Plant extractionPlant extraction

IntroductionThe use of plant-derived medicinal dates back many

centuries although it is still under estimation in modern

medicine.

Plants remain the most important source of natural drugs.

- More than 30% of prescription drugs are natural products.

- More than 60% of anticancer and anti-infective drugs are natural products.

The main sources of drugs are as follows:

1- Natural substances:

From plants, microorganisms, animals,- etc. (totally obtained from nature).

2- Semisynthetic substances:

These are drugs that are manufactured by partial synthesis.

3- Synthetic substances:

These are drugs which are manufactured by total synthesis

To carry phytochemical screening the following points must be fulfilled:

1- Selection of promising plant materials.

2- Proper collection of selected plants.

3- Authentication of plant material.

4- Drying of plant materials.

5- Grinding of the dried plants.

6- Garbling of the dried plants

7- Packing, storage and preservation

8- Extraction and fractionation of constituents.

9- Methods of separation and purification.

10- Methods of identification of isolated compounds (structure elucidation e.g. UV, IR, MS, H-NMR and C-NMR).

1.Selection of promising plant materials:

Before investing time, effort and money in phytochemical screening it is very important to select a promising plant.

The choice of promising plant depends upon the following: 1- A plant which have a biological activity. 2- A plant used in folk medicine. 3- A plant which show a particular toxicities.

2. Proper collection of selected plants

Drug may be collected from: 1- Wild plants. 2- Cultivated plants.

Wild plantWild plantCultivated plantCultivated plant

DisadvantageDisadvantageAdvantageAdvantage

1- Scattered in large or1- Scattered in large or unlimited areaunlimited area

Present in limited area.Present in limited area.

2- Difficult to reach2- Difficult to reachEasy to reachEasy to reach

3- The collector must be highly 3- The collector must be highly skilled botanistsskilled botanists

The collector must not be skillful The collector must not be skillful person person

4- Deficiency may occur due to4- Deficiency may occur due to continuous collection continuous collection

Continuous supplyContinuous supply

The following precautions should be considered during stage of collection:

a- The proper time of the day, time of the year and maturity stage of collection is particularly important because the nature and quantity of constituents may vary greatly in some species according to the season and time of collection.

b- The collected plant should be free from any contamination.

c- Collecting plants which are free from diseases (i.e. which are not affected by viral, bacterial, fungal infection).

3- Authentication of plant material

This may be confirmed by:

1- Establishing the identity by a taxonomy experts.

2- Collection of a common species in their expected habitat by a field botanist.

3- By comparing the collecting plant with a voucher specimen ( herbarium sheet)

4- Drying of plant materials

Drying is done in: -Shade and in sunlight (Natural drying). - Hot air drying or by freeze-drying (Artificial drying).

Aim of drying:

1- Ease of transport. 2- Ease of grinding 3- Inhibit the growth of microorganisms. 4- Preservative of active constituents.

Changes may occur during the drying:

1- Size and weight

2- Shape and appearance

3- Color

4- Odor

5- Taste

6- Active constituent

5- Grinding of the dried plants

6- Garbling of the dried plants

7- Packing, storage and preservation

8- Extraction and fractionation of constituents

There is no general (universal) method for the extraction of plant materials.

The precise mode of extraction depends on:

1- The texture of the plant material. 2- The water content of the plant material. 3- The type of substances to be extracted or nature of active constituents.

Extraction: is the separation of medicinally active portion of plants or animal tissues through the use of selective solvent and suitable methods extraction.

The principal methods of extraction are:

1- Maceration 2- Percolation 3- Infusion 4- Decoction 5- Digestion 6- Continuous hot extraction technique (Soxhlet extraction process). 7- Liquid-liquid extraction

8- Distillation

Continuous hot extraction technique (Soxhlet extraction process)

Reflux is the process of boiling reactants while continually cooling the vapor returning it back to the flask as a liquid. It is used to heat a mixture for extended periods and at certain temperatures.. A condenser is attached to the boiling flask, and cooling water is circulated to condense escaping vapors. One should always use a boiling stone or a magnetic stirrer to keep the boiling solution from "bumping."

The temperature of a reaction in a refluxing mixture will be approximately the boiling point of the solvent used for the reaction.

Sonication is the process of converting an electrical signal into a physical vibration that can be directed toward a substance. Sonicators are vital lab equipment and are used for a number of purposes. Sonication is usually performed to break apart compounds or cells for further examination. The vibration has a very powerful effect on solutions, causing their molecules to break apart and cells to rupture. A prime example is in DNA testing, where the cells that may contain DNA information are subjected to sonication to break them apart and release the DNA proteins so they can be tested.

Sonicator

Rotary vacuum evaporatorRotary vacuum evaporator

Dimethyl sulfoxideDimethyl sulfoxide boils boils at 189 °C at at 189 °C at atmospheric pressureatmospheric pressure. . Under vacuum, it Under vacuum, it distills at 70 °Cdistills at 70 °C

Choice of solventChoice of solvent As a general empirical rule:

Non polar solvents (petroleum ether and hexane) will dissolve non-polar compounds (fats and waxes).

While polar solvents (methanol, ethanol and water) dissolve polar compound (alkaloid salts and sugars).

(that mean like dissolve like)

The affinity of the solute for the organic phase may be

greatly increased by using mixture of solvents instead

of single ones (sometimes used mixtures of solvent to

increase the solubility).

Should readily dissolve substance to be Should readily dissolve substance to be extractedextracted. .

Should not react with the substance to be Should not react with the substance to be extractedextracted. .

Should not react with or be miscible with water Should not react with or be miscible with water ((the usual second solventthe usual second solvent( ( ..

Should have a low boiling point so it can be Should have a low boiling point so it can be easily removed from the producteasily removed from the product. .

Common extraction solvents are diethyl ether Common extraction solvents are diethyl ether and methylene chlorideand methylene chloride. .

Phytopharmacological screening:

● Antimicrobial activity Cinnamomum verum القرفة (Eugenol) Thymus vulgaris الزعتر (Thymol) Lavendula officinalis الخزامى (Linalool)

● Antineoplastic activity Catharanthus roseus (Vincrestine, vinblastin) Taxus brevifolia (Taxol)

● Antimalarial: Cinchona succirubra (Quinine) Artemisia annua (Artemisinin)

● Hypoglycemic: Garlic (Allium sativum) (Allicin)

● Cardiotonic Digitalis purpurea (Digoxin and Digitoxin) Strophanthus kombe (K-strophanthoside) ● Antiarrythemic Cinchona succirubra (Quinidine)

Phytopharmacological screening:

Removal of waterRemoval of water: : ((Although the criteria states Although the criteria states that the organic solvent chosen should not be that the organic solvent chosen should not be miscible with water, some solvents dissolve a small miscible with water, some solvents dissolve a small amountamount. . Thus, water must be removed before Thus, water must be removed before separating the organic product from the organic separating the organic product from the organic solvent or else the product will be contaminated solvent or else the product will be contaminated with waterwith water. . A drying agent must be usedA drying agent must be used. . There are There are a number of drying agents available to the organic a number of drying agents available to the organic chemistchemist: : we will be using we will be using sodium sulfatesodium sulfate and and magnesium sulfatemagnesium sulfate. . Placing the organic solvent Placing the organic solvent containing the dissolved product in contact with the containing the dissolved product in contact with the chosen drying agent will allow the agent to absorb chosen drying agent will allow the agent to absorb any dissolved waterany dissolved water. . The agent can then be The agent can then be removed from the solvent and then the product can removed from the solvent and then the product can be isolatedbe isolated. .

KirbyKirby--Bauer antibiotic testingBauer antibiotic testing ) ) KB testingKB testing oror disk diffusiondisk diffusion antibiotic sensitivity testing antibiotic sensitivity testing ( (

is a test which usesis a test which uses antibioticantibiotic--impregnated wafers to test impregnated wafers to test whether particularwhether particular bacteriabacteria are susceptible to specific are susceptible to specific antibioticsantibiotics. . A known quantity of bacteria are grown onA known quantity of bacteria are grown on agaragar plates in the presence of thin wafers containing plates in the presence of thin wafers containing relevant antibioticsrelevant antibiotics. . If the bacteria are susceptible to a If the bacteria are susceptible to a particular antibiotic, an area of clearing surrounds the particular antibiotic, an area of clearing surrounds the wafer where bacteria are not capable of growing wafer where bacteria are not capable of growing ((called called a zone of inhibitiona zone of inhibition).).

This along with the rate of antibiotic diffusion are used to This along with the rate of antibiotic diffusion are used to estimate the bacteria's sensitivity to that particular estimate the bacteria's sensitivity to that particular antibioticantibiotic. . In general, larger zones correlate with smallerIn general, larger zones correlate with smaller minimum inhibitory concentrationminimum inhibitory concentration ( (MICMIC) ) of antibiotic for of antibiotic for that bacteriathat bacteria. . This information can be used to choose This information can be used to choose appropriate antibiotics to combat a particular infectionappropriate antibiotics to combat a particular infection..

Minimum inhibitory concentrationMinimum inhibitory concentration

(MICs)(MICs) are defined as the lowest are defined as the lowest concentration of an antimicrobial that will concentration of an antimicrobial that will inhibit the visible growth of a inhibit the visible growth of a microorganism after overnight incubationmicroorganism after overnight incubation

and minimum bactericidal concentrations and minimum bactericidal concentrations (MBCs)(MBCs) as the lowest concentration of as the lowest concentration of antimicrobial that will prevent the growth of antimicrobial that will prevent the growth of an organism after subculture on to an organism after subculture on to antibiotic-free media. antibiotic-free media.

Kirby-Bauer antibiotic testing

Disk-diffusion method for determing the activity of antimicrobials

well diffusion methodwell diffusion method

Mechanisms of Antimicrobial Drugs