Plant extraction

Introduction  The 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 plant Cultivated plant DisadvantageAdvantage 1- Scattered in large or unlimited area unlimited area Present in limited area. 2- Difficult to reach Easy to reach 3- The collector must be highly skilled botanists skilled botanists The collector must not be skillful person 4- Deficiency may occur due to continuous collection continuous collection Continuous 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 evaporator Dimethyl sulfoxide boils at 189 °C at atmospheric pressure. Under vacuum, it distills at 70 °C Dimethyl sulfoxide boils at 189 °C at atmospheric pressure. Under vacuum, it distills at 70 °C Dimethyl sulfoxide Dimethyl sulfoxide

Choice 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 extracted. Should readily dissolve substance to be extracted. Should not react with the substance to be extracted. Should not react with the substance to be extracted. Should not react with or be miscible with water (the usual second solvent(. Should not react with or be miscible with water (the usual second solvent(. Should have a low boiling point so it can be easily removed from the product. Should have a low boiling point so it can be easily removed from the product. Common extraction solvents are diethyl ether and methylene chloride. Common extraction solvents are diethyl ether and 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 water: (Although the criteria states that the organic solvent chosen should not be miscible with water, some solvents dissolve a small amount. Thus, water must be removed before separating the organic product from the organic solvent or else the product will be contaminated with water. A drying agent must be used. There are a number of drying agents available to the organic chemist: we will be using sodium sulfate and magnesium sulfate. Placing the organic solvent containing the dissolved product in contact with the chosen drying agent will allow the agent to absorb any dissolved water. The agent can then be removed from the solvent and then the product can be isolated. Removal of water: (Although the criteria states that the organic solvent chosen should not be miscible with water, some solvents dissolve a small amount. Thus, water must be removed before separating the organic product from the organic solvent or else the product will be contaminated with water. A drying agent must be used. There are a number of drying agents available to the organic chemist: we will be using sodium sulfate and magnesium sulfate. Placing the organic solvent containing the dissolved product in contact with the chosen drying agent will allow the agent to absorb any dissolved water. The agent can then be removed from the solvent and then the product can be isolated.

Kirby-Bauer antibiotic testing ) KB testing or disk diffusion antibiotic sensitivity testing ( is a test which uses antibiotic-impregnated wafers to test whether particular bacteria are susceptible to specific antibiotics. A known quantity of bacteria are grown on agar plates in the presence of thin wafers containing relevant antibiotics. If the bacteria are susceptible to a particular antibiotic, an area of clearing surrounds the wafer where bacteria are not capable of growing (called a zone of inhibition). is a test which uses antibiotic-impregnated wafers to test whether particular bacteria are susceptible to specific antibiotics. A known quantity of bacteria are grown on agar plates in the presence of thin wafers containing relevant antibiotics. If the bacteria are susceptible to a particular antibiotic, an area of clearing surrounds the wafer where bacteria are not capable of growing (called a zone of inhibition).antibioticbacteria agarantibioticbacteria agar This along with the rate of antibiotic diffusion are used to estimate the bacteria's sensitivity to that particular antibiotic. In general, larger zones correlate with smaller minimum inhibitory concentration (MIC) of antibiotic for that bacteria. This information can be used to choose appropriate antibiotics to combat a particular infection. This along with the rate of antibiotic diffusion are used to estimate the bacteria's sensitivity to that particular antibiotic. In general, larger zones correlate with smaller minimum inhibitory concentration (MIC) of antibiotic for that bacteria. This information can be used to choose appropriate antibiotics to combat a particular infection. minimum inhibitory concentration minimum inhibitory concentration

Minimum inhibitory concentration (MICs) are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation (MICs) are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation and minimum bactericidal concentrations (MBCs) as the lowest concentration of antimicrobial that will prevent the growth of an organism after subculture on to antibiotic-free media. and minimum bactericidal concentrations (MBCs) as the lowest concentration of antimicrobial that will prevent the growth of an organism after subculture on to antibiotic-free media.

Kirby-Bauer antibiotic testing Disk-diffusion method for determing the activity of antimicrobials

well diffusion method

Mechanisms of Antimicrobial Drugs