Extraction of Plant Material Pharmacognosy I Duhok University/ Faculty of Pharmacy L.A. Dilbreen Barzanji

Extraction of plant material The commonly employed technique for separation of active substance from crude drugs is called ‘Extraction’ which involves the use of different solvent. Whether samples are plants, microbes, marine animals or insects they are referred to as biomass. All plant material used should be properly authenticated, as much time and money can be wasted on the examination of material of doubtful origin.

Dried materials are usually powdered before extraction, whereas fresh plants (e.g. leaves, etc) can be homogenized or macerated with a solvent such as alcohol. (Homogenizing is particularly useful for stabilizing fresh leaves by dropping them into the boiling solvent). Dried biomass is ground into small particles using either a blender or a mill. Plant material is milled twice, first using a course mill and then a fine mill to generate a fine powder. The grinding process is important as effective extraction depends on the size of the biomass particles; large particles will be poorly extracted, whereas small particles have a higher surface area and will therefore be extracted more efficiently.

Selection of the solvent extraction approach is very important. If a plant is under investigation from an ethnobotanical perspective, then extraction should mimic the traditional use. For example; if indigenous people use a specific extraction protocol such as a water extract, a cold/heat tea, alcohol or alcohol-water mixtures, then an identical or at least a very similar method should be used in the laboratory so that the same natural products are extracted.

Failure to extract biomass properly may result in loss of access to active compounds. Additionally, using an inappropriate extraction method, such s strong heating of biomass with a solvent, may result in degradation of natural products and consequent loss of biological activity.

The choice of extraction procedure depends on the nature of the plant material and the components to be isolated. Alcohol is general solvent for many plant constituents (most fixed oils are exception) and as such may give problems in the subsequent elimination of pigments, resins, etc. Water-immiscible solvents are widely used; Light petroleum (essential and fixed oil, steroids), Ether and chloroform (alkaloids, quinines). The extraction of organic bases (e.g. alkaloids) usually necessitates basification of plant material if a water-immiscible solvent is to be used; for aromatic acids and phenols acidification may be required.

Special method for volatile oil , such as Clavenger and sometimes Enfleurage process.( enflourage: is the process of extracting fragrance from flowers by using odorless fats or oils to capture the essential oils. The perfumes of plants like jasmine could only be extracted by enfleurage).

Ultrasound may enhance the extraction process of some plant materials. Ultrasound present several advantage several advantages in term of: shortening the time of process, decrease the volume of the solvent, increasing the yield of the extract in comparison with conventional methods.

cold extraction Extraction itself may be performed by repeated maceration with agitation, percolation or by continuous extraction (e.g. in a Soxhlet extractor). Numerous extraction methods are available, the simplest being cold extraction (in a large flask with agitation of the biomass using a stirrer) in which the ground dried material is extracted at room temperature sequentially with solvents of increasing polarity: first hexane (or petroleum ether), ethyl acetate, acetone, methanol and finally water.

The major advantage of this protocol is that it is a soft extraction method as the extract is not heated and there is little potential degradation of natural products. The use of sequential solvents of increasing polarity enables division of natural products according to their solubility (and polarity) in the extraction solvents. This can greatly simplify an isolation process. Cold extraction allows most compounds to be extracted, although some may have limited solubility in the extracting solvent at room temperature.

hot percolation In hot percolation, the biomass is added to round-bottomed flask containing solvent and the mixture is heated gently under reflux. Typically, the plant material is ‘stewed’ using solvents such as ethanol or aqueous ethanol mixtures. The technique is sometimes referred to as total extraction and has the advantage that, with ethanol, the majority of lipophilic and polar compounds is extracted.

An equilibrium between compounds in solution and in the biomass is established, resulting in moderate extraction of natural products. Heating the extracts for long periods may also degrade labile compounds; therefore a pilot experiment should first be attempted and extracts assessed for biological activity to ascertain whether this extraction method degrades the bioactive natural products. Care should be taken, as extraction is never truly total; for example, some highly lipophilic natural products are insoluble .

decoction The decoction is used for active ingredients that doesn´t modify with temperature. In this process the drug is boiled in water for 15 to 60 minutes depending on the plant or the active ingredient to extract.

Supercritical fluid extraction Supercritical fluid extraction utilizes the fact that some gases behave as liquids when under pressure and have solvating properties. The most important example is carbon dioxide which can be used to extract biomass and has the advantage that, once the pressure has been removed, the gas boils off leaving a clean extract. Carbon dioxide is a non-polar solvent but the polarity of the supercritical fluid extraction solvent may be increased by addition of modifying agent, which is usually another solvent (e.g. methanol or dichloromethane).

Soxhlet extraction The most widely used method for extraction of plant natural products is Soxhlet extraction. This technique uses continuous extraction by solvents of increasing polarity. The biomass is placed in a Soxhlet thimble constructed of filter paper, through which solvent is continuously refluxed. The Soxhlet apparatus will empty its contents into the round-bottomed flask once the solvent reaches a certain level.

As fresh solvents enters the apparatus by a reflux condenser, extraction is very efficient and compounds are effectively drawn into the solvent from biomass due to their low initial concentration in the solvent. The method suffers from the same drawbacks as other hot extraction methods (possible degradation of products), but it is the best extraction method for the recovery of a big yields of extract. Moreover, providing biological activity is not lost on heating, the technique can be used in drug lead discovery.

In general terms, regardless of the extraction method used, extracts are of two types: lipophilic (‘fat-loving’), resulting from extraction by non-polar solvents ( e.g. petroleum, ethyl acetate, chloroform, dichloromethane) and hydrophilic (‘water-loving’), produced by extracting biomass with polar solvents (e.g. acetone, methanol, water). The value of using solvents of different polarities is that the chemical complexity of the biomass is simplified when taken into the extract, according to the solubility of the components. This can greatly simplify the isolation of an active compound from the extract.

Additionally, certain classes of compounds may have high solubilities in a particular solvent (e.g. monoterpens in hexane), which again can simplify the chemical complexity of an extract and help with isolation process. Regardless of the extraction technique used, extracts are concentrated under vacuum using rotary evaporators for large volumes of solvent (> 5 ml) or ‘blown down’ under nitrogen for small volumes (1-5 ml), ensuring that volatile components are not lost.

Removal of solvent should be carried out immediately after extraction, as natural products may be unstable in the solvent. Aqueous extracts are generally freeze-dried using a lyophilizer. Dried extracts should be stored at -20 0C prior to screening for biological activity as this will decrease the possibility of degradation of bioactive natural product.

Sublimation Sublimation may sometimes be possible on whole drug, as in the isolation of caffeine from tea or for the purification of materials present in a crude extract. Modern equipment employs low pressures with a strict control of temperature. Freeze-drying or lyophilization requires a first step where the temperature of the extract (solutes and solvents) is reduced until they are frozen, followed by a sublimation step carried out via a very high vacuum which sometimes accompanied by heating. Freeze drying is widely applied whenever a thermolabile substance is of interest. It forms part of extraction process of several products, for example; antibiotics, antioxidants, hormones, etc.

Distillation Fractional distillation has been traditionally used for separation of the components of volatile mixtures; in phytochemistry it has been widely used for isolation of components of volatile oils. On a laboratory scale it is not easy by this method to separate minor components of a mixture in a pure state and gas chromatography is now routinely used. Steam distillation is much used to isolate volatile oils and hydrocyanic acid from plant material.