Rodger W. Griffin Jr.
New College Sarasota, Florida 33578
Natural Products An independent study project
There are relatively few graduation requirements for students of New College, where the chemistry curriculum minimizes the number of courses and maximizes the amount of time that a student spends in research. However, the requirement of four independent study projects and a senior thesis, taken with the fact that there are only three faculty members in chemistry, dictates that in this context an organic chemist cannot be a narrow specialist, but must offer a wide variety of problems in the area. For some time there has been increased student interest in natural products and biochemistry. This paper provides leadine references and describes in detail an independent study project suitable for students with at least one term of general chemistry, although a full year is preferable. It is suggested that a high school student, given adequate library and laboratory support might develop an excellent science fair project in this area. At New College one month is set aside for students to engage in Independent Study. The program described here was undertaken by six first-year students who had studied one term of general chemistry (no lab). The immediate objective was to screen local flora for three classes of natural products: alkaloids, terpenes, and flais to develop vonoids. The long-range goal of this structure determination problems in the area of natural products for senior theses. Student activities for this period can be summarized by the followine cateeories. each of which is discussed below in detail: cgemistry lectures, botany (field collection and ~ l a n identification), t laboratory screening procedures and isolation of large quantities, library work, and presentation of results as oral and written reports. Chemrstry. At the appropriate points, 1-2 hr lectures were given on the following topics: brief introduction to organic chemistry including bonding, aromaticity, and functional groups as well as specialized lectures on terpenes 1 - 6 , flavonoids (7-12), and alkaloids (13-17). Thin-layer chromatography was explained in a lecturedemonstration using the dipping method with both microscope slides and 5 x 20-cm glass plates. The use of the library and Chemical Abstracts was explained and a faculty member was on hand in the library for the first 3-5 hr. in order to answer general questions about the procedures and to clarify particular points concerning the chemistry. ~
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In addition to lectures on distillation, extraction, and spectroscopy, students also heard talks by more advanced students who were carrying out research in synthetic and mechanistic organic chemistry in the same laboratory. Botany. Although it would be preferable to have a professional botanist collaborate in the early stages of this work, one of our graduating seniors took charge of this aspect of the program. Students first collected and examined flowering plants available on campus. After instruction on using botanical keys (18, 19) and review of the parts of a flower, students proceeded to identify 3 or 4 plants they collected on Longboat Key. Herbarium samples were prepared, mounted, labelled, and have been preserved. Screening. Each of the plants was separated into flowers, leaves, stems, roots, etc. and screened for alkaloids, terpenes, and flavonoids (20). In addition, students prepared extracts of known materials: alkaloids (tobacco and black pepper in 3.5% HCI), flavonoids (citrus peels in 2 N HCI), and terpenes (citrus peels, ground cloves, and other spices in low-boiling petroleum ether). The alkaloids were detected by Mayer's reagent and the terpenes and flavonoids by thin-layer chromatography on Silica Gel-G using various sprays. (See experimental section.) Libraw. of the identified s ~ e c i e swere suhiected to " Two an intensive literature search using the subject indices of Chemical Abstracts. The extent of the known literature of these species varied considerably and students chose one nlant for which little or nothine was known and proceeded to carry uut a larye-scale field collection of that species. l\olutron It rook 5-10 hr of tedious work fur students ro separate the various plant parts for the species to be studied. Only one of the three natural products classes was studied, but the flowers, leaves, etc. were examined separately. Students collected 3-10 kg of plant material and usually extracted 0.2-2.0 kg of each part with the appropriate solvent. In general, a single species, once separated into its botanical components and extracted, gave much more chemical material than students could thoroughly study in the time allotted and many students expressed a desire to continue working on the problem throughout the year. Presentations. Students wrote final research reports (20-30 pages) using the current journal style in chemistry.
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These reports indicated clearly the direction their particular problem should take in the future. Students also gave a talk on their work, concentrating on the "tips and hints" which they found useful in their experience. Experimental Alkaloids. The plant material was either ground in a mortar and pestle (3.5% HCI) with a small amount of sand or simply allowed to stand at room temperature for -48 hr. The supernatent was then tested with Mayer's Reagent, prepared by dissolving 1.358 g. of HgCI2 in 50-60 ml water, mixing this with a solution of 5.0 g of KI in 10 ml water, and diluting the mixture to a final volume of 100 ml (21). Although Mayer's Reagent is reported to be extremely sensitive, often giving a positive test where alkaloids are absent, it was found that Wagner's Reagent (21) was too insensitive, giving a negative test with known alkaloid solutions. Terpenes. The plant material was either ground or the finelydivided material was simply allowed to stand -48 hr at room temperature in law-boiling petroleum ether. The essential oil was also obtained by steam distillation, followed by ether extraction, drying, and concentration. Silica G e l 4 plates were made by dipping two clean microscope slides or two 5 x 20-cm glass plates into a slurry of 35 g of Silica Gel-G in a mixture of 35 ml CHIOH and 65 ml CHCls. After drying, these were activated at 110-120'C for 15-20 min. Plates were spotted with the solutions to be tested and developed with either (1) hexane, fallowed by redevelopment in CCI, or (2) 15% EtOAc in hexane, followed by redevelopment in 10% EtOAe in hexane. Various sprays were used, the most general being 0.05% fluorescein in water followed hy exposure of the dry plate to Bn vapor. Terpenes are detectable as yellow spots on a pink background; uv visualization often shows additional spots (20). Flavonoids: Although the plant material was treated with 2 N HCL, as described above for the terpenes, it became clear that tlc on Silica Gel-G, development with Forestal solvent (CHaCO0H:concd HCl:HeO, 30:3:10) or TAW solvent (toluene:CH3COOH:Hz0. 4:1:5), followed by acid or base spray (uv) was inadequate for the formation of distinct spots. Suggestions given in reference (10) are worthy of further study in this regard.
Acknowledgment
I a m grateful for the initial impetus provided by Lynwoad Sawyer a t a time when natural products research a t
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New College was only in the discussion stage, and to Barbara Beaman for her conscientious efforts in the botanical aspects of the research. T h e success of this project is due to the hard work of those first-year students who undertook this research, each of whom has read and contributed t o this paper: Jeff Prior, Martin Schwartz, June Bronfenhrenner, Mary Hill, Beth Brown, and Leslie Nelson.
Literature Cited (1) Guenther. E., "The Eesentisl Oils." Van Nasfrsnd, New York. Vois. I-VI, 19481952. (21 Pinder, A. R., "The Chemistry of tho Terpensr: John Wilay and Sons, Inc., New Yark. 1960. (31 deMayo. P.. "The Chwistry of Natural Produefs: Voi. n. Mono- and Senguitewnoids." Wiley-Infcrscirnrr. New York. 1959. (41 deMayo, P.. "The ChemirWy of Natural Pmducts: Vol, US. The Higher Terpenoids," Wiley-Interseimec, New York, 1959. (51 Ourisson, G., Crabbe, P., and W i g , 0. R.. "Tetrscyelie Triferpenes." HoldcnDay, Ine.. San Fmncisco. 1964. Goodwin. T. W. (Editor,, '-Aapwta of Terponoid Chemistry and Biochemi~try: Phyto~hemiealSocief~ Symposium 1970,'AeademicPrees, NewYork. 1971. Gcissman. T. A. !Editor), "The Chemistry of the Flavonoid Compaunds,' The Maemillan Co., New "--L ,'-* Wdham. J. 9. (Edit", Q.
N e w Ymk, 1967. Mabry. T . J.. Markham, K. R., and Thomas. M. B., "The Systematic Idenfificst i m of Flavanoids."Springer-Verlag.New York. 1970. Gmduln. T. W., Themistry end Biochemistry of Plant Pigments." Academic P r e s , New Yark. 1965. Harbocne. J. 8.."Biochemistry of Phenolic Compound%" Academic P r e s , New York. 1964. (131 Manske, R. H. F., and Holmos, H. L.. "The Alkaloids: Chemistry and PhyaioloNew York. Vols. I-XIV. 1950-1973. k/."Academie RFSI, (I41 Benfley, K,W.. '"The Chemistry of Natural Productsi Vol. I. The Alkaloids,' WileyInterseienec,Ine.. N&York. 1957. (15) Pellotier. S. W. (Editor), "Chemistry of the Albloids." Van Nostrand Rhoinhold P^
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1161 Rsffsuf. R. F.. "A Hsndbmk of Alkaloids and Alksloid-confainina Plants," Wilev~ Inferscience, N e w York. 1970. (171 Shamma, M.. "The lsoquinoline Alkaloids: Chemistry and Phamacolopy," AcsdemiePrees, Now York, 1972. (18) Long, R. W., and Lakela. 0.. "A Flora of Tropical Florida." U,of Miami Prora, Corsl Gables. 1971. (191 Conard. H. S.. "Plants of Cenwal Florida: Ridge Audubon Society. Odando, 1971. (20) A gmd general referenee for laboratory work in this area is kan. R., "Natural Products: Alaboratory Guide." Academic Press, NFU~YO*,1969. (21) Wrast. R. C. (Editor), "Handboak of Chemirwy m d Physics." Chemical Rubber Publishing Co., Cleveland, Val. 51, 1970-71. p, DS9.
