Use of Brown Algae to Demonstrate Natural Products Techniques Lee A. Porter Jacksonville University. Jacksonville, FL 3221 1 As one becomes involved in teaching the techniques of any modern scientific discipline, one becomes aware that the students take for granted every hard-won body of knowledge or method. They have no perspective of what went hefore, much less any feeling for the range of what is possible. With the aim of demonstrating some of what is known about the compounds in marine plants and tvoes .. of ~roceduresrequired for their isolation, two examples are offc;ed helow from the area of marine natural products chemistrv. Both of these examples have been taken from the brown algae. The first describes the isolation of the primarv metabolite. D-mannitol: the second one describes the isolagon of cholesterol from group of closely related sterols (secondary metabolites) ( I ) . The first isolation is quite atypical. I t describes a case in which, for a small expenditure of time and chemicals, a very large amount of a pure material can be obtained from a rather easily collected and identified plant. For comparison, the serond isolation is far more typiral of what is usually found. It desrribes a procedure that requires considerably greater relative amounts of time, skill, chrmicals and equipment to ohtain H very small amount of a material from a plant tbat is much harder for professionals, much less the novice, to collect and identify. Either or both of these procedures may be performed as individual or group experiments by undergraduates or beeinnine- " eraduate students. Thev are narticularlv suited for use a t locations with strong marine science programs since thev combine in sinele the disciolines of marine - exneriments . hot&, biochemistry, physiology, and marine natural products organic chemistrv. In addition.. exneriments of this sort are es&cially good learning experiences since they combine many needed skills in a single procedure. The first procedure is quite short and involves only a few skills such as extraction, recrvstalliition, and derivation formation combined with the plant collection and identification. The second isolation, however, is more lenethv and involves several more skills. such as column, thm.layer, and preparative-layer chromatographv. Since it deals with such small amounts of final i~rodurt.it requires some skill and experience and is someahat more difficult. The study of these procedures, whether or not actually performed in the laboratory, will provide students a brief lwk a t some much-needed historicaiperspective. They will also provide an introduction to some of the skills required and the knowledge that is available as well as an appriciation of the often considerable time and effort needed to acquire tbat knowledge. In addition, the fmt of these procedures can provide a small taste of the role serendioitv can olav in science. since the unusual ease with which ihe-~-mannkolcan be bbtained was stumbled upon in the course of another isolation in this author's laboratory. It may also be used to demonstrate a typical "Mur~hv'sLaw" corollarv, .. viz... if it crvstallizes out of the crude-extract in nearly pure form, you really do not need or want it. The second procedure and its references can also provide a brief introduction into the subiect of chemical taxonomv. since particular secondary metabolites of this type can frk:
a
A
quently be shown to he class-, order-, family-, and occasionally genus- or subgenus-related (2). Discussion Brown algae contain free D-mannitol, frequently in a high concentration that may be based as much as 25%on dry weight of plant material (3).This makes it unusually easy to isolate D-manitol from brown algae and provides an especially suitable introductory undergraduate experiment. D-mannitol is the primary repository of photosynthetic energy in brown algae and can be thought of as analogous to the corresponding mono- and oligosaccharides of higher plants (4). Compounds sucb as D-mannitol are of interest to researchers investigating the biochemistry and physiology of plants, especially the processes of ~hotosvnthesis.as well as to chemists interested in pure carbohydrate chemistry in plants. The alga used in the D-mannitol procedure was Sargassurn nntans, which is one of the two common pelagic (free-floating) Sarzassum species of the tropical north Atlantic. the Gulf coast, the aha am as, ~ e r m u d aand ; the Caribbean sea. However, any of several other hrown aleae includine.. Sarzassum .. fluitans, the other common pelagi~.~orgos.wn species, and 1.orninorio s u . , have sufficientlv hieh concentrations of 1)mannitol a t %st times of the yearto make this procedure feasible (5). The physical appearanre of both S. fluitans and S. natons is quite distinctive, and with the aid of some excellent drawings and descriptions available, e.g., in the marine plant taxonomy by Taylor (6), i t is possible even for inexperienced students t o collect and identify either one. Sargassurn natans or S. fluitans can be obtained most easily by collecting i t a t the high-tide line along the beach. This collection was made on the beach of a small island in the Exumas, Bahamas. I t is recommended that beach collections and identifications be made on material that has not yet dried out. This is best done shortly following an early morning high tide. Collections can also be made from a boat by sorting through the materials found floating in occasional windrows on the open ocean. The nlant material must be freed from the usual oreanic soluble materials by extraction with methylene chloride &fore it is extracted with methanol. Direct extraction with methanol causes the D-mannitol to he lost in the resulting dark greenblack extract from which it cannot be isolated readily. The scale on which this extraction was originally done is mentioned here not only as an example of what is possible, but also because the use of sucb a scale produces an impressively large number of crystals. The preliminary extraction with methylene chloride, which removed the usual organics and most of the chlorophvlls, was followed bv an extaction with methanol using a iarge Soxhlet extractor fitted with a 12-1 round-bottom flask. After cooling and standing undisturbed for several days a t room temperature, the flask was found to contain 8 1 of a pale green (residual chlorophyll) methanol solution containing fibrous needles and blades of D-mannitol which filled most of the liquid volume. The procedure for carrying out the experiment on one-tenth the original scale is given in the experimental section. Volume 62 Number 7 Julv 1985
635
T h e alga used in the cholesterd procedure was Grariloria foliif~ru.It is a more typiral brown alga in that it requires a hard or rocky substrate for attachment. It is found along the coasts of the North Atlantic Ocean from North Carolina and Bermuda to Florida, along the roar1 of the Gulf of Mexico and along the coasts in and around the Caribbean Sea and the non hem coast of South America. Althoueh Tavlor's taxonomv (6)describes G. foliifera a s being common on rocks and shells in quiet, shallow water, t h e plants used i n this isolation were collected in Northeast Florida on natural rocks in the surf and on man-made "iettv rocks. both of which are subject t o h e a w wave action. Sterols in plants. such as t h e one described below, are generally cokideredto be secondary metabolites ( I ) , whether this designation is always functionally correct or not. I n addition to providing more information on the problem of what purpose these secondary metabolites serve i n t h e organism, they are of interest to natural products chemists for their novel structures or raritv or their occurrence in unusual places. For example, for years cholrsterol was thought t o he exclusively the product of animal metabolism. Hecentlv. however it has bee; found t o occur in the marine algae rathi; commonly (7). Manv secondarv metabolites also have verv strong .and often usefil pharmachogical activities. T h e isolation orocedure described for cholesterol is one that will obtain neariy all t h e cholesterol residues present in the nlant, whether i n free form, esterified, or t h e like. As such it is a typical procedure for the isolation of the nonsaponifiable lioid fraction in plants. T h e basic hvdrolysis used will free the cholesterol moikty from most attached groups. Different orocedures must be used if one wants only free cholesterol or glycosides or esters in unchanged form. Unlike steryl esters, steryl glycosides are not hydrolysed in hase a n d thus they occur in the general nonsaponifiahle lipid fraction. However, they appear in a different fraction chromatographically.
-
-
Experimental After collection,the Sorgassum natons, S. fluitons, or Gracilaria foliifera should he carefully freed of foreign matter, both plant and animal, and air dried immediately. For those who do not have ready access to the ocean to make their own collections, upon request and for a fee, Carolina Biological Supply Company1will collect and identify useful amounts of material, if given sufficient advance notice. However, this necessarily removes one of the multidiseiplinaq aspects of these experiments.
bolation of 0-Mannitol A sample of 85 g2 of dried, ground3plant material is extracted exhaustively in a 65-mm-i.d. Soxhlet extractor4 with 11of methylene chloride5to remove the organic soluble materials. This will require about 48 h, more or less, depending on the distillation rate. After removal of the methylene chloride residues, the ground plant material is extracted exhaustively (approximately 48 h) with 11of methanol. The cold methanolextract is allowed to stand undisturbed for several days, after which the hrownish-white, fibrous-looking solid which forms is filtered from the methanol extract. A typical yield of crystals is 11%of the dried plant material. The melting point will he in the ranre of 152-167'. P hv reervstallizstion of about 1 - k i e s t i o n is aceomolished -~~~~ - e of the ,~~~~ crude n-mannitol from 51)O ml of methanol. It will he necessary to filter off somr rnaoluhle material while sill hot.The pure D-mannitol crystallizes as silky, white needles and blades, mp 16616S0 (lit. mp 166168') (8,s). The hexacetate (lit. mp 119-120°) (8) and the hexabenzaate (lit. bv standard methods (8). The mn 149-150') (8) mav. he oreoared . . h;xaacrtete preparation is straighrfdnvard. The hexahenroate is difficult to crystallize and requires nomc skill and patienre. ~~~~
~~
.~ -
~
~
~
~~
~~~~
~
-~
Journal
of Chemical Education
~
~
~
ldno -="
.
This material is dissolved in 1ml of benzene:ether, 45, and applied in a narrow strin to the oriein of a freshlv..oreoared TLC . nrenaratlve . . plate (10 g silica gel with hinder. 20 cm X 20 cm X 1 mm). The plate is eluted with the same solvent. The plate is visualized with both long and shon-wavelength uv light I366 nm and 251 nm, respectively) and an IzICHC13spray (using a mask that produces three narrow sprayed strips arranged verticalli, one along each side of the plate andme up the middle). After elution, each band is numhered starting from the bottom or oriein. There will he cholesterol and the sterol of hieher - RI. value on the plate, plus, in most cases, a hand at the origin and a faint band near the solvent front. The first hand ahove the origin is cholesterol (as verified by comparison with authentic material). It is scraped off (the three zones sprayed with 12/CHC13are left on the plate) and boiled for about 10 s with 10 ml of methanol and filtered hot. The SiOz in the filter funnel is washed once with hot methanol. The filtrate and washings are comhined and evaporated. A typical vield is 20 me of nearlv white crvstalline material:. a twical .- meltineboint of the ;earl~ .auk . cholest&ol is 145-149". Further purification in nruomplished by recrystalli7arion from a mixture of2S1ml of diorane, 1.5 ml of methanol end enough water added dropwise to produce a slight turbidity at the boiling point (1.0 ml). A typical yield is 15mp of shiny white flakes melting at 14M4g0 (lit. mp~i48.5') (10). The acetate (lit. mp 115-116°) (10) (11) and the dihromide (lit. mp 114V. 1121 . . mav. be oreoared . . bv standard methods (11)(13). Both preparations are straightforward. ~
~
Llterature Cited (1) Geiasrnan,T.A..andCmut,D. H. G.,"OrganieCherniatryof Seeondsry PlantM=tabolism." heernan. Caoper andCornpany, Ssn Franciaeo, 1969. PP. 3-8. (2) Ref ( I ) , pp. 11-19:Irwin.M. A.,and Gciaam8r.T. A.,PhytorhmGtry, 12,849,(1973):
Geiaaman, T A., andSaitoh,T., Phylochamistry, 11,1157(1972). (3) Perciual. E., and MeDowiell, R. H., "Chemistry aod Enzymology of Mariae Algal Polyaaccbariden." Academic PIPSB, New York, 1967,p. 5. (4) Krernor. B. P.,PhytochmGtry, LZ,M)9(1973). (5) Black. W.A. P., J MorineBiolAssoc..29,45(1950J. (6) Taylor, W. R,"Marine Algae ofthe Eastern Tropical and Subtropical Coastr of the Amerieas,"Unive.aityof Michigan Press,Ann Arbor, MI, 1967,p. 231. (7) Smith, L. L., et 81.. Phytochemistry. 12, 2121 (1913); Fattoru~ao,E., et el., Phytyla. ehemiafrv. 14.1519 119151:Kabore. S. A..Phrtochsmi~trv.22.1239 l1983):Le%n.
(11) ~iese..L.F.,andW i l l i m ~ ~ t ~L.,"OrganieExperirnatr,"~thed..D. ,K. C. Haathand Company, kxington, MA, 1979,p. 129.
112) Fieser,L. F.,andFiear.M., "Stemida,"ReinholdPublishingCorp..NwYork, 1959,
" 44
~
Isolation of Cholesterol A samplr ofC?g2ofdried, ground3 plant material is extracted exhaustively in a 65-mm-i.d.Soahlet extractd with 1 I of petroleum ether (hp RO-60') for about 4d h, more or less, depending on the dis.
636
tillation rate. The resulting extract is concentrated on a rotary evaporator to a dark green, oily semisolid. This material (twicallv .. . about 2 e) is dissolved in 15 ml of a 10% solution of potassium hydroxide in 50°0ethanol-water.The solution is rrfluxed for 3 h. The reaction mixture is then cooled to room tem. peratureand cxtrarted withether 13 X 5 ml).Theetherextractsare comtinrd, dried and concentrated on a rotary evaporator lo a dark, aemirolid residue A typ~rnlyield at this stage i s 0 2 g. The sticks residur is then disrulved in a few milliliters (1-3) of benzene:eth&, 4:1, placed on a silica-gel column (25 g, 1cm ~ 1&),5 and eluted with 250 ml of the same solvent..takine..15mlfractions. The fract~onsarrmonitored by 11.C wing a cholesterol standard, thesame elutmg solvent and 12 chamher viwali7ation. Fractions 7 to 10, contninmg the chuleetrrol and another sterol of higher Rr value, are combined and evaporated to give a nearly white crystalline solid. A typical yield at this point is 50 mg; a typical melting point is 10%
Main Office and Laboratories, Burlington. NC 27215. It should be remembered when collecting that algae are iyplcally m r e than 90% water by weight. Thus the amount of dried algae indicated requires the collection of 10 times that amount of fresh algae. A word of caution should be added here. Any metal parts of equlpment, e.g.. a Wiley Mill, used to grind the dried algae, that come in contact with the plant material, must be thwoughly washed, dried. and oiled immediately to prevent severe salt corrosion. Sargent-Welch Scientific Co., Catalog #S-31265. In a recent note in C&E News (August 3. 1981) inhalation of methytene chloride vapars has been shown to pose no significanthealth hazard when used within established guidelines.
'
