The University of Arizona From Plant to Drug Stefani D. Hines & Dr. Susanne Valcic The University of Arizona Center for Toxicology - Self Introduction - At the Center we have people doing research trying to find new medicines to help fight cancer and other diseases. - One place they are looking for these medicnes is from plants. - Today I am going to share with you why biodiversity is importantto this type of research - and then I will share with you some of the basic processes used to get from plant to drug

Source Material = Biomass Plants Microorganisms bacteria, fungus Marine organisms Animal products - Almost half of all presecription drugs used in the US were originally made from living things. Such as Plants, microorganisms (bacteria), marine organisms, animal products - Today, those same basic chemicals contained in the plants or animals are used, just many have been synthesized. - Synthetic drugs - the active chemical has been identified and then copied. - aspirin - willow - sometimes saves a lot of $ (when you do your experiment you will see why) - Some of pharmeceutical drugs are also sythesized or created from scratch - designer drugs. - But plants & other living organisms continue to be a very important resource for new medicines.

Role of Biodiversity in Medicine 1.75 million known species of animals, plants, fungi, & micro-organisms Estimated to be 10 million species on earth Each species contains 100’s - 1,000’s of chemicals 1 billion - 1 trillion chemicals Biodiversity is important for many reasons, but today we are focusing on why biodiversity is important to medical research - there are ~ 1.75 million known species - estimated to be 10,000 - each species contains 110s to 1000s of chemicals. Some of which we are familiar with, many of which we know nothing about. - So there is a very rich resource of chemicals in nature - 1 billion to 1 trillion -, some of which may help us fight diseases and health problems.

Loss of Biodiversity ~ 100 species/day is lost Loss of potential new medicines - Now, the loss of biodiversity could end up hurting us because we are losing potential medicinal sources. -It is estimated that ~100 species/day is lost due to destruction of ecosystems like forests

Biological Activity Major Targets Infectious Diseases Cancers Pesticides

Biomass Crude Extract Extract Fractions Pure Compounds Extraction Crude Extract Determination of Biological Activity Separation Extract Fractions Isolation - Once you have collected some plants there are a series of steps you use to extract , separate & identify the bio-active compounds - The reason you want to do this is because there are so many chemicals in each plant type, some of which may be harmful to us. We want to get rid of the chemicals that do nothing, or have a toxic effect, and keep the good ones. - Here are the steps 1. Once you’ve collected the biomass, you want to extract all of the chemicals & separate it from the plant matter. 2. This mess of chemicals is called the crude extract. 3. You then want to see if that extract contains any bio-active compound of interest. Keep in mind usually screening for only a few applications, can’t look for everything. 4. If crude extract has some bioactive compounds, you want to separate them from the rest of the mess. This leaves us with the extract fractions. Then you test agin to see which fractions contains the chemical of interest. 5. You continue separation & retesting processes until you have isolated the pure compound, which you then try to identify its chemical structure & give it a name. Active Lead Compound Pure Compounds Identification

Collection Strategies ecological strategy ethno-medicinal strategy environmental strategy taxonomical strategy random strategy - “bio-active compounds” - looking for biological activity to help with health issues like infectious diseases & cancers - “bio-active” compounds are also used for things like pesticides.

Biomass Extraction

Extraction Methods Solvent extraction at room temperature or higher flowing or non-flowing - There are a couple of ways to initially extract the chemicals from the plants to make the crude extract. - You always use a solvent of some sort. - a solvent is a substance that can dissolve another substance - water is a common solvent, so is alcohol - sometime the chemicals will come out of the plant at room temperature - sometimes it has to be done at a higher temperature (like when you wahs your clothes on hot to help remove certain stains). - Sometimes you have the solvent flow through the plant material other times you let it sit in the solvent.

Crude Extract A complex mixture of unknown components obtained from the biomass Separation of the mixture done by the differences in physical or chemical properties - Now we are left with the crude extract - Crude extract is a complex mixture of unknown components - Lets say we identified that there is a bio-active compound in the crude extract, now we want to further separate the mixture. The techniques that are used for separation depend on the physical or chemcial properies of the extract.

Biomass Crude Extract Extraction Determination of Biological Activity (in vitro) Separation

Separation Methods Centrifugation Distillation Crystallization Filtration Chromatography Separation is done by any number of methods. Often multiple methods are used because one technique works better than another depending on the chemicals. Centrifugation- spin the heck out of the sample. Separating chemcials from blood are often done this way. How many people have ridden the carnival ride where you spin & the floor drops out? What happens to your skin? If you did it long enough & fast enough your skin & muscles would separate from your bones, your brain would ooze out if it weren’t encased in your skull. You would be separating body parts. Distillation - You’ve heard of distilled water? Basically what they do when they distill water is boil it & then catch the steam(condense) Minerals & other chemicals in the water are left behind. Chrystalization - works if the compound you are interested in will chrystallize out of the solution. Filtration - get the chemical to solidify in the solution and filter it out (like coffee) Chromatogrphy - This is the separation method I am going to focus on today and is what you will be doing in the lab.

Chromatography - Basic requirements Stationary Phase paper, glass, plastic, gel Mobile Phase water, other solvent - In chromatography you have a stationary phase - which is basically something solid like paper, glass, or plastic, on which you do the separation. - In the activity you will do next, you will use paper - Then you have a mobile phase, which carries is the exract you want to separate. This can be water, alcohol, or some other solvent (a solvent is - In today’s activty you will use water.

- This is an example of what you will be doing in the lab today. - Can you describe to me what you see? (different chemicals separating) - Any ideas about why this happens? (size, weight, charge)

Gingko biloba Extract There are also big machines that do chromatography and they give you information that looks like this.

Bioactivity-Guided Separation Extraction Plant Extraction Crude Extract Bioassay Chromatography - Ok, so here is the step0by-step process up to this point bioassay - test to see if bioactive compound is in the liquid Repeat Pure Compound Bioassay

Complex Mixture to Single Compound This is what it looks like. You start off with the crude extract (left) which has a bunch of differenr chemicals in it, And you keep separating the chemicals until you isolate the one that you are interested in.

? = Active Compound Determine Molecular Structure Now, that you have found the active compound, you want to find out what it is. It could be a chemical we already know about, so you would try to match it. - But if it is an unknown chemical, you want to determine its molecular structure. That way you can name it and maybe make it. Or you can test other plants to see if its present. Determine Molecular Structure

From Plant to Drug = Plant Crude Extract Pure Compound Molecular Structure = Biological Activity