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Satyajit D. Sarker and Lutfun Nahar (eds.), Natural Products Isolation, Methods in Molecular Biology, vol. 864, DOI 10.1007/978-1-61779-624-1_13, © Springer Science+Business Media, LLC 2012
Chapter 13
Extraction of Plant Secondary Metabolites
William P. Jones and A. Douglas Kinghorn
Abstract
This chapter presents an overview of the preparation of extracts from plants using organic solvents, with emphasis on common problems encountered and methods for their reduction or elimination. In addition to generally applicable extraction protocols, methods are suggested for selectively extracting specifi c classes of plant-derived compounds, and phytochemical procedures are presented for the detection of classes of compounds encountered commonly during extraction, including selected groups of secondary metabolites and interfering compounds. Successful extraction begins with careful selection and preparation of plant samples and thorough review of the appropriate literature for suitable protocols for a particular class of compounds or plant species. During the extraction of plant material, it is important to minimize interfer- ence from compounds that may co-extract 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.
Key words: Plant extracts , Plant secondary metabolites , Percolation , Maceration , Extraction artifacts , Interfering compounds , Phytochemical detection methods
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 eco- logical roles, mainly as chemical messengers and defensive com- pounds ( 5, 6 ) . Thus, researchers from a variety of scientifi c disciplines confront the challenge of extracting plant material with solvents, often as a fi rst step toward isolating and identifying the specifi c compounds responsible for biological activities associated with a plant or a plant extract. This chapter covers broadly various aspects of plant extraction:
1. Introduction
(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 specifi c 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 recog- nize and avoid common pitfalls and to overcome specifi c problems that may be encountered during the extraction of plant secondary metabolites.
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 iden- tifi cation of plant material directly affect the reproducibility of phytochemical research, and carelessness at this stage of an investi- gation may greatly reduce the scientifi c value of the overall study.
Plant secondary metabolites often accumulate in specifi c 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 (see Note 1 ). Specifi c secondary metabolites also vary both quantitatively and qualitatively among closely related species, within a single species, and among members of a popula- tion ( 7, 8 ) . Caution should therefore be exercised when making broad inferences about the presence or absence of specifi c com- pounds in a species under investigation, and when recollecting samples with the intention of isolating more of a specifi c metabo- lite (see Note 2 ). Many logistical considerations related to the collection of plant material from the fi eld have been addressed elsewhere ( 2, 7, 9, 10 ) .
2. Materials
(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 (see Note 3 ).
(b) It is advisable to attempt fi eld identifi cation of the samples col- lected (at least to the level of genus) (see Note 4 ), and voucher specimens (including reproductive organs, when feasible) should be prepared and deposited in herbaria.
(c) For the convenience of other investigators, herbarium speci- mens should be deposited in a local herbarium in the source country, if applicable, and in one or more major institutions elsewhere. A notation affi xed to the voucher specimen should include pertinent observations, such as local uses of the spe- cies, its habitat, microenvironment (e.g., shaded vs. sunny location of collection), state of overall health, stage in the reproductive cycle as well as other facts that may be useful for future investigations.
(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 ) (see Note 5 ). For drug discovery from plants, samples may be selected using a number of 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 ) (see Note 6 ).
(b) Biodiversity-based sources: Procure samples by random or sys- tematic 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 scientifi c literature (including chemical ecology, toxicology, and veterinary reports). Databases may be used in selecting species that meet one or a combination of specifi ed criteria ( 16 ) .
(a) Dried plant material: Plant material should be dried at tem- peratures below 30°C and away from sunlight to avoid chemi- cal degradation of heat-labile or UV-sensitive constituents.
2.1. Sourcing Plant Materials: General Considerations
2.2. Plant Material Selection Approaches
2.3. Common Plant Material Forms
To prevent the buildup of heat and moisture, air circulation around the plant material is essential, so it should not be com- pacted during drying, and it may be necessary to use a fan or other means to provide air fl ow around or through the drying sample (see Note 7 ).
(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. Alternatively, methods that keep the material in a relatively fresh state, such as freezing, preserving in alcohol, or other methods can be used.
(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 fi re and explosion when using and storing highly fl ammable solvents and solvents that tend to form explosive peroxides (such as diethyl ether). Care should be taken to protect the investigator and other people in the vicinity from exposure to chemical haz- ards and to reduce environmental contamination (see Note 8 ).
Before using an unfamiliar solvent or reagent, the material safety data sheet (MSDS) should be reviewed, and appropriate per- sonal 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 stand- point that they are more easily concentrated, whereas water and n -butanol are more diffi cult to remove.
(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 informa- tion 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 (or closely related taxa) under investigation should be reviewed to become familiar with the compound classes to expect and the solvents and procedures that may be used for purifi cation.
2.4. Solvents: General Considerations
2.5. Solvents Selection
In addition, it may be worthwhile performing several trial extractions using different solvents and techniques, and com- paring total extract and appropriate secondary metabolite yields, or the potency of a selected biological activity in a bioas- say, to indicate which method gives the optimal results.
(a) Drying equipment: Drying equipment selection depends on sample size, the number of samples to be dried, and the infra- structure available, among other considerations. Effective dry- ing can be carried out with minimal equipment (see Note 7 ), 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 (with sometimes the addition of a small amount of sand to aid in the process). Milling of large quanti- ties of plant material is usually best carried out using heavy- duty comminution equipment (see Note 9 ).
(a) Equipment for percolation: Percolation is an effi cient 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 fl ow (see Note 10 ). Glass or nonporous ceramic labware 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.
(The fl asks can be covered with Parafi lm or aluminum foil to prevent evaporation of solvent). Sonication or shaking tables may be needed (see Subheading 3 ). 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.
2.6. Drying and Milling Equipment
2.7. Extraction Equipment
(d) Filtration of samples: Suitable fi ltration devices depend on the sample size and the form (see Subheading 3 ).
(e) Equipment for concentration of extracts: Standard laboratory equipment, such as rotary evaporators, centrifugal vacuum concentrators, and lyophilizers can be used for most applica- tions. For large-scale projects or other specialized applications, appropriate equipment will have to be identifi ed on a case-by- case basis.
A range of techniques, varying in cost and level of complexity, may be used for extraction of plant material. For most applications, rela- tively simple techniques, such as percolation and maceration are effective and economical. Some specifi c applications, however, require the use of more sophisticated (and sometimes costly) extrac- tion technology, such as supercritical-fl uid extraction (see Chapter 3 ), accelerated solvent extraction (or pressurized solvent extrac- tion) (see Chapter 4 ), microwave-assisted extraction (see Chapter 5 ), ultrasound-assisted extraction equipment (see Chapter 18 ), and large-scale steam distillation apparatus. Full discussion of these methods is beyond the scope of this chapter, and the interested reader is encouraged to consult the specialized literature for further information (see Note 11 ).
1. Regardless of the extraction technique used, the resulting solu- tion should be fi ltered to remove any remaining particulate matter. Small volumes of extracts can be fi ltered through fi lter cartridges, and larger volumes can be fi ltered through solvent compatible membranes using vacuum fi ltration 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.
3. Extracts can be concentrated at reduced pressure (on a rotary evaporator, centrifugal vacuum concentrator, or similar equip- ment), or dried under a stream of nitrogen. If a rotary evapora- tor is used, it is advisable to keep the water bath temperature below 40°C 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.
3. Methods
3.1. General Consideration
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 fl ow slowly into a collecting vessel. The fl ow 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.
1. Soxhlet extraction, using commercially available devices, is a convenient method for extraction of small to moderate vol- umes of plant material. Instructions specifi c 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. Suffi cient volume of the selected solvent is added to the collection fl ask, being careful not to overfi ll (consult equip- ment specifi cations).
3. Extraction: The extraction is carried out by the fl ow of refl uxed solvent through the sample. The exact amount of time to com- plete extraction depends on many factors, but usually can be standardized by the number of times the extraction chamber fi lls 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 com- monly used extractors, however, the heat needed to drive the extraction will likely cause heat-labile constituents to form arti- facts or decomposition products.
1. Loading of plant material: As with percolation, the solvent- wetted plant material is loosely and evenly packed into the con- tainer. Selected solvent is added to the container to cover the sample.
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 sol- vent, 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 fi lter, and fresh solvent added to the fl ask.
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.
3.2. Percolation
3.3. Soxhlet Extraction
3.4. Maceration
5. Methods for accelerating the extraction process and additional methods: Sonication of the macerating sample or gentle swirl- ing on a fermentation broth table is sometimes used to reduce the time needed for thorough extraction. For relatively small sample sizes, extraction in sealed tubes can be carried out using a shaking mixer. Experience indicates that after three solvent changes, the plant material is almost completely exhausted (see Note 12 ).
When a large number of extractions are carried out, it is not feasi- ble 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 3.5. Sample
Preparation for Large-Scale Biological Screening
Fig. 1. General procedure for preparing extracts representing a range of polarities, includ- ing a partially tannin-free chloroform extract, adapted from the literature ( 18 ) .
extract is essentially free of vegetable tannins (plant polyphenols), 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 nonpolar compounds that tend not to be promising candidates for drug development ( 17, 18 ) .
1. Extraction with methanol (MeOH): The samples are macer- ated three times with MeOH.
2. Concentration of MeOH extract: The pooled batches are con- centrated under a vacuum (at a temperature not more than ca.
40°C to avoid thermal artifact formation). The concentrated extract should then be reconstituted in 90% MeOH (MeOH:water = 9:1). As a general rule, enough solvent mix- ture 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 petro- leum ether and methanolic extracts are used for each partition.
Usually, the defatting step is repeated twice for a total of three batches. The pooled “lipid extracts” can be concentrated, tested for biological activity, or submitted to chromatographic procedures, as desired.
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 chlo- roform. Equal volumes of water and chloroform are usually effective. After separation, the lower (organic solvent) layer is collected, and this process is repeated for a total of three batches. (The pooled organic layers can be concentrated to reduce the total volume).
6. Partial removal of tannins: The resulting organic extract is par- titioned with 1% NaCl in water (w/v) ( 18 ) . The organic layer is then concentrated to dryness.
1. When a particular phytochemical constituent or compound class is to be the target of an investigation, specifi c extraction procedures may be employed to produce enriched extracts. In some instances, the polarity of a solution may be modifi ed to cause particular compound classes to precipitate, leaving unwanted compounds in solution.
3.6. Preparation of Phytochemically Enriched Extracts:
General Considerations
2. Compounds containing primary, secondary, or tertiary amines, carboxylic acids, lactones, and phenols may be extracted selec- tively using pH modifi cations to manipulate the polarity/solu- bility 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.
Figure 2 shows a procedure for isolating mixtures of crude saponins (i.e. , steroidal or triterpene glycosides) based on a method described in the literature ( 19 ) .
1. The plant material is defatted with hexane, and extracted with MeOH, ethanol (EtOH) or EtOH/water.
3.6.1. Isolation of Mixtures of Crude Saponins
Fig. 2. General fractionation procedure to obtain a precipitate of crude saponins from plants, adapted from the literature ( 19 ) .
2. The alcoholic extract is concentrated under vacuum, and suspended in water (presaturated with n -butanol) and partitioned with n -butanol.
3. Diethyl ether is added to the n -butanol partition to precipitate the saponin fraction.
Plant sterols (including sapogenins, bufadienolides, and cardiac glycosides) can be extracted using chemical manipulations and solvent/solvent partitioning, for example a method using revers- ible chemical reactions has been described elsewhere ( 20 ) , and is outlined here.
1. Separation of non-alcohols from alcohols: The sample is parti- tioned between aqueous phthalic anhydride and organic solvent. The alcohols partition into the aqueous layer as half- phthalates and can be regenerated by the treatment with sodium methoxide in MeOH.
2. Separation of ketone-containing sterols from non-ketones: The extract is partitioned between the organic and aqueous layers with Girard’s hydrazide reagents (H 2 N·NH·CO·CH 2 ·NR 3 + Cl − ).
Ketones can be regenerated by acid hydrolysis ( 20 ) . As with all chemical processes, one should be sure to follow safe protocols.
Alkaloids containing basic amines can be extracted selectively using a modifi ed version of the classic “acid–base shakeout” method (Fig. 3 ). As a general rule, mineral acids and strong bases should be avoided in extracting alkaloids (and plant material in general) when the target compounds are unknown or potentially acid labile because of the risk of artifact formation ( 21, 22 ) .
1. Initial extraction: A defatted MeOH extract may be prepared as described above, or alternatively a MeOH extract can be obtained from defatted plant material (extraction with hexane or petroleum ether).
2. Acid partition: The MeOH extract is extracted under vacuum, and partitioned between dilute tartaric acid (titrated to pH 5) and EtOAc. The resulting EtOAc partition should contain non-alkaloids and neutral compounds.
3. Base partition: The pH should be carefully adjusted using a solution of sodium carbonate to a pH of about 10. Then, the basic solution is partitioned with EtOAc. This second EtOAc extract will contain most primary, secondary, and tertiary amines (which will be in the neutral form at this pH). The resulting aqueous solution will contain quaternary amines and various polar constituents, including alkaloid N -oxides as well as sugars and other non-alkaloids.
3.6.2. Selective Extraction and Fractionation of Plant Sterols
3.6.3. Selective Extraction and Fractionation of Alkaloids
4. Alternative method with stepwise pH adjustment: This extraction scheme can also be modifi ed for use with a series of partitioning steps, employing increasingly basic solutions for extraction of neutral (or mildly acidic) to increasingly basic alkaloids.
5. Additional alkaloid methods: Alkaloids can also be extracted with 10% acetic acid in EtOH, followed by concentration under vacuum to one-quarter the original volume and precipi- tation of the crude alkaloid fraction by drop-wise addition of NH 4 OH ( 23 ) . Alternatively, the plant material may fi rst be wetted with a dilute base solution, followed by percolation or maceration with a nonpolar organic solvent.
1. Extraction of carboxylic acids: Many compounds containing carboxylic acid functional groups can be selectively extracted by partitioning between organic solvent and basic aqueous solutions. The resulting organic solution should contain mostly neutral compounds, and the aqueous solution will contain many organic acids (ionic form in basic solution). Adjusting the pH of the aqueous solution with acid (to several pH units below the pK a the acid(s) of interest), then partitioning with organic solvent can be used to prepare an organic extract enriched in acids.
3.6.4. Use of pH Modifi cation to Extract Non-alkaloids
Fig. 3. General procedure to obtain alkaloidal extracts from crude plant material ( 21 ) .
2. Similarly, polyphenols can often be removed from an organic extract by partitioning with aqueous strong base (about pH 12). Basic conditions also increase the hydrophilic charac- ter of fl avonoid aglycones that possess free phenolic groups (see Chapter 18 ).
1. Although traditional medicines are often prepared by water extraction as infusions (steeping in hot or cold water) or decoc- tions (extracting in boiling water), 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-sol- uble constituents using conventional isolation methods, and the relative diffi culty of concentrating water extracts on a rotary evaporator (because of the relatively high boiling point of water and common tendency to foam or “bump”).
2. Water is a “green solvent” and can be used not only for the extraction of polar compounds, but also for extracting slightly nonpolar 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 85°C yielded propor- tionally more of the polar constituents, taxifolin and silychris- tin, whereas water at 100°C, 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 concentrat- ing water extracts by other methods.
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 proce- dures may also result in artifact formation. In addition to the spe- cifi c examples given in the notes (see Note 13 ), Middleditch ( 26 ) has compiled a detailed treatment of the subject of analytical arti- facts in chromatography.
1. Avoiding artifact-prone procedures: In general, mild condi- tions should be employed during the isolation process, unless it is known beforehand that the compounds of interest are sta- ble under the specifi c conditions of a proposed extraction method.
2. Recognizing artifacts: It is useful to be familiar with the type of artifacts that might form during a specifi c extraction protocol.
One should be vigilant for structural clues (such as racemiza- tion or apparent adduct formation) that indicate a compound isolated under certain conditions may be an artifact.
3.6.5. Water Extraction:
Challenges and Opportunities
3.7. Avoiding
Extraction of Artifacts
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 nonreactive solvents and mild conditions, and the resulting extract analyzed using LC-MS/MS to determine if the com- pound is present in the original plant material ( 27 ) .
Many naturally occurring compounds may interfere with the results of bioassays performed on an extract or fraction that contains these compounds. In addition, compounds from sources other than plants (including several synthetic contaminants, such as phthalate esters and silicone grease) are frequently found in extracts, espe- cially when less than optimal procedures are used (see Note 14 ).
1. Lipids: Lipids can be partially removed by defatting the plant material or during the extraction process. However, this is not always feasible or completely effective. Fatty acids and other
“greasy” constituents are usually extracted with solvents of low polarity, but they may co-extract when polar solvents are used.
Fatty acids have been found to give false-positive results in cer- tain receptor-binding, enzyme-inhibition, and radiometric assays ( 28– 30 ) . Lipids can be selectively separated from more polar constituents using appropriate solid-phase extraction or reversed-phase chromatographic steps.
2. Plant pigments: Carotenoids have been reported to interfere with electron-capture gas chromatography detection, and a method for removing them by fi ltering over silver nitrate impregnated alumina has been described ( 31 ) . Chlorophylls can often be suffi ciently removed using solvent–solvent parti- tion between hexanes or petroleum ether and 90% MeOH (Fig. 1 ). Solid-phase extraction or other chromatographic methods can be used to achieve more complete removal.
Chlorophylls can also be removed by passage over (or standing with) activated charcoal ( 32 ) , although this carries the risk of loss of important active constituents.
3. Tannins: Many vegetable tannins give false-positive results in various biological assays, usually because of their tendency to form nonselective complexes with proteins (including enzymes, receptors, and structural proteins) through multipoint hydro- gen bonding ( 33 ) . Aqueous and organic extracts containing tannins may nonselectively inhibit topoisomerases 1 and 2 (T-1 and T-2), viral reverse transcriptase, and other enzymes, leading to false-positive results ( 18, 33– 35 ) . To remove most of the tannins from a chloroform extract, the extract may be washed with an equal volume of 1% aqueous NaCl, with the upper phase being discarded, and the chloroform phase then dried with anhydrous Na 2 SO 4 . Other tannin removal methods have 3.8. Recognizing and
Avoiding Common Interfering Compounds
been reported ( 36– 38 ) . Among the more convenient methods, the extracts can by passed over polyvinyl pyrrolidone (PVP) or polyamide ( 36, 37 ) . PVP or polyamide may be used in batches, or packed in a small column through which the extract is passed, with monitoring of the supernatant or eluate by the ferric-chlo- ride (FeCl 3 ) reaction to determine whether the tannins have been removed, although this method is not without a risk of loss of important nontannin phenolic constituents.
The phytochemical screening reagents and procedures presented in this section are suitable for use without chromatographic separa- tion, but many of them are also used in visualizing spots on TLC plates ( 39, 40 ) . General issues related to phytochemical screening have been discussed in detail elsewhere ( 41 ) . A positive reaction should not be taken as proof of the presence of a certain type of secondary metabolite because other compound types may give false-positive reactions. Nevertheless, these detection methods are often effective for generating hypotheses about what types of sec- ondary metabolites may be present in a mixture of “unknowns,”
and for monitoring the presence of compounds of interest. In addi- tion to these colorimetric procedures, the use of HPLC in the anal- ysis of plant extracts is widespread, both for metabolite profi ling studies and for dereplication of active constituents (see Note 15 ).
1. Methods for detecting alkaloids: Aliquots of alkaloid extracts should be analyzed using one of the many methods that have been developed for alkaloid detection, for instance, TLC chromatography using appropriate spray reagents (Dragendorff reagent, iodoplatinate, etc.) or LC-MS meth- ods (see Note 16 ). In addition, some modifi ed procedures can be used to test for alkaloids in extracts (without the need for chromatographic separation). Dragendorff reagent —As with other reagents, prepared kits are available, and there are many variations, including the following, which is useful for testing extracts directly. Solution I: Dissolve 8.0 g bismuth subnitrate [Bi(NO 3 ) 3 ·H 2 O] in 30% w/v HNO 3 . Solution II:
Dissolve 27.2 g KI in 50 mL water. Procedure: Slowly com- bine the solutions and let stand for 24 h, fi lter, and dilute to 100 mL with deionized water. In acid solutions, an orange- brownish precipitate will appear. The alkaloids may be recov- ered by treatment with Na 2 CO 3 and subsequent extraction with solvents immiscible with aqueous solutions. Wagner reagent —Solution: Dissolve 1.27 g I 2 (sublimed) and 2 g KI in 20 mL water, and make up with water to 100 mL.
Procedure: A brown precipitate in acidic solutions suggests the presence of alkaloids. Some alkaloids that might be pres- ent in a plant extract may not give a positive reaction because of structural idiosyncrasies. When these reagents are used for 3.9. Techniques
for Detection of Phytochemical Groups in Extracts
phytochemical screening, it is often desirable to use at least two different reagents to reduce the risk of false-positive and false-negative results ( 41– 43 ) .
2. Methods for detecting sesquiterpene lactones and cardiac gly- cosides: Compounds containing α , β -unsaturated lactone func- tional groups can be detected using the following methods ( 20, 39, 40 ) . Kedde reagent —Solution I: Dissolve 2% of 3,5-dinitrobenzoic acid in MeOH. Solution II: 5.7% aqueous KOH. Procedure: Add one drop of each solution to 0.2–
0.4 mL of the sample solution, and a bluish to purple color will appear within 5 min. The solution should not contain acetone, which gives a deep bluish color. Alkaline trinitrophenol test solution (Baljet reagent) —Solution I: Dissolve 1 g picric acid in 100 mL ethanol. Solution II: 10 g NaOH in 100 mL water (be sure to use due caution when handling these reagents).
Procedure: Combine solution I and II (1:1) before use and add two to three drops to 2–3 mg of sample; a positive reac- tion is indicated by an orange to deep red color. Legal reagent — Solution I: Dissolve 0.5% of a recently prepared sodium nitroprussiate in water. Solution II: 0.2 N NaOH. Procedure:
Dissolve 2 mg of sample into pyridine (two to three drops), add one drop solution I, and four drops solution II (one at a time). Extracts containing cardiac glycosides will produce a deep red color, and α , β -unsaturated lactones and some β , γ -lactones will produce a pink color. (These types of com- pounds may isomerize in alkaline solution, and so the extracts or compounds should be stored under controlled pH).
3. Methods for detecting fl avonoids: Certain fl avonoids may cause false-positive results in assays using 3-(4,5-dimethylthi- azol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) for the quantifi cation of cell viability. For example, kaempherol was found to directly reduce MTT to a colored product in a cell- free system, constituting a “positive” result that could be inter- preted as indicating the presence of living cells ( 44 ) . The reagents below are generally described in published mono- graphs ( 41, 45 ) . Shinoda test —General method: To a few mil- liliters of an alcoholic solution of the sample, add a small amount of magnesium powder and a few drops of concentrated HCl. Before adding the acid, it is advisable to add t -butyl alco- hol to avoid accidents from a violent reaction; the colored compounds will dissolve into the upper phase. Flavones, fl a- vonols, the corresponding 2,3-dihydro derivatives, and xan- thones produce orange, pink, red to purple colors with this test. By using zinc instead of magnesium, only fl avanonols give a deep-red to magenta color; fl avanones and fl avonols will give weak pink to magenta colors, or no color at all. Sulfuric acid — Procedure: Flavones and fl avonols dissolve into concentrated
H 2 SO 4 , producing a deep yellow colored solution. Chalcones and aurones tend to produce red or red-bluish solutions and fl avanones give orange to red colors.
4. Methods for detecting polyphenols: Vegetable tannins are loosely defi ned as polyphenolic compounds that precipitate protein. The following detection procedures are described in the literature ( 18, 46 ) . Ferric chloride —Solution: Dissolve 5%
(w/v) FeCl 3 in water or ethanol. Addition of several drops of the solution to an extract produces a blue, blue-black, or blue- green reaction in the presence of polyphenols. This is not a specifi c reagent for tannins, as other phenolic compounds will also give a positive result. “Wall” test —Procedure: For the detection of tannins in solution, 10 mg of an extract is dis- solved in 6 mL of hot deionized, distilled water (fi ltering if necessary), and the solution is divided between three test tubes.
To the fi rst is added a 1% solution of NaCl, to the second is added a 1% NaCl and 5% gelatin solution, and to the third is added a ferric chloride solution. Formation of a precipitate in the second treatment suggests the presence of tannins, and a positive response after addition of ferric chloride to the third portion supports this inference ( 18 ) .
5. Methods for detecting sterols: As with most colorimetric tests, the following may give positive responses with compounds other than the target compounds. Additional sterol-detecting reactions have been described in the literature ( 41, 47 ) . Liebermann-Burchard test —Solution: Combine 1 mL of anhy- drous acetic acid and 1 mL of chloroform and cool to 0°C, and add one drop of concentrated sulfuric acid. Procedure: When the sample is added, either in the solid form or in solution in chloroform, blue, green, red, or orange colors that change with time will indicate a positive reaction; a blue-greenish color suggests the presence of Δ 5 -sterols, with maximum intensity at 30 min. (This test is also applicable for certain classes of unsat- urated triterpenoids). Salkowski reaction —Procedure: Dissolve 1–2 mg of the sample in 1 mL of chloroform and carefully add 1 mL of concentrated sulfuric acid, forming two phases, with a red or yellow color indicating the presence of sterols and meth- ylated sterols.
6. Detection of saponins: Owing to their surface-active proper- ties, when shaken, saponin-containing aqueous solutions tend to produce foam, which is stable for approximately 15 min. An additional test for saponins makes use of their tendency to hemolyze red blood cells ( 19, 46 ) , although this tendency may be inhibited by the presence of tannins in the extract, presum- ably because tannins cross-link surface proteins, thereby reduc- ing the cell’s susceptibility to lysis ( 48 ) .
1. Preliminary bioassay or analytical-scale chromatographic evalu- ation or “dereplication” of separate parts may suggest high pri- ority parts for further investigation. In studies of plant-derived anticancer agents in the laboratory of one of the authors (ADK), it has been the practice to collect up to four separate anatomical plant parts, since different phytochemical profi les may occur ( 49– 51 ) .
2. For maximum assurance that a recollected sample will contain the same constituents as the original collected plant material, recollections should as far as possible be carried out in the same location, on the same plant part, at the same time of the year.
However, care should be taken not to destroy all of the speci- mens growing at a particular collection location.
3. The International Union for Conservation of Nature and Natural Resources maintains a database that lists many species considered to be endangered or threatened, and may be searched online ( http://www.iucnredlist.org/ , The IUCN Red List of Threatened Species , accessed September 2010).
4. In most cases, it is necessary to collaborate with an experienced fi eld botanist, since reproductive organs are often missing at the time of collection, and sterile features, such as leaf mor- phology, smell, and bark characteristics, must be used for iden- tifi cation. Nonetheless, even skilled botanists may only be able to complete a partial identifi cation for some species, in which case specimens should be sent to taxonomic experts specializ- ing in the family or genus in question.
5. It is the responsibility of investigators to ensure that the neces- sary permits and agreements have been obtained and that the stipulations therein are followed. Examples of the types of documentation that may be required include offi cial permits for collecting in a national park, proof of prior-informed con- sent when interviews are conducted as part of the selection and collection process, and benefi t-sharing agreements between offi cial representatives from a collaborating institu- tion or government agency in the source country and from the investigator’s institution (and potentially other legal parties).
There is a broad consensus that disregard for laws governing plant collection and transport, intellectual property rights, and privacy rights is unacceptable, constituting a breach of scien- tifi c integrity.
6. If herbal medicines are being studied, the decision must be made whether they will be collected from the wild, purchased from a local store or wholesaler, grown in a greenhouse or research
4. Notes
farm, or obtained through collaboration with a manufacturer.
Regardless of the source, ensuring the authenticity of samples is essential. Often, it is possible to purchase minimally processed material, such as whole or coarsely milled root or leaf, which can be authenticated using macroscopic and microscopic character- istics, in addition to chemical characteristics ( 39, 40 ) . Monographs published by the American Herbal Pharmacopoeia (PO Box 66809, Scotts Valley, CA 95067) and other organiza- tions (including the United States Pharmacopeia-National Formulary) are available for a growing list of botanicals. If the purpose of the study is to compare the levels of specifi c active constituents or marker compounds in commercially available products, it may only be feasible to identify the commercial source of the products. In the absence of the deposit of a voucher specimen in a herbarium, some scientifi c journals (including the Journal of Natural Products and Planta Medica ) require proof of the identity of herbal remedy through the presentation of a standard HPLC chromatogram showing the presence of known marker compounds.
7. An effective technique is to place plant material in bags made of loose-weave muslin or synthetic mesh with drawstring clo- sures ( 10 ) . The use of such bags aids in labeling samples and prevents accidental mixing of samples, and the porous fabric allows air circulation, speeding the drying process and prevent- ing the buildup of heat and moisture and the growth of mold.
These bags can be conveniently moved under shelter, sus- pended from hooks, and transported as necessary.
8. Solvents that produce toxic vapors should be manipulated in a fume hood or other approved area. Although odor is sometimes a reliable indicator of unsafe air concentrations of solvents, the safe exposure limits of some solvents, includ- ing chloroform, can be considerably exceeded without being detected by smell ( 52 ) .
9. Various mills suitable for milling of plant material are commer- cially available. In a typical arrangement, plant material is intro- duced into a chamber that contains a set of rotating knives, which chop the plant material and traject it against a screen.
The degree of milling is controlled by selection of a screen that will give the desired particle size. Screen size is expressed as the number of holes per linear inch (termed “mesh size”), or as the diameter of the holes in mm. The product of milling is material of a relatively uniform particle size. Milling improves the effi - ciency of extraction by increasing the surface area of the plant material and decreasing the amount of solvent needed for extraction. Although it might seem that milling plant material to a very fi ne powder would be the most ideal, if the particles are too fi ne, solvent cannot fl ow easily around them.
Furthermore, the friction of milling generates heat (the fi ner the particle produced, the more heat), potentially causing vol- atile constituents to be lost and heat-labile components to degrade and oxidize.
10. Simple conical glass percolators are useful for amounts of plant material of a kilogram or less. Stainless steel percolators are useful for larger sample sizes. Deagen and Deinzer ( 53 ) have described a percolator system using 55-gallon drums, a solvent pump, electronic fl ow regulation, and parallel ion-exchange columns for large-scale extraction of pyrrolizidine alkaloids.
11. Steam distillation is used when the compounds of interest are volatile, mainly in the preparation of fragrance and fl avoring agents ( 54 ) . Supercritical fl uid extraction (SFE) using carbon dioxide as the extraction solvent shows great promise as a
“green alternative” to conventional extraction methods, because it uses an essentially nontoxic solvent, exhibits mini- mal potential for artifact formation, and CO 2 can be obtained in high purity suitable for production of food-grade extracts.
The addition of polarity modifi ers such as ethanol, and the development of SFE equipment capable of producing pres- sures in excess of 600 bar has made possible the extraction of some compounds of intermediate polarity, but polar com- pounds, including those with phenolic and glycosidic groups, are still poorly extracted ( 55 ) . For this reason, SFE may be too selective for use in general extractions of plant material for bio- assay-guided isolation. Pressurized solvent extraction (also called accelerated solvent extraction) uses optimized condi- tions of temperature and pressure to modify the extraction power of solvents. This technology promises to extend the range of compounds that can be effi ciently extracted with green solvents, such as water as well as other solvents ( 56, 57 ) . Reviews of microwave assisted extraction, ultrasound-assisted extraction, and other specialized methods have been presented elsewhere ( 58 ) .
12. For a simple test of whether the extraction is complete, an ali- quot of each successive extract may be dried and compared. If a relatively large amount of residue remains after drying (or if chemical detection methods indicate that a signifi cant amount of a compound or compound class of interest is present), then the extraction is not complete, and additional extraction may be warranted, or it may be necessary to switch to a more suit- able solvent.
13. Artifacts form under a range of conditions and from a wide range of natural product classes. The following is not meant to be an exhaustive list, but rather to provide examples of the types of problems that can occur.
(a) Alkaloid artifacts from chlorinated solvents: Alkaloids appear to be particularly susceptible to artifact formation.
Phillipson and Bisset ( 59 ) reported that brucine and strychnine formed bromochloromethane and dichlo- romethane adducts during extraction of a species of Strychnos , and concluded that traces of HCl and diha- lomethanes in the chloroform used for extraction reacted with the alkaloids to form artifacts.
(b) Artifacts due to phosgene: Phosgene (COCl 2 ), a reactive and toxic compound that rapidly forms from the decom- position of chloroform in the presence of air and light, is known to react with alkaloids, particularly those with sec- ondary amino groups ( 60 ) , and phosgene may combine with alcohols, such as MeOH, EtOH, and isopropanol, and react with amines to form methyl, ethyl, or isopropyl carbamates ( 61, 62 ) . Phosgene can build to dangerous concentrations in unsealed bottles of chloroform ( 63 ) . Although ethanol or other stabilizers are usually added to chloroform, they are not entirely effective. Distillation of chloroform removes a portion of the stabilizer, but is not effective in removing phosgene. Passing chloroform over activated alumina or letting stand overnight over Ca(OH) 2 removes phosgene ( 61 ) . Partitioning with aqueous H 2 SO 4 , followed by drying over anhydrous CaCl 2 (overnight) and distillation of the chloroform removes both phosgene and ethanol ( 64 ) . Chloroform thus treated should be pro- tected from extended exposure to light and air.
Dichloromethane does not readily form phosgene; how- ever, dichloromethane extraction of tablets of cyprohepta- dine hydrochloride was found to produce an N -chloromethyl adduct, suggesting the potential for an analogous reaction to occur with other compounds in plant extracts ( 65 ) .
(c) Artifacts due to MeOH: MeOH may directly form meth- oxy group-containing artifacts by acting as a nucleophile with compounds containing α , β -unsaturated carbonyl groups, as in the case of a number of minor ring-A meth- oxylated withanolide artifacts from the aerial parts of Physalis philadelphica (tomatillo) ( 27 ) . Ammonium hydroxide with acetone may produce artifacts that give false-positive responses in alkaloid screening tests ( 66 ) . (d) Artifacts in compounds with acidic protons at chiral cen-
ters: Some chiral compounds are prone to base-induced racemization, even under relatively mild basic conditions.
A classic example is that of hyoscyamine under the usual
“acid–base shakeout” conditions, but other classes of com- pounds are also susceptible to base-catalyzed modifi cation.
For instance, lignans containing strained lactone rings may epimerize under basic conditions, as has been noted for the conversion of podophyllotoxin to picropodophyllin and for other lignans associated with Podophyllum in the presence of organic or inorganic base ( 67 ) .
14. Plasticizers and grease can be introduced at different stages in the extraction process. When some grades of MeOH are used in bulk for extraction, followed by concentration of the extract, plasticizers can become a signifi cant portion of the extract ( 68 ) .
(a) Plasticizers may contaminate solvents, fi lter papers, plastic apparatus, and chromatographic stationary phases stored in plastic containers. Plasticizers can be eliminated or greatly reduced by distilling solvents used for extraction and chromatography. Instructions for constructing a dis- tillation apparatus suitable for laboratory-scale purifi cation of solvents can be found in practical chemistry texts ( 69 ) . Alternatively, high purity solvents stored in glass bottles can be used.
(b) Phthalate esters are the plasticizers perhaps most likely to be encountered. Dioctylphthalate sometimes contami- nates extracts from plants. Pure dioctylphthalate is an yellow oil that exhibits discernible cytotoxic activity for P-388 murine lymphocytic leukemia cells.
Diethylhexylphthalate, reportedly isolated from Aloe vera , was found to induce apoptosis in several human cancer cell lines ( 70 ) . On TLC plates dioctylphthalate shows a pink- violet spot when sprayed with concentrated sulfuric acid or concentrated sulfuric acid/acetic acid (4:1), and heated at 110°C for 5 min with R f = 0.4 (petroleum ether:EtOAc = 19:1). The following phthalate-identifying spray reagent combination is described in the literature ( 71 ) . Solutions: Spray solution I: add zinc powder to a 20% ethanolic resorcinol solution. Spray solution II: 2 M sulfuric acid. Spray solution III: 40% aqueous KOH solu- tion. Procedure: Spray with I, heat for 10 min at 150°C, spray with II, heat 10 min at 120°C, and spray with III.
Phthalate esters will appear as orange spots on a yellow background. Spectroscopic data: UV l max 275 nm (log ε 3.17), shoulder at 282 nm; 1 H NMR ( δ , CDC1 3 ) 7.70 (2H, dd), 7.52 (2H, dd), 4.20 (4H, dd), 1.2–1.8 (14H, m), 0.90 (12H); EIMS ( m / z ) 279 , 167, and 149 (100%) ( 68 ) . Common ions due to plasticizers detected in positive-ion electrospray and APCI mass spectra include m / z 391, rep- resenting [M + H] + for dioctylphthalate, and m / z 550, 522, 371, and 282 for various other plasticizers from poly- ethylene and other sources ( 72 ) .
(c) Grease: Silicone grease is used as a lubricant in ground- glass joints in extraction apparatus and in stopcocks in col- umns and vacuum lines, although it is becoming less common as Tefl on is supplanting glass fi ttings. Nonetheless, silicone grease may contaminate plant samples and can be recognized by the following mass spectrometric fragmen- tation ions: m / z 429, 355, 281, 207, and 133. When hydrocarbon grease is used, the ion pattern shows losses at 14 mass unit intervals because of the degradation of the aliphatic chains ( 68 ) .
15. The following are a few practical tips and considerations to protect the HPLC equipment when analyzing crude or semi- pure plant extracts. The sample should be fi ltered prior to HPLC analysis (e.g., dissolved in the mobile phase, or another suitable solvent, and fi ltered through a 0.45 μ m fi lter mem- brane, and/or passed over a solid-phase extraction cartridge with the same stationary phase as the column to be used for HPLC).
16. As a secondary screening procedure, duplicate TLC plates can be prepared and sprayed with two different alkaloid reagents, such as Dragendorff and iodoplatinate. Electrospray-ionization mass spectrometry in the positive mode can be used for confi r- mation of the presence of alkaloids, since most alkaloids (those with an odd number of nitrogen atoms) will produce a strong even-integer [M + H] + ion, distinguishing them from other nat- ural product classes, which generally give an odd-integer ion.
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