. dioxane. alcohols.

Multiple extractions Water extract (1) Parameters: Decoction Powdered plant in water Centrifugation 1-Temperature 2-Time Decoction in water Water extract (3) Centrifugation Precipitate Decoction in water Precipitate Centrifugation Precipitate Water extract (2) .

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 . Problem: the extracts are diluted . -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.

Water extract concentrate by liophylization Ethanol tandem extract evaporation Ethanol extract We can obtain a powder from different extracts .

solution-based so a spectrophotometer could be used for quantification.g. .Problem: Standardised or quantified extracts The method must be simple. simple phenolics. Colorimetric methods used for other plant secondary metabolites (e. amendable to the simultaneous analysis of multiple samples. anthocyanins.. and carotenoids ) -Total polyphenol contents. -Total limonoids compounds estimated with colorimetric method -Chromatographycs methods (HPLC) to estimate the concentration of leader components. rapid. flavonoids (flavanones . estimated by Folin–Ciocalteu method -Characterization and measurement of anthocyanins by UV-visible spectroscopy -Carotenoids (analytical methods). and able to incorporate a commercially available standard or established extinction coefficient.

COMBINED ACTIVE EXTRACTS .ACTIVE EXTRACTS .THERAPEUTIC ACTION OF NATURAL PRODUCTS .ACTIVE COMPOUNDS .

require large amounts of material. -suitable for screening large number of samples in Drug Discovery programs. less selective. In-Vivo Bioassays More selective. but diagnostic. Animal model.ADVANTAGES AND DISADVANTAGES In-Vitro Bioassays -Rapid. less expensive. -suitable for minute amounts of materials. . hardly could guide the fractionation of extracts or mixture of compounds. expensive. mostly Time Consuming.

Medicinal Plant Extraction Crude Extracts Determination of Biological Activity Separation Extract Fractions Isolation Active Lead Compound Pure Compounds Identification .

-Active fractions are further fractionated to isolate the active compound(s) in pure form. . -Characterization of structure. -Only BioActive extracts are fractionated.METHODS OF DRUG DISCOVERY FROM NATURAL PRODUCTS BIOACTIVITY APPROACH: -Extracts are screened for a specific medicinal activity (an in-vitro bioassay).

2. The PHARMACODYNAMIC phase It is related to the pharmacological activity of the drug (interaction with specific molecules…) . removal of the drug.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 drug’s action is performed through three different phases: 1. Every step influences the drug half-life. metabolism. The PHARMACOKINETIC phase It is related to the distribution. The BIOPHARMACEUTICAL phase It is related to the drug release from the pharmaceutical dosage form. the drug solubilization and the drug absorption through specific cellular compartments in order to reach the systemic blood circulation. 3.

Distribution: Transport / equilibration between the blood and the rest of the organism 3. Absorption: Uptake of the drug from the compartment of application into the blood 2.‘Pharmacokinetics’ Or: Does the drug actually reach its site of action? This is governed by three factors: 1. Elimination: Filtration and secretion in the kidneys. chemical modification in the liver Determine the time needed to reach the target Determines the time available .

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Pharmacokinetic Toxicokinetics Site of Action Effects Pharmacodynamics Toxicodynamics .Dosage Plasma Concen.

Pharmacokinetics Amount adsorbed Clearance Plasma concentration • Onset: Time to reach minimum effective plasma concentration • Therapeutic window: Range of plasma concentrations where the drug is effective but not toxic • Duration: Time within the therapeutic window Maximum safe concentration Minimum effective concentration Time .

a more complete analysis by HPLC or LC-MS to identify individual metabolites and establish their relative concentrations .The analyses are generally conducted in a two-step process 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.

capsules. PHARMACEUTICAL TECHNOLOGY It is the science that studies how to chose an appropriate pharmaceutical dosage form*** to administer and delivery a drug or plant extracts at appropriate rate and site of action. any systems that enables drug or extracts administration. What’s a pharmaceutical dosage form? Tablets. . injectables.The BIOPHARMACEUTICAL phase.

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bulking agents) -DRUG DELIVERY (from molecular packaging to targeting) . Suspensions) (Fillers. diluents.PHARMACEUTICAL TECHNOLOGY -PREPARATION OF STERILE PRODUCTS -INJECTABLE DOSAGE FORMS -FREEZE DRYING (LYOPHILIZATION) STORAGE ( Advantages Disadvantage) -THE ORAL ROUTE (Dosage forms for the oral route administration :Tablets. Capsules. Solutions. Scirops.

The PHARMACODYNAMIC phase It is related to the pharmacological activity of the drug (interaction with specific molecules…) Interaction with isolated Biochemical systems (specific enzymes. receptors…..factors.) or cells. .

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-ANTIVIRAL AGENTS . -IMMUNOMODULATORY. -ANTICARCINOGENIC.DRUGS TO BE DISCOVERED FOR EXAMPLE: IF PRIORITY GIVEN TO: -ANTITUMORS.

Curiosity COSMECEUTICAL AND PHYTOPHARMACEUTICAL PRODUCTS FROM MEDICINAL PLANTS .

-NEXT STEPS FOR THERAPY DEVELOPMENT -VALIDATION OF TRADITIONAL MEDICINE -QUALITY CONTROLS -CONSERVATION and SUSTAINABLE USE of MEDICINAL PLANTS .

Thanks for your attention .