Extraction of Natural Products

Introduction
Plants form the foundation of traditional medicine pharmacopeias, and are a proven source of pharmaceutical drugs (1 4). There is a growing body of research that many of the secondary metabolites of organisms, including plants, serve important biological and ecological roles, mainly as chemical messengers and defensive compounds (5,6). Thus, researchers from a variety of scientific disciplines confront the challenge of extracting plant material with solvents, often as a first step toward isolating and identifying the specific compounds responsible for biological activities associated with a plant or a plant extract. This chapter covers broadly various aspects of plant extraction: (a) Selecting, collecting, processing, and documenting plant samples; (b) Procedures for extraction of plant material; (c) Techniques to eliminate the most common nuisance compounds; (d) Extraction protocols to suit specific purposes; (e) Common sources of contamination by extraction artifacts; (f) Simple methods for detection of selected classes of plant secondary metabolites; (g) Methods for recognizing and avoiding common interfering compounds. Emphasis is placed throughout on practical means to recognize and avoid common pitfalls and to overcome specific problems that may be encountered during the extraction of plant secondary metabolites.

Materials used in plant extraction

The procedures for the extraction of plant material represent a series of apparently simple steps. The ultimate success of this type of research project, however, depends on the care devoted to each aspect of the work. The methods employed in the selection, collection, and identification of plant material directly affect the reproducibility of phytochemical research, and carelessness at this stage of an investigation may greatly reduce the scientific value of the overall study. Plant secondary metabolites often accumulate in specific plant parts. Unless it is known which part contains the highest levels of the compound or compounds of interest, it is prudent to collect multiple plant parts or the whole plant, to ensure the extracts prepared are representative of the range of secondary metabolites produced by the plant. Specific secondary metabolites also vary both quantitatively and qualitatively among closely related species, within a single species, and among members of a population (7, 8). Caution should therefore be exercised when making broad inferences about the presence or absence of specific compounds in a species under investigation, and when recollecting samples with the intention of isolating more of a specific metabolite. Many logistical considerations related to the collection of plant material from the field have been addressed elsewhere (2, 7, 9, 10).

i) Sourcing Plant Materials: General Considerations

Extraction of Natural Products

(a) For a biodiversity-based collection, taxa endemic to the region are of high priority, while pandemic weedy species are probably of little interest, and rare or endangered species are to be strictly avoided. (b) It is advisable to attempt field identification of the samples collected and voucher specimens should be prepared and deposited in herbaria. (c) For the convenience of other investigators, herbarium specimens should be deposited in a local herbarium in the source country, if applicable, and in one or more major institutions elsewhere. (d) In selection of plant material for study, it may be immediately evident that phytochemical considerations will play a major part in much of the decision-making process, but what may not always be obvious is that ethical and legal issues associated with intellectual property are at least as important, particularly when plant material or extracts will cross international borders (11, 12). ii) Plant Material Selection Approaches (a) Ethnobotanical sources: Investigation of plant species based on traditional use by humans for food, medicine, or poison based on review of the literature or interviews conducted as part of the investigation (2, 13) (b) Biodiversity-based sources: Procure samples by random or systematic collection of a biodiverse set of plant samples, typically from an ecological region that is comparatively uncharted as regards secondary metabolite production (3, 14). (c) Chemotaxonomic sourcing: Select samples based on botanical relationship to a species known to produce a compound or compound class of interest (15). (d) Literature-based approaches: Investigate the chemical basis for reports of biological activity in the scientific literature (including chemical ecology, toxicology, and veterinary reports). Databases may be used in selecting species that meet one or a combination of specified criteria (16). iii) Common Plant Material Forms (a) Dried plant material: Plant material should be dried at temperatures below 30C and away from sunlight to avoid chemical degradation of heat-labile or UV-sensitive constituents. To prevent the buildup of heat and moisture, air circulation around the plant material is essential. (b) Fresh plant material: The constituents of freshly collected material are susceptible to decomposition, and should be extracted as soon as possible. Field extraction with solvents will inactivate enzymes that may be present in the plant. iv) Solvents: General Considerations

Extraction of Natural Products (a) Factors that should be considered when choosing a solvent or solvent system for extracting plant material include polarity/ solubility of the target constituents, safety, ease of working with the solvent, potential for artifact formation, and the grade and purity of the solvent. (b) Safety: Precautions must be taken to minimize the risk of fire and explosion when using and storing highly flammable solvents and solvents that tend to form explosive peroxides. Before using an unfamiliar solvent or reagent, the material safety data sheet (MSDS) should be reviewed, and appropriate personal protective equipment should be employed. (c) Ease of use: Solvents with relatively low boiling points [e.g., acetone, dichloromethane, ethyl acetate (EtOAc), and hexane/ petroleum ether] are generally easier to use from the standpoint that they are more easily concentrated, whereas water and n -butanol are more difficult to remove. v) Solvents Selection (a) Solvent(s) for extraction of known compounds: A solvent or solvent mixture should be selected based on a consideration of the substance(s) intended to be extracted. Solvents should be used that are indicated from the literature to be appropriate for the compound class under investigation. Where such information is unavailable, a rule of thumb is that the solvent used should have a similar polarity to the compound(s) to be extracted. (b) Solvent(s) for extraction of material when the compound of interest are not known: The literature related to the species under investigation should be reviewed to become familiar with the compound classes to expect and the solvents and procedures that may be used for purification. In addition, it may be worthwhile performing several trial extractions using different solvents and techniques, and comparing total extract and appropriate secondary metabolite yields, or the potency of a selected biological activity in a bioassay, to indicate which method gives the optimal results. vi) Drying and Milling Equipment (a) Drying equipment: Drying equipment selection depends on sample size, the number of samples to be dried, and the infrastructure available, among other considerations. Effective drying can be carried out with minimal equipment and often improvised apparatus will prove to be serviceable. However, for certain applications, such as drying large amounts of material or liquids, commercial drying chambers or freeze drying equipment may be necessary. (b) Milled plant material: Small quantities of plant material can be ground using an electric blender, coffee or spice mill, or with a mortar and pestle. Milling of large quantities of plant material is usually best carried out using heavy duty comminution equipment. vii) Extraction Equipment

Extraction of Natural Products (a) Equipment for percolation: Percolation is an efficient method of extraction, suitable for bench-scale to pilot-scale batches. A variety of different vessels can serve as percolators. The main requirements are that they have a wide opening at the top to accommodate addition and removal of plant material, and a valve at the base to regulate solvent flow. Glass or nonporous ceramic lab ware may be used as weights for compressing plant material. (b) Equipment for Soxhlet extraction: Commercial sources of Soxhlet extraction equipment should be consulted for the equipment that best suits the application. Most systems use a glass apparatus, condenser, flasks, and a heating mantle. A common system uses cellulose thimbles to hold the plant material. The size of the equipment to use depends on the amount of material to be extracted, and can be estimated by measuring the volume of the material and comparing to the thimble volumes. Usually, Soxhlet systems are suitable for bench-scale extraction. (c) Equipment for maceration: Maceration can be conveniently carried out in vials, Erlenmeyer fl asks, or in larger containers. Sonication or shaking tables may be needed. Large samples may also be macerated, usually in a large container with a tap at the base, as with large-scale percolation, except that the solvent is changed in batches. (d) Filtration of samples: Suitable filtration devices depend on the sample size and the form. (e) Equipment for concentration of extracts: Standard laboratory equipment, such as rotary evaporators, centrifugal vacuum concentrators, and lyophilizers can be used for most applications. For large-scale projects or other specialized applications, appropriate equipment will have to be identified on a case-by case basis.

Methods used in plant extraction

A range of techniques, varying in cost and level of complexity, may be used for extraction of plant material. For most applications, relatively simple techniques, such as percolation and maceration are effective and economical. Some specific applications, however, require the use of more sophisticated extraction technology, such as supercritical-fluid extraction accelerated solvent extraction, microwave-assisted extraction ultrasound-assisted extraction equipment and large-scale steam distillation apparatus. i) General Consideration 1. Regardless of the extraction technique used, the resulting solution should be filtered to remove any remaining particulate matter. Small volumes of extracts can be filtered through filter cartridges, and larger volumes can be filtered through solvent compatible membranes using vacuum filtration systems. 2. Plant extracts should not be stored in solvent for periods or more than a day or two at room temperature, or in sunlight, because of the accompanying increased risk of artifact formation and decomposition or isomerization of extract constituents.

Extraction of Natural Products

3. Extracts can be concentrated at reduced pressure or dried under a stream of nitrogen. If a rotary evaporator is used, it is advisable to keep the water bath temperature below 40C to prevent decomposition of heat-labile components. 4. Throughout the following paragraphs, several methods and procedures are discussed. If one is not familiar with the use of the procedures mentioned, or the equipment to be used, a knowledgeable person should be consulted for guidance. ii) Percolation 1. Loading and soaking of plant material: The solvent-wetted plant material is loosely and evenly packed into the container leaving room for expansion. Selected solvent is added to the top of the material and allowed to soak for several hours or overnight, with solvent being added as needed to keep the plant material covered. 2. Extraction: After soaking, the percolator valve is opened slightly to allow solvent to flow slowly into a collecting vessel. The flow rate is regulated to ensure that the solvent exiting is nearly saturated with solute, and fresh solvent is added at the top of the percolator to replace that lost from the bottom. iii) Soxhlet Extraction 1. Soxhlet extraction, using commercially available devices, is a convenient method for extraction of small to moderate volumes of plant material. Instructions specific to the equipment should be followed. 2. Loading of plant material: As with percolation, the solvent wetted plant material is loosely and evenly packed into the container. Sufficient volume of the selected solvent is added to the collection fl ask, being careful not to overfill. 3. Extraction: The extraction is carried out by the flow of refluxed solvent through the sample. The exact amount of time to complete extraction depends on many factors, but usually can be standardized by the number of times the extraction chamber fills and empties (cycles). 4. Because the extraction takes place in a closed system in which the solvent is continually recycled, the amount of solvent needed for Soxhlet extraction is minimal. In the most commonly used extractors, however, the heat needed to drive the extraction will likely cause heat-labile constituents to form artifacts or decomposition products.

iv) Maceration 1. Loading of plant material: As with percolation, the solvent wetted plant material is loosely and evenly packed into the container. Selected solvent is added to the container to cover the sample. 5

Extraction of Natural Products

2. Extraction: The sample with solvent is allowed to stand and extract. The container should be covered to prevent loss of solvent. As a rough guideline, after each addition of fresh solvent, the plant material should be left to macerate overnight. 3. Decanting: At the end of the extraction period, the solvent should be decanted through a screen or filter, and fresh solvent added to the flask. 4. Replacing solvent: After saturated solvent is removed, the sample is then mixed with the fresh solvent by stirring or swirling, and left to macerate again. 5. Methods for accelerating the extraction process and additional methods: Sonication of the macerating sample or gentle swirling on a fermentation broth table is sometimes used to reduce the time needed for thorough extraction. v) Sample Preparation for Large-Scale Biological Screening When a large number of extractions are carried out, it is not feasible to extract each plant sample with a different tailored solvent system. A general procedure must be developed and validated, giving consideration to the rate of detection of active extracts (hits) obtained using several extraction methods, and followed by analysis of the rate of false-positive responses. This approach has been used to develop a general extraction protocol to be used in extracting plant constituents for in vivo or in vitro biological screening (17, 18) (Fig. 1). The resulting chloroform-soluble extract is essentially free of vegetable tannins and may be used in primary screening against a variety of cell lines, in vivo systems, and enzyme-based assays. For these test systems, it has been determined that the chloroform extract prepared in this manner retains most of the biological activity of a plant sample, except for activity due to vegetable tannins or highly polar or non polar compounds that tend not to be promising candidates for drug development ( 17, 18 ) . 1. Extraction with methanol (MeOH): The samples are macerated three times with MeOH. 2. Concentration of MeOH extract: The pooled batches are concentrated under a vacuum. The concentrated extract should then be reconstituted in 90% MeOH. As a general rule, enough solvent mixture should be added to dilute to 10% of solids or less. 3. Defatting the methanolic extract: In a suitable separatory funnel, the reconstituted extract is then partitioned against petroleum ether or hexane to separate most of the lipophilic components. As a general guide, equal volumes of the petroleum ether and methanolic extracts are used for each partition.

Extraction of Natural Products

Fig. 1. General procedure for preparing extracts representing a range of polarities, including a partially tannin-free chloroform extract 4. The defatted methanolic extract should be concentrated under a vacuum following the usual precautions to avoid excessive bubbling. 5. Partitioning to remove the most polar constituents from the organic extract: Reconstitute in water and partition with chloroform. Equal volumes of water and chloroform are usually effective. After separation, the lower layer is collected, and this process is repeated for a total of three batches. 6. Partial removal of tannins: The resulting organic extract is partitioned with 1% NaCl in water (w/v) (18). The organic layer is then concentrated to dryness. vi) Preparation of Phytochemically Enriched Extracts: General Considerations

Extraction of Natural Products

1. When a particular phytochemical constituent or compound class is to be the target of an investigation, specific extraction procedures may be employed to produce enriched extracts. In some instances, the polarity of a solution may be modified to cause particular compound classes to precipitate, leaving unwanted compounds in solution. 2. Compounds containing primary, secondary, or tertiary amines, carboxylic acids, lactones, and phenols may be extracted selectively using pH modifications to manipulate the polarity/solubility of the compounds of interest, although acidic and basic extraction conditions should be employed with caution because of the potential to chemically alter the naturally occurring secondary metabolites during the extraction process. 3. It is advisable to test the stability of the target compounds on a small scale prior to submitting a major portion of the plant sample or crude extract to one of these potentially damaging techniques. vii) Water Extraction: Challenges and Opportunities 1. Although traditional medicines are often prepared by water extraction as infusions or decoctions many investigators prefer not to work with aqueous extracts. This is due, at least in part, to the added challenges associated with isolation of water-soluble constituents using conventional isolation methods, and the relative difficulty of concentrating water extracts on a rotary evaporator. 2. Water is a green solvent and can be used not only for the extraction of polar compounds, but also for extracting slightly non polar compounds under the right conditions, both because of co-solubility issues and because the polarity of water decreases somewhat at high temperatures. Silybum marianum seeds extracted by maceration in water at 85C yielded proportionally more of the polar constituents, taxifolin and silychristin, whereas water at 100C, extracted proportionally more of the less polar compounds, silybinins A and B (24). 3. Aqueous extracts may be freeze-dried and re-extracted with a series of solvents in the order of increasing polarity (25). This partially overcomes the problems associated with concentrating water extracts by other methods. viii) Avoiding Extraction of Artifacts Many solvents used in extraction of plant material have at least occasionally been implicated in artifact formation, either directly, or because of impurities in the solvent. Certain extraction procedures may also result in artifact formation. Middle ditch (26) has compiled a detailed treatment of the subject of analytical artifacts in chromatography. 1. Avoiding artifact-prone procedures: In general, mild conditions should be employed during the isolation process, unless it is known beforehand that the compounds of interest are stable under the specific conditions of a proposed extraction method. 8

Extraction of Natural Products

2. Recognizing artifacts: It is useful to be familiar with the type of artifacts that might form during a specific extraction protocol. One should be vigilant for structural clues that indicate a compound isolated under certain conditions may be an artifact. 3. Analytical methods: A sample should be retained of the original plant material for future analysis, in case one of the isolates should later be suspected of being an artifact of the extraction method used. The reference material can be extracted using non reactive solvents and mild conditions, and the resulting extract analyzed using LCMS/MS to determine if the compound is present in the original plant material (27).

Conclusion
Plant secondary metabolites are currently the subject of much research interest, but their extraction as part of phytochemical or biological investigations presents specific challenges that must be addressed throughout the solvent extraction process. Successful extraction begins with careful selection and preparation of plant samples, and thorough review of the appropriate literature for indications of which protocols are suitable for a particular class of compounds or plant species. During the extraction of plant material, it is important to minimize interference from compounds that may coextract with the target compounds, and to avoid contamination of the extract, as well as to prevent decomposition of important metabolites or artifact formation as a result of extraction conditions or solvent impurities. This discussion presents an overview of the process of plant extraction, with an emphasis on common problems encountered and methods for reducing or eliminating these problems. In addition to generally applicable extraction protocols, methods are suggested for more or less selectively extracting specific classes of compounds, and phytochemical methods are presented for detection of classes of compounds commonly encountered during plant extraction, including selected groups of secondary metabolites and interfering compounds.

References
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6. Wink M (1999) Function of secondary metabolites. In: Wink M (ed) Functions of plant secondary metabolites and their exploitation in biotechnology. Annual plant reviews, vol 3. Academic, Sheffi eld, UK, pp 116 7. Barclay AS, Perdue RE Jr (1976) Distribution of anticancer activity in higher plants. Cancer Treat Rep 60:10811113 8. Ayres DC, Loike JD (eds) (1990) Lignans: chemical, biological and clinical properties. Phillipson JD, Ayres DC, Baxter H (ser eds) Chemistry and pharmacology of natural products. Cambridge University Press, Cambridge, UK 9. Perdue RE Jr (1976) Procurement of plant materials for antitumor screening. Cancer Treat Rep 60:987998 10. Soejarto DD (1993) Logistics and politics in plant drug discovery: the other end of the spectrum. In: Kinghorn AD, Balandrin MF (eds) Human medicinal agents from plants, vol 534, ACS Symposium Series. American Chemical Society, Washington, DC, pp 96111 11. Baker JT, Borris RP, Carte B et al (1995) Natural product drug discovery and development: new perspectives on international collaboration. J Nat Prod 58:1325 1357 12. Appendino G, Fontana G, Pollastro F (2010) Natural products drug discovery. In: Verpoorte R (ed) Comprehensive natural products chemistry II, vol 3. Elsevier, Oxford, UK, pp 205236 13. Soejarto DD (1996) Biodiversity prospecting and benefi t-sharing: perspectives from the fi eld. J Ethnopharmacol 51:115 14. Calderon AI, Angerhofer CK, Pezzuto JM et al (2000) Forest plot as a tool to demonstrate the pharmaceutical potential of plants in a tropical forest of Panama. Econ Bot 54:278294 15. McKee TC, Covington CD, Fuller RW et al (1998) Pyranocoumarins from tropical species of the genus Calophyllum : a chemotaxonomic study of extracts in the National Cancer Institute collection. J Nat Prod 61:12521256 16. Loub WD, Farnsworth NR, Soejarto DD et al (1985) NAPRALERT: computer handling of natural product research data. J Chem Inf Comput Sci 25:99103 17. Statz D, Coon FB (1976) Preparation of plant extracts for antitumor screening. Cancer Treat Rep 60:9991005 18. Wall ME, Wani MC, Brown DM et al (1996) Effect of tannins on screening of plant extracts for enzyme inhibitory activity and techniques for their removal. Phytomedicine 3:281285

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Extraction of Natural Products 19. Hostettmann K, Hostettmann M, Marston A (1991) Saponins. In: Charlwood BV, Banthorpe DV (eds) Terpenoids. Dey PM, Harborne JB (ser eds) Methods in plant biochemistry, vol 7. Academic, San Diego, pp 435471 20. Klyne W (1957) The chemistry of the steroids. Wiley, New York 21. Cordell GA (1981) Introduction to the alkaloids: a biogenetic approach. WileyInterscience, New York 22. Hesse M (2002) Alkaloids: natures curse or blessing? Wiley-VCH, Weinheim, Germany 23. Harborne JB (1998) Phytochemical methods: a guide to modern techniques of plant analysis, 3rd edn. Chapman and Hall, New York 24. Barreto JF, Wallace SN, Carrier DJ et al (2003) Extraction of nutraceuticals from milk thistle: I. Hot water extraction. Appl Biochem Biotechnol 105108:881889 25. Vedenskaya IO, Rosen RT, Guido JE et al (2004) Characterization of fl avonols in cranberry ( Vaccinium macrocarpon ) powder. J Agric Food Chem 52:188195 26. Middleditch BS (1989) Analytical artifacts: GC, MS, HPLC, TLC, and PC, Journal of Chromatography Library, vol 44. Elsevier, New York 27. Gu JQ, Li WK, Kang YH et al (2003) Minor withanolides from Physalis philadelphica : structures, quinone reductase induction activities, and liquid chromatography (LC)-MS-MS investigation as artifacts. Chem Pharm Bull 51:530539

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