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Ethnobotanicals

John Norton

Objective:

This is a newly designed thematic unit which will explore the medicinal values of local plants. It is designed as a generic unit that will allow for regional differences. The specific areas to be addressed will include:
1. Literature/Cultural research into local plants of medicinal value
2. Identification of candidate plants for testing for biologic activity
3. Design of a scientific protocol for testing for biologic activity
4. Preparation of plant samples for testing
5. Bacteriologic lab activities to assess biologic activity
6. Protozoal lab activities to assess biologic activity
7. Arthropod lab activities to assess biologic activity
8. Chromatographic techniques to isolate active fractions from plant extracts
9. Testing for activity of isolates
10. Characterization of isolates


1. Literature and cultural research
What to do:
Our school librarian will gather books from other school and public libraries and put them on a cart I can use in my classroom. I supplement these texts with books I have in my classroom library. Try to get 15 sources on plants of your area ( multiple copies of a given source are OK). Have students work in pairs to research local plants that have already been identified as having medicinal value. For those of you interested in using Internet resources I suggest that you have students do a keyword search using Veronica or Archie. I think that most of us can find a cultural resource for information as well. In my area we have a large population of Tlingit Indians as well as smaller numbers of Haida, Aleut, Eskimo, Athabascan, Navajo, and others. There is often reluctance on the part of young people to validate their ethnicity so be persistent in looking for these connections. Each pair of students is responsible for submitting five (5) 5x7 index card(s) with the following information:

Names of students on Team
Date
Common name of plant
Scientific name of plant (Family, Genus, species)
Description of medicinal activity
Reference / source of info.

2. Identification of local plants for testing for biologic activity
This section of the project is designed to narrow the field of candidate plants to a reasonable number for your students to work with. This section differs from the preceding in a number of ways.
  1. defining the criteria for selecting candidate plants.
    a. anti-bacterial (Perhaps the easiest place to start)
    b. anti-fungal (only a few drugs are available, usually with side effects)
    c. anti-viral (like anti-fungals, few exist)
    d. known for medicinal value other than above
    e. unknowns, wild-cards
    f. cultural history
    g. availability in local area
    h. ease of culture or domestication
    i. poisonous in large / small doses
    j. additional characteristics of interest (seasonality, persistence, etc.)
    k. roots,shoots, stems, leaves?

  2. Use a class decision-making structure to select specific candidate plants for initial study. This should be a fairly large group of potential candidates (10 or more). The screening process will eliminate some of these. Those that remain after the initial tests will become the focus of all researchers.

  3. Physical procurement of specimens and identification. Mastery of identification, common name, genus and species names, medicinal value of plants selected in #2 above. Quiz on these characters.

What to do:
  1. Break students into groups of 4.
    Product: list of criteria for selecting candidate plants
    Class product: Consolidated list of criteria

  2. Ask student groups to put their 5 x 7 cards up on the wall so that all students can review them. Ask students to rank their top 6 (1 plant for every 4 students) choices based on selection criteria from step #1 above. Then compile results of the class ranking. Review the rankings to see if these are truly the plants that should be looked at closely. Verify by class vote that these will be the candidate plants. Make a class poster board of 5 x 7 cards of chosen plants. Hang poster in prominent location.

  3. Assign groups of 4 students to work as team in initial analysis. Establish an equitable method for assigning plants to each group. Students procure specimens.

3. Design of a scientific protocol for testing for biologic activity
This is a very fun and critical part of the process. This is your opportunity to teach the philosophy of the scientific method.

What to do
Ask the class to help you list the key parts of the scientific method on the board / overhead. Discuss why this methodology is so important to decision making / gaining knowledge. Steps: (these are not cast in stone but are generally accepted).
Define the problem
Gather information
Form a hypothesis
Design experiments to test the hypothesis
Carry out experiments: observe, record data
Analyze data
Conclusions

Define the problem: In our case this is the need for new therapeutic drugs, esp. in light of resistance to current pharmaceuticals. Discuss "Flesh eating bacteria", AIDS, resistant forms

What to do: Get students into research groups. Assign roles of Moderator, Reporter, Recorder, Procurer.
Moderator drives the group, insuring participation by all members
Reporter reports the group decisions to the class
Recorder keeps notes of deliberations, data, outcome
Procurer gets needed materials, like butcher paper, marker, labware, references, assistance.

Have each group write their problem definition statement on the blackboard. Use class discussion to come to agreement on a single definition.

Collect information:
This is a great area to use communications technology. Use your school library, do an Internet keyword search, seeking advice from scientists both locally and distant, talk to other schools involved in this project.

What to do:
a. Do a detailed literature / cultural search on the plants selected by the class for study. If there is a local person who is knowledgeable in medicinal plants, now is a good time to have them come in to speak.
b. Collect information on how to prepare plants for assessment. Can you freeze them? How do you extract fluids? Can you dry the plants and assess later? What solvents are used in extractions (alcohol, water)? etc.
c. Collect information on how to culture test organisms. If you intend to test for anti-bacterial activity, get information on bacterial culture and select a culture type (I suggest that as a group we decide on a few strains that can be purchased from biologic supply companies).
d. Read one of the popular press articles on antibiotic resistance. TIME recently had a good article that is readable by high school students.

Form a hypothesis
This is a formal step in which each research group designs a specific statement for their specific plant.

What to do: Have student research groups construct their statement. Require each group design a poster regarding their plant. Include the 5 x 7 card of initial plant information and their hypothesis. Leave room for other information as it develops, e.g. illustration of plant, characteristics, historical data, data tables, graphs, analysis, conclusions.

Design experiments to test the hypothesis

The above section was meant to give you some idea of what a bacteria lab looks like and to help you in the planning of your experiments. Planning this process with your class is an important part of teaching them to be scientists. They need to know prior to doing the lab all of the expectations and anticipated outcomes. Do this planning stage with them so they feel like they have taken part in the design of the lab work. They will then be ready to actually do the lab.

For illustrative purposes I will assume that my project uses Plantain (Plantago sp .) which has been reported to have been used in a poultice for boils and infection. This is a common lawn invader and is very cosmopolitan. It would be available for all AE Fellows.

Experimental Design
Weekend Homework Assignment: Procure plant for assay

Initially I will be screening for anti-bacterials. However I expect that as the unit evolves I will screen for anti-fungals and anti-virals. There is a report in the literature of a plant that local Native people used to treat warts, so possibly it has anti-viral capabilities. Might be other things too. This week (Sept 7, 1994) I will instruct my students in the technique of growing bacteria. We will initially work with local strains picked from the gym, phones, and water fountains around our school. I can generally recognize strep and staph from simple Gram stain so this is how it will look for me.

Monday : Pour agar into 150 x 15 petri dishes as class activity. You will need two petri dishes for each pair of students. One dish will be inoculated today. The other will be used later this week and will have to be refrigerated till then. Have nutrient agar in 150 ml Ehrlenmeyer flasks in a hot water bath so students can learn to pour with sterile technique. Set petri dishes on desks for approx. 15 min. to cool. While agar is cooling use time to give notes about types of bacteria, nutritional needs, Gram stain characteristics,shape, nomenclature. Allow students last 7-10 minutes of class to travel the school with their petri dish, inoculating in interesting locations. I issue cotton swabs as applicator sticks to do the inoculations. Some people like to use tape, some use toothpicks.

Tuesday : Other work

Wednesday : Gram Stain and Nutrient broth inoculation. Prepare six nutrient broth tubes to be inoculated at the end of the class period. Instruct your class in the proper technique for doing Gram Stains. Tell them you are particularly interested in finding strep or staph. The class goal is to find six forms of bacteria that look like they might be of interest in the plant extract tests. These six candidate forms will be used to inoculate the nutrient broth tubes. Keep in mind that we have not "Streaked for isolation" so we are being hopeful that the colony is homogeneous. Have students make slides of colonies of interest. Slides showing heterogeneous bacteria populations will be disqualified. Draw the class together in the last 10 minutes of class and arrive at a consensus on the six colonies that will be used to inoculate the tubes. Teach sterile "loop" technique and inoculate tubes. Incubate tubes over-night.

4. Preparation of plant and plant extracts for testing .

Thursday
This step can include several protocols. I would expect some help on this from collaborating scientists (Genentech folks I hope) but at a minimum I would expect that this would involve grinding a given quantity of plant material to be used as an initial test of anti-microbial activity. Grinding would be done with a clean mortar and pestle.

If mortars and pestles are not readily available you will need to improvise. I have gone to Salvation Army and gotten old coffee cups and use chopsticks as pestles. Raw, ground plant would be tested. In the event that additional fluid was necessary for grinding I will used distilled water in amounts necessary to just moisten the plant material. In the first test I will use small quantities of "ground" plant material. Other protocols for preparation of plant material will probably include extraction of fluids using alcohol, acetone, petroleum ether and of course water.

5. Bacteriologic lab activities to assess biologic activity

Bring out the petri dish that you had refrigerated from Mondays' pour. Instruct each student pair in proper technique to fully inoculate the petri dish with 5ml of broth culture. Before any broth is poured you will need to have the class design how they will use the six species of bacteria to test the plant samples that you have selected. In a class of 24 students you will have 12 petri dishes, therefore each species will be poured onto 2 petri dishes. These two dishes will have small samples of each plant placed on them. If you have ever done an antiseptic sensitivity lab this will be familiar.

The idea is that any plant material that has anti-bacterial properties will become evident as a zone of inhibition will easily be seen around that plant sample.Wax pencil must be used to mark the bottom of the petri dishes so that species of bacteria are known as well as the types of plant samples and their location on the petri dish. Caution students about the need for sterile technique. Make sure that all student pairs label things well as it will get complicated from here on. Poor labeling will negate all data for a petri dish.

After properly labeling the bottom of each petri dish, and prior to inoculating the petri dishes you will need to have students prepare their plant samples. Instruct students to take a 1cm square section of plant and grind it into a paste. Add small amounts of distilled water as needed to make a paste as mentioned above. A portion of the paste will be placed on each species of bacteria.

Pour nutrient broth into petri dishes, fully spread broth, place plant samples in their respective locations on the dish. Incubate for 24-48 hours.

Friday: Read and interpret data.

Carry out experiments: observe, record data
I hope that the description given above gives a good enough picture of how to carry out an experiment. It is not meant to be a definitive method but rather is for illustrative purposes. If you have a better way of doing things please feel free to do so.

Observation and Recording of Data
The evidence for anti-bacterial activity of your plant material is a zone of inhibition seen around a plant sample on the broth inoculated petri dish. Students will generate a class data sheet in which the student name, species of bacteria, plant specimen, positive or negative response, and zone of inhibition (in millimeters) is recorded.

Analyze data
The data analysis is best done by making an overhead acetate of your class data sheet. I use our your Zerox machine to do this. Have students write an analysis of the data. The analysis should list those plants with no apparent anti-bacterial activity and those that look interesting and should be carried forward for further testing.

Conclusions
This is a summary statement which briefly tells what was attempted and what was learned. Attach this information to the poster each group has begun for their plant.

6. Techniques to isolate active fractions from plant extracts
It is now time to return to begin to focus on "plants of promise". These are plants that have shown some interesting properties in your initial screening.

Millipore filtration: There are a number of ways to isolate the various components within a mixture. The simplest way to begin this process is to use filters to cut the sample into fractions. Millipore makes an apparatus that does the job well. If you have access to this equipment in your school I suggest using it.

This is really not intimidating science but it is involved. There are a number of places where biologic activity can disappear or suddenly appear. Your experimental design must account for these. This is a great place to discuss "experimental design" again. Points of interest will be
  1. solvents
  2. dilution of active ingredient
  3. controls
You will have to use solvent in sufficient quantity to be able to have fluid wetting the filter discs and carrying molecules through as a filtrate. How much is enough but not too much? You'll have to try it once with only solvent to see what works.

Once you have used the Millipore apparatus to filter your "crude fraction" you will use a three hole punch to cut small discs from the filter. These discs will be used on a nutrient broth agar petri dish to check for zones of inhibition.
If you have only one or two Millipore apparatuses available it might be a good idea to have your G/T or more capable students use these while the rest of the class uses paper chromatography as their methodology.

Paper Chromatography
I would imagine that most of you are familiar with paper chromatography. E-mail me if you are uncertain how to proceed and I will send you a copy of a lab I do with a variety of plants to separate out various chlorophylls. The problem for this project is with maintaining biologic activity through the separatory process. The choice of solvent will most likely be critical as will the effect of dilutions. Since this is terra incognita for me I will merely be making suggestions at this time.

I would suggest that you have at least two (2) student teams of 4 working on each plant specimen of promise. The redundancy is necessary to broaden your data base. Each plant specimen should be run through several solvent systems in order to determine which solvent will work best. I suggest that you use these solvents as a starting point :
Water
Paint thinner
80% acetone / 20% petroleum ether

Thin layer chromatography
This technique is very similar to paper chromatography conceptually. It has greater resoultion and greater cost. If you have access to these materials I suggest you use them.

7. Preparation of samples:
Samples must be crushed and molecules brought into solution. Place three 2 x 6 cm wide strips of plant material in a test tube. Add 4ml ethanol (90%). Place the test tube in a hot water bath for 30 seconds (500 ml beaker on a hot plate will work fine). Remove the test tube. Use a glass rod to crush the plant material. Return the test tube to the water bath for another 30 seconds and remove, crush. Repeat this 6 times or until the plant connective tissue appears white in color and most pigments are removed. Add additional ethanol if necessary. Remove test tube from water bath. Sample is now ready for spotting on chromatography paper.

I will assume that you are familiar with how to set up a paper chromatography chamber using a large test tube. Run the chromatography using the solvent you have chosen.

8. Testing for activity of isolates
At this point you should now have a strip of chromatography paper (Millipore filter disc) on which various molecules have been spread out by the solvent. Use a three-hole punch to create discs from each section of the chromatography strip. These discs will then be tested for biologic activity by placing them on a petri dish covered with inoculated nutrient broth, just as you did in the initial assay for activity. Active fractions will be evident by their zone of inhibition. Be certain to include a control disc to test your solvent system.

9. Characterization of isolates
Assuming that you have found something of interest that you have been able to isolate, it is then time to try to characterize it. This area is really beyond the scope of what most of us will want to do, however you may want to continue on this. Characterization of active molecules includes describing its' response to heating, freezing, treatment with enzymes, molecular size range, response to dilution, and classification as protein, carbohydrate or lipid. Other characteristics can be added to this list. I can be of some help here, so if you are interested send me a note. I would expect though, that some of your scientific contacts can be much more helpful than I.

10. Other assay organisms : fungi and viruses
Anti-fungal and anti-viral drugs are in great demand. I f you and your class are interested in these avenues of research you will be moving into areas I am not familiar with. If you have a methodology for the cultivation of these organisms it would be worthwhile to post them for any AE Fellows who might want to go in those directions. As I mentioned earlier, local native peoples used a plant found here for reducing warts, perhaps indicating anti-viral properties. It would be fun to look at it.

Protozoal lab activities to assess biologic activity
If you would like to look for a broad spectrum effect I would suggest that you begin looking for anti-protozoal effects using Paramecium sp. I would suggest growing paramecium to high density and introducing a drop of your culture into a depression slide. A variety of amounts of crushed plant material of interest can then be added to the slides and the reaction of the protists can be observed under 10x or a dissecting microscope. It may turn out that looking at paramecium behavior will be important to understanding potential toxic affects. Normally paramecium will swim with a left hand spiral. They normally reverse swimming at random intervals in their search for food. Pin-wheeling, continued reverse swimming, and altered swimming patterns all indicate disruption of normal ciliary function. Ciliary function is due to receptor mediated trans-membrane calcium fluxes. Unusual swimming patterns are indicative of alteration of normal function. This may be a good way to get a hook into studying protists, membranes, drug toxicity and simple behaviors.

Summary
I hope you will forgive me any spelling or grammatical errors. I have been a bit rushed to get this out to you for your comment and use. This document is a first draft and should improve with revisions. Most of the lab work I have suggested here has been done in my Bio. 1 class of sophomores. I will be happy to send along lab instruction sheets for the bacteria and chromatography labs I have used in past years and data sheets for this unit as I develop them this Fall. I have already begun this unit with my students and they would like to exchange data with other schools. Let me know if you are interested. Good Luck.
John Norton AEJNorton@aol.com Juneau, Alaska Sept. 5, 1994

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