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Chemistry (including Biochemistry)/Identifying the active ingredient of a herb/plant

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Question
Dr. Robichaud,
I'm a student in AUTH school of medicine(6th year) and I'm keen on devoting time on clinical pharmacology and research on new possible drugs. I have a basic question. This is a random example.
Let's say we have a plant/herb(X). And we have observed that X makes blood pressure drop, for instance, or increases saliva production.
My first question is how can we find which ingredient/compound of this plant is the one that is responsible for this biological result? What kind of techniques or what kind of device is needed for this?

Answer
Hello Yannis!

This is a lot of work. There are natural products labs and papers entirely devoted to this process. :)

1) Develop and optimize an in vitro assay that is reasonably cheap and simple to do, so that we can 'grade' how well unknown X works to drop blood pressure. (Or another readout to tell us how awesomely we do that thing we want.)

2) Obtain several samples of plant X. Using small amounts of freeze-dried, powdered plant, test different solvent mixtures to make extracts of plant X. (Methanol, Ethanol/Water, Methylene Chloride, etc.) Dry down the extracts under vacuum. Mass out dried extract with high precision, then compare extract methods to see which version is the best at assay 1. (most efficacy / least concentration)

3) Using the best extraction method in phase 2, scale up the proceeding for a lot of powdered plant. (How much is 'a lot'? At least 500g after drying.)

4) Retest extract in vitro to make sure the season hasn't changed, no more ants, more sun, less sun, soil content etc. for the bulk plant harvest doesn't mess things up and you still have the active compound present in the live plants you harvested. (sigh)

5) Take dried extract and solubilize it in solvent. Run extract out on silica gel plates for thin layer chromatography. Note how many spots develop after UV treatment and/or heat treatment with sulfuric acid / ethanol. Ideally your compound is just one of those spots. Usually it looks like a smear at this point.

6) Depending on the polarity conditions determined by trial and error, plan a silica gel test HPLC gradient. I used a Biotage SNAP column system, though you can pack a glass cylinder with silica gel and hook it up to any pumping system that will give you an adjustable gradient. Resolublize a small amount of the plant extract (consult manual for how much extract a given column volume can handle) and dry it onto silica gel under vacuum. Pack this silica gel on top of your column. Run column from most nonpolar solvent (e.g. methylene chloride) to a middle solvent (e.g. ethyl acetate) to most polar (methanol), and collect fractions in test tubes. In a perfect world, you'd be hooked up to a UV detector. Then you'd have one test tube for each UV peak. (220nm? Check manual for best wavelength.)

7) Evaporate solvent off of fractions under vacuum. Mass fractions. Use assay 1 to compare fraction activity to crude activity. Ideally you will find a fraction that is more active by mass than the crude. Run fractions out on thin layer chromatography plates. Hope that only one fraction is active and that it has only one spot. (thing in it)

8) When your active fraction has more than one spot, either repeat the silica column (if you have a lot of mass, over 1g) or run a reverse-phase HPLC column (C18 is good) with 20mg runs per preparatory column. Collect peak fractions, evaporate away solvent, test for activity compared to crude and active pool fraction. Run these peaks on TLC and hope for a single spot. (It is often helpful to run out multiple samples of 20mg each on the same solvent gradient such that these can be combined later.)

9) If you have access to one, it is handy to submit HPLC purified samples for LC-MS, IR, or NMR analysis by an experienced chemist. LC-MS will tell you the molecular weights of the compounds in your fraction, as well as how many there are and which is predominant. NMR analysis is helpful if you have a pure fraction of one compound; it will tell you the relative orientation and bonding patterns of the atoms in your proto-drug. An experienced chemist can look at an NMR spectra and instantly know what the structure is. There are also computer software packages to aid with this.

10.) Obtain proto-drug structure from plant X. Run it in Chemspider to see if it is a presently known drug. Talk to the intellectual property lawyers to see if your discovery is patentable. Cross fingers. Apply for more grant funding to see if drug obtained from plant X and characterized by this work actually has the effects in mice or tissue culture cells that you think it will have.

And that's just the beginning!

Hope this helps. It certainly tells the story of where I've been for the last 3 years!

Chemistry (including Biochemistry)

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Trista Robichaud, PhD

Expertise

No homework questions, especially ones copied and pasted from textbooks. I will answer questions about principles or give hints, but I do not do other's homework. I'm comfortable answering basic biochemistry, chemistry, and biology questions up to and including an undergraduate level of understanding. This includes molecular biology, protein purification, and genetics. My training/inclination is primarily in structural biology, or how the shapes of things affect their function. Other interests include protein design, protein engineering, enzyme kinetics, and metabolic diseases such as cancer, atherosclerosis, and diabetes. My chemistry weaknesses are that I do not know organic or inorganic synthesis well, nor am I familiar with advanced inorganic reactions. I will attempt quantum mechanics and thermodynamics questions, but primarily as they relate to biological systems. Furthermore, I cannot tell you if a skin photograph is cancerous, or otherwise diagnose any disease. I can tell you how we currently understand the basic science behind a disease state, but I cannot recommend treatment in any way. Please direct such questions to your medical professional.

Experience

I hold a PhD in Biomedical Science from the University of Massachusetts Medical School in Worcester. I specialize in Biochemistry, with a focus on protein chemistry. My thesis work involved the structure and functions of the human glucose transporter 1. (hGLUT1) Currently I am a postdoc working in peptide (mini-protein) design and enzymology at the University of Texas Health Science Center in San Antonio, Texas. I am in Bjorn Steffensen's lab (PhD, DDS), studying gelatinase A and oral carcinoma.

Organizations
2001 American Association for the Advancement of Science
2007 American Chemical Society
2007 Protein Society
2011 UTHSCSA Women’s Faculty Association


Publications
Levine KB, Robichaud TK, Hamill S, Sultzman LA, Carruthers A. Properties of the human erythrocyte glucose transport protein are determined by cellular context. Biochemistry 44(15):5606-16, 2005. (PMID 15823019)
Robichaud TK, Appleyard AN, Herbert RB, Henderson PJ, Carruthers A “Determinants of ligand binding affinity and cooperativity at the GLUT1 endofacial site” Biochemistry 50(15):3137-48, 2011. (PMID 21384913)
Xu X, Mikhailova M, Chen Z, Pal S, Robichaud TK, Lafer EM, Baber S, Steffensen B. “Peptide from the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) inhibits membrane activation of matrix metalloproteinase-2 (MMP-2)” Matrix Biol. 2011 Sep;30(7-8):404-12. (PMID: 21839835)
Robichaud TK, Steffensen B, Fields GB. Exosite interactions impact matrix metalloproteinase collagen specificities. J Biol Chem. 2011 Oct 28;286(43):37535-42 (PMID: 21896477)

Poster Abstracts:
Robichaud TK, Carruthers. A "Mutagenesis of the Human type 1 glucose transporter exit site: A functional study." ACS 234th Meeting, Boston MA. Division of Biological Chemistry, 2007
Robichaud TK, Bhowmick M, Tokmina-Roszyk D, Fields GB “Synthesis and Analysis of MT1-MMP Peptide Inhibitors” Biological Chemistry Division of the Protein Society Meeting, San Diego CA 2010
Robichaud TK; Tokmina-Roszyk D; Steffensen B and Fields GB “Catalytic Domain Exosites Contribute to Determining Matrix Metalloproteinase Triple Helical Collagen Specificities” Dental Science Symposium. UTHSCSA 2011
Robichaud TK; Tokmina-Roszyk D; Steffensen B and Fields GB “Exosite Interactions Determine Matrix Metalloproteinase Specificities” Gordon Research Conference on Matrix Metalloproteinase Biology, Bristol RI 2011


Education/Credentials
Oakland University, Auburn Hills MI BS, Biochemistry 1998
University of Massachusetts Medical School, Worcester MA PhD, Biochemistry & Molecular Pharmacology 2001-2008
University of Texas Health Science Center, San Antonio TX Postdoc, Biochemistry 2009-Present


Awards and Honors
1998 Honors College Graduate, Oakland University
2009 Institutional National Research Service Award, Pathobiology of Occlusive Vascular Disease T32 HL07446
2011 1st Place, Best Postdoctoral Poster, Dental Science Symposium, UTHSCSA, April 2011


Past/Present Clients
Invited Seminars:
Robichaud TK, Fields GB. “Synthesis and Analysis of MTI-MMP Triple Helical Peptide Inhibitors” Pathology Research Conference, University of Texas Health Science Center San Antonio Pathology Department (June 18th, 2010)
Robichaud TK & Hill, B “How To Give A Great Scientific Talk” Invited Lecture, Pathobiology of Occlusive Vascular Disease Seminars, UTHSCSA (Nov 11th 2010), Cardiology Seminar Series, Texas Research Park (Feb 21st, 2011)
Robichaud TK; Tokmina-Roszyk D; Steffensen B and Fields GB “Exosite Interactions Determine Matrix Metalloproteinase Specificities” Gordon-Keenan Research Seminar “Everything You Wanted to Know About Matrix Metalloproteinases But Were Afraid to Ask” Bristol, RI (Aug 6th, 2011)

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