Preparation and extraction

Whether samples are plants, microbes (fermented or solid phase), marine animals (corals, slugs, tunicates) or insects they are referred to as biomass. In the case of plants, following their identification and classification by a field botanist into a species and family, samples are collected from the aerial parts (leaves, stem and stem bark), the trunk bark and roots or, in the case of large trees, the heartwood (sometimes referred to as timber). These samples are then gently air-dried, although this can be problematic in highly humid environments such as rainforests and coastal regions. Better control is achieved in the laboratory using drying cabinets or lyophilizers (freeze-driers), although biomass must be dried quickly to avoid degradation of components by air or by microbes. Care must be taken with lyophilizers as they utilize a high vacuum, which can remove volatile components that may have interesting biological activities.

Once biomass has been dried, it is ground into small particles using either a blender or a mill. Plant material is milled twice, first using a coarse 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 the extraction should mimic the traditional use. For example, if an indigenous people use a specific extraction protocol such as a water extract, a cold/hot 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 as strong heating of biomass with a solvent, may result in degradation of natural products and consequent loss of biological activity.

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), then chloroform (or dichloromethane), 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.

In hot percolation, the biomass is added to a 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 in polar solvents (e.g. the monoterpenes).

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 a modifying agent, which is usually another solvent (e.g. methanol or dichloromethane).

The most widely used method for extraction of plant natural products is Soxhlet extraction (Fig. 7.1). 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 solvent enters the apparatus by a reflux condenser, extraction is very efficient and compounds are effectively drawn into the solvent from the 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 big yields of extract. Moreover, providing biological activity is not lost on heating, the technique can be used in drug lead discovery.

Fig. 7.1 Soxhlet extraction apparatus. Generally used to give greatest yield of extract when extracts are stable in hot solvent.

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. petrol, 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. the monoterpenes in hexane), which again can simplify the chemical complexity of an extract and help with the 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 °C prior to screening for biological activity as this will decrease the possibility of bioactive natural product degradation.

If it is known that certain classes of compounds, such as acids or bases, are present in the biomass, they can be extracted using a tailored protocol. The most common group of natural products that are extracted in this manner are the alkaloids (see Chapter 6 and below), which are often present in plant material as salts. A brief outline of how these basic compounds may be extracted is as follows:

This extraction method generates a mixture of alkaloids that are essentially free of neutral or acidic plant components and is specific for compounds that are basic (able to form free bases). However, care should be taken with alkaloid extractions as the acids and bases employed may destroy active natural products that have functional groups which are readily susceptible to degradation (e.g. glycosides, epoxides and esters). Additionally, the stereochemistry of a molecule may be affected by the presence of these strong reagents. The most important factor to consider is: is biological activity retained following the extraction protocol?

Partitioning

Possibly the simplest separation method is partitioning, which is widely used as an initial extract purification and ‘clean up’ step. Partitioning uses two immiscible solvents to which the extract is added; this can be sequential by using immiscible organic solvents of increasing polarity. Typically, this may take place in two steps: (1) water/light petroleum ether (hexane) to generate a non-polar fraction in the organic layer; (2) water/dichloromethane or water/chloroform or water/ethyl acetate to give a medium-polar fraction in the organic layer. The remaining aqueous layer will contain polar water-soluble natural products. This is a soft separation method and relies on the solubility of natural products and not a physical interaction with another medium (e.g. adsorption on silica gel in thin-layer chromatography, TLC; see below). Partitioning may give rise to excellent separations, particularly with compounds that differ greatly in solubility; for example, monoterpenes are easily separated from phenolics such as tannins.

Gel chromatography

Assuming that the extract is still active, the next step is chromatography. A procedure that is widely used as an initial clean-up is gel chromatography, also known as size exclusion chromatography. This technique employs a cross-linked dextran (sugar polymer) which, when added to a suitable solvent (e.g. chloroform or ethyl acetate), swells to form a gel matrix. The gel contains pores of a finite size that allow small molecules (< 500 Da) to be retained in the matrix; larger molecules (> 500 Da) are excluded and move quickly through the gel. This gel is loaded into a column and the extract is added to the top of the column. Large molecules are the first to elute, followed by molecules of a smaller size. This is an excellent method for separating out chlorophylls, fatty acids, glycerides and other large molecules that may interfere with the biological assay. Different sorts of gels are available which may be used in organic solvents (e.g. LH-20) or aqueous preparations such as salts and buffers (e.g. G-25). Therefore both non-polar and polar natural products can be fractionated using this technique. Additionally, compounds are not only fractionated according to size but there is also a small amount of adsorption chromatography occurring, as the dextran from which the gel is made contains hydroxyl groups that interact with natural products, facilitating some separation according to polarity.

This is a non-destructive ‘soft’ method with a high recovery (compounds are rarely strongly adsorbed) and a high quantity of extract (hundreds of milligrams to grams) may be separated. A further benefit of this technique is that many different gels are available with a variety of pore sizes that can be used to separate compounds from 500 to 250,000 Da. This is the method of choice for large molecules, in particular proteins, polypeptides, carbohydrates, tannins and glycosides, especially saponin and triterpene glycosides.

Ion-exchange chromatography

The separation of small polar compounds, in particular ionic natural products, is often problematic. It is possible to separate these metabolites from larger molecules (using gels) but they are generally very strongly adsorbed with normal-phase sorbents such as silica or alumina, and, even with the use of polar solvents and modifiers (e.g. acid and base), efficient separations may not be achievable. Additionally, these compounds are not retained on reverse-phase sorbents such as C₁₈ or C₈. These natural products possess functional groups, such as CO₂H, -OH, -NH₂, that contribute to the polarity of the molecule, and this may be used to develop a separation method using ion-exchange chromatography.

This technique is limited to natural products that can carry charge on their functional groups. The sorbent or stationary phase has charged groups and mobile counter ions which may exchange with ions of the functional groups present in the natural product as the mobile phase moves through the sorbent. Separation is achieved by differences in affinity between ionic components (polar natural products) and the stationary phase. These ion-exchange sorbents or resins are divided into two groups: cation exchangers, which have acidic groups (CO₂H, -SO₃H) and are able to exchange their protons with cations of natural products, and anion exchangers, which have basic groups (-N⁺R₃) that are incorporated into the resin and can exchange their anions with anions from the natural product. These ion-exchange resins may be used in open column chromatography or in closed columns in applications such as high performance liquid chromatography (HPLC).

An example of the technique is shown in Fig. 7.2. 2,5-Dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP) from Lonchocarpus sericeus (Fabaceae) is a nematocidal polyhydroxylated alkaloid (PHA), and also inhibits insect α- and β-glucosidases. Compounds of this type are bases and form cations in acidic solutions. When added to a cation exchanger [e.g. Amberlite CG-120, which has a sulfonic acid bound to the resin which can exchange its proton (cation)], the DMDP cations are retained (bound) by the cation exchanger and protons are displaced. If the cation exchanger is then eluted with a solution containing a stronger cation such as (e.g. from 0.2 M NH₄OH), then the DMDP cation is desorbed from the exchanger and is unbound and mobile. This affinity can be used to separate such alkaloids from acidic (anionic) or neutral components which would not be retained by the cation exchanger and may be washed from the resin by water.

Fig. 7.2

Plant extracts that contain DMDP are used as a nematocide against infected crops (bananas) in Costa Rica and are licensed by the National Institute of Biodiversity. This is an example of a renewable resource as the extracts may be prepared from the seeds of the plant and DMDP is ecologically friendly as it is biodegradable.

Biotage™ flash chromatography

Biotage™ flash chromatography may be used for quick efficient separations. This employs pre-packed solvent-resistant plastic cartridges (Fig. 7.3), which contain the sorbent (silica, alumina, C₁₈, HP-20, or ion exchange resin). These cartridges are introduced into a radial compression module (the metal cylinder in Fig. 7.3), which pressurizes the cartridge and sorbent radially. This results in a very homogeneous packed material (sorbent), reduces the possibility of solvent channelling when the system is run and minimizes void spaces on the column head.

Fig. 7.3 Biotage flash chromatograph. Fast separations are achieved with good resolution.

Using this technique, milligrams to tens of grams can be separated. The bioactive extract can be dissolved in solvent and loaded onto the column directly; solvent is then pumped through the column and fractions are collected, resulting in a rapid separation of extract components. This is a rapid method; 10 g of extract can be fractionated into 12 fractions of increasing polarity in 30 min using a step gradient solvent system. There are a number of benefits to this, particularly that speed minimizes contact with reactive sorbents (e.g. silica) and that hazardous sorbents such as silica, which when free may cause silicosis, are contained in the cartridges. Additionally, the cartridges may be re-used, reducing the cost of the bioassay-guided process. The high flow-rates employed by this technique (20–250 ml/min) retain ‘band-like’ movement of the components through the column, resulting in a high resolution. Compounds eluting from the column may be detected by TLC (of fractions) or the eluant may be passed through a UV detector so that compounds that absorb UV light can be detected as they elute from the column. Some laboratories run several of these flash columns simultaneously, resulting in a high number of fractionated extracts having sufficient mass for further purification of the active components.

Thin-layer chromatography

Thin-layer chromatography (TLC) is one of the most widely used and easiest methods for purifying a small number (2–4) of components, typically following a Biotage flash separation. This method employs glass or aluminium plates that are pre-coated with sorbent (e.g. silica gel) of varying thickness dependent on the amount of material to be loaded onto the plates. The coating on analytical plates is generally of 0.2 mm thickness; preparative plates may have a coating 1–2 mm thick. The compound mixture is loaded at 1–2 cm from the bottom edge of the plate as either a spot or a continuous band. The plate is then lowered into a tank containing a predetermined solvent which will migrate up the plate and separate the compound mixture according to the polarity of the components.

In analytical use, micrograms of material may be separated using this technique and samples such as drugs of abuse (e.g. cannabis resin) may be compared with standards (e.g. tetrahydrocannabinol) for quick identification.

Sorbent-coated plates often incorporate a fluorescent indicator (F₂₅₄) so that natural products that absorb short-wave UV light (254 nm) will appear as black spots on a green background. Under long-wave UV light, certain compounds may emit a brilliant blue or yellow fluorescence. Both UV absorbance and fluorescence properties may be used to monitor the separation of compounds on a TLC plate.

Preparative scale TLC has great use and loadings of 1–100 mg can readily produce enough purified material for biological assays and structure elucidation. It is rapid and cheap and has been the method of choice for separating lipophilic compounds. Preparative plates are available from suppliers as pre-coated plates of 1–2 mm thickness in silica, alumina or C₁₈. However, home-made plates offer greater flexibility by allowing the incorporation of modifying agents into the sorbents (e.g. silver nitrate for separation of olefinic compounds – known as argentation TLC), use of other sorbents (ion exchange, polyamide, cellulose) and the addition of indicators and binders.

The scale-up from analytical to preparative mode is crucial, as an increase in the sample load may drastically change the separation of the components. Normally, the method developed on the analytical scale must be modified, generally with a reduction of solvent system polarity. Preparative TLC is used as a final clean-up procedure to separate 2–4 compounds. The sample is dissolved in a small volume of solvent and applied as a thin line 2 cm from the bottom of the plate and dried. The plate is then eluted in a suitable solvent and UV-active compounds are visualized at 254 or 366 nm. Natural products that are not UV-active will need development using a suitable spray reagent such as vanillin-sulphuric acid, Dragendorff’s reagent, phosphomolybdic acid or antimony trichloride. In this case, an edge of the plate is sprayed with the reagent (taking care that only a small area of the plate is covered) and separated compounds are visualized as coloured bands. The bands containing pure natural product are scraped off the plate and the natural product is desorbed from the sorbent. This desorption may be carried out by placing the compound-rich sorbent into a sintered glass funnel and washing with a suitable solvent followed by collection and concentration of the filtrate. The purified ‘band’ should then be assessed for purity by analytical TLC.

There are a number of advantages of this method for the analysis and isolation of biologically active natural products:

The major disadvantages of TLC are that:

High-performance liquid chromatography

The final separation technique discussed in this section is high-performance liquid chromatography (HPLC). This method is currently in vogue and is widely used for the analysis and isolation of bioactive natural products. The analytical sensitivity of the technique, particularly when coupled with UV detection such as photodiode array (PDA), enables the acquisition of UV spectra of eluting peaks from 190 nm to 800 nm. The flow-rates of this system are typically 0.5–2.0 ml/min and sample loading in the analytical mode allows the detection and separation of tens to hundreds of micrograms of material. With PDA UV detection, even compounds with poor UV characteristics can be detected. This is especially useful in the analysis of natural products such as terpenoids or polyketides which may have no unsaturation or chromophores that give rise to a characteristic UV signature.

HPLC is a highly sensitive technique when coupled to electronic library searching of compounds with a known UV spectrum. Modern software enables the UV spectra of eluting peaks to be compared with spectra stored electronically, thereby enabling early identification of known compounds or, usefully, the comparison of novel compounds with a similar UV spectrum, which may indicate structural similarity. It is also possible to increase the size of these electronic libraries and improve the searching power of the technique. HPLC is a powerful technique for fingerprinting biologically active extracts and comparisons can be drawn with chromatograms and UV spectra stored in an electronic library. This is currently very important for the quality control of herbal medicines for which appropriate standards in reproducibility of extract quality must be met.

HPLC can be run in fully automated mode and with carousel autosamplers it is possible to analyse tens to hundreds of samples. These HPLC systems are computer-driven and not only run samples, but may be programmed to process data and print out chromatograms and spectra automatically. Radial compressed column technology can also be made use of in HPLC and, as with Biotage flash chromatography, can access highly varied column technology, including standard sorbents such as normal phase (silica) and reverse-phase (C₁₈ and C₈) and more ‘exotic’ stationary phases such as phenyl, cyano, C₄, chiral phases, gel size exclusion media and ion exchangers. This versatility of stationary phase has made HPLC a highly popular method for bioassay-guided isolation.

HPLC is a high-resolution technique, with efficient, fast separations. The most widely used stationary phase is C₁₈ (reverse-phase) chromatography, generally employing water/acetonitrile or water/methanol mixtures as mobile phase. These mobile phases may be run in gradient elution mode, in which the concentration of a particular solvent is increased over a period of time, starting, for example, with 100% water and increasing to 100% acetonitrile over 30 min, or in isocratic elution mode, in which a constant composition (e.g. 70% acetonitrile in water) is maintained for a set period of time.

HPLC is also used preparatively and, with the aid of computer-controlled pumping systems, very accurate mixing of solvents can be achieved leading to superb control of elution power. Many preparative columns employ radial compressed column technology as these columns have a long column life, few void volumes, homogeneous column packing with little solvent channelling and excellent ‘band flow’ of components as they flow through the column. As with TLC, an analytical HPLC method is developed for scale-up to preparative HPLC; flow-rates of 50–300 ml/min are common. Detection in preparative HPLC generally utilizes a UV detector that has been optimized to detect the natural product of interest (e.g. at 254 nm) by knowledge of the spectra acquired by analytical PDA HPLC. A large sample loading of tens of milligrams to grams of material can be achieved and rapid isolation can be facilitated by the use of intelligent fraction collectors that can ‘peak collect’ compounds as they elute from the column by receiving input from the UV detector.

The technique can be used for the majority of natural products that are soluble in organic solvents and can be adapted to ion exchange for the isolation of highly polar compounds. HPLC is the method of choice for the pharmaceutical industry because of its excellent separating power, speed and reproducibility. The major disadvantage of this technique, however, is its expense, as analytical instrumentation may cost upwards of £20,000, and preparative HPLC may be £30,000. Consumables for this technique are also expensive, especially preparative columns (£2000), which may have a short life and at high solvent flow-rates there is a high cost for purchase and disposal of high-purity solvents.