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Traditional, Analytical, and Preparative Separations of Natural Products

Traditional, Analytical, and Preparative Separations of Natural Products

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Natural Products from Plants, Second Edition

8.4.4

8.4.5

8.4.6



Gas Chromatography ...................................................................................................... 294

Size-Exclusion Chromatography .................................................................................... 294

Case Studies: Examples of Chromatography Protocols................................................. 297

8.4.6.1 Purification of Proteins Using Ion-Exchange Chromatography ..................... 297

8.4.6.2 Extraction and Bioactivity-Directed Separation of the Bark of an

Undescribed Salacia Species from Monteverde, Costa Rica.......................... 298

8.5 Electrophoresis ............................................................................................................................. 301

8.5.1 The Basics ....................................................................................................................... 301

8.5.2 Agarose Gel Electrophoresis........................................................................................... 301

8.5.3 Polyacrylamide Gel Electrophoresis (PAGE) ................................................................. 302

8.5.3.1 SDS-PAGE ....................................................................................................... 302

8.5.3.2 Nondenaturing PAGE....................................................................................... 303

8.5.3.3 Two-Dimensional Gel Electrophoresis............................................................ 303

8.5.3.4 Staining Techniques ......................................................................................... 304

8.5.4 Capillary Electrophoresis ................................................................................................ 305

8.5.4.1 Basic Modes of Capillary Electrophoresis...................................................... 307

8.5.4.2 Selecting the Mode of Capillary Electrophoresis ........................................... 311

8.6 Conclusions .................................................................................................................................. 313

References .............................................................................................................................................. 313



8.1



Introduction



It was estimated that about 80% of all the world’s medicines are originally derived from plant sources,

especially those found in tropical regions. However, many of the plants within these often remote regions

of the world have yet to be identified as species, and only about 15% of the known angiosperm species

in these regions were examined for their medicinal potential. Therefore, there are most definitely a large

number of plant-derived medicines and other useful compounds that have yet to be discovered and

characterized around the world. While we discuss the social problems of environmental and plant

conservation in other chapters (see Chapters 14 and 15), the aim of this chapter is to give the reader an

understanding of how one begins to determine what plants have useful compounds by looking at the

process of plant collection, methods of extracting compounds, and general methods of separating plant

compounds within crude extracts. In Chapters 9 and 10, we go on to explore how isolated compounds

are characterized, with a focus on nuclear magnetic resonance (NMR) techniques and bioassays for the

characterization of potential medical benefits.



8.1.1



Traditional Methods



Much of the knowledge about plants that have useful properties comes from native populations of people

living in each given area. Natural medicines have been used for centuries in many parts of Asia, such

as China and India. In particular, Native Americans had a profound influence on the natural medicines

of today (Ody, 1993). For centuries, such native peoples passed on this knowledge from generation to

generation, making use of techniques that they had available to them to perform plant extractions. Such

traditional methods used for the separation of metabolites from plant materials include the use of hot

water extracts to make teas or natural plant dyes (e.g., the monoterpenes from mints to make mint tea).

Salves and decoctions are often made from a single plant source (e.g., the oleoresin terpenes from pitch

of balsam fir used directly to treat burns). However, more complicated mixtures of plants are also used

(e.g., many commercial herbal teas that utilize chamomile, mint, bee balm, lavender, and other plants

or upregulators of the immune system, such as Echinacea and goldseal). The important idea here is that

such extracts or preparations rely on the synergistic action of several plant metabolites that are more

effective than any one alone (see Chapter 13). An excellent discussion of these traditional methods is

found in Penelope Ody’s book, The Complete Medicinal Herbal (1993), but the point remains that plant

extractions in even a traditional sense can be diverse due to the complexity of the mixtures of compounds

resulting from crude extraction processes. Consequently, during the technological advances of the last



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century, contemporary methods were developed to better separate the specific compounds within crude

plant extracts, allowing for better characterization of the usefulness of each compound.



8.1.2



Contemporary Methods



Modern methods of isolation of natural products, in contrast to traditional methods, utilize principles of

extraction that are based on the polarity (relative solubility in organic solvents), solubility in water, and

various alterational solubilities based on salts and pH (relative acidity or alkalinity) (see Chapter 1).

These contemporary methods are meant to complement the traditional methods. Yet, they provide a

greater degree of resolution of the types of metabolites present in plant samples (see Chapter 2). They

also allow one to quantitate their levels and to study how genetic and environmental factors regulate

their synthesis (see Chapter 3). The basic methods for extraction and quantitation of the metabolites

present are described in detail in this chapter and in Chapter 23 of Cseke et al. (2004).



8.2



Collection, Storage, and Vouchering of Plants



The first steps in isolating natural products are to be able to identify the plants of interest and to preserve

the compounds within them. During discovery projects, it is not normally known which of the plants

actually have useful compounds. Therefore, it is essential to be able to keep complete records of all

collected specimens. The following sections offer suggestions to follow, based on our experience.



8.2.1



Collection of Plants in the Field: Do’s and Don’ts



When collecting plants in the field for natural product extractions, it is important to be properly prepared.

We recommend the following:

















Wear field clothes, and cover yourself head to toe if collecting is to be in the cold of winter

or when mosquitoes or flies are in abundance.

Take along a notepad (Rite in the Rain® waterproof paper is excellent) and pencil to record

information about the collecting site location, soil conditions, ecological habitat, date of collection, plant identity, and who collected the plant(s).

If possible, take along a portable global positioning system (GPS) unit/altimeter for recording

geographical positions and elevations.

Take soil samples from each site so as to get information later on soil nutrients, soil pH, and

soil type where each plant grows.

Take along a pocket-size field guide (with photos, drawings, and good, usable identification

keys) to the local flora and a hand lens to help you identify each plant.

Take a good-quality digital camera to keep a photographic record of each plant.



Unfortunately, during field collections, the availability of optimal storage equipment is often not an

option due to the remote collection locations. Depending on weather, the trip will be short or long. With

available storage equipment nearby, we recommend the following:





If you are collecting live plants on a short trip (1 or 2 h) and have access to liquid nitrogen,

take along a large thermos full of liquid nitrogen and some aluminum foil. The thermos should

have a good handle on it to avoid spilling liquid nitrogen on your skin. The samples can be

collected, wrapped in aluminum foil pockets, labeled with a Sharpie® pen, and dropped into

the liquid nitrogen. This is by far the best method for preserving compounds within the plant

that may be easily degraded or chemically altered. Remember not to close the cap on the

thermos too tightly, or the container will explode.



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8.2.2



Natural Products from Plants, Second Edition

If you are collecting live plants on a longer trip, or you do not have access to liquid nitrogen,

take some Ziploc® plastic bags of various sizes in which to put the samples after collecting

plants on site. Such bags keep the plant materials alive until they can be frozen. Slips of recycled

waterproof paper or flagging tape are good to have so you can place notes on plant identity with

your collected specimens that match up with your field notes about the respective collections.

In all cases, when you collect plants for extracts, it is important to get representative samples

of all parts available: roots, vegetative shoots, bark from stems (if woody plant), flowers, fruits,

and seeds (if mature).

When collecting plants in the field, do not take every last plant in the population, especially if

the plant is rare, threatened, or endangered.

In the process of collecting herbaceous perennial plants (plants that come from the same mother

plant year after year), leave some of the original plant intact where it is growing so that it can

reproduce during the current and following years. Many of these plants take years to produce

even a small amount of new biomass every year.

If you are collecting mushrooms or puffballs in the field, wrap the fruiting bodies in wax paper

and place them in a collecting basket or other suitable container where they will not become

squashed. This will help for later identification and for making spore prints from the fruiting

bodies. This is impossible with giant puffballs (Calvatia gigantea); these can be collected intact

and placed in large paper shopping bags. Some of these mushrooms attain a diameter of 0.5 m.

As the Native Americans do, thank the plant for providing you with substrate for your extracts.

While this may not seem important to some, we all do this in various ways when we collect

plants in the home garden for food or for aesthetic purposes, or when we collect wild edible

plants in the field.



Storage of Plants at Low Temperatures



As noted above, one of the most important aspects of plant collection for the purpose of extracting many

natural plant products is the preservation of the compounds within the collected tissue. By far the best

way to do this is by quickly freezing the tissue in liquid nitrogen. Such samples can then be stored long

term in a –80°C commercial freezer. This technique prevents almost all degradation of the plant material

or any enzymatic changes that alter or degrade naturally occurring metabolites. Such techniques are

especially important for molecular biological studies, because once a plant is damaged by being picked,

it undergoes a drastic defense response that can greatly alter the composition of plant compounds, such

as RNA and proteins. Also, many additional stress compounds are produced once the plant perceives

itself as having been “attacked” during the collection process. So, one must work quickly to keep the

natural components of the plant intact. Even if liquid nitrogen is not available, it is a good idea to freeze

the specimens that will be used for extraction immediately in a freezer at –20°C or in a commercial

freezer held at –80°C. However, some of the collected plant material should be kept unfrozen for the

vouchering process (see below).

On the other hand, natural drying of the plant material can also be done if yield of metabolites is not

critical. This is usually the case for plant material used for dyeing fibers. This also works well when

using seeds for extraction. Seeds are usually dried to a low moisture content to prolong seed viability.

If the drying process is slow and the temperature is at ambient level, very little degradation of stored

metabolites in the seeds occurs. However, for extraction of medicinal compounds from plants, the use

of dried plant material is not desirable due to degradation of naturally occurring metabolites during the

drying process. Rather, it is best to rely on the use of frozen plant material.



8.2.3



Vouchering of Plants Collected in the Field



The vouchering process involves keeping detailed records of the collected plant tissue. It may not be

known for some time if a particular collected tissue will contain compounds of interest. This is especially



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true when searching for new medicinal compounds. So, accurate and lasting records are absolutely

essential. We recommend using the following combination of approaches.



8.2.3.1



Keep a Good Logbook



One should always keep a logbook of the plants and the tissues that were collected and frozen. This

includes information about the collecting site location, soil conditions, ecological habitat, environmental conditions, as well as the date of collection, plant identity, and who collected the plant. Many

times, the environmental conditions help control the biosynthesis of various compounds; so, a record

of the collection conditions may help to avoid confusion when comparing multiple collections of the

same type of plant. For similar reasons, we recommend taking soil samples and later recording

information on soil nutrients, soil pH, and soil type where each plant grows. It is also important that

proper labels be kept on samples stored in the freezer. Slips of paper work well inside Ziploc plastic

bags at –20°C, but these bags do not work well at –80°C. In these conditions, it is better to store the

tissue inside aluminum foil packets labeled with a Sharpie pen. In addition, a backup inventory on

the computer is a good idea. Why is this important? Sometimes, labels with collected plant material

become lost. Sometimes, one needs to examine lists of collected plants quickly, without having to go

through all of the frozen material. And sometimes, one needs to send such lists to others involved in

the project.



8.2.3.2



Photographic Records of Plants Collected



It is often desirable to keep photographic records of plants that were collected, either for plant identification or for re-collection of a particular plant. We recommend taking digital photographs of the live

plant in the field in addition to performing high-resolution scans (using a flatbed computer scanner)

back in the laboratory. The newer digital cameras provide high-resolution photos with special built-in

features, such as macro-lenses that provide surprising detail and good-quality flashes for low-light

conditions. Most of the computer scanners available can scan images in excess of 1200 dpi, and such

detailed photographic records augment any herbarium voucher specimens (see below and example shown

in Figure 8.1).



FIGURE 8.1 Digital photograph and scan of Calyptranthes pallens (Myrtaceae), spicewood, collected from Abaco Island,

the Bahamas.



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8.2.3.3



Preparation of Dry Specimens



Dried plant specimens are prepared in order to have them available at any time as voucher specimens

representing typical plants that were collected in the field and used for plant extracts. They are also called

herbarium specimens. Dried plant specimens are prepared in the traditional way by placing the collected

plant between single newspaper sheets and then placing this in a sandwich consisting of a dry blotter above

and below the newspaper sheets. A piece of corrugated cardboard (with air spaces present) is then placed

above and below each blotter. Successive sandwiches are placed atop one another and then compressed

between two wood-slatted frames and tied together tightly with straps. The entire assembly is then placed

upright on its side over a heat source, such as a radiator or a plant drier, with the heat on a moderate temperature

(e.g., 35 to 40°C). The specimens are allowed to dry this way for 48 h or longer. If plant specimens are very

high in water content, it is a good idea to replace the blotters with dry ones in the middle of the drying process.

Rapid drying ensures that plant pigments are well preserved; if the drying process is slow, chlorophylls

will degrade, and the leaves will appear yellow; flower pigments also fade badly with slow drying. For

this reason, newer methods making use of a microwave oven have also been developed. This works

especially well for the preservation of flower color. The tissue is placed between two sheets of absorbent

paper and compressed between two blotters, similar to the above technique. The final sandwich (pressed

in a microwave-safe frame) is then heated in a microwave oven for 1 to 5 min, depending on the type

of tissue. In a similar technique, plant tissues can be submerged in silica gel crystals and heated in a

microwave. However, such samples are not flat, making them more difficult to store.

Once plant specimens are dry, they can be mounted flat (with glue or cement) on heavy paper of

sufficient size to accommodate the specimen and a label with information about the plant, location of

the collecting site, collector, date of collection, genus and species of the plant, and the family to which

the plant belongs. The label may be placed in the lower right-hand corner of the sheet of heavy paper.

To avoid damage to the dry, mounted specimen, the entire sheet can be covered with Saran™ Wrap or

even laminated if the equipment is available. Dried sheets with plants mounted on them are usually

stored in an airtight cabinet in which paradichlorobenzene or naphthalene flakes are kept to deter insects

that can damage the specimens. If use of such chemicals is not desirable due to their harmful effects to

humans if inhaled continuously, natural insect repellents, such as dried lavender (Lavandula officinalis)

or neem (Azadirachta indica), or other less toxic commercial repellents can be used.



8.2.3.4



Living Plant Specimens



In our experience, we found it to be a good idea to collect seeds and living specimens of the plants that

are to be used for the extraction of natural products. We do this in order to have the living plants on hand

(e.g., medicinal plants, dye plants, or culinary herb garden or in a greenhouse) to be used for later extractions

or experimental treatments to enhance metabolite biosynthesis; this is especially important when access

to the original collecting site is not possible or convenient. A good example of this is with the tree of joy

(Camptotheca accuminata), which is the source of the drug camptothecin, used to treat patients with

prostate cancer. Our original seed came from China, and it would not be convenient to travel to China

each time that we need tissue for further experiments. Thus, the seed used in our greenhouse experiments

came from progeny from the original Chinese seeds that were grown in Louisiana. These seeds were

kindly provided by Mr. Tracy Moore, President of Xylomed Research, Inc., in Monroe, LA (Figure 8.2).

Using records that came from the naturally growing tree of joy, we were able to simulate the growing

conditions in our greenhouse, thereby being able to reproduce natural levels of camptothecin. The results

from these studies are provided in the research essay in Chapter 3, written by Atul Rustgi and Ashish Goyal.



8.3

8.3.1



Grinding and Extraction Protocols

General Extraction Protocols for Biologically Important Compounds



The primary ways to extract organic molecules of interest to biologists and medical investigators involve

breaking open the cells of the organism under investigation. Cell rupturing is accomplished in a variety



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FIGURE 8.2 (Left to right) Dr. Stanley Carpenter, Louisiana State University (LSU); Dr. Zhijun Liu, postdoctoral research

fellow at LSU; Tracy Moore, president of XyloMed Research, Inc.; and Dr. John D. Tarven, LSU, while visiting Beijing,

People’s Republic of China, in November 1996 to collect seed from different native populations of tree of joy ( Camptotheca

accuminata), the source of the drug, camptothecin, used to treat cancer patients. (Photo courtesy of Tracy Moore.)



of ways. The method used depends on the type of organism being considered and the type of tissue

used. In this section, we aim to give the reader a general understanding of such protocols. In the following

sections, we examine in more detail traditional and contemporary extraction methods that have proven

particularly useful for subsequent natural product separations.

It is important to note here that many compounds are rapidly degraded once the extraction process

has begun. For example, there are many proteases located inside of each cell. Normally, these proteases

are sequestered into specific regions of the cell, where they do little damage to important proteins.

However, once the cells are ruptured, the proteases are released, and they begin to destroy potentially

important proteins. To maximally inhibit those proteases, it is important to keep the temperature low

(from 0 to 4°C), and protease inhibitors are commonly added to lysis solutions or buffers used for

protein/enzyme isolation. There is also a wide variety of different solutions used for compound extraction.

It is strongly recommended that specific buffer conditions be maintained during the extraction and

purification of water-soluble compounds because the structures or activities of many compounds are

sensitive to pH changes. Using the example of proteins, it is essential to use proper buffering, because

some proteins or enzymes may be degraded or lose their enzymatic activities. Such buffer conditions

need to be optimized for specific cells, tissues, and protein types. In addition, extraction and purification

of membrane proteins usually requires detergents to help release them from the membranes (see Section

8.3.7.4), and many proteins require specific reducing agents, salts, and metal ions to remain active.

Therefore, the choice of rupturing procedure and extraction solution is often critical for the extraction

of specific plant products. A good discussion of these methods is found in Cseke et al. (2004).



8.3.1.1



Rupturing Bacterial Cells



Bacterial cells, like plant cells, have very strong cell walls that often make them difficult to extract.

Modern protocols for the extraction of water-soluble compounds make use of specific enzymes that are

able to cleave the protein components of the bacterial cell wall. In such protocols, cell cultures are

pelletized by spinning them in a centrifuge. The pellet is then resuspended in lysis buffer, to which an

enzyme, such as lysozyme, is added. After an incubation period, the suspension becomes very viscous

due to large amounts of released DNA from inside the cells. This can be reduced through the addition

of other enzymes, such as DNase I, to chop up the DNA and reduce the viscosity of the final extract

(see Cseke et al., 2004). Traditional bacterial extractions make use of a French press so as to break open

the cell walls. This involves using a heavy cylinder with high pressure applied to a piston that compresses

the cells in an extraction solution into a successively smaller volume within the free cylinder. As the

cells leave the cylinder, the rapid drop in pressure causes the cells to lyse and release their components.



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8.3.1.2



Natural Products from Plants, Second Edition

Rupturing Plant Cell Suspension Cultures



Like the bacterial procedures discussed above, similar procedures are used for plant cells grown in

suspension culture or for plant callus tissue. Specific enzymes, such as cellulase or pectinase, can be

added to lyse the walls of cells in aqueous solutions, or the cells can be passed through a French press.

Many plant cells grown in culture can simply be ruptured with a glass tissue homogenizer. However,

another good method for disrupting cells in suspension involves the use of special equipment called a

sonicator. In this case, the sonicator releases repeated high-frequency pulses of ultrasonic vibrations

that rupture the cell membranes. For heat-sensitive compounds, such as proteins or RNA, it is especially

important to keep the sample on ice, because the sonicator vibrations generate a great deal of heat. This

is why repeated pulses are used instead of a continuous discharge; this procedure allows the sample to

cool between pulses. Many of these methods, however, assume that water-soluble compounds are being

extracted, and they may not be applicable to many compounds. In these cases, the culture can be spun

down in a centrifuge to yield a pellet that can be used as described below.



8.3.1.3



Rupturing Whole Plant Tissues



Probably the best way to extract highly lignified or silicified plant tissues within organs such as leaves,

stems, roots, seeds, or fruits is to freeze the tissue and pulverize it using liquid nitrogen in a mortar and

pestle (Figure 8.3). This frozen powder can then be added to an appropriate extraction solution and



A



B



FIGURE 8.3 (A) A typical container used for the storage of liquid nitrogen. (B) Gregg Roslun, undergraduate student at

the University of Michigan, grinding a frozen plant sample with liquid nitrogen and a ceramic mortar and pestle, in

preparation for analysis of products of medicinal value in this sample. (Photo by David Bay.)



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processed for final extraction (see Section 8.3.2). On the other hand, fresh tissue can simply be finely

chopped using a food processor or finely milled using a Wiley mill, and softer plant tissues can be

ground in a small volume of buffer in a mortar, using whitewashed sand and a pestle to rupture the cells.

The bottom line here is that the more finely ground the material is, the better the final extraction of

compounds will be.



8.3.1.4



The Extraction Process



Once the cells are ruptured, the actual extraction is performed using techniques that depend on the

chemical properties of the compounds of interest. Water-soluble compounds and proteins are extracted

in water or buffers. Water-insoluble compounds are extracted with organic solvents. For example, because

taxanes are miscible in methanol, this solvent is often used as the extraction reagent. This is by no means

the only solvent that will work (see Section 8.3.2). Some compounds, such as cell-wall constituents,

have no need to be solubilized for extraction, because they can be obtained in pellet form by filtration

or centrifugation followed by washing with buffer solutions. It is worth noting that integral membrane

proteins often require the use of strong detergents, such as Triton X 100, to be extracted from the

membranes (see Section 8.3.7.4).

Two steps are usually critical for good extraction. First, ruptured cells should be ground or homogenized in the extraction solvent, depending on the cell-rupturing technique chosen. For example, taxanes

(terpenoid compounds derived from plants) are extracted by grinding the plant tissue in organic solvent

(methanol) in the same mortar and pestle that is used for the liquid nitrogen to rupture the cells. Waxes,

on the other hand, can be removed from the aqueous phase coming from a French press by homogenizing

and partitioning with chloroform and methanol. Second, once ground or homogenized, the extraction

mixture should be allowed to stand undisturbed for 0.5 to 24 h at a temperature that will not allow

degradation of the compound(s) or protein(s) of interest to occur (e.g., 4°C). This is done simply to

allow time for the extraction solvent to penetrate all parts of the ruptured cells.

After these two critical steps, the resulting slurry is filtered to obtain a filtrate free of particulates,

or it is centrifuged in order to obtain a cell-wall or membrane pellet fraction and a cell cytosolcontaining supernatant fraction. Such crude fractions can be used directly for enzyme reaction

assays, or they can be subjected to further purification and cleanup procedures in order to separate

and identify the compounds of interest. For example, C18 Sep-Pak™ columns can be used to remove

chlorophylls, which interfere with subsequent analysis of taxane extractions (compare Figure 8.7A

and B).

For the extraction of natural products of potential medicinal or other value in plant samples, the

protocols in the following sections can be employed. These are the conventional counterparts based on

the more traditional methods described above.



8.3.2



Aqueous and Organic-Solvent Extraction Methods



Extraction methods to be chosen should be based on knowledge of several physicochemical properties

of the compound of interest. These include partition coefficients in water or organic solvents, relative

polarity of the molecule, stability of the molecule in light or dark, as well as the temperature employed

during the extraction process. So, if the compound of interest is highly soluble in water, we employ hot

or cold water to obtain an aqueous extract. If, on the other hand, the compound is highly soluble in a

particular organic solvent, we employ that solvent to obtain an organic-solvent extract.



8.3.2.1 Aqueous Extraction of Compounds

8.3.2.1.1 Traditional Methods of Aqueous Extraction

The preparation of herbal remedies based on traditional methods of water extraction utilizes two

different approaches: if extracting herbaceous tissues of leaves, roots, and flowers, or soft-textured fruits

with a relatively high water content (in the range of 60 to 95% water) with hot water or cold water,

relatively mild physical conditions are used to obtain what is called an infusion. However, for woody,



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highly lignified tissues with relatively low water content (in the range of 5 to 50% water), such as roots,

barks, twigs, and some dry fruits, we need to employ more vigorous physical extraction procedures,

using longer extraction times and boiling water, to obtain what is called a decoction. There is an almost

endless list of methods for preparing infusions and decoctions, but all are based on the same principles.

An example of each type of traditional extraction is described in the following sections.

The preparation of hot water infusions can be done as follows:

1. Begin with a standard quantity of tissue to be extracted, such as 30 g dried herb (either freezedried or dried on newspaper over several days in the dark, as with rose hip tea [from fruits of

Rosa spp.], lemon balm tea [from leaves of Melissa officinalis], bee balm tea [from leaves of

Monarda didyma], chamomile tea [from flowers of Matricaria spp.], mint tea [from leaves of

Mentha spp.], green tea [from shoot tips of Camelia sinensis], or rhubarb tea [from leaf petioles

of Rheum palmatum]). Fermented dried herb can also be used in similar amounts (as with black

tea, derived from fermented shoot tips of Camelia sinensis). Generally, more tissue weight is

used for fresh herbs due to the water content in the tissue. Fresh herbs are best collected from

young leaves and shoot tips.

2. Place the herb in a teapot or water-boiling kettle that includes a tight-fitting lid.

3. Pour hot water (preferably from a reliable bottled water source or water rendered potable

[drinkable] by boiling and filtering) over the herb and allow to infuse for 10 min.

4. Pour the ambient hot water extract through a nylon sieve or strainer.

5. Use the infusion immediately, as you would for a cup of hot tea. Such infusions can also be

stored in a brown bottle away from the light in a cool place, like a refrigerator, and must be

used after no more than one year because of degradation of the active constituents. If bacterial

or fungal contamination occurs, the infusion should be thrown out immediately.

The preparation of cold water infusions can be done as follows:

1. Place several bags of black tea (obtained from Camelia sinensis) in a clear glass jar or bottle

filled with cold water.

2. Cover the jar to exclude particulate matter, such as dust or insects.

3. Allow to stand in the sun outdoors for a single day.

4. Chill by storing in a refrigerator at 4°C.

5. Drink when cold. The product here is often called sun-made tea.

The preparation of a decoction can be done as follows:

1. Place the quantity of herb (described above) in a saucepan, and add cold water. Again, the

tissues requiring the preparation of a decoction are often woody or highly lignified tissues.

2. Bring the water to a boil (100°C).

3. Allow to simmer for up to 60 min or until the volume has been reduced by one-third.

4. Strain the mixture through a nylon sieve into a bottle or jar.

5. Store in a cool place, such as a refrigerator, at 4°C. If bacterial or fungal contamination occurs,

the decoction should be thrown out immediately.

The above and other methods of preparation of plant extracts for medicinal purposes (like tinctures,

syrups, hot and cold oil infusions, creams, ointments, powders and capsules, compresses, and poultices)

are described and beautifully illustrated by Penelope Ody in The Complete Medicinal Herbal (1993,

pp. 116–125).



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Laboratory Methods of Aqueous Extraction



In contrast with the traditional methods of aqueous extraction described above, laboratory methods for

aqueous extraction rely on the use of more stringent quantitative procedures and more sophisticated

equipment. This is evident in the following methods typically used to prepare lab-type hot water extracts

that allow for compounds of a more polar nature to be obtained (see Chapter 1):

1. Weigh out a 0.5 g sample of a given plant that was previously placed in a deep freeze at –80°C.

2. Grind frozen plant tissues to a fine powder using small amounts of liquid nitrogen in a ceramic

mortar and grinding with a ceramic pestle.

3. Place 0.5 g of the powder into a 20 ml Corex® or Pyrex® centrifuge tube containing 10 ml of

hot (80°C) water, and place the tube into a hot-water bath for 10 min.

4. Centrifuge the extract at 3000·g for 10 min. By centrifuging it, all of the particulate plant

materials from the grinding get pelleted at the bottom of the tube, leaving a relatively clear

liquid (the supernatant) containing the water-soluble compounds of interest.

5. Filter the supernatant using a 40 μm filter disk in the filtration apparatus to make sure that no

plant particulates remain in the filtrate.

6. Freeze-dry/lyophilize the filtrate. It may require 2 to 12 h to lyophilize the filtered extract so

as to remove all moisture.

This yields a powdered residue that can then be used to separate and identify the kinds and amounts of

compounds present by means of high-performance liquid chromatography/mass spectrometry

(HPLC/MS) or other appropriate procedures. Some of these characterization procedures are described

later in this chapter as well as in Chapter 9.

Hot Water Extraction Protocol for Natural Product Screens

Weigh 0.5 g samples of plant tissue from tissue stored at –80°C in a deep freeze



Grind the sample in liquid N2 to a fine powder



Place tissue in 10 ml of hot (80°C) water for 10 min



Centrifuge at 3000 × g and take supernatant



Filter supernatant through a glass filter



Lyophilize the sample to dryness



2976_C008.fm Page 274 Wednesday, May 31, 2006 10:30 AM



274



Natural Products from Plants, Second Edition



FIGURE 8.4 Soxhlet extractor. (From AM Glassware Ltd. catalog.)



8.3.2.2



Laboratory Methods of Organic-Solvent Extraction of Compounds



If the compounds of interest are not soluble in water because of their non-polar nature (see Chapter 1),

select an organic solvent (e.g., acetone, methanol, ethanol, chloroform, diethyl ether, methylene chloride, or

a combination of more than one organic solvent) to carry out the extraction. The temperature of this extraction

depends on the boiling point of the solvent chosen and must be carefully watched due to the special equipment

that is used. One can use a Soxhlet extractor, which is basically a specialized glass refluxing unit that is

used for such organic-solvent extractions (Figure 8.4). In the following example (using a 1:1 mixture of

methylene chloride/methanol), the temperature must be maintained at 40°C for 8 h in order to obtain complete

extraction of each sample. If the temperature falls below this, extraction will be slow. If the temperature

goes above this, the risk of degrading the compounds of interest becomes great. With the presence of higher

temperatures, there is always the risk of degrading some of the active compounds; thus, low-boiling-point

solvents, such as dichloromethane or diethyl ether, are usually the best choice. The basic procedure is outlined

below. Preliminary cleanup procedures are usually necessary before samples are analyzed by HPLC or by

any of the other chromatographic techniques (see Section 8.3.7.1 and Figure 8.7).

The example we use here employs methylene chloride. It is highly toxic, and therefore, needs to be

used in a fume hood to avoid breathing the toxic fumes. It should also be handled carefully, using latex

gloves so that none of this solvent comes in contact with the skin. The extraction methods are as follows:

1. Weigh out a 0.5 g sample of plant tissue that was previously stored in deep freeze at –80°C.

2. Grind frozen plant tissues to a fine powder using small amounts of liquid nitrogen in a ceramic

mortar and grinding with a ceramic pestle.

3. Weigh out a 0.5 g sample powdered tissue in a cellulose thimble in a Soxhlet extractor (see

Figure 8.4) containing 100 ml of a 1:1 mixture of methylene chloride/methanol.

4. Reflux the sample for 8 h at 40°C using a condenser (with running cold water) attached to the

top of the Soxhlet. This condenser drops the temperature quickly, allowing the solvent to

condense on the sides of the glass and drop back into the cellulose thimble.

5. Allow the solvent to cool to room temperature, and filter with a 40 μm filter to remove any

particulate matter.



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