Journal of EthnopkarmacoIogy, 8 (1983) 127-147 127

Elsevier Scientific Publishers Ireland Ltd.

Review Paper
-
THE PHARMACOLOGICAL EVALUATION OF NATURAL PRODUCTS -.
GENEBAL AND SPECIFIC APPROACHBS TO SCREENING
ETHNOPHARMACEUTICALS*

MARVIN H. MALONE

Pkys~ology-Ph~rnaco~o~ Unit, School of Pharm~y, Uniue~jty of the Pacific, Stockton,

CA 95207 (U.S.A.)
(Accepted November 29‘1982)

Summary

Natural product research is most efficiently conducted in a systematic

fashfon concentrating on ten critical questions which must be answered in
sequential fashion by volubly trained professionals experienced in such
research. The minimum ethnopharmacology research team should consist
of a botanist, a chemist and a pharmacologist with each carrying the respon-
sibility for answering certain of the critical questions. The screening pro-
cedures used for the ev~uation of synthetic medieinals generally are not
appropriate for guiding the extraction of active principles from crude drugs
and establishing extraction efficiency; therefore, specialized techniques
unique to ethnopharmacology must be used.

Introduction

Although this author was introduced to natural product research some

27 years ago by Drs. O.P. Wintersteiner, B. Rubin, P.A. Diassi and F.L.
Weisenbom of the Squibb Institute for Medical Research, and although he
has done a wide variety of other pharmacological research in addition to
ethnopharmacology since that time, he still believes that natural product
research has the best chance fol: discovering new prototype drugs that will
be useful in the clinic.

*This paper was prepared for the VII Symposium on Pharmacognosy held October 10,
1980 at the Vrije Universiteit Brusael and is reprinted here, with permission, from
Ondenoek naar Biolo~che Aktiviteit vun ~a~u~ioffen: Proceedings wn het VII
Symposium voor ~a~~o~osie, C. van Violon, V. Maes and A. Vercruysse (Eds.), Vrije
Universiteit Brussel, Brussels, Belgium (1981). pp. 3-28.

0378-8741/83/$06X0 o 1983 Elsevier Scientific Publishers Ireland Ltd.

Published and Printed in Ireland
128

A prototype drug is defined here as one that has a wholly different chemi-
Cal StrtiCture from existing agents and wholly different medical applications.
Every time such a drug is discovered, there will be major changes in the
practice of medicine; consider the impact of curare on the art of surgery, the
impact of penicillin G on mortality from infectious disease, the impact of
reserpine on the treatment of mental disease. These three drugs alone have
dramatically altered our life expectancy and the quality of life on earth. In
addition, plant principles do not even need to be drugs to be important;
consider the impact of the plant toxicons, muscarine and nicotine, on the
development of theoretical physiology and pharmacology.
The odds for the discovery of a new prototype drug in a natural product
program based on sound ethnopharmacologic principles are significantly
better than the odds for the discovery of a new prototype drug in an all-
synthetic program (see Conclusions). However, in the United States pharma-
cological natural product research (especially in regard to the higher plants)
is now at an all time low (Farnsworth and Bingel, 1977). Why is this so, since
only a fraction of the world’s plants has been studied?
There are significant differences in procedure and philosophy between
natural product research and research dealing with synthetic medicinals.
At the present time, there are at least 14 books available which deal with the
problems associated with the pharmacological evaluation of synthetic drugs,
but there are no texts dealing with the fundamentals of natural product
pharmacology. Recently a European publisher expressed interest in the
production of a book on the methods of natural product pharmacology;
this undoubtedly reflects the increased interest of European pharmacologists
in natural product research.
To illustrate the differences betwen the pharmacological evaluation of a
natural product and the evaluation of a synthetic medicinal, one can consult
Fig. 1, which lists the first eight steps in a classic ethnopharmacologic study.
For a synthetic program, Stages 7 and 8 merge to become Stage I and
Stage 6 becomes Stage II. The other stages are missing.
In the United States, in industry, in academia, and in governmental
research, there have been several instances of large and expensive drug plant
projects being assigned to individuals who were reputable and capable
chemists or pharmacologists but who had never worked with natural pro-
ducts before. These projects invariably failed and usually for embarrassingly
simple reasons. Since the projects that this author is thinking of were widely
publicized when they were started, their failures were also widely discussed
by chemists and pharmacologists. This has led to a rather widely held belief
in the United States that good natural product research is impossible to do
and that natural product research should be avoided for the sake of one’s
professional reputation. This author has never found a laboratory animal
capable of lying; therefore the failure of a research project must always
rest on the one who designed and carried out the experiments or on the
one who organized the project. Certainly the plants cannot be blamed.
 129

The moral here is a simple truth that applies to all scientists and all
research projects - never assume responsibility for a research project or part
of a research project unless you know what you are doing. Working with
someone who is experienced is the best way to learn especially if there are
no textbooks available. Natural product research is more complicated than
projects working with synthetic medicinals but not necessarily more expen-
sive. A simple technical error on the part of one person can ruin a natural
product research project while an equivalent error in a synthetic program
will create only minor delays and expenses.

Common problems in natural product research

If everyone on an ethnopharmacologic research project is reasonably well

schooled in the classic techniques and principles of botany, chemistry and
pharmacology, what are the common mistakes made in natural product
research? This author will now attempt to define these problems and
indicate how they may be avoided. The topics to be discussed may seem
too simple to warrant mention; however, readers must realize that the
author is being very realistic - it is nearly always a simple technical error
or a simple oversight that rums a natural product research project. Generally,
these problems and oversights reflect a lack of practical experience on the
part of one of the research team.
The research projects that go easiest to completion are those where every-
one on the project is a chemist - or where everyone is a pharmacologist -
or where everyone is a botanist. In such instances, everyone has a common
educational background and a common technical language. Beyond some
personal rivalry, it is easy to work together.
This happy situation is impossible in natural product research because a
multidisciplinary team is necessary if the project is to be both efficient
and successful. The minimum natural product team consists of three very
different professionals: a botanist, a chemist and a pharmacologist. The
training for each of these professionals is different, their technical vocabu-
laries are different, and their responsibilities to the project are very differ-
ent. Because of these differences, professional jealousy can develop unless
individual responsibilities to the project are defined clearly and agreed upon
mutually before the project is launched.
If one individual of the natural product research team does not do his/
her job, then the work of the others becomes worthless. Therefore, every
project must have a project leader who has ultimate responsibility for
getting the research done correctly on schedule and in a co&effective
manner. Logically, there are two professionals who could function as the
project leader: (i) a natural product pharmacologist, since the activity of
the plant principles determines in large part the degree of involvement of
the other two professionals; or (ii) a natural product chemist or chemically-
oriented pharmacognosist, since this individual tends to be a middleman in
 130

Estab/Is& te&atfve IdeatiKhtka

Ethnobotonist Field Botanist * Decision # 2 :
Genus? S@eciest

Iliad Scfeea tii Whle Matefial Actfv,y

Field Botanist Pharmacologist* Decision# 3:
Dose -reqmmse oclivi&
of /g/Kg or less ?
Absokte ?Isnt kdsrtn/crikor
Field Botanist .Decision #4 :
4fee 0~) genus and
species ?

Quaafftative Extfactfve/.iaf c Assays

C/rssk Hafmae&gkal Evaluat&a

Decision # 6 :
Chemist Pharmacologist*
Unique activity ?

Classk Structure Detefmfuthu

Structure * Decision # 7:
Chemist
Chemist Uflique nw/ecde ?

Synthesis aad Stfmtufe Akodnrkcrt/av

Fig. 1. Illustration of a functional scheme for ethnopharmacological research based on a

sequential answering of eight critical questions. An asterisk (*) indicates the research
professional respohsible for answering the critical question posed for each stage of
research. Arrows indicate the routes of interaction between researchers.
 131

Stages 5 and 6 (Fig. 1) between the botanist and the pharmacologist and
(if the project is successful) also the middleman in Stages 6-8 between the
pharmacologist and the structure-determination and synthesis chemists,
Although the author is a pharmacologist, he believes that the natural
product chemist is the most effective project leader. This attitude un-
doubtedly goes back to his early training at the Squibb Institute for Medical
Research, where Dr. O.P. Wintersteiner was an inspirational example. Later
work with Drs. V.E. Tyler, L.R. Brady and A.E. Schwarting has been equally
rewarding.
While the project leader keeps track of each plant being tested and co-
ordinates the research, he does not carry responsibility for some of the most
critical decisions. It is easy to predict failure for a project where the botanist,
the chemist, and the pharmacologist vie for leadership and work indepen-
dently from one another, but it is equally important to realize that many
projects have failed because there was a single project leader who assumed
an authoritarian role over the other two professionals. It is unrealistic for
the project leader to make all the critical decisions necessary for good
natural product research. Figure 1 illustrates the first eight stages of research
in the average ethnopharmacologic project set up to discover new prototype
medicinals. Natural product research is best conducted in a systematic
fashion concentrating on certain critical questions which should be answered
in a sequential fashion by appropriately trained and experienced profes-
sionals. In the scheme presented here, the validity of the decisions at each
stage depend upon the validity of the decisions made at each preceding stage.
One must not try to do two or three or all stages simultaneously. Each stage
has a designated decision-maker who reports to the project leader who
co-ordinates the sequential efforts of all individuals. The present writer has
always found that those who will make decisions at each stage respond
magnificently to this responsibility once they clearly know,tIiat it is their
responsibility (not that of the project leader) and that others are depending
on them. In this scheme of research, there are no professionals working as
paid technicians - all carry some decision-making responsibility for the
project.

Stage 1: The ethnobotanist as a decision-maker

Medicine men simply do not give up their professional secrets on

request - especially to foreigners. Consequently a good ebhnobotanist
is a person native to the area being studied and who has some sound
botanical training. He should like the local people, like conversation, and
respect plants (roughly in that order of importance). If not already a
medicine man in his own right, he should study up before he makes any
contacts so that he knows certain of the medicinal plants of the area by
sight and knows how they can be prepared for medicinal use and what kinds
of disease they can be used to treat. This allows him to “shop-talk” with a
132

practicing medicine man on an almost equal “professional” footing. Stage 1

must be conducted in a relaxed manner without deadlines.
It is the duty of a capable ethnobotanist never to believe in the usefulness
of a recommended plant until he can get an independent, unprompted con-
firmation from at least one other medicine man. The two medicine men
should not be friends or from the same area. The validity of two similar
claims for a single plant increases directly proportional to the distance
between the areas of operation of the two medicine men consulted. Possible
plants for investigation can be gleaned from library screening of folk
medicine texts, and this is much cheaper than carefully done Stage 1
research. However, many of the attributions found in such books are based
on single reports by untrained visitors to an area. Rarely have the following
procedures been done for the cited plants.
The capable ethnobotanist must always visit the areas where the plants
are growing and collect voucher specimens. Interviews with medicine men
should be taped if at all possible. A handwritten day-book should be main-
tained by the ethnobotanist which summarizes the important information
collected each day. Day-books are easily lost so carbon copies should be
made and carried separately from the day-book itself. The day-book must
contain the medicine man’s name, his practice name (frequently different),
the plant’s local names, plant part used, what used for, how prepared,
dosage and route of administration, how frequently taken, and other plant
drugs taken simultaneously. Very frequently, it turns out that one of these
“other plant drugs” is the truly active plant. When collection is accomplished,
the day-book should show where the plant was located (latitude, longitude,
altitude), voucher specimen number, and observations about the soil and
growing climate.

Stage 2: The field botanist as a decision-maker

A field botanist is one who is a fully trained professional interested in the

flora of a particular area. A capability in chemistry, pharmacology and/or
medicine is not necessary. If the field botanist is a native of the area being
studied, it is possible that he can double as the ethnobotanist in Stage 1;
however, this ideal situation is very very rare.
The field botanist and the ethnobotanist return together to the area where
the interesting plants are located. Agreement is made as to the exact plant to
be acquired and a very careful collection is made. Only one species is placed
in a collection sack. Once two different plants are mixed (even for a short
time), it is difficult and sometimes impossible to separate them. A mold- and
insect-resistant card is placed in the collection sack containing: local plant
name, presumed genus and species, date of collection, area of collection
(latitude, longitude, altitude, local landmarks), relevant field notes,
collector’s name, collection number, plus volume and page of the field
botanist’s day-book recording the collection. The sack is securely tied with
 133

mold- and insect-resistant cord and a card attached indicating collection

number, collector’s name, plus the volume and page of the day-book
documenting the acquisition. The day-book, in this instance, is maintained
by the field botanist and again carbons are made, removed each day, and
carried separately from the day-book.
In making collections, it is a general rule that if one finds one plant of a
species in an area, there are usually more of that species close by and fre-
quently in abundance. This aids collection certainly. However, there are two
cautions and these cautions are the reasons for the field botanist’s presence
in Stage 2.
If one species grows abundantly in a certain locale, then closely related
species frequently will also find that area desirable for growth. This means
that closely related species can grow in very close proximity and they can
look very similar to an untrained eye. Stage 2 collections must be made by
the field botanist or directly supervised by him. The amount collected is
relatively small (l-2 kg) since many plants are usually collected on one trip
for efficiency, and portability can quickly become a problem. Less than
100 g will be needed to complete Stage 3 screening, but it is always desirable
to hold the remainder as a reference reserve. A definitive voucher specimen
is collected by the field botanist for each plant at the time and place of
sample acquisition.
No matter how abundant or poor the collection site, the field botanist
must be sure that no more than half of the plants in a single area are
harvested. If a l-kg sample will require harvesting all the plants, none of
the plants should be collected. The site should be noted and the search
continued for a more abundant collection site. This action guarantees that
plants can be collected for Stage 5 research from the Stage 2 collection site.

Stage 3: The pharmacologist as a decision-maker

Frequently, newcomers to natural product pharmacology will decide to

skip the general screening of Stage 3 since the specific testing of Stage 6
appears to them to accomplish the same thing. Historically, the elimination
of Stage 3 testing has wasted the time of many chemists and has led to more
pharmacological mistakes than any other item this author cbuld mention.
Stage 3 is set up for four important reasons.
(i) To detect and define both rapid-onset and delayed-onset physiological
activity in an unbiased manner through the use of blind testing of the
plant under a code name.
(ii) To set up an efficient assay system that can be used to guide the chemist
as he sets up extraction schemes for isolation of the active ingredient (or
ingredients) in Stage 5.
(C)To predict directions for specific pharmacologic testing in Stage 6.
(iv) To predict the toxic potential of the active ingredient(s), so that Stage 6
research does not ignore this very important aspect.
134

When it is time for Stage 3 research to begin, everyone concerned with the
project has some sort of preconceived idea about what the plant can or
cannot do. Medicine men are frequently wrong about how their plant drugs
work. Moreover, they are not always good diagnosticians of disease. As
mentioned earlier, a “minor” drug plant used to make a mixed-drug decoc-
tion frequently carries all the useful activity. It will be useful to cite here an
illustration that will offend no living researcher.
The midwives of England knew absolutely that digitalis was a diuretic and
good for dropsy. They also knew quite well (but did not admit publically)
that foxglove did not work for all dropsy patients. It took the unbiased
scientific approach of William Withering (1785) and John Ferriar (1799) to
show that digitalis primarily affects the heart and is a good “diuretic” only
when fluid retention is secondary to cardiac decompensation.
The blind general screening of Stage 3 clearly will indicate that both
Digit& hnata and purpurea are not diuretics and that both of these plants
have qualitatively similar, rather selective cardioactive principles and that
Stage 6 testing should focus primarily upon cardiac pharmacology and toxi-
cology. On the other hand, if one were to believe the English midwives and
skip Stage 3 testing and set up a number of specific diuretic experiments,
one would collect a large amount of data indicating that digitalis is without
merit as a diuretic, generally uninteresting, and rather toxic. Further work
on the plant principles would undoubtedly be stopped.
It is useful to remind readers at this point that the goal of ethnopharma-
cologic research is to find new prototype drugs - molecules with new
mechanisms of action that will change the course of therapeutics. If we
search for only known types of activity (Stage 6 testing), we not only stand
a good chance of missing activities that are different, but we may also
actually reject unique activity because it is so different. The latter is all too
frequently the case - the author can cite the example of Rauwolfiu serpen-
tina, where the plant principles had been isolated and investigated in the
United States before the Swiss pharmacologists became interested in the
plant’s reputation. Reports of Indian clinicians and native doctors for anti-
psychotic activity were discounted in the U.S.A. and the research was
shelved because the pharmacologic activity of the reserpine-like molecules
was so different. In fact, there was more therapeutic interest in Rauwolfia’s
yohimbine-like molecules - presumably because they represented the more
familiar cr-adrenolytic profile of pharmacologic activity.
It is not the purpose of this manuscript to detail the simple observational
technique (hippocratic screening) used to conduct Stage 3 research (Malone
and Robichaud, 1962). Those unfamiliar with the technique will be surprised
to learn that only crude plant material is used. Extractives are not tested for
one major reason -by definition, extracts exclude certain materials found in
the whole plant material. No individual at this stage knows what chemical
type will account for the activity.
Plant material as specified by the medicine men (leaves, bark, roots, whole
 135

plant, etc.) is dried, chopped and run through a hammer mill. That product
is then placed in a ball mill until all materials will pass a 200-mesh sieve. The
resulting powder is about the fineness and consistency of a high-quality face
powder. It has been shown that such finely divided powders (suspended by
trituration in an inert dosing vehicle, i.e. sterile 0.25% agar solution in
doubledistilled water) can be injected intraperitoneally into rats and the
active principles will mobilize from the peritoneal cavity into the blood
almost as fast as if they had been injected in a soluble form (Malone et al.,
1961; Malone and Robichaud, 1962; Malone et al., 1962,1967). Only rats
can be used for hippocratic screening of crude natural products because they
effectively resist both infection and peritonitis.
The manner of drying the plant material should follow that used by the
medicine men - sundried or shade-dried can make differences in biological
activity. If the medicine man advocates the use of fresh plant material, then
fresh material is obtained and quickly freeze-dried. In all cases, what is
injected is a 200-mesh powder in 0.25% agar.
Per plant sample, 5-15 unanesthetized intact rats are used for the hippo-
cratic evaluation. Intact animals are used to parallel the clinical situation
since plant principles can affect any or all organ systems. This approach is
very realistic since the natural homeostatic systems of the intact unanesthe-
tized body can counteract the effects of agents that look very promising in
in vitro screens.
A log-dose series of intraperitoneal injections are made ranging from
1 g/kg downwards - the goal being to document the effects of one lethal
dose, one ineffective or near-ineffective dose, and 3-4 equally spaced log-
doses between those two. The presence or absence of 63 symptoms are
recorded using a standardized record sheet (see Fig. 2) with observations
required at + 5,10,15,30 and 60 min, + 2,4,6 and 24 h, and at + 2,4 and
7 days. It is just as important for the evaluation to record the lack of drug-
induced symptoms as it is to record the presence of symptoms. Adding up
positive scores at the various time intervals will allow one to plot sigmoid
dose-response curves for a single symptom (Malone et al., 1961). If one is
interested in a more complex analysis, one need not add up the scores
(as just specified) but use a computer to get a three-dimensional dose-
response plot of effect versus log-dose versus time. However, if a computer
is available, one might as well strive for a matrix analysis of all 63 symptoms
at once (effect vs. log-dose vs. time). The result is a unique pharmacologic
“fingerprint” for each drug class; within each drug class, individual drugs
vary only in potency. Such “fingerprint” analyses are useful in telling the
investigator without bias whether or not a new prototype drug has been
discovered (Malone and Carrano, 1970; Malone, 1977).
Virtually all known drug types can be detected as active by the hippo-
cratic screen conducted in rats with the exception of the various chemo-
therapeutic drugs (e.g. the antibiotics). Even some of these agents can be
detected because of their dramatic toxic effects.
136

Date. Notebook No._pp._

Hippocratic Quolitotive/Semiquontitative Screen ond Toxicity Report of:

Lot/ Identification No.

Dosage : mg/ kg; Dosage Vehicle: ;Conc. : __mg /ml

Fig. 2. The standardized hippocratic work-sheet used to record drug-induced symptoms

of unanesthetized rats. To be valid, each blank on the sheet must be filled in with a
numerical measurement, a rating, or requested data. The blank reverse of this sheet is
(continued opposite)
 137

Project Code: ;Reoder Signature :

Test Animol: ; Fasted/ Nonfasted : ; Sex: -I .Weight:_g

Mark: -;Dye Color:-;Cage No. ; Special Treatment:

Injection Volume:.-ml; Injection Route: ;Clock Time : --
Response Roting (O-+4) or Measurenwnt
PARAMETERS Con- +5 +I0 +lS’+3ol+60 + 2 + + +24+48+ +7
trol min. min. min. min. min. hrs hrs. hrr. hrs. hrs. daysdovs *
Chromodacryorrhea
i

used to record all other observations (each associated with the onset and termination
times for the specified symptom) plus the necropsy notes (taken at the time of drug-
induced death or at the time of killing at + 7 days). For details and samples of filled-
out sheets, see Malone and Robichaud (1962), Malone and Carrano (1970) and Malone
(1977).
138

Since all samples are coded and tested in a blind fashion, the technicians
operating the screen are kept honest and interested by the supervisor
introducing coded known drugs at random intervals (e.g. the addition of a
cardiac glycoside to a known inert plant drug). While this practice keeps the
technicians conscientious, it also allows one to build up a library of data
regarding known drugs with known mechanisms of action and known mecha-
nisms of toxicity. Using this library of known drug effects as a reference, a
competent pharmacologist can apply his knowledge of physiology and phar-
macology and predict (without the aid of a computer) the organ systems
being affected by an active plant drug being tested. If the screening program
is large, routine library matching can be done by a computer (Malone and
Carrano, 1970; Malone, 1977); however, the final decision as to whether
interesting dose-response activity exists must always be made by a pharma-
cologist.
If clearly defined dose-response effects are not seen at doses less than
1 g/kg, the plant should not be considered worthy of future study. In
practical terms, this means that the plant principles have such a low potency
or occur in such low concentrations that the plant cannot be regarded as a
commercially viable source. However, if results with the powdered drug are
equivocal and if the ethnic uses are especially interesting, one should
consider testing a 70% ethanol extract of the powdered plant material. All
the alcohol and water are removed, the gravimetric yield of extract noted,
and the residue is suspended in 0.25% agar and tested as if it were whole
plant material. It is the author’s experience that a 70% ethanol extract
appears to carry with it all plant principles with drug-favorable Meyer-
Overton ratios.

Stage 4. The classic botanist as a decision-maker

If the coded plant material has sufficient potency to clear the dose-
response requirements of Stage 3 and if its profile of induced symptoms
predicts interesting activity, it is then worthwhile to confirm the identity of
the plant in an unbiased fashion before proceeding with further research.
In several instances where this was not done and commercial crude drug
suppliers (e.g. Meer Corporation, S.B. Penick & Company) were contracted
to supply the large amounts needed for Stages 5 and 6, a different plant was
correctly delivered. Such a situation creates considerable confusion and a
crisis in confidence.
The herbarium specimen collected by the ethnobotanist in Stage 1 and the
definitive specimen collected by the field botanist in Stage 2 and any other
requested specimens are presented to an expert classic botanist who has
hitherto not participated in the project. Like Stage 3, this determination is
conducted in a blind fashion with all specimens delivered with code identifi-
cation numbers. If there is disagreement as to identity (a common occur-
rence when dealing with obscure species from remote areas and especially
 139

when dealing with the higher fungi), the field botanist and the hired expert
botanist must get together to key out the specimens and seek agreement.
The hired botanist, who has been hired and paid as an authority, should have
the final decision.

Stage 5. The chemist as a decision-maker

Once identity of the plant has been resolved to the satisfaction of the
project leader, it is wise to secure a relatively large amount of authentic plant
material since it is desirable to handle Stages 5-7 from a single batch. The
amount needed depends upon the potency estimates made in Stage 3.
The goal of Stage 5 is to use bioassay to guide the chemist in isolating out
the active principles. If done without bioassays to monitor the process,
the isolation becomes a hit-or-miss process where the activity can pro-
gressively vanish because of inappropriate or inefficient extraction
schemes or because the extraction procedures result in degradation of
the active chemicals.
The pharmacologist selects as bioassay parameters one or more of the
63 symptoms used for hippocratic screening in Stage 3. More animals per
dose are added to produce statistically valid dose-response assays. If
another animal will respond equally well or better, one is not restricted
to the use of rats and the intraperitoneal route of injection. Parameters
that the author has used to create monitor assays have been the following,
among others: (i) blepharoptosis or the downward displacement of the
eyelid (Rubin et al., 1957); (ii) salivation (Malone et al., 1961); (iii)
lacrimation and chromodacrorrhea (Malone et al., 1962); (iv) blood coagula-
tion time (sampling by cardiac puncture is lethal while sampling from the
optic sinus will not harm either the mouse or rat) (Khanna et al., 1965);
(v) blood glucose (Kocialski et al,, 1972); (vi) relative exophthalmos/
enophthalmos (Chilton et al., 1973); (vii) blood pressure (plethysmographi-
tally using rat tail or by direct puncture of the carotid artery) (Rubin et al.,
1957); (viii) the number of blood vessels that can be counted in the ear
(a good measure of peripheral constriction or vasodilation); (ix) pupil size
(swiftly and very accurately measured using a matching-dot method under
a constant light); (x) rectal temperature (conducted in constant-temperature
cold or hot rooms); (xi) the healing rate of open full-skin-thickness wounds
(Morton and Malone, 1972). Certain of the parameters just listed are not
used in hippocratic screening because they take too much time to do con-
sidering the time constraints or because they harm or excite the test
animal. Nevertheless all represent physiological measurements that will
change in a dose-response fashion when certain drugs are administered.
Even if the parameter selected for the assay represents the side or toxic
effects of the plant principle being isolated, such side/toxic effects are
still real measures of a molecule’s intrinsic activity.
Several extraction schemes should be attempted, not merely those well
140

established for alkaloids, organic acids, glycosides, etc. The important thing
is that for each extraction step, the yields for both the extractive and the
mart (extraction residue) should be calculated. If this is done conscienti-
ously, then one can account for 100% of the activity and note the effici-
ency of each extraction step. Some of the classic extraction schemes are
not very efficient; each milligram that ends up discarded in a mart contri-
butes to this inefficiency. Historically, chemists and pharmacologists have
always focused their attention on the extractive and automatically dumped
the marts which were presumed to be inactive. If one follows the procedures
described here, mams (reduced to a 200-mesh powder and suspended in
0.25% agar) can also be assayed.
Assays can be conducted in two ways: (i) in a rather traditional manner
where the extracts and marts are assayed relative to the starting material
which acts as the primary reference standard of activity; or (ii) where the
starting material initially is assayed relative to a secondary standard (pref-
erably a pure chemical) and then all extracts and marts are assayed relative
to this secondary standard. An example of the first instance is illustrated in
Table 1 where 70% ethanol has been used to extract muscarine-like princi-
ples from Inocybe nupipes Lange (starting materials, extract and mart
courtesy of the Drug Plant Laboratory, University of Washington, Seattle,
WA 98195, U.S.A.). Although the mart in this illustration still possesses
measurable activity, this one-step primary extraction has been remarkably
efficient. However, if several extraction schemes are to be attempted and if
each consists of multiple steps, this approach can consume significant
amounts of the precious starting material in a rather wasteful fashion. Conse-
quently, the present author recommends the use of a secondary standard,
provided a commercially available chemical can be found which will produce
the symptoms being used as the assay parameter. This secondary standard
need not be a constituent of the plant material being studied (e.g. metha-
choline chloride has been used as a secondary standard for the assay method
cited in Table l), but the slope of its dose-response curve must parallel that
seen with the starting material.
Only rarely does one find quantitative reports in the literature of instances
where secondary standards have been used to check the efficiency of isola-
tion procedures (Malone et al., 1961). Table 2 summarizes some unpublished
data (B. Rubin, Squibb Institute for Medical Research, 1952) relating to the
efficiency of certain extractions from Rauwolfiu serpentina Benth. This
example may not be the best illustration to use here since the secondary
standard used (reserpine) is found in the starting material; however, the
situation presented is a fairly complex one; other reserpine-like molecules
are present (e.g. rescinnamine) in significant amounts and a number of
yohimbine-like molecules are also present which lack intrinsic ptotic activity
but which potentiate the ptotic activity of the reserpine-like principles
(Malone and Roth, 1962). In Table 2, the efficiencies of two different
 141

TABLE 1

CALCULATED RECOVERY OF MUSCARINE-LIKE ACTIVITY IN THE EXTRAC-

TION OF INOCYBE NAPIPES USING THE RAT SALIVATION ASSAY TO MONITOR
EXTRACTION EFFICIENCY

Identification Dry yield Lacrimation Percent of Tally of

from carpo- potency carpophore activity
phore (X) (95% C.L.) total activity (%)

Inocybe napipes
reference batch 1 100.0 1.00 100.0 100

19W61200A, 70%
ethanol extractive 40.2* 2.39* 96.1
(1.60-3.58)
97
19W61-200B, mart 593* 0.0114* 0.7
(0.0086-0.0150)

* Values followed by an asterisk are experimentally obtained figures. All others are
calculated in the following manner: percent of carpophore total activity = lacrimation
potency x percent dry yield; tally of activity = percent activity of extract + percent
activity of mart. Assay method is that of Malone et al. (1961) with test materials
administered intraperitoneally in a vehicle of 0.25% agar. Only 5 rats per dose; each
assay N = 30; assay lambda (s/b) = 0.23 (extract) and 0.22 (mart).

is faced with a rather non-exciting task - the routine production of large

amounts of the pure plant principle by extraction so that Stage 6 can
the acetic acid extraction (78% of total activity) although somewhat more
expensive in terms of the solvents used. In addition, the alkaline ethylene
dichloride extractive has much less bulk than the acetic acid extractive,
and this predicts that fewer steps will be needed to isolate the active
principles. With 21% of the total ptotic activity present, it would be
wasteful to discard the acetic acid mart. The entire extraction scheme
cannot be presented here, but DII-75E and DII-75F represent the extractive
and mart respectively, from one of the steps following the alkaline ethylene
dichloride extraction. Note that this step apparently is not an efficient one
in that 22% of the activity can be found in the supposedly inert mart. What
has happened at this stage of the extraction needs to be debated by both the
chemist and the pharmacologist, since the splitting of activity may be useful
to the extraction scheme.
Eventually pure crystalline materials are isolated and this achievement
allows the chemist to answer a very important question from the physical
data -has a new plant principle been isolated? If a unique active molecule
has been discovered, the chemist is always eager to get on with Stage 7 and
Stage 8 research and this is a very natural reaction. Logically, however, he
142

TABLE 2

CALCULATED RECOVERY OF RESERPINE-LIKE ACTIVITY IN THE EXTRACTION

OF RA UWOLFIA SERPENTZNA USING THE MOUSE BLEPHAROPTOSIS BIOASSAY
TO MONITOR EXTRACTION EFFICIENCY

Identification Dry Blepharoptotic potency Percent Tally of

yield of whole activity
from Reserpine = Whole root (%)
root 1.0 root = 1.0 total
(%) activity

Rauwolfia serpentina 100.00 0.00104* 1.00 100.00 100

reference batch 3

DII-66A, alkaline 2.76* 0.0402* 38.6 106.5

ethylene dichlor-
ide extractive <113
DII-66B, mart 97.24* <0.00007* <0.0673 ~6.5

DII-75E, fat-free 0.73* 0.126* 121.2 88.5

neutral subfraction 110
DII-‘75F, mart 1.25* 0.0182* 17.5 21.9

DII-IOSA, acetic 23.35* 0.00345* 3.32 77.5

acid extractive 99
DII-102B, mart 76.65* 0.00029* 0.279 21.4

* Values followed by an asterisk are experimentally obtained figures. All others are
calculated in the following manner: potency relative to whole root = potency of
sample relative to reserpine/potency of whole root relative to reserpine (e.g. for
DII-66A: 0.0402/0.00104 = 38.6); percent of whole root total activity = potency
relative to whole root x percent dry yield (e.g. 38.6 x 2.76 = 106.5); tally of
activity = percent activity of extract + percent activity of mart (e.g. 106.5 t 6.5 =
113). All test materials were administered orally in a vehicle of 0.25% agar and ptosis
was read 5 h after dosage using the method of Rubin et al. (1957).

primary extraction methods are compared. Of these, the alkaline ethylene

dichloride extraction is much more efficient (106% of total activity) than
proceed efficiently. Only if the pharmacological activity is unique (Stage 6),
can the costs of Stages ‘7 and 8 be justified. Historically, this principle has
been demonstrated many times.

Stage 6. Classic pharmacological evaluation

Perhaps the major problem encountered by beginners in ethnopharma-

cology arises from the injection of imperfectly soluble extracts or solvent-
solubilised materials into what should be a purely aqueous, isotonic
pharmacologic system (e.g. muscle bath studies, perfusion experiments,
intravenous injections into animals, etc.). Results obtained with insoluble
 143

materials or in the presence of organic solvents and/or ionic shifts can be

erratic and unreliable. Using the research scheme presented here (Fig. l),
pure plant principles have beeri isolated by the time that Stage 6 research
begins. This means that water-soluble forms of the plant principle can be
prepared in an intelligent fashion. In addition, molecular weight will have
been determined by the time Stage 6 research starts and this knowledge
allows more meaningful drug-receptor studies to be conducted.
Before starting specific Stage 6 testing based on the premises made in
Stage 3 and reinforced by hippocratic screening of the pure plant
principle itself in rats, this author believes that one more standardized,
multidimensional screen should be performed; one conducted in a non-
rodent species and one designed to cover certain aspects that could not
be documented using unanesthetized rats. The screen proposed by the
author for this purpose has been published in detail (Morton and Malone,
1967; Malone and Carrano, 1970; Malone, 1977) and it is not the purpose
of the present article to discuss that procedure. Since the screen involves
physiologically significant drugdrug interactions (test compound vs.
epinephrine, acetylcholine and histamine in addition to its effects on the
response to dual carotid occlusion), it has been termed a pharmaco-
dynamic screen. Economically conducted in one ethyl carbamate-
anesthetized intact dog, the results allow one to plan a specific program
for Stage 6 research with some confidence.
After hippocratic and pharmacodynamic screening of the pure plant
principle, one can usually focus subsequent research into one (or at most,
two) of the 11 inter-related specialty areas listed in Fig. 3. Pharmacological
research literally never stops in regard to prototype drugs, e.g. valid research
papers are still being written as to the mechanisms of action of the digitalis
glycosides. However, the goal of Stage 6 is not to establish the mechanism(s)
of action of the test compound in a definitive fashion, instead one works
in the classic manner to prove that the newly isolated principle is either
pharmacologically and toxicologically similar to existing drugs or qualita-
tively unique. This limited answer can usually be obtained in a relatively
short period of time, e.g. the six basic experiments that this author
believes are necessary to define the uniqueness of a drug affecting nerve
or skeletal muscle are noted in Fig. 3. Other experiments may be necessary
to establish uniqueness, but to omit one of the basic six experiments in this
instance would be most unwise. In the process of conducting such basic
experiments in classical pharmacology, one automatically acquires much
information that will allow him to propose pharmacologic and toxicologic
mechanisms of action. Further research along this line (specialized, indi-
vidually-designed experiments) can keep the pharmacologist busy and
happy while Stage 7 research is being conducted, and the results may be
important for Stage 8 research dealing with synthesis and molecular
modification.
144

Hippocratic Observational
Screen f Rats 1
Specialty
--- Areas :

CNS depressants
Anoigesics
Pharmacodynamic Screen Curdiowscu&
with Drug Cumulated Skeletal muscfe/nerve
Au fonomics
until Death (Dog) Aniioutacoid
Aniiin flomm fffory
Salihwter Moffce
~e~obo~i&~~rrn~~~

I 723xicons
CA6 stimu/ants

Skeletal ~uscle/~efve
Essential Minimum of Specialized Screening :

C/ossfic Phormoco~ogy
lso/ored phrenic ~erve~d~~~ro~rn lrff#j
/so/ailed sciofic nerve /frog)
Drua - Recgptor Khettii fxperiments

I
/so/o/cd recfus /frog)
Bokzted jejvnum (rofl

Intact - Animal Nerve - MuMe Preporotihns

with Dru9 Cumu/afed until Death
Anterior tibia/is /cot I
Gosfrocm?m&s lcbickenl

Continuing ‘&cademic Very spec&&?ed, j~djvid~o~~y-

Research &signed experihenfs I
Fig. 3. Schematic sequence for Stage 6 research as it progresses from multi-dimensional
procedures to standardized basic procedures in a specialty area and finally to individually
designed experiments,

As mentioned earlier, there are at least 14 texts available which discuss

the classic pharmacologic research that constitutes Stage 6. Those by Ther
(1949), Laurence and Bacharach (1964), Turner (1966), Turner and
Hebborn (1971), and Domer (1971) may be the most useful. Although
relatively rare, articles can be found in the scientific literature which address
certain general problems (Janssen et al., 1965; Long and Chiou, 1970). It
 145

should be apparent by now that Stage 6 research does not require the special
skills and patience of the natural product pharmacologist, any conscientious,
well-trained pharmacologist will suffice.

Stages 7 and 8: The attempt to secure a patent

Under United States patent law, it is virtually impossible to secure an

effective patent for a naturally occurring drug chemical as soon as it has been
isolated and characterized. How can one prove “unique discovery” if the
chemical already exists in nature? How can one prove “unique application”
if the chemical has been correctly used by primitive cultures for centuries?
On the other hand, a synthesis patent for a wholly synthetic drug is
relatively easy to secure and this is why the United States drug industry
prefers all-synthetic programs over natural product research.
In Stage 6, research proceeds very much as if the test chemical had been
produced by an all-synthetic program. In reality, the structure is not known;
therefore, in Stages 7 and 8 the burden shifts from the natural product
chemist to a classic type of synthesis chemist and a classic type of structure-
modification chemist, who jointly must come up with a synthesis patent for
a clinically acceptable version of the natural plant principle. These goals can
be facilitated if the accomplishments of the natural product research team
are not published immediately. Such reports are known to excite ambitious
chemists, and a rival group can take a “crash” approach tddetermining the
structure and an efficient synthesis procedure. If the rival group is successful
first, the accomplishments of the original group are significantly diminished
and the patent situation becomes infinitely muddled. The plant principle or
some chemical analog may never come to be released commercially as a drug.
The goal of ethnopharmacologic research is, of course, to find new proto-
type drugs and to make them available for the treatment of disease, and a
secure patent is a very important requirement for the release of a new drug.
since it alone protects the company’s considerable investments in research
and clinical/toxicological studies.
If secrecy is imposed, the natural product team (Stages l-5) may see
some recognition in print 5-10 years after completion of their work. In
reality, there is a strong possibility that the pharmacologists of Stage 6 and
the chemists of Stages 7 and 8 will be the first to have papers in print. This
may be another reason why there are so few natural product pharmacologists
and chemists in the United States today; it is a rare scientist who does not
want priority credit for productive original research.
Stage 9 is not illustrated in Fig. 1, but deals with the acute and chronic
toxicological evaluation of the compound selected for the patent. Stage 10 is
devoted to documenting the clinical effectiveness. Both Stages 9 and 10
must be conducted in line with current government regulations for a new
drug application and lie outside the expertise of the natural product research
team which is the focus of this paper.
146

Conclusions

The discussion just presented presumes a pharmacologic search for unique

medicinal agents using plants with a documented history of folk use. From
the author’s experience, roughly one out of ten plants selected in the manner
described will possess active medicinal agents in commercial amounts and
one out of ten such active plants will contain a unique medicinal principle.
While this discussion has centered on pharmacological research rather than
chemotherapeutic research (antimicrobials, antitumor agents, insecticides,
etc.), it appears that these 1: 100 odds also apply to chemotherapeutic
research. For example, a program emphasizing chemotherapeutic screening
was set up by Eli Lilly and Company in 1956, and by 1976 the screening of
400 plants had yielded vin blastine and vincristine (both from Catharanthus
roseus), 9-methoxyellipticine (from Ochrosia maculatu), and acronycine
(from Acmnychiu baueri) (Farnsworth and Bingel, 1977). These are very
good odds relative to the all-synthetic screening programs. Irwin (1962)
notes that in 1958 (a time of peak activity), approximately 114,600 chemi-
cals were synthesized by the pharmaceutical industry for testing. Of these
1900 went for clinical trial (1: 60 odds), but only 44 were placed on the
market (1: 2600 odds). The present author’s survey of the new drugs
released in the decade following 1958 indicates that (on average) no more
than five drugs were released per year that could be considered to be proto-
type drugs as defined here (1: 22,900 odds).
The present author prefers working with plants from areas where civiliza-
tion has had the least influence - an area such as the upper Amazon region.
In such areas, the natives must rely on effective plants for their drug needs.
No person willingly will take an inactive drug when he or she is sick. No
human will patronize an ineffective medicine man when he or she is ill.
In consequence of this natural selection, most of the plant drugs used in
primitive areas have some real benefit and relatively little toxicity. In
essence, the clinical trials for these drugs have all been run before the
scientist arrives and all the ethnopharmacology team must do is to isolate
and define that useful activity in an orderly way.
In the United States there is an attitudinal barrier that discourages natural
product research. This is best expressed by a common phrase that one hears
again and again on television, in popular magazines, in newspaper editorials,
and in professional journals - it is that we are now “living better through
the magic of synthetic chemistry.” It is a deeply believed phrase. The
public needs to be educated to the fact that plants are truly miraculous in
their synthetic capacity and that they take their energy from the sun rather
than from petrochemicals. Their demands for raw materials are exceedingly
modest (again not requiring petrochemicals) and they do not have to be
paid the wages of a chemist. The world is now energy conscious for many
good reasons, so it is now time to educate the peoples and the governments
of the world about the economical wonders of sun-powered natural product
chemistry,
 147

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