Ethnopharmacology:
An Interdisciplinary Science

Since the earliest times known to man, naturally occurring substances found in plants, animals, and minerals have been utilized as a source of medicine, and of these three, it has been plants that have made the largest contribution. Natural plant molecules "account for the active ingredients of 25% of all prescriptions dispensed in pharmacies in the U.S.A., and this does not include those synthetic compounds which were derived via a knowledge of natural product molecules (Phillipson and Anderson, 1989)". Indigenous people have provided leads for the discovery of many of these naturally derived drugs because most of them were first used in a traditional context as medicines or poisons (Etkin, 1993).

Ethnopharmacology (also termed ethnobotany, ethnopharmocognosy, ethnopharmacokinetics) is a relatively new interdisciplinary science. It has really only been twenty-five to thirty years that western medicine has truly been interested in ethnopharmacology as a science. Its goal is to obtain a broad perspective on the traditional use of crude drugs and poisons by combining efforts from a variety of different disciplines such as cultural anthropology, pharmacology, toxicology, chemistry, botany, and medicine (de Smet and Rivier, 1989). It makes sense to look at plants that have already been used for many, sometimes hundreds or thousands of years, by an indigenous group of people for medicinal purposes. If field research can be done that shows us what a specific plant’s known effects are, we can more accurately test its potency and screen for chemical activity.

So, which plants are important to look at? The search for new active compounds can be done through random screening of plants, or by screening plants that have been traditionally used by indigenous people to treat illnesses and diseases (this is different from random selection because we choose to study plants already thought to be medicinally active). The first of these methods, random selection and screening, is not the method of ethnopharmacology. Even so, random screening of plants is being conducted by some major companies that have the resources to "blindly" look for active compounds in plants. This requires so much money that very few research teams can do this for a substantial amount of time. One group that has had some success in a form of specialized random plant screening is the National Cancer Institute. It is a specialized random screening because NCI’s researchers have been testing solely for antitumor compounds in their randomly chosen plants. By keeping the screening specific, they cut back on time and cost. This is how Taxol, an antitumor drug, was discovered. So it would not be fair to say that random plant screening amounts to insignificant discoveries. The argument to be made by several studies, though, is that the "hit rate" for medicinally active compounds in plants is higher when plants are selected according to traditional use, rather than random screening (Canigueral, 1999).

Once a plant has been selected, the first step is the evaluation of its biological activity (Verpoorte, 1989). Either a general screening or a specific screening is done to guide in the isolation of an active compound. A general screening (not having specific properties in mind when screening) used to be the most common method used, but this requires relatively large quantities of plant extracts, skilled and experienced technicians, and expensive facilities. Through this method, a single plant extract can be screened for many different biological activities. Presently, screening for specific activities is more common; such as, antiviral and antimicrobial activity. Only a small amount of plant extracts or fractions are needed to conduct these specific screenings because the chemists have in mind what they are looking for.

One of the first problems for all plant screening methods is the choice of the extraction process. For accurate spectroscopic identification and full characterization of a new compound, purified natural products are required. Purified materials are also needed for pharmaceutical standards, testing, and patents. Synthesis of a natural compound is later done through researching the purified plant compound. Not much has changed in the processes of extraction methods. Although, there has been greater efficiency during the last ten years in laboratories that have received enough funds to have costly high pressure extraction devices. Many ethnopharmacologists, though, are suggesting that there is still a need for chemists to perform systematic studies in the field of extraction of plant material and fractionation so that this step can become even more efficient.

The method of extraction chosen decides what types of compounds will be present in the final extract. It is mainly the choice of solvents (also termed eluents) which determines whether the sample will be prepared correctly. In other words, various extraction methods pull different compounds out of the complicated chemical matrix (in this case, a plant). "Initial extraction with low-polarity solvents yields the more lipophylic (hydrophobic) components, while alcoholic solvents give a larger spectrum of apolar and polar material. If a more polar solvent is used for the first extraction step, subsequent solvent partition allows for a finer division into different polarity fractions (Hostettmann and Martson, 1998)." Also, the actual extraction procedure can be performed in various ways. Stirring, percolation (for example, soxhlet extraction), and pressurized solid-liquid extraction are utilized by chemists in ethnopharmacology labs. In pressurized solid-liquid extraction, the eluent is pumped through a column filled with plant material. This last method is a very efficient method, but the instrument of choice, the ASE 200 (Accelerated Solvent Extraction), is very expensive.

Usually, chemists desire to extract alkaloids, a secondary metabolite, but there are many other compounds that are plant derived and of medicinal interest; mostly other secondary metabolites. Secondary metabolites play a major role in the protection of plants from their environment, predators, and competition directed from other plants for limited growing space and nutrients. They are highly reactive substances in low doses. Alkaloids include literally thousands of nitrogen containing bases found throughout the plant kingdom. Usually, they have one or more rings of carbon atoms with a nitrogen atom in the ring. The position of the nitrogen atom in the carbon ring varies with different alkaloids and with different plant families. Sometimes even, the nitrogen atom is not in a carbon ring. They are bitter tasting and usually in the form of white solids (nicotine is an exception; brown liquid). Alkaloids are usually insoluble in water and soluble in alcohols. They combine with acids without the loss of a water molecule to form water-soluble salts. In plants, they may exist in the free state, as salts, or as N-oxides. Alkaloids produce striking physiological effects in low doses. These effects vary greatly depending on the specific alkaloid. For instance, some cause stimulation of the nervous system or paralysis, elevation or lowering of blood pressure, or pain relief. Others are tranquilizers or antibiotics. Some specific examples of alkaloids are morphine, strychnine, nicotine, quinine, ephedrine, and caffeine (these all end in —ine to indicate that they are amines). Please refer to Figure I for two examples of alkaloid structures.

Nicotine	Morphine
Figure I. Two examples of alkaloids and their structures (Solomons, 1997).

Once an interesting compound has been separated through chromatography and is isolated, its structure needs to be identified. In the past 40 years, natural product research has become more efficient in determining structures of unknown compounds.

Figure II. Structure of strychnine, an alkaloid respiratory stimulant (Bisset, 1996).

For example, strychnine (see Figure II), which is found in some arrow and dart poisons and has been used medicinally as a respiratory stimulant, was first isolated in 1819. Although, its correct structure was not known and documented until 1948 (129 years later!). Now there are numerous non-degradative spectral methods to help determine relatively quickly a compound’s structure (Bisset, 1996). Some of these methods are UV-, IR-, CD-, ORD-, ¹H-NMR-, and ¹³C-NMR-spectroscopy. Again, unlike the past, only 1-10mg of purified compound is sufficient for determining the structure of active compounds (Verpoorte, 1989). Mass-spectroscopy is the only degradative method, but like the others, a very small amount of the compound in question is sufficient for structure determination. These techniques not only help to determine the atoms and functional groups found in an unknown compound, they also unveil the absolute stereochemistry of the molecule. This is important in medicine because different enantiomers and isomers can create various reactions in the body. The wrong enantiomer could have adverse effects in humans. For example, (S)-penicillamine "is a highly potent therapeutic agent for primary chronic arthritis," but (R)-penicillamine has no therapeutic action, and it is highly toxic (Solomons, 1997).

In ethnopharmacology, ¹³C-NMR is the most popular of all the forms of spectroscopy mentioned above because it gives direct information about the carbon skeleton very efficiently. But there is still one method that is considered superior to all others for determining and confirming the structure of an isolated compound; X-ray crystallography (Verpoorte, 1989). The one problem with this method, is that the compound of interest must be able to crystallize, and not all compounds can in a suitable form. Even though X-ray crystallography is accurate, it usually is not the first method applied when determining a compound’s structure because of the cost, expertise needed, and time investment involved.

X-ray crystallography is able to give a very detailed picture of the arrangement of atoms and ions because the X-rays produced when high-speed electrons hit atoms, are approximately the same length as the distance between atoms. It is possible "to study the diffraction effects produced when X-rays pass through a crystal, and build up an image of the structure by calculation (Bunn, 1986)." Electron density maps are created because it is the electrons in atoms which are responsible for the diffraction of X-rays. Different types of atoms can be identified because they have different electron densities. A couple of these "contour maps" taken at different angles allow the creation of an accurate model of the compound’s structure through various mathematical calculations (Bunn, 1986).

Once a biologically active compound that is derived from a plant is isolated and purified, more specific laboratory tests can be done to understand its stability and help standardize the amounts needed to produce a desired medical effect. Most pharmaceutical companies will want to know how to alter the structures of these active compounds in order to increase their activity or decrease their toxicity. "Such molecules will then be candidates for strong patent positions that are necessary to recoup the costs of development (Farnsworth, 1994)." Also, companies do not intend to rely on the plants as their source for the medicines; complete synthesis is the ultimate goal.

It can be admitted that the number of useful new medicines that are discovered through ethnopharmacology is small when compared to the amount of new drugs that are created synthetically. Pharmaceutical companies seem to be more interested in synthetic compounds than natural ones because they are easily patented in comparison. If more finances were directed to ethnopharmacological research, and patents were more readily attainable on natural compounds, then perhaps we would see a rise in the amount of plant derived medicines (de Smet, 1989). Scientists need to be reminded that the search for new compounds is only a small part of ethnopharmacology. Chemists are needed and are becoming more involved in isolating, purifying, and synthesizing medicinally active compounds so that these plant derived drugs may be patentable and marketable (Prance, 1984).

An exciting part about ethnopharmacology is the multidisciplinary collaboration of scientists from around the globe to promote this relatively new science. This vast and ever growing network is designed to coordinate and pull together ethnopharmological research efforts. There have been several journals dedicated solely to ethnopharmacology and ethnobotany now for almost two decades. These began as small efforts to educate people, but now have grown large and are helping to connect interested scientists from all fields. In the early 1990’s, the formation of the International Society for Ethnopharmacology reflected a growing interest in the area of natural product medicine (Houghton, Soejarto, Verpoorte, Watanabe, 1986), (de Smet and Rivier, 1989). If ethnopharmacology continues to grow and gain the respect of scientists from all fields, we could witness large medical breakthroughs in the next few decades that could change people’s lives forever.