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Essential Oils and Their Constituents XX
Research ArticlesEssential Oils and Their Constituents XX Detection and Estimation of Menthofuran in Mentha arvensis and Other Mint Species by Coupled Gas-Liquid-Thin-Layer Chromatography By ISHWAR C. NIGAM* a n d LEO LEV1 The occurrence of menthofuran in Mantba arvnrtis is reported for the first time. From 0.01 to 0.04 per cent of the heterocycle was determined b means of coupled gas-liquid-thin-layer chromatography in enuine essential oils okined from plants grown in Argentina, Brazil, Formosa, Infin, Japan, and South Africa. Its presence at such low concentrations could not be detected by conventional chemical tests, eg., ~ l - ~ ~ k ireaction, g e r ~ ~or by modern instrumental techni ues, ecludin ultraviolet and infrared specuophotometry applied to analyaA. amens Lectly. The classical distincuon between Mentba piperita (pe p e r d r m d M. a t w m a s (mint), ostensibly based on the presence or absence ofmenthofuran, is therefore no longer justifiable. Other mint s ies, including spearmints (Mentba cadiaca Gerard ex Baker cultivar Scotch or Kghland spearmint, Mentba spicata L. cultivar common or native American spearmint, and Mentba viridi), pennyroyal (Mentba L and Hdmnap~kgiokZes ) Pers.), bergamot mint (Mcntba citrata E r M w t b a sylveswis L., and Man& tvtmd#ol& contained trace amounts of menthofuran also. The experimental data suggest a reap r a i s a l of presentl acknowledged concepts regarding both biogenesis and %emotaxonomy o f the genus Mentba.
the liquid develops a blue color, which on continued heating deepens and shows a copper fluorescence, then fades, leaving a golden yellow this sequence of acetienitric acid solution (1). In 1890, Polen- solution (6). With M.a&s, ske suggested that the phenomenon was due to color changes is not observed, the test solution the presence of a volatile, nonnitrogenous con- attaining merely a light yellowish tinge which stituent in the essential oil (2). Thirtv years undergoes no marked change during the 5-minute later the compound. menthofuran, was isolated heating period. Wering a valuable criterion for distinguishing by Carles as an “ether-oxide” from the flower oil of Italian peppermint (3). Its constitution readily two closely allied and commercially most was established soon afterwards by Wienhaus important products of the genus Mcntha, the and Dewein (4). In 1936, Treibs synthesized reaction was soon considered and still is an the heterocycle and demonstrated that its official specification (7, 8). As a spot test properties were identical with those of the natural requiring but 1 drop of material, it was later utilized by Zolotovich for the examination of M . isolate (5). As a criterion of identity for M. ~perit0, amensis as well as M.Piperita oils and many of Flkkiger’s test came to be applied widely and theirhybrids (9). Studies camed out recently in our laboratory with only slight modifications. It is generally carried out as follows. Three drops of the es- on products of different geographical origins scntial oil are mixed in a dry test tube with 5 confirmed the specificity generally ascribed to rnl. of a solution of 1 vol. of nitric acid in 300 the reaction (10). Yet, not always were exvol. of glacial acetic acid, and the tube is placed perimental data entirely satisfactory, and the in a beaker of boiling water. Within 5 minutes investigation was continued. It is the purpose of this paper to report the results that we obReceived October 2, 196t. from the Food and Drug Directained using coupled gas-liquid-thin-layer chrotorate. Department of National Health and Welfare, Ottawa, Ontario Canada. matography as the method of analysis. Aceeited for publication November 5, 108.3. years ago Fliickiger A observed that peppermint oil gave a characteristic color reaction when heated in glacial LMOST ONE-HUNDRED
The authors are indebted t o W. Skakum for technical assistance. Previous paper: Pcrfumrry Esscnl. Oil Record. 54, 814 (1‘383).
*
National Research Council of Canada Postductoral Fellow, 1962-1963.
EXPERIMENTAL Apparatus and analytical procedures were previously described (11).
Vol. 53, No. 9, September 1964 Materiala EaaenW m.41of mint (M.u ~ s i s was ) provided through the courtesy of Dr.Juan-Carlos Tuja, Cultivos Industriales. S.A.. Buenos Aires, Argentina,Drs. M.T.MagalhHes and 0.R. Gottlieb,
20
1009 Instituto de Quimica Agricola, Rio de Janeiro, Brazil; Dr.T. S. Chou, Bureau of Commodity Inspection and Quaranthe, Republic of China, Taipei, Taiwan; Dr. I. C. Chopra, Regional Research Laboratory, Jammu-Tawi, Kashmir State, India;
I--
TIME [MINUTES)
TIME (MINUTES)
Fig. l.-Gas chromatograms of M.urvcnsis oils. Analytical peaks reproduced in heavy lies. Column-SAIB (20%)on Chromosorb W (acid-washed), 60-80 mesh; temperature, 170" C.; helium, i 5 ml./minute; sample volumes, 2rl.
Journal of Pharmaceutical Sciences
1010 Dr. Teikichi Hiraizumi, Takasago Perfumery Co., Ltd., Tokyo; Dr. Sumio Shiimizu, Laboratory of Agricultural Chemistry, Shinshu University, InaNagano Ken; Dr. Nagamori Ikeda. Laboratory of Plant Breeding, Okayama University, Tsushima, Okayama City, Japan; Dr. D. H. S. Horn, Council for Scientific and Industrial Research, National Chemical Research Laboratory, Pretoria, South Africa; and Dr. F. J. Cramer, A. M. Todd Co., Kalamazoo, Mich. Oil of spearmint ( M . cardiaca, M . spicatu, M . Gridis) was supplied through the courtesy of Dr. F. J. Cramer, A. M. Todd Co., Kalamazoo, Mi&, and Dr. C. Scholtens, N. V. Chemische Fabriek “Flebo,” Hoogezand, The Netherlands. Oil of pennyroyal (M. pulcgium L. and Hedcoma pulegioides (L.)Pers.) was provided through the courtesy of Dr. E. Guenther, Fritzsche Brothers, Inc.. New York. N. Y.; Dr. F. J. Cramer, A. M. Todd Co., Kalamazoo, Mich.; Dr. J. M. Quigg, Compagnie Parento, Ltd., Toronto, Ontario, Canada ; Destilaciones Bordas Chinchurreta, S.A., Seville, Spain. Oil of bergamot mint ( M . cityah Ehrh.) and oil of M. sylvcslris L. was supplied through the courtesy of Dr. F. J. Cramer. A. M. Todd Co.,Kalamazoo, Mich. Oil of M. rotundifolia was provided through the courtesy of Dr. Sumio Shimizu, Laboratory of Agricultural Chemistry, Shinshu University, InaNagano Ken, Japan. Essential Oil Constituents.-Menthofuran (ex M . piperitu, freshly distilled for use as reference = 6,750. d-Pulegone (ex standard); eE2RSl9 M.pulegizrm L.) was provided through the courtesy of Dr. F. J. Cramer. A. M.Todd Co., Kalamazoo, Mich . Isolation of Menthofuran Fraction from Two Grams of Brazilian Oil of M. arvensis Sample aliquots of 50 pl. each were subjected to gas chromatography (column: SAIB (2073,) on Chromosorb W; temperature. 170’ C.; helium: 75 ml./minute) (12) and efauents emerging just prior to menthone (retention time 4 . 5 to 7.5 minutes) trapped in carbon tetrachloride. Following evaporation of solvent, the residue obtained was examined by infrared and ultraviolet spectrosropy, thin-layer chromatography, and Fliickiger’s classical color test. Coupled Gas-Liquid-Thin-Layer Chromatograms of Essential Oils Samples ranging from 25-100 pl. were injected into the column and fractions emerging within 6.5 to 7.5 minutes (see Fig. 1) deposited directly on chromatoplates mounted a t the vapor exit as previously reported (11). n-Hexane was applied for the development of condensates thus spotted, and a 5% solution of vanillin in concentrated sulfuric acid served as spray reagent. Identification and Estimation of Menthofuran in Analytical Specimens To 10-Gm. samples of an authenticated Japanese oil of M. atvcnsis (Fliickiger test negative) were added accurately weighed amounts of menthofuran and preparations containing from 0.10 to 1.00% of the heterocycle made up as reference standards. They served as controls for compound identification and the appraisal of product recovery. Following
deposition of 0.5 to 1-pl. aliquots of these mixtures on chromatoplates, development in n-hexane for about 45 minutes, and treatment with the 5y0 vanillin-sulfuric acid reagent, characteristic deep orange-red spots were readily observed. On prolonged standing, they developed a bluish-purple hue. RESULTS AND DISCUSSION Characterization of Menthofuran FractionObtained from Brazilian M. urwfisis Infrared analysis of the isolate, prepared in accordance with the procedure described and spread as a thin film between two salt plates, showed weak yet distinct bands characteristic of menthofuran at 1680, 1640, 1566. and 735 an.-’ Marked absorptions throughout the 1200 and 920 cm.-* regions indicated admixture with menthone and linalool, respectively. A Perkin-Elmer double beam, model 221 spectrophotometer equipped with prism grating interchange and automatic gain control was used to make the measurements. It was operated at fivefold scale expansion and flushed with dry nitrogen during the experiment. Although the analytical specimen was of heterogeneous composition, purification by rechromatography was not attempted for fear of recovering insufficient material to carry out further tests. The crude isolate was therefore subjected to direct ultraviolet analysis and its U.V. characteristics compared with those of the essential oil from which it had been obtained. Taken up in 25 ml. of ethyl alcohol it exhibited peak absorbance ( e = 2.1) at 220 mp, indicative of the presence of approximately 0.03% of menthofuran in the essential oil. Similar examination of a number of M . arvensis oils showed invariably the occurrence of strong maxima or shoulders throughout the 220-240 mp region; the specimen which had served as starting material for preparation of the menthofuran concentrate displayed a maximum a t 231 mp (E::m. = 43.9). Hence the presence of trace amounts of
1
M. atynSZS
2
M.
spicatu
3
M.
cardiaca
4
Menthofuran
5 Isolate from M. amensis
Fig. 2.-CGT-Chromatograms of analytical peaks from 1, M.amnsis; 2. M.spicata; 3. M. cardiata; and TL-Chromatograms of 4, menthofuran standard, and 5. menthofuran isolate from M. atvcnsis as d m b e d under Expe~imntal.
Vol. 53, No.9, Scptcmber 1964
1022
menthofuran in these oils escaped recognition by direct ultraviolet analysis. Thus, the addition of
as much as 1% of the heterocycle to genuine preparations could not be detected with any degree of reliability. It is because of such relatively intense absorption by nonmenthofuran constituents throughout the analytical wavelength range that M.piperdu oils fail, likewise, to submit to menthofuran determinations by direct ultraviolet spectrophotometry. Following recovery of the concentrate from its ethanolic solution, a 1-MI. aliquot was examined along with a 1-pl. aliquot of reference standard (0.1% menthofuran) by thin-layer chromatography using n-hexane as developing solvent. Deep orange-red colorations of identical Rj values were observed following treatment of the chromatoplate with the spray reagent (see Fig.2). The remainder of the analytical sample was used
TABLB I.-TLC
for observing Fliickiger’s reaction. The characteristic sequence of colorations was seen clearly and a strong positive test obtained within 1 minute. Thus, each of the analyses had supplied ample evidence for the presence of menthofuran in M . a m i s , and its occurrence in this species can now be considered d n e d beyond doubt. The most convenient and perhaps also the most convincing of the criteria of identity established were those assembled by thin-layer chromatography carried out under the experimental conditions specified. Of a great many terpene compounds examined similarly in our laboratory, none showed the migration characteristics plus color response typical of menthofuran. Pertinent chemical and chemotaxonomic applications of the technique were reported (13). and further data illustrativeof its scope for the analysis of essential oils and their constituents are to be published elsewhere.
AM) CGTC OF ESSENTIAL OILS OF TBE GENUSMeutha TLC
Sam Ic Vol.,
Essential Oil
8 4.
CGTC
Sample Vol.,
Menthoforan,
+ ++ + +
0.01 0.02 0.01 0.03 0.04
++
0.04
26-100 #I.
%
Mint
M.arvensis L. Argentina Bradl APPA-MA-1 APPA-MA4 Formosa India
Specimen obtained
from Japanese mint plants raised and processed in State of Kashmir Japan “Sanbi” “Hakubi” south Africa strain of imported Japanese mint spearmint M.carGerard ex Baker dtivar Scotch or Highland M. s@katacdtivar common or native American M.mdk Pennyroyalo M.p&gium L. Spanish Moroccan Hcdcoma p&gioides (L.) Pas. Oregon race
M.sykrcshis L.. M.ci&& Ehrh.. M. rotuu&fo&ao French strain, Main constituent neo-neo-isopulegol Italian strain, Main constituent piperitenone oxide
+
0.02 0.03
0.02 0.01 0.02
++ +-
+ + -
++ + + + + + +
0.30 0.25 0.05 0.02 0.05 0.07
0.06 0.02
Specimens stored prior to analysis under nitrogen and at S°C. tor periods up to 3 years. Menthofuron contents of freshly distilled oils probably hipher than -ported. 0
Journal of Phurmaceutical Sciences
1012 Coupled Gas-Liquid-Thin-Layer Chromatograms
analyzedsome of them produced from authenticated strains cultivated under carefully controlled conditions-the possibility of the presence of menthofuran Oia admixture should also be ruled out. Fig. 1. Qualitatively their profiles are similar. Yet Contamination with peppermint can likewise be quantitative correlations of peaks observed for discounted, for M. pipcrita is not grown in Brazil, biochemically related constituents assures classifica- Formosa, and Japan. Only experimental plantation of these oils according to geographical origin tions exist at this time in Argentina and India. Finally, and equally important is the fact that (10). In each of the chromatograms a minor peak or commercially exploited M. anmsis fields in the small shoulder precedes the emergence of menthone. various producing countries are carefully supervised Shown in heavy lines it illustrates the d u e n t ex- at all times to insure that only essential oils of amined by coupled gas-liquid-thin-layer chromatog- highest quality and uniformity are reaching the raphy as described under Experimental. Codeposi- world’s markets. Menthofuran is, evidently, a tion of linalool and menthone as well as other con- genuine constituent of the species itself. stituents emerging during collection of the d u e n t hnsitivitp of Analytical Techniques does not interfere with the analyses because of the Examination of samples by the glacial aceticselectivity of the solvent system and spray reagent nitric acid reaction proceeded as expected for M. applied. In each instance color intensities observed amnsis oils. Faintly yellowish solutions formed were compared with those produced by accurately without intermediate colorations being observed. measured aliquots of menthofuran (O.~OJO)-M. Similar analyses of menthofuran-M. alvcnsis caliamnsis calibration standards examined under the bration standards proved that pasitive tests were same experimental conditions. The presence of obtained only at menthofuran concentrations 0.01 to 0.04yoof menthofuran as a genuine constitu- equal to or greater than 0.4% by weight. Thinent of M.a m s i s was thus established (see Table I). layer chromatography yielded distinctly positive reCorrelation of these results with those reported sults when applied to 1-PI. aliquots of menthofuranin previous investigations (10, 11, 14, 16) illustrates M. crrocnsis reference standards containing as little the occurrence of a given constituent at vastly as 0.1% of menthofuran by weight. M. pipmifa dderent concentrations in various species belonging oils examined similarly gave strongly positive tests to a given genus. Such findings will make impera- with less than 0.5 fil. sample aliquots necessary for tive revisions of our present concepts concerning the analysis. Having at our disposal reliable calibration biogenesis of essential oils and their constituents standards, we screened conveniently menthofuran and aid in the development of more reliable methods of plant classifications by physicochemical techniques. Because of its insensitivity, Fliickiger’s test, as this study has shown, may still be considered of distinct practical value to the industry. Yet its fundamental biogcnetic significance and chcmotaronomu importance, as implied in the literature throughout the years, no longer exist. One of the M. arumsis oils assayed, a commercial sample obtained from England. gave a positive menthofuran test (o.14yO)when 2-4. aliquots were spotted for direct thin-layer chromatography. Since adulteration of oils of M. a m s i s with oils of M. piper& would be unprofitable, the observation is probably indicative of genuine compositional variations to be encountered. Quite likely, menthofuran concentrations up to 0.2% are still true criteria of M.alvcnszs authenticity. For a time it was thought that contamination of M. anmsis with wild growing plants might account for the experimental observations. Accordingly, a Brazilian oil from such a species,’ grown experimentally in the region of Colorado (Parand State), was subjected to similar analyses. It contained 0.12% of menthofuran. (Pulegone content -SO%.) Comparable data were obtained for an American M. a m s i s oil’ produced from plants grown experimentally in Wisconsin. It would thus appear that the presence of menthofuran in M. a m s i s cannot be ascribed to contamination with wild growing mints. Since, further1 2 3 4 more, and for the purpose of clarifying this point, Fie. 3.-Thin-layer chromatograms of 1. M. a m essential oils of different geographical origins were sis (presence of menthofuran not detected); 2, M. piperdu (Italo-Mitcham)-menthofuran 6% ; 3. 1 Courtesy of Dr. M.T. M hPes, Institute de QdmiCP M. piper& (English-Mitchamementhofuran Ag~iwla,Rio de Jsoeiro, B - 9 2.6%; and 4, M. pi itCr (USA.-Yakimaj-menCourtesy of Dr. F. J. Cramer, A. M. Todd Co., galama-, Mi&. thofuran 9%. smpFm1ume-o.6pl.
The gas chromatograms of six M. a r m s i s oils of different geographical provenance are shown in
~
f
Vol. 53, No. 9, September 1964
1013
TABLEI I . S B N S I T I V I P Y OF VARIOUSTECHNIQUESgenated p-menthanes in a given specieswasquestioned and formation of one or the other of the Limit of three types considered exclusive (20). Subsequent Method of Analysis Sensitivity workers acknowledged and endorsed this classificaUltraviolet spectroscopy > 1o/b tion (21, 22). Recently, however, the Occurrence Fliickiger reaction 0.40j,m of trace amounts of 3-oxygenated terpenoids, Differential infrared menthone and isomenthone, in spearmints was ob0.25YO spectroscopy Thin-laver served in this laboratory (14). The present study chromatography o.05Y0 ( 0 . 5mcg./pl.) demonstrates the existence of a cyclic1constituent. CGTC o.olyob” menthofuran, in M. citrata. Furthermore, the occurrence of pulegone, a possible precursor to menthoa Sensitivity io solutioo slightly better thnn that reported furan formation. was likewise established in this by Zolotovich (0.6%) for spot t u t carried out on filter paper with but 1 drop of oil and reagent. respcstively (9). Sub- species by the CGTC technique. Small amounts ject to further refinement through increase of sample v o l u m a of the cyclic ketone occurred also in M. sylwsfris csed for noalyris. Such re6ocment does oot app’y to thinlayer chromatography which as a rule prlarger and M. rofundqolia. Its presence in M. c a r d k a , complex sam l a , lean efficiently than smaller ones. Thus, nooc of the h. mornsis oils listed in Table I pnve podtivc M. spicakr, M. viridis, and M. awensis was previmenthofuran tests when 1 0 - d . ali uots were spotted for ously reported (10, 14). analysis. The feature ia n d e c i d J advantage of CGTC over TLC (see Table I) and outweighs the disadvantage These observations appear to confirm that similar of material Iwhich might occur during chromastereospecific biochemical processes are taking tography. place in most, if not all, species of the genus Menfha, contents of peppermint oils available in our collec- but a t very different rates (23). A Critical biogetion, as shown in Fig. 3. netic scheme correlating the experimental data reAn infrared method for the estimation of menthoported with others now assembled will make the furan in M. pipcrikr oils was reported by Naves. subject of a forthcoming publication (24). Based on absorption intensities observed at 736 This study has shown how a delicate constituent an.-’it permitted detection of 1% of the hetero- not readily identified by direct application of cycle in genuine products. M. a m s i s oils ex- classical chemical as well as modern instrumental amined comparably failed to show the analytical methods of analysis may be detected and estimated band (16). We applied infrared spectroscopy in the various species of a given genus by means of the likewise during the course of this study and con- CGTC technique. In this regard it serves as a h e d Naves’ results. None of the essential oils further example of the value of this method for the displayed infrared absorptions suggesting the pres- more thorough exploration of specific compositional ence of menthofuran. Only when using differential regions comprising a gas chromatogram, for the techniques (17, 18) and operating the instrument at isolation and determination of a minor constituent fivefold scale expansion was it possible t o detect in a complex natural material, and hence for the the addition of 0.25% of menthofuran t o authenti- establishment of new, sensitive criteria of product cated preparations. With menthofuran concentra- authenticity. tions in M. amensis below this level d i r e infrarrd REFERENCES procedures would presently appear to be inadequate (1) Plflckiger. F. A,, Pharm. J . . 3. (No. 1) 682: ibid.. methods for such analyses. 12) 32111871) ~ ~ .~ ~ ~ (2 Polenske. E.,Pharm. Zfg., 35. 647 1880) The sensitivity of the various techniques applied (31 Carla,, H . . Parfum. M o d . . 22, 615[1929): is summarized in Table 11. (4) Wienhnus, H..nnd D c w a n . H.. A n g m . Chem., 47, A 1 K I 10741 Examination of Essential Oils from Other Mint (5 Trcibs. W., Bn.,70B,M(1937). Species (61 Guenther. E . , “The Essential Oils,” Vol. 1, D.Van Nostrand Co., Ioc., New York. N. Y.. 1948. p. 343. CGTC-analyses based on the experimental tech(7) “British Pharmncopoda,” Pharmaceutical Press, London, Eoglnod. 1968, niques described were also carried out on oil of (8) :‘United Stntea 8h:%copeia.” 16th rev., Mack spearmint ( M .cordiota, M. spicah, M. mridas), Publishin Co., Ea*oo, Pa., 1960, p. 613. (9) d o t o v i c h . 0..C a P f . Rend. Acad. Bulg. S c i . . 13, oil of pennyroyal ( M . pukgium and Hedcoma pule(No.3) 343(1880). gcoidcs), oil of bergamot mint ( M . cilrata), oil of M. (10) Smith, D. M.,and Levi, L., J . Agr. Food Chrm.. 9, sylocstris, and oil of M.rotundifolia. The presence m n i 1M I (11) N i w m . I. C.. SohoarPbudhc, M., and Levi, L., Can. of menthofuran in each of these species was thus J . C h m . , 41, 1 6 3 6 ( 1 W ) . (12) Smith. D. M., W e t , J. C.. nod Levi. L.. Anal. likewise established for the first time (see Table I). >-,
BIOCHEMICAL SIGNIFICANCE OF EXPERIMENTAL RESULTS The specificity of the metabolic pathways governing formation of carvone derivatives in spearmints and of menthone derivatives in peppermints was first pointed out by Kremers (19). Later, Reitsema substantiated and expanded these observations on the basis of a series of profound and extensive studies, grouping mint oils into the spearmints, peppermints, and lemon mints. Spearmints were characterized by the presence of 2-xygenated p menthanes, (camone group), peppermints (including M.aroensis and M.pukgium) by the presence of 3-oxygenated p-menthanes (menthone group), and lemon mints by the presence of acyclic compounds. The coexistence of both 2- and 3-0xy-
Chon., 32, 668(1960). io,’,l~mN~m I. ,C..,and L e d . L.. “Abstrpcts.” 144th meetcao Chermcd S a i e t y . L m Angelu. Calif., April 6, 1963 MA. (14) ’AthD. , M.. %turn. W., and Levi. L.. J . A i r . Food C h . . 11. 276(1963). (16) Airth, J. M.,strlllpa, B.,Skakum, W . , and Levi. L., 1. Assoc. OiAc. A r r . Ciknnisk, 45,476(1962). (16) Naves, Y. R . , Bull. Soc. Chim. France. 1954, 657. (17) Roo. G . S. K . , Staturn,, W . , nod Levi. L.. “Infrared Examination of Ylaog-Ylmg 011,” The American Perfumer. ChicaKo. Ill., 1882. (18) R a o 0. S . K . Nigam I . C. Bartlet J. C. and Levi, L., Proc. S A . s e d . TAM & A&., 313, d(ima). (19) Kremers. R . E.,J . Bid. C h m . , 50, 31(1922). (20) ReitJema. K. H.,THIS JOURNAL, 43, 414(1964); ibid.. 47, 265, 267(1958). (21) BPttde. J.. and Loomis, W. D., Biachim. Biophys. Acla. 51. M6(1961). (22) Rdtsemn. R. H..C m m a , P. W y , N . J., and Choroey, W.. TEISJOUI~NAL, 50, l S ( l k 1 ) . (23) ,Hands. K. L . , Smith,, D. M., and L e d . L.. “ 4 b atmcts, 141st mutrng, AmencDn Chemical Sodcty. Washington, D. C.. March 29, 1962, p. 14A. (24) Hands. K . L., N i p m . I. C . , Smith, D. M., M d Levi, L.,T m s J O V ~ N A L ,in press.
.
the liquid develops a blue color, which on continued heating deepens and shows a copper fluorescence, then fades, leaving a golden yellow this sequence of acetienitric acid solution (1). In 1890, Polen- solution (6). With M.a&s, ske suggested that the phenomenon was due to color changes is not observed, the test solution the presence of a volatile, nonnitrogenous con- attaining merely a light yellowish tinge which stituent in the essential oil (2). Thirtv years undergoes no marked change during the 5-minute later the compound. menthofuran, was isolated heating period. Wering a valuable criterion for distinguishing by Carles as an “ether-oxide” from the flower oil of Italian peppermint (3). Its constitution readily two closely allied and commercially most was established soon afterwards by Wienhaus important products of the genus Mcntha, the and Dewein (4). In 1936, Treibs synthesized reaction was soon considered and still is an the heterocycle and demonstrated that its official specification (7, 8). As a spot test properties were identical with those of the natural requiring but 1 drop of material, it was later utilized by Zolotovich for the examination of M . isolate (5). As a criterion of identity for M. ~perit0, amensis as well as M.Piperita oils and many of Flkkiger’s test came to be applied widely and theirhybrids (9). Studies camed out recently in our laboratory with only slight modifications. It is generally carried out as follows. Three drops of the es- on products of different geographical origins scntial oil are mixed in a dry test tube with 5 confirmed the specificity generally ascribed to rnl. of a solution of 1 vol. of nitric acid in 300 the reaction (10). Yet, not always were exvol. of glacial acetic acid, and the tube is placed perimental data entirely satisfactory, and the in a beaker of boiling water. Within 5 minutes investigation was continued. It is the purpose of this paper to report the results that we obReceived October 2, 196t. from the Food and Drug Directained using coupled gas-liquid-thin-layer chrotorate. Department of National Health and Welfare, Ottawa, Ontario Canada. matography as the method of analysis. Aceeited for publication November 5, 108.3. years ago Fliickiger A observed that peppermint oil gave a characteristic color reaction when heated in glacial LMOST ONE-HUNDRED
The authors are indebted t o W. Skakum for technical assistance. Previous paper: Pcrfumrry Esscnl. Oil Record. 54, 814 (1‘383).
*
National Research Council of Canada Postductoral Fellow, 1962-1963.
EXPERIMENTAL Apparatus and analytical procedures were previously described (11).
Vol. 53, No. 9, September 1964 Materiala EaaenW m.41of mint (M.u ~ s i s was ) provided through the courtesy of Dr.Juan-Carlos Tuja, Cultivos Industriales. S.A.. Buenos Aires, Argentina,Drs. M.T.MagalhHes and 0.R. Gottlieb,
20
1009 Instituto de Quimica Agricola, Rio de Janeiro, Brazil; Dr.T. S. Chou, Bureau of Commodity Inspection and Quaranthe, Republic of China, Taipei, Taiwan; Dr. I. C. Chopra, Regional Research Laboratory, Jammu-Tawi, Kashmir State, India;
I--
TIME [MINUTES)
TIME (MINUTES)
Fig. l.-Gas chromatograms of M.urvcnsis oils. Analytical peaks reproduced in heavy lies. Column-SAIB (20%)on Chromosorb W (acid-washed), 60-80 mesh; temperature, 170" C.; helium, i 5 ml./minute; sample volumes, 2rl.
Journal of Pharmaceutical Sciences
1010 Dr. Teikichi Hiraizumi, Takasago Perfumery Co., Ltd., Tokyo; Dr. Sumio Shiimizu, Laboratory of Agricultural Chemistry, Shinshu University, InaNagano Ken; Dr. Nagamori Ikeda. Laboratory of Plant Breeding, Okayama University, Tsushima, Okayama City, Japan; Dr. D. H. S. Horn, Council for Scientific and Industrial Research, National Chemical Research Laboratory, Pretoria, South Africa; and Dr. F. J. Cramer, A. M. Todd Co., Kalamazoo, Mich. Oil of spearmint ( M . cardiaca, M . spicatu, M . Gridis) was supplied through the courtesy of Dr. F. J. Cramer, A. M. Todd Co., Kalamazoo, Mi&, and Dr. C. Scholtens, N. V. Chemische Fabriek “Flebo,” Hoogezand, The Netherlands. Oil of pennyroyal (M. pulcgium L. and Hedcoma pulegioides (L.)Pers.) was provided through the courtesy of Dr. E. Guenther, Fritzsche Brothers, Inc.. New York. N. Y.; Dr. F. J. Cramer, A. M. Todd Co., Kalamazoo, Mich.; Dr. J. M. Quigg, Compagnie Parento, Ltd., Toronto, Ontario, Canada ; Destilaciones Bordas Chinchurreta, S.A., Seville, Spain. Oil of bergamot mint ( M . cityah Ehrh.) and oil of M. sylvcslris L. was supplied through the courtesy of Dr. F. J. Cramer. A. M. Todd Co.,Kalamazoo, Mich. Oil of M. rotundifolia was provided through the courtesy of Dr. Sumio Shimizu, Laboratory of Agricultural Chemistry, Shinshu University, InaNagano Ken, Japan. Essential Oil Constituents.-Menthofuran (ex M . piperitu, freshly distilled for use as reference = 6,750. d-Pulegone (ex standard); eE2RSl9 M.pulegizrm L.) was provided through the courtesy of Dr. F. J. Cramer. A. M.Todd Co., Kalamazoo, Mich . Isolation of Menthofuran Fraction from Two Grams of Brazilian Oil of M. arvensis Sample aliquots of 50 pl. each were subjected to gas chromatography (column: SAIB (2073,) on Chromosorb W; temperature. 170’ C.; helium: 75 ml./minute) (12) and efauents emerging just prior to menthone (retention time 4 . 5 to 7.5 minutes) trapped in carbon tetrachloride. Following evaporation of solvent, the residue obtained was examined by infrared and ultraviolet spectrosropy, thin-layer chromatography, and Fliickiger’s classical color test. Coupled Gas-Liquid-Thin-Layer Chromatograms of Essential Oils Samples ranging from 25-100 pl. were injected into the column and fractions emerging within 6.5 to 7.5 minutes (see Fig. 1) deposited directly on chromatoplates mounted a t the vapor exit as previously reported (11). n-Hexane was applied for the development of condensates thus spotted, and a 5% solution of vanillin in concentrated sulfuric acid served as spray reagent. Identification and Estimation of Menthofuran in Analytical Specimens To 10-Gm. samples of an authenticated Japanese oil of M. atvcnsis (Fliickiger test negative) were added accurately weighed amounts of menthofuran and preparations containing from 0.10 to 1.00% of the heterocycle made up as reference standards. They served as controls for compound identification and the appraisal of product recovery. Following
deposition of 0.5 to 1-pl. aliquots of these mixtures on chromatoplates, development in n-hexane for about 45 minutes, and treatment with the 5y0 vanillin-sulfuric acid reagent, characteristic deep orange-red spots were readily observed. On prolonged standing, they developed a bluish-purple hue. RESULTS AND DISCUSSION Characterization of Menthofuran FractionObtained from Brazilian M. urwfisis Infrared analysis of the isolate, prepared in accordance with the procedure described and spread as a thin film between two salt plates, showed weak yet distinct bands characteristic of menthofuran at 1680, 1640, 1566. and 735 an.-’ Marked absorptions throughout the 1200 and 920 cm.-* regions indicated admixture with menthone and linalool, respectively. A Perkin-Elmer double beam, model 221 spectrophotometer equipped with prism grating interchange and automatic gain control was used to make the measurements. It was operated at fivefold scale expansion and flushed with dry nitrogen during the experiment. Although the analytical specimen was of heterogeneous composition, purification by rechromatography was not attempted for fear of recovering insufficient material to carry out further tests. The crude isolate was therefore subjected to direct ultraviolet analysis and its U.V. characteristics compared with those of the essential oil from which it had been obtained. Taken up in 25 ml. of ethyl alcohol it exhibited peak absorbance ( e = 2.1) at 220 mp, indicative of the presence of approximately 0.03% of menthofuran in the essential oil. Similar examination of a number of M . arvensis oils showed invariably the occurrence of strong maxima or shoulders throughout the 220-240 mp region; the specimen which had served as starting material for preparation of the menthofuran concentrate displayed a maximum a t 231 mp (E::m. = 43.9). Hence the presence of trace amounts of
1
M. atynSZS
2
M.
spicatu
3
M.
cardiaca
4
Menthofuran
5 Isolate from M. amensis
Fig. 2.-CGT-Chromatograms of analytical peaks from 1, M.amnsis; 2. M.spicata; 3. M. cardiata; and TL-Chromatograms of 4, menthofuran standard, and 5. menthofuran isolate from M. atvcnsis as d m b e d under Expe~imntal.
Vol. 53, No.9, Scptcmber 1964
1022
menthofuran in these oils escaped recognition by direct ultraviolet analysis. Thus, the addition of
as much as 1% of the heterocycle to genuine preparations could not be detected with any degree of reliability. It is because of such relatively intense absorption by nonmenthofuran constituents throughout the analytical wavelength range that M.piperdu oils fail, likewise, to submit to menthofuran determinations by direct ultraviolet spectrophotometry. Following recovery of the concentrate from its ethanolic solution, a 1-MI. aliquot was examined along with a 1-pl. aliquot of reference standard (0.1% menthofuran) by thin-layer chromatography using n-hexane as developing solvent. Deep orange-red colorations of identical Rj values were observed following treatment of the chromatoplate with the spray reagent (see Fig.2). The remainder of the analytical sample was used
TABLB I.-TLC
for observing Fliickiger’s reaction. The characteristic sequence of colorations was seen clearly and a strong positive test obtained within 1 minute. Thus, each of the analyses had supplied ample evidence for the presence of menthofuran in M . a m i s , and its occurrence in this species can now be considered d n e d beyond doubt. The most convenient and perhaps also the most convincing of the criteria of identity established were those assembled by thin-layer chromatography carried out under the experimental conditions specified. Of a great many terpene compounds examined similarly in our laboratory, none showed the migration characteristics plus color response typical of menthofuran. Pertinent chemical and chemotaxonomic applications of the technique were reported (13). and further data illustrativeof its scope for the analysis of essential oils and their constituents are to be published elsewhere.
AM) CGTC OF ESSENTIAL OILS OF TBE GENUSMeutha TLC
Sam Ic Vol.,
Essential Oil
8 4.
CGTC
Sample Vol.,
Menthoforan,
+ ++ + +
0.01 0.02 0.01 0.03 0.04
++
0.04
26-100 #I.
%
Mint
M.arvensis L. Argentina Bradl APPA-MA-1 APPA-MA4 Formosa India
Specimen obtained
from Japanese mint plants raised and processed in State of Kashmir Japan “Sanbi” “Hakubi” south Africa strain of imported Japanese mint spearmint M.carGerard ex Baker dtivar Scotch or Highland M. s@katacdtivar common or native American M.mdk Pennyroyalo M.p&gium L. Spanish Moroccan Hcdcoma p&gioides (L.) Pas. Oregon race
M.sykrcshis L.. M.ci&& Ehrh.. M. rotuu&fo&ao French strain, Main constituent neo-neo-isopulegol Italian strain, Main constituent piperitenone oxide
+
0.02 0.03
0.02 0.01 0.02
++ +-
+ + -
++ + + + + + +
0.30 0.25 0.05 0.02 0.05 0.07
0.06 0.02
Specimens stored prior to analysis under nitrogen and at S°C. tor periods up to 3 years. Menthofuron contents of freshly distilled oils probably hipher than -ported. 0
Journal of Phurmaceutical Sciences
1012 Coupled Gas-Liquid-Thin-Layer Chromatograms
analyzedsome of them produced from authenticated strains cultivated under carefully controlled conditions-the possibility of the presence of menthofuran Oia admixture should also be ruled out. Fig. 1. Qualitatively their profiles are similar. Yet Contamination with peppermint can likewise be quantitative correlations of peaks observed for discounted, for M. pipcrita is not grown in Brazil, biochemically related constituents assures classifica- Formosa, and Japan. Only experimental plantation of these oils according to geographical origin tions exist at this time in Argentina and India. Finally, and equally important is the fact that (10). In each of the chromatograms a minor peak or commercially exploited M. anmsis fields in the small shoulder precedes the emergence of menthone. various producing countries are carefully supervised Shown in heavy lines it illustrates the d u e n t ex- at all times to insure that only essential oils of amined by coupled gas-liquid-thin-layer chromatog- highest quality and uniformity are reaching the raphy as described under Experimental. Codeposi- world’s markets. Menthofuran is, evidently, a tion of linalool and menthone as well as other con- genuine constituent of the species itself. stituents emerging during collection of the d u e n t hnsitivitp of Analytical Techniques does not interfere with the analyses because of the Examination of samples by the glacial aceticselectivity of the solvent system and spray reagent nitric acid reaction proceeded as expected for M. applied. In each instance color intensities observed amnsis oils. Faintly yellowish solutions formed were compared with those produced by accurately without intermediate colorations being observed. measured aliquots of menthofuran (O.~OJO)-M. Similar analyses of menthofuran-M. alvcnsis caliamnsis calibration standards examined under the bration standards proved that pasitive tests were same experimental conditions. The presence of obtained only at menthofuran concentrations 0.01 to 0.04yoof menthofuran as a genuine constitu- equal to or greater than 0.4% by weight. Thinent of M.a m s i s was thus established (see Table I). layer chromatography yielded distinctly positive reCorrelation of these results with those reported sults when applied to 1-PI. aliquots of menthofuranin previous investigations (10, 11, 14, 16) illustrates M. crrocnsis reference standards containing as little the occurrence of a given constituent at vastly as 0.1% of menthofuran by weight. M. pipmifa dderent concentrations in various species belonging oils examined similarly gave strongly positive tests to a given genus. Such findings will make impera- with less than 0.5 fil. sample aliquots necessary for tive revisions of our present concepts concerning the analysis. Having at our disposal reliable calibration biogenesis of essential oils and their constituents standards, we screened conveniently menthofuran and aid in the development of more reliable methods of plant classifications by physicochemical techniques. Because of its insensitivity, Fliickiger’s test, as this study has shown, may still be considered of distinct practical value to the industry. Yet its fundamental biogcnetic significance and chcmotaronomu importance, as implied in the literature throughout the years, no longer exist. One of the M. arumsis oils assayed, a commercial sample obtained from England. gave a positive menthofuran test (o.14yO)when 2-4. aliquots were spotted for direct thin-layer chromatography. Since adulteration of oils of M. a m s i s with oils of M. piper& would be unprofitable, the observation is probably indicative of genuine compositional variations to be encountered. Quite likely, menthofuran concentrations up to 0.2% are still true criteria of M.alvcnszs authenticity. For a time it was thought that contamination of M. anmsis with wild growing plants might account for the experimental observations. Accordingly, a Brazilian oil from such a species,’ grown experimentally in the region of Colorado (Parand State), was subjected to similar analyses. It contained 0.12% of menthofuran. (Pulegone content -SO%.) Comparable data were obtained for an American M. a m s i s oil’ produced from plants grown experimentally in Wisconsin. It would thus appear that the presence of menthofuran in M. a m s i s cannot be ascribed to contamination with wild growing mints. Since, further1 2 3 4 more, and for the purpose of clarifying this point, Fie. 3.-Thin-layer chromatograms of 1. M. a m essential oils of different geographical origins were sis (presence of menthofuran not detected); 2, M. piperdu (Italo-Mitcham)-menthofuran 6% ; 3. 1 Courtesy of Dr. M.T. M hPes, Institute de QdmiCP M. piper& (English-Mitchamementhofuran Ag~iwla,Rio de Jsoeiro, B - 9 2.6%; and 4, M. pi itCr (USA.-Yakimaj-menCourtesy of Dr. F. J. Cramer, A. M. Todd Co., galama-, Mi&. thofuran 9%. smpFm1ume-o.6pl.
The gas chromatograms of six M. a r m s i s oils of different geographical provenance are shown in
~
f
Vol. 53, No. 9, September 1964
1013
TABLEI I . S B N S I T I V I P Y OF VARIOUSTECHNIQUESgenated p-menthanes in a given specieswasquestioned and formation of one or the other of the Limit of three types considered exclusive (20). Subsequent Method of Analysis Sensitivity workers acknowledged and endorsed this classificaUltraviolet spectroscopy > 1o/b tion (21, 22). Recently, however, the Occurrence Fliickiger reaction 0.40j,m of trace amounts of 3-oxygenated terpenoids, Differential infrared menthone and isomenthone, in spearmints was ob0.25YO spectroscopy Thin-laver served in this laboratory (14). The present study chromatography o.05Y0 ( 0 . 5mcg./pl.) demonstrates the existence of a cyclic1constituent. CGTC o.olyob” menthofuran, in M. citrata. Furthermore, the occurrence of pulegone, a possible precursor to menthoa Sensitivity io solutioo slightly better thnn that reported furan formation. was likewise established in this by Zolotovich (0.6%) for spot t u t carried out on filter paper with but 1 drop of oil and reagent. respcstively (9). Sub- species by the CGTC technique. Small amounts ject to further refinement through increase of sample v o l u m a of the cyclic ketone occurred also in M. sylwsfris csed for noalyris. Such re6ocment does oot app’y to thinlayer chromatography which as a rule prlarger and M. rofundqolia. Its presence in M. c a r d k a , complex sam l a , lean efficiently than smaller ones. Thus, nooc of the h. mornsis oils listed in Table I pnve podtivc M. spicakr, M. viridis, and M. awensis was previmenthofuran tests when 1 0 - d . ali uots were spotted for ously reported (10, 14). analysis. The feature ia n d e c i d J advantage of CGTC over TLC (see Table I) and outweighs the disadvantage These observations appear to confirm that similar of material Iwhich might occur during chromastereospecific biochemical processes are taking tography. place in most, if not all, species of the genus Menfha, contents of peppermint oils available in our collec- but a t very different rates (23). A Critical biogetion, as shown in Fig. 3. netic scheme correlating the experimental data reAn infrared method for the estimation of menthoported with others now assembled will make the furan in M. pipcrikr oils was reported by Naves. subject of a forthcoming publication (24). Based on absorption intensities observed at 736 This study has shown how a delicate constituent an.-’it permitted detection of 1% of the hetero- not readily identified by direct application of cycle in genuine products. M. a m s i s oils ex- classical chemical as well as modern instrumental amined comparably failed to show the analytical methods of analysis may be detected and estimated band (16). We applied infrared spectroscopy in the various species of a given genus by means of the likewise during the course of this study and con- CGTC technique. In this regard it serves as a h e d Naves’ results. None of the essential oils further example of the value of this method for the displayed infrared absorptions suggesting the pres- more thorough exploration of specific compositional ence of menthofuran. Only when using differential regions comprising a gas chromatogram, for the techniques (17, 18) and operating the instrument at isolation and determination of a minor constituent fivefold scale expansion was it possible t o detect in a complex natural material, and hence for the the addition of 0.25% of menthofuran t o authenti- establishment of new, sensitive criteria of product cated preparations. With menthofuran concentra- authenticity. tions in M. amensis below this level d i r e infrarrd REFERENCES procedures would presently appear to be inadequate (1) Plflckiger. F. A,, Pharm. J . . 3. (No. 1) 682: ibid.. methods for such analyses. 12) 32111871) ~ ~ .~ ~ ~ (2 Polenske. E.,Pharm. Zfg., 35. 647 1880) The sensitivity of the various techniques applied (31 Carla,, H . . Parfum. M o d . . 22, 615[1929): is summarized in Table 11. (4) Wienhnus, H..nnd D c w a n . H.. A n g m . Chem., 47, A 1 K I 10741 Examination of Essential Oils from Other Mint (5 Trcibs. W., Bn.,70B,M(1937). Species (61 Guenther. E . , “The Essential Oils,” Vol. 1, D.Van Nostrand Co., Ioc., New York. N. Y.. 1948. p. 343. CGTC-analyses based on the experimental tech(7) “British Pharmncopoda,” Pharmaceutical Press, London, Eoglnod. 1968, niques described were also carried out on oil of (8) :‘United Stntea 8h:%copeia.” 16th rev., Mack spearmint ( M .cordiota, M. spicah, M. mridas), Publishin Co., Ea*oo, Pa., 1960, p. 613. (9) d o t o v i c h . 0..C a P f . Rend. Acad. Bulg. S c i . . 13, oil of pennyroyal ( M . pukgium and Hedcoma pule(No.3) 343(1880). gcoidcs), oil of bergamot mint ( M . cilrata), oil of M. (10) Smith, D. M.,and Levi, L., J . Agr. Food Chrm.. 9, sylocstris, and oil of M.rotundifolia. The presence m n i 1M I (11) N i w m . I. C.. SohoarPbudhc, M., and Levi, L., Can. of menthofuran in each of these species was thus J . C h m . , 41, 1 6 3 6 ( 1 W ) . (12) Smith. D. M., W e t , J. C.. nod Levi. L.. Anal. likewise established for the first time (see Table I). >-,
BIOCHEMICAL SIGNIFICANCE OF EXPERIMENTAL RESULTS The specificity of the metabolic pathways governing formation of carvone derivatives in spearmints and of menthone derivatives in peppermints was first pointed out by Kremers (19). Later, Reitsema substantiated and expanded these observations on the basis of a series of profound and extensive studies, grouping mint oils into the spearmints, peppermints, and lemon mints. Spearmints were characterized by the presence of 2-xygenated p menthanes, (camone group), peppermints (including M.aroensis and M.pukgium) by the presence of 3-oxygenated p-menthanes (menthone group), and lemon mints by the presence of acyclic compounds. The coexistence of both 2- and 3-0xy-
Chon., 32, 668(1960). io,’,l~mN~m I. ,C..,and L e d . L.. “Abstrpcts.” 144th meetcao Chermcd S a i e t y . L m Angelu. Calif., April 6, 1963 MA. (14) ’AthD. , M.. %turn. W., and Levi. L.. J . A i r . Food C h . . 11. 276(1963). (16) Airth, J. M.,strlllpa, B.,Skakum, W . , and Levi. L., 1. Assoc. OiAc. A r r . Ciknnisk, 45,476(1962). (16) Naves, Y. R . , Bull. Soc. Chim. France. 1954, 657. (17) Roo. G . S. K . , Staturn,, W . , nod Levi. L.. “Infrared Examination of Ylaog-Ylmg 011,” The American Perfumer. ChicaKo. Ill., 1882. (18) R a o 0. S . K . Nigam I . C. Bartlet J. C. and Levi, L., Proc. S A . s e d . TAM & A&., 313, d(ima). (19) Kremers. R . E.,J . Bid. C h m . , 50, 31(1922). (20) ReitJema. K. H.,THIS JOURNAL, 43, 414(1964); ibid.. 47, 265, 267(1958). (21) BPttde. J.. and Loomis, W. D., Biachim. Biophys. Acla. 51. M6(1961). (22) Rdtsemn. R. H..C m m a , P. W y , N . J., and Choroey, W.. TEISJOUI~NAL, 50, l S ( l k 1 ) . (23) ,Hands. K. L . , Smith,, D. M., and L e d . L.. “ 4 b atmcts, 141st mutrng, AmencDn Chemical Sodcty. Washington, D. C.. March 29, 1962, p. 14A. (24) Hands. K . L., N i p m . I. C . , Smith, D. M., M d Levi, L.,T m s J O V ~ N A L ,in press.
.