Name_______________________________
Section______________________________
Lab 8
Bioactive Drugs
Many of the most useful bioactive chemicals-those that exert effects upon the physiology of
organisms-have been isolated from plants, and many of these have been identified as alkaloids.
Alkaloids are organic compounds containing rings of nitrogen, and as their name implies, they are
alkaline, or basic, in solution. Alkaloids have a bitter taste, and according to standard terminology,
most are named to end with the suffix -ine. Alkaloids often act upon the nervous system by
mimicking the effects of neurotransmitters, natural chemicals of the nervous system. The action of
alkaloids varies with the drug and the dosage. They may be stimulants, depressants, or even
hallucinogens. Some common examples of plant alkaloids are cocaine, morphine, nicotine,
caffeine, and quinine.
A second group of physiologically active substances from plants are the terpenes. Terpenes
belong to the largest group of plant chemicals and serve a variety of functions in plants. They are
the principal ingredients in plant-derived perfumes, soaps, flavorings, dyes, and many medicines.
The lactones are a group of terpenes that have drawn the attention of researchers because they are
often the active ingredient in many herbal preparations. Glycosides are another grouping. The
defining characteristic of glycosides is the presence of a sugar group (usually glucose), but it is the
compound attached to the sugar that defines the biological activity of a glycoside. Glycosides may
be classified as cyanogenic, cardioactive, or saponins. Cyanogenic glycosides release cyanide.
Plants containing this type of glycoside are usually considered poisonous rather than therapeutic.
Cardioactive glycosides affect the circulatory system, specifically the heart. Depending upon the
dosage, preparations from plants with this type of glycoside can poison by causing heart failure or
act as a medicine by strengthening the contractions of a weakened heart. Saponins have a steroidal
component. Steroids are physiologically important because many hormones are steroids. The
saponins from some plants have been used as precursors for creating synthetic hormones in the
laboratory. Examples of glycosides include salicin, the base for aspirin, and aloin, the collective
name for many of the principal ingredients in aloe vera.
In this laboratory topic, you will observe the actions of several plant-derived bioactive
drugs upon a test animal that demonstrates the effects of these chemicals on human physiology.
After completing this laboratory topic, students should be able to:
1. Recognize a variety of bioactive drugs derived from plants.
2. Know the value of an animal model system to test and to predict the effects of bioactive drugs on
human physiology.
3. Use the scientific method and practice experimental design.
The Blackworm Model to Test Bioactive Drugs
To test the actions of bioactive drugs derived from plants, you will work with a segmented worm,
or annelid. In these worms, the body is organized into repeated rings or segments. Annelids can be
found in terrestrial, freshwater, and marine environments. There are three classes of annelids, based
on the relative number of bristles, or setae on the body wall. You will be studying a species within
the oligochaetes, annelids that have few bristles-in this case, only four per segment.
Our experimental animal is the California blackworm, or mudworm (Lumbriculus
variegatus). It is found throughout North America at the edge of fresh- water ponds, takes, and
marshes. This free-living animal survives by ingesting organic debris and microscopic algae. It is
a small worm, only 2.5-5.0 cm in total length. It has a closed circulatory system, much like our
own in that the blood is contained within blood vessels. Two major blood vessels, the ventral and
dorsal vessels, run lengthwise from head to tail, and several lateral vessels connect the two in the
anterior segments. The dorsal (along the backside) blood vessel acts as the heart, pumping the
blood through wavelike contractions of its muscular walls. The blood of the worm is also like ours
in that it is bright red due to the presence of a hemoglobin-like pigment (erythrocuruorin) that
transports oxygen. Unlike our circulatory system, this pigment is not contained in red blood cells
but is dissolved in the plasma.
Since the body wall of the worm is transparent, you will be able to observe the blood
directly as it pulses through the dorsal vessel. By immersing the worms in various plant extracts,
you will observe the bioactive effects of these drugs on the worms' circulatory systems. It is
expected that the pulse rate of the California blackworm will be affected by the plant drugs just as
our circulatory system is affected by these same drugs. Please note that the worms will not be
harmed by this testing.
Materials Needed
California blackworms (Lumbriculus variegatus)
Compound light microscope
Kimwipes
Marker pen
Observation chamber
Paper cups, 5-oz size
Pan of spring water for fresh worms
Pan of spring water for used worms
Pasteur pipette, large bore
Test solutions of nicotine, caffeine, kava kava, valerian root, and ephedrine
Spring water
Worm wrangling tools
Watch or clock with second hand
Procedure
1. You will need a lab partner for this exercise. One student observes and counts the
pulsation rates of the test worms, while the other student acts as a timer and recorder of the
data.
2. Decide upon a bioactive plant extract or drug to test. Label one paper cup as the control
and three other paper cups with the appropriate concentrations of the drug or extract
solutions. Obtain a compound microscope, observation chamber, Pasteur pipettes (one for
each solution and concentration), and a worm wrangling tool.
3. With the Pasteur pipette, select four worms from the culture pan and place them in a cup
of spring water. Make sure the worms are approximately the same length. Do not pick
worms that have recently regenerated tail segments. Regenerated segments will be
unpigmented.
4 .The next step is to determine the baseline
pulse rate in spring water (before treatment)
for each of the four worms. Using the
pipette, place one worm in the observation
chamber (fig on right). Add enough spring
water so that the worm will not dry out, but
do not over- fill or the worm will leave the
confines of the trough. The water level
should just fill the trough. Absorb any
excess water with a kimwipe. Do not add a
coverslip. Let the worm crawl around and
adjust to its new surroundings for a couple
of minutes
Observation Chamber
5. Place the observation chamber under the low power (10 x objective) of the compound
light microscope. Use the lowest possible setting of the lamp so as not to dry out or "cook"
the worm.
6. Using the worm wrangling tool, lightly touch the worm to coax it into a position that
allows you to view the dorsal blood vessel. Now you are ready to determine the basal pulse
rate of your worm.
7. Count the pulse rate from a mid-body location by watching the wave of blood flow in a
single body segment. As the timer keeps track of time, count the number of pulses for at
least 15 seconds and, if possible, 30 seconds. Repeat for two additional readings. Convert
each pulse rate to beats per minute, and record it in worksheet at the end of this laboratory
topic. Return the control worm to a cup of spring water for 15 minutes, and then redo the
pulse determination.
8. For each drug treatment, you will take pulse readings at three concentrations. Before the
worms are immersed in the treatment concentration, a baseline pulse rate must be taken in
springwater as in step 7. After the baseline pulse rate is determined, immerse the treatment
worm in a cup with the particular drug concentration for 15 minutes. At the end of the
exposure time, place the drug-exposed worm in the observation chamber. Fill the
observation chamber with spring water (not the drug solution!). Redo the pulse
determination as before and record in worksheet at the end of the exercise write up.
9. After each drug treatment, rinse the worm briefly in spring water and then place the
worm in the pan marked "used worms”. It is very important to prevent contamination by
rinsing the observation chamber thoroughly with spring water after a pulse reading is taken
on a drug-treated worm.
10. Calculate the means for both before and after treatment, and analyze the data in
worksheet.
11. Record the results of other teams using different drugs.
Plant or Drug tested:
Drug
Initial Pulse Rate (BPM)
Concentration
Trial 1 Trial 2 Trail 3
Mean
Water Control
Class Results
Drug and
concentration
Initial Pulse Rate
(BPM)
Final pulse rate (BPM)
Trial 1
Final pulse rate
(BPM)
Trail 2
Trail3
% increase or
decrease
1. In what way does the California blackworm? (Lumbriculus variegatus) make an ideal
experimental animal for testing bioactive drugs?
2. Why have so many plants that have been identified as toxic been used medicinally?
3. What is the expected effect of a stimulant upon pulse rate? Of a depressant?
4. Why is it important to test drugs or extracts at various concentrations?
5. Why is it important to measure the pulse rate of a worm in spring water?
Mean