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Essential Oils From Herbs And Spices Grown In Alberta
Essential Oils From Herbs And Spices Grown In Alberta Peppermint oil, Mentha piperita var. Mitcham, L.* M. B. Embong**, L. Steele, and D. Hadziyev Department of Food Science University of Alberta Edmonton, Alta. T6G 2N2 and
S. Molnar Alberta Horticultural Research Center Brooks, Alta. TOJ OJO * Previous report: Sage oil. Salvia officinalis. L (Labiatae). Can. lost. Food Sci. Techno!' J.. 10:201-207 (1977) ... Recipient of Canadian Commonwealth Fellowship. Permanent address: Universiti Kebangsaan Malaysia, Kuala Lumpur. Malaysia.
Abstract
Introduction
parts of the flowering plants, when steam distilled, give the mint oil. This oil is one of the indispensable items in the food flavoring industry, with the major market being chewing gum and toothpaste production. The fresh or dried herb, itself, has very limited practical use for flavoring. Mentha arvensis, L. (synonymous to Mentha canadensis) is found among the native wild flora of the Prairies and Rocky Mountains, especially near banks of irrigation ditches, sloughs, lakes, and streams. Growing along with it are numerous species of this genus, as well as some mint garden 'escapees' such as citrata, pulegium, rotundifolia, spicata, or even piperita. According to Canadian Food and Drug Regulations (B.1O.019), peppermint oil must be obtained from the leaves and flowering tops of Mentha piperita, L., and/or Mentha arvensis, var. piperascens, Holmes. In addition, either oil must contain not less than 50% of free and combined menthol. The main commercial growing regions of mint are in the United States, mostly in the Midwest States of Michigan and Wisconsin, and in Oregon, Washington and Idaho. Their total area of peppermint harvested in 1976 is estimated at a record 29,000 ha, with an average oil yield of 61.5 kg/ha (USDA Statistical Reporting Service, June 1976). Mint is not grown commercially in Canada at present, although, preliminary research in Manitoba (Dorrell, 1972), and in Saskatchewan (Hamon and Zuck, 1972) indicated that good quality mint oil can be produced. The current domestic requirement of the oil is met by imports exceeding 45 tons per year (DBS, 1971-1975). . In this investigation the performance of black MItcham mint (Mentha piperita Huds., var. officinalis, forma rubescens Camus) grown in Southern, Central, and Northern Alberta (latitudes of 50° 33',53° 42', and 56°, respectively) was assessed during five seasons. Determinati.oOS were made of the effects of irrigation and harvest t1m.e upon the yield of oil and the amount of the major and mInor oil constituents.
Several species of mint are grown, but the most common for commercial use are peppermint (Mentha piperita, L.), and spearmint (Mentha spicata, L.). The above ground
(a) Plant material. In the spring of 1969 certified Mentha
This study was undertaken to indicate whether or not a satisfactory peppermint oil could be produced in the Alberta environment with its short growing season. The horticultural performance of the solid mint stand over three consecutive seasons was rated as good for both central Alberta and southern Alberta, where irrigation was also used. Satisfactory maximum yields, near 7100 kg/ha of the ~plant and 86 kg/ha of the oil (1.2% of dried herb), were obtained at the full bloom stage. TL- and G L-chromatography and mass spectral analysis were used to determine the major and minor oil constituents. The oil composition, which changed with time, had its most ideal profile at 20% bloom in southern Alberta, and at full bloom in central Alberta. At these stages the respective percentages for southern and central oils were: total menthol (53.1, 54.6), free menthol (47.5. 41.2), menthyl esters (4.2,3.8), menthone (27.8, 25.8), and menthofuran (2.8, 1.7). The central Alberta oils, at all stages of plant development, were lower in free menthol, menthyl esters, and, especially, in menthofuran. Alberta oils could be differentiated from oils of other geographical origins through compositional analysis. Finally, the quality of Alberta oils was assessed relative to a standard quality peppermint oil from the United States which is used by the domestic food industry.
Resume On a etudie la possibilite de produire de I'huile de menthe dans les regions d'Alberta ou la saison de croissance est courte. La performance horticole de la menthe en semis dense a ete bonne, au cours de trois saisons consecutives, dans les regions du centre et du sud d'Alberta ou I'on pratique I'irrigation. Des rendements maximums satisfaisants, 7100 kg/ha de plante, et 86 kg/ha d'huile (1.2% de I'herbe, base seche), ont ete obtenus au stade de pleine floraison. On a determine les constituants majeurs et mineurs de l'huile a I'aide de chromatographies en couche mince et gaz-liquide et de spectrometrie de masse. La composition de I'huile, qui a varie avec Ie temps, a eu son meilleur profil au stade de 20% de floraison dans Ie sud d'Alberta, et au stade de pleine floraison dans Ie centre d'AIberta. Aces stades, les compositions (%) des huiles ont ete respectivement pour les regions du sud et du centre comme suit: menthol total (53.1, 54.6), menthollibre (47.5,41.2), esters menthyliques (4.2, 3.8), menthone (27.8, 25.8), et menthofuran (2.8, 1.7). Les huiles du centre d'Alberta, a tous les stades de developpement de la plante, ont ete plus faibles en menthol libre, en esters menthyliques, et surtout, en menthofuran. II est possible, par la composition, de diffhencier les huiles d' Alberta des huiles en provenance d'ailleurs. On a enfin evalue la qualite des huiles d'Alberta par rapport a une huile de menthe standard des Etats-Unis qui est utilisee en industrie alimentaire au Canada.
247
Materials and Methods J. Inst. Can. Sci. Technol. Aliment. Vol.
10. No.4. October 1917:1 ::1
piperita L., var. black Mitcham roots were obtained from Oregon and were planted and propagated in a greenhouse. On the 15th of May, 1970, they were transplanted into field plots in rows 90 em apart with 30 cm between plants. Plots were at Beaverlodge in Northern, Two Hills in Central, and Brooks in Southern Alberta. Before planting, 335 kg/ha of 11-48-0 N:P:K were applied to the sandy loam (pH 7.9, low in organic matter and lime). In June 220 kgl ha of 34-0-0 were applied as a side dressing about 8-10 cm along both sides of the row, and slightly below the surface (Gretskaya, 1973; Franz, 1972; Nelson et al., 1971a,b). In Southern Alberta irrigation equivalent to 30-45 cm was applied four times per season using furrows placed 90 cm apart (Kruepper et al., 1968). Field care during the first season consisted of rototilling between the rows to remove stolons, roots, and weeds. Weeds within the rows were removed by hand. Harvesting was done at various stages of bloom in August and early September, with a total of seven to ten harvests about 10 days apart. Over the winter the plots were either covered by straw, or left unprotected. The following season the same N:P fertilizer as before was applied in early spring and incorporated by a shallow rototilling of the entire soil surface, while the N fertilizer was side banded in June at the same rate as before. The selective control of annual and perennial weeds within the established field was achieved by applying TREFLAN (a,a,a-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) at a rate of 0.55 to 0.85 kg/ha, and/or TERBACIL (3-tert-butyl-5-chloro-6-methyluracil) at a rate of 0.9 to 1.8 kg/ha. Both herbicides were incorporated in the soil by preemergence broadcast plus rototilling. In the third season the plants were allowed to completely fill in between the rows, and they had done so by the middle of August. Subtreatments with fertilizer and herbicides were continued for two years. The essential oil was obtained from plants which were cut close to the ground, and then partially air dried for two days prior to steam distillation (Watson and St. John, 1955). The resultant oil was separated from the water, and dried over anhydrous sodium sulfate. It was stored in sealed tubes at 4°C to await chemical analysis. (b) Chemicals. Pure compounds used as standards in TLchromatography, GL-co-chromatography, and infrared and mass spectral analysis were obtained from Aldrich Chern. Co. Inc., Milwaukee, Wisc., K & K Labs. Inc., Plainview, N.Y., and Eastman Org. Chern., Rochester, N.Y. Isomenthol and neoisomenthol (from isomenthone) and neomenthol (from menthone) were synthesized by us USing aluminum isopropoxide and applying the MeerweinPonndorf-Verley reduction procedure. Neomenthyl- and ~eoisomenthyl acetate were also prepared by us by refluxIn~ the appropriate alcohols in xylene with glacial acetic aCId, acetic anhydride and fused sodium acetate. A sample of standard quality essential oil from peppermint, representative of those supplied to the food industry, and one which meets the standards of the United States and other Pharmacopoeia, was obtained from H. G. Hotchkiss Essential Oil Co., Lyons, Wayne County, N.Y. (c) Chemical analysis. Preparative TL-chromatography Was done using silicic acid as adsorbent, with a solvent syst~m ofbenzene:ethyl acetate, 95:5 (v/v). The separation of oIl constituents into hydrocarbon, oxide, ester, carbonyl, Can. Ins!. Food Sci. Technol. J. Vol. 10. No.4, October 1977
and alcohol classes was based on the differences in th . polarity. The compounds within each band were recover~~ In ethyl acetate and further analyzed by G LC. GL-chromatography was performed on a Bendix M 2500 gas chromatograph equipped with 1.8 m X 3 mm I.D. glass columns packed with 15% EGS on Chromosorb P, AW, 100/120 mesh. Operating conditions were: injection temperature 200°C, N 2 carrier gas flow rate 30 mIl min, and temperature programming from 60-150°C at I ° I min. A flame ionization detector was used. A Varian Aerograph 1400 with a TC detector was used in separations for mass spectral determinations. Two GLC columns, designated 'fresh' (one which was freshly prepared), and 'aged' (one which had been in use for at least four months) were used in combination to achieve an effective separation of the major and minor oil constituents. G LC runs were usually preceded by separation of the oil constituents into classes by preparative TLC. As an alternative to a detailed analysis, GL-chromatogram computations were limited to total menthol (1.menthol + its stereoisomers + menthyl esters), L-menthol, menthyl acetate, total menthone (menthone + isomenthone), menthofuran, and pulegone.
Results and Discussion Generally, the conditions of the Alberta environment were sufficiently conducive to the growing of mint. For example, the soil in Southern Alberta was a sandy loam with available nutrients in kg/ha of N:27, P: 124 and K:750, and low sulfates at 3.8 ppm. Over the second to fifth years of growth the average precipitation (cm) and daily bright sunshine (hr) were: May 16-31 (l.8, 143), June (6.9, 312), July (2.3,350), August (3.0,328), and September 1-15 (1.2, 118). Similarly, in Central Alberta, precipitation and daily bright sunshine were: May 16-31 (1.6, 137), June (9.6, 260), July (7.6,280), August (6.6,282), and September 1-15 (1.8, 93). Daily temperature data are available from the Atmospheric Environment division of Environment Canada. The major drawback of the Alberta environment was the short growing season and the cold and long winters. Results of horticultural observation. Morphologically the mints grown in southern, central, and northern Alberta did not differ. Under the cultural treatments of strip row, and, later, solid stand growing, the over-all horticultural performance of the plant was rated as 4 (good) for southern and central Alberta, and 3 (medium) for northern Alberta. When compared to other special crops, this rating was lower than those given to parsley, sage, or spearmint, all rated as 5 (very good), and, except for northern Alberta, was better than anise, which was rated as 3. Winter kill of the irrigated southern Alberta stand was 30% with snow fencing or straw cover, while it was 70% for non-irrigated stands in central and northern Alberta which were not protected by straw cover or wind breaks. Mint hay and oil yielas. Dry matter and oil yields at each stage of plant ontogeny are given in Table 1. Yields for northern Alberta were low, and, therefore, are not listed. The solid stands gave hay yields which increased with time until the beginning of September. The lowest yields were obtained before bloom at the vegetative stage of the plant. The highest yields were found at full bloom, which occurred near the end of August, but was a week 248
Table I. Yields of herb and oil of peppermint grown in solid stands in Alberta and harvested at different stages of plant ontogeny. Southern Alberta
Central Alberta Yield (kg/ha) Stage of ontogeny Vegetative Bud forming Beginning of bloom Bloom, 5% flowerettes 20% bloom 50% bloom 75% bloom 80% bloom Full bloom End of bloom
Yield (kg/ha)
Oil yield (wt % of herb)
Oil yield (wt %of herb)
Date of harvest
dry herb'
oil'
dry
fresh
Date of harvest
dry herb"
oil'
dry
fresh
Aug. I
3,167
41.50
1.31
0.24
July 5 July 27
4,043 4,408
41.44
0.93 0.94
0.18 0.18
Aug. 5
3,920
47.10
1.20
0.24
Aug. I
4,377
56.90
1.03
0.21
Aug. 7
4,357
52.28
1.20
0.24
Aug. 15 Aug. 22
5,384 7,012
54.91 79.24
1.02 1.13
0.24 0.24
Aug. 29 Sept. 4
7,112 7,045
85.34 71.16
1.20 0.99
0.26 0.22
Aug. 9 Aug. 14 Aug. 20 Aug. 27 Aug. 29 Sept. 3 Sept. 12
5,883 5,951 6,776 6,913 7,007 7,134 6,832
65.86 68.43 81.98 84.34 86.88 87.75 71.05
1.12 1.15 1.21 1.22 1.24 1.23 1.04
0.20 0.22 0.27 0.26 0.27 0.27 0.23
'Samples of harvested fresh mint were dried at 70°C in an air circulating oven. The dry matter of the herb at various stages was: 18.2%, vegetative; 20.0%, bud forming; 20.1-20.9%, dunng bloom; 21.8-22.5%, full bloom. Results m thiS and followmg tables are an average of three consecutive growing seasons. 'Hay was cured by air drying for two days prior to the distillation. During curing, the moisture loss was 30% at early stages of plant ontogeny, and close to 50% at later stages.
earlier in central than in southern Alberta. The results obtained after irrigation and one application of 225 kg N/ha indicated that yields in southern Alberta were slightly higher than in Washington state with row cultivation, but were lower than with solid stand (Nelson et al., 197Ia). Yields in Alberta were greater than those in Manitoba (Dorrell, 1972). Hamon and Zuck (1972) found increases in the number of leaves and stolons, and greater height and bushiness as a result of fertilization and irrigation under Saskatchewan conditions. Average oil yields were found to depend on the stage of plant development. The highest yields, 87.7 and 85.3 kg/ha, respectively, were experienced at full bloom in southern and central Alberta. This high yield was only possible over a narrow span of time. Seven days earlier, at 75% bloom, the oil yield was less, while six days later, at the end of bloom, a dramatic drop was experienced. Under similar cultivation conditions, Washington yields were 98.0-117.6 kg/ha for early harvests, and 132.7 for late harvests timed between the 17th to 31st of August (Nelson et al., 1971a). In central Alberta higher oil yields per plant were obtained until 5% bloom. During subsequent stages of blooming, and especially after 50% bloom, the southern Alberta yields were higher. In Washington Nelson et al. (l97Ia) reported yields of 2.3% (July 13), 1.8% (August 3), 1.69% (August 17), and 1.55% (August 31). Their results for solid stand did not differ from those of strip row cultivation but higher yields of oil per plant were gained from the addition of only 112 kg N/ha, along with irrigation. Treatment at the level of 112 kg N/ha was not done in Alberta. However, results of a 140 kg N/ha application with irrigation on a mint strip row are available from Saskatchewan (Hamon and Zuck, 1972). Their yields of oil in kg/ha were 44.0 for early harvest and 70.2 for late harvest, with a dramatic drop to 33.6 at the end of bloom. Similar yields were reported for Manitoba (Dorrell, 1972), for which the average yield of oil on a dry matter basis was 0.94%, with a peak yield of 1.15% that was recorded in the 249
middle of August. Somewhat similar results were obtained in central Alberta when no fertilizer was applied. The yield of oil was only 33.6kg/ha, and was 0.95% on a dry matter basis for mint hay. These results confirmed the suggestion that higher oil yields on the Prairies can be expected only after fairly heavy application of N fertilizer. Thin layer chromatography. The TL-chromatogram of peppermint oil and some of its associated pure compounds is illustrated in Figure I. The Rf values and tentative identities of the spots, and the number of constituents found in each spot are given in Table 2. As seen from Figure I, the TLC method was able to Rf SOLVENT
VALUE
-+
,-
,
FRONT
functional group
.73__ .66~-- hydrocorbon .59 _-OCH 3
.
47
constituents
oil
,
n 10
o
- -OCCH3 ::;CO
.28
t-
-OH
.12
f-
START,OO t-
Fig. I.
spot number
TL-chromatogram of peppermint oil and its associated pure compounds. Adsorbent: 300 p. of MN-Kieselgel N (Macherey Nagel & Co.); spot detection: spraying 1% vanillin in cone. sulfuric acid and heating in an oven at 105° for 10 min. Volume of oil applied: I p.1 (a band of 50 /.II was applied in preparative runs). J. Inst. Can. Sci. Technol. Aliment. Vol. 10. No.4. October 1977
Table 2. Thin layer chromatography data for peppermint oils from Alberta. Spot Number
Rf Value
Tentative Identity
Identity Confirmed by Preparative TLGL Chromatography, MS and IR Analyses
Numberof Constituents in the Spot
I 2
0.13 0.20
? Menthol
2
3 4
0.26 0.29
Piperitone Neomenthol
5 6 7 8 9
0.33 0.35 0.38 0.46 0.53
10
0.67
1,8-Cineol Pulegone Isomenthone Menthone Menthyl Acetate Menthofuran
II
0.72
Minor constituents 38 & 39a Menthol (32), 3-0ctanol (16), Isomenthol (33), and minor or trace constituents 7,15,17-19,&25. Piperitone (4) Neomenthol (27), Neoisomenthol (31), and linalool (26) I ,8-Cineol (10) Pulegone (35) Isomenthone (24) M~nthone (22), and trace constituent (II) Menthyl acetate (30), and a minor constituent, neoisomenthyl acetate (29) Menthofuran (20), and trans-sabinene hydrate (21) a-thujene (I), a-pillene (2), camphene (3), {3-pinene (4), sabinene (5), {3-myrcene (7), limonene (8), {3-phellandrene (9), cisocimene (12), p-cymene (13), y- terpinene (14), {3-caryophyllene (28), and minor and trace constituents 23, 34, 36, 37 (Germacrene D), 41-46.
Terpene Hydrocarbons
9 I 3 I I I '2 2 2 22
aGLC chromatogram peak numbers (see Figure 2).
separate a great number of oil constituents, and, partIcularly, the menthone related constituents (Petrowitz, 1960) into menthol (Rf 0.20), neomenthol (0.29), isomenthone (0.38), menthone (0.46), menthyl acetate (0.53), and menthofuran (0.67). However, menthol was not well separated from isomenthol, nor was neomenthol from neoisomenthol, or menthyl acetate from neomenthyl acetate. Nevertheless, TLC combined with GLC separation was proven to be an effective method for analysis of most of the hydrocarbon, oxide, ester, alcohol, and carbonyl constituentsof the oil. Gas liquid chromatography. Both columns were used for each oil analysis (Figures 2 and 3). On a fresh column myrcene was resolved from limonene, and pulegone from sesquiterpenes. If a preliminary TLC separation was omitted, then 1,8-cineol was eluted along with peak 11, menthofuran with trans-sabinene hydrate, and menthyl acetate with neoisomenthol. On an aged column the chromatogram showed that 1,8-cineol was well resolved from peak 11, and menthofuran from sabinene hydrate, while pulegone appeared just after ,8-caryophyllene, and piperitone was eluted along with two sesquiterpenes. The separation of menthone and menthol stereoisomers was best on freshly prepared columns. The elution order was menthofuran, menthone, isomenthone, neomenthol, neoisomenthyl acetate, menthyl acetate, neoisomenthol (only the latter two have a trend to inverse on an aged column), menthol, isomenthol, pulegone, and piperitone. This elution sequence is different from that on Carbowax 20M, or sucrose diacetate hexaisobutyrate, SAIB (Handa et al., 1964). Gillen and Scanlon (1972) indicated that the limiting factor on a number ofliquid phases for the separation of menthone and menthol stereoisomers was the resolution of neoisomenthol either from neomenthol or menthol. In our analyses this was the case only on the aged column, while on the fresh column it was the Can. Inst. Food Sci. Techno!. J. Vol. 10, No.4. October 1977
resolution of neoisomenthol from its own acetate, and from menthyl acetate. In this study TL- and GL-chromatography had to be used in combination in order to get the best separation of peppermint oil constituents. This technique, as proven by Nigam et al. (1963) and Nigam and Levi (1964), offers advantages in chemical and taxonomical characterization of oils. However, recent analysis of peppermint oil by using liquid-liquid-chromatography for preliminary separation of hydrocarbons from oxygenated constituents, followed by preparative GLC using capillary columns (BelAfi-Rethy et al., 1973), appeared even more advantageous when an oil analysis that includes all trace constituents is required. Also superior was a combination of alkali extraction, fractional distillation, and subfractionation by alumina and silver nitrate-alumina column chromatography, which was then followed by capillary and preparative G LC (Lawrence et al., 1972). The latter authors characterized a total of 99 constituents in oil from the Wilamette Valley, Oregon. Nevertheless, a fast and accurate characterization of major and minor oil constituents, that reflects the oil's origin, quality, and degree of purification, can be achieved by a simple GLC separation (Hefendehl and Ziegler, 1975). Mass spectral analysis. Additional confirmation of the identities of peppermint oil constituents was provided by a combination of TL- and GL-chromatography, and mass spectral analysis. Data on some menthone-related oil constituents are provided in Table 3. The fragmentation patterns of most oil components were compatible with those of standard compounds, or with literature data (e.g., menthols and theIr acetates, Thomas and Willhalm, 1966; menthone and isomenthone, Willhalm and Thomas, 1965). Further discussion is restricted to four major oil constituents.
250
22
®
20,21
Alberta oil fresh column
32
10
8
26,27 24
36,37
11 18 1 '} 3
4 5 67
9
12
15
17
23 19
42 43
44 45
46
22
®
32
29
30 31
Commercial oil from United States 10 27
Fig. 2.
GL-chromatogram of peppermint oil on a freshly packed column. (a) Alberta oil. (b) Commercial oil from the United States. Peak designations: a-thujene (I), a-pinene (2), camphene (3), {3pinene (4), sabinene (5), {3-myrcene (7), limonene (8), {3-phellandrene (9), 1,8-cineol (10), trans-2-hexanal (II), cisocimene (\ 2), p-cymene (13), y-terpinene (14), aliphatic alcohols (\5-19, with 3-octanol at peak 16), menthofuran (20), trans-sabinene hydrate (21), menthone (22), pentadecane (23), isomenthone (24), a sesquiterpene (25), linalool (26), neomenthol (27), {3-caryophyllene (28), neoisomenthyl acetate (29), menthyl acetate (30), neoisomenthol (31), menthol (32), isomenthol (33), a sesquiterpene (34), pulegone (35), sesquiterpenes (35-39, with Germacrene D at peak 37), piperitone (40), and sesquiterpenes (42-46).
The principal fragment from menthol, C.H,O+ at ml e 71, arose from cleavage of the 3,4-bond of the molecular ion, followed by loss of C6 H 13 • Removal of OH and H from the parent ion to give various ions at ml e 138 was followed by further breakdown to cyclic species at ml e 95 (C 7 H" +), and mle 81 (C 6 H g ). The fragment at mle 82 (C 6 H lO +) arose 251
from cleavage of an mle 138 ion. Acetic acid was eliminated from menthyl acetate to give the ion at mle 138 (C IO H 18 +). This occurred either by 1,2-elimination, or by loss along with a H from the isopropyl side chain. Further fragmentation was similar to that of menthol. J. Insl. Can. Sci. Technol. Alimenl. Vol. 10. No.4, October 1971
Alberta oil
"
aged column
31 '0
J2
22
" 20 36,37,40
28
~
L Fig. 3.
~
l
GL-chromatogram of Alberta peppermint oil on the aged column. Peak identities as in Figure 2. Table 3. Mass spectral data for some peppermint oil constituents. GLC Parent Peak Numbera Peak [M.]b 20 22 24 30 32 35 40
150 154 154 198 156 152 152
Base Peak 108 112 112 95 71 81 82
Compound Identity
mle'
Menthofuran Menthone Isomenthone Menthyl Acetate Menthol Pulegone Piperitone
15079109779115180 6941551391547056 694155139154 III 8113812367698296 81 95 138 82 123 55 41 96 1526710982436942 110951371094115254
aGLC separation data as on Figure 2. bA parent peak in the range ofmle 204 - 212 was ascribed to sesquiterpenes. Some sesquiterpenes of peppermint oil were determined by Vlahov et al. (1967), while a number of their fragmentation patterns were described by Moshonas and Lund (1970). 'In decreasing order of relative abundance. Table 4. Oil composition of peppermint plants grown in a solid stand in Central Alberta, and harvested at different stages of plant ontogeny. Stage of ontogeny
Total menthol
L-Menthol
Menthyl ester
Total menthone
Menthofuran
Pulegone
39.6a 40.0 40.4 44.9 46.5 49.2 54.6 53.1
30.2 31.8 32.3 36.3 39.2 40.3 41.2 44.2
4.4 4.0 3.1 3.4 3.5 3.7 3.8 3.8
43.3 40.0 36.1 32.5 28.1 29.4 25.8 24.0
0.8 0.8 1.0 l.l l.l 1.2 1.7 2.9
0.7 0.7 0.8 1.1 0.9 0.8 l.l 0.9
vegetative vegetative bud forming 5% bloom 50% bloom 75% bloom full bloom end of bloom aResults in percent.
The main fragmentation of menthone to give the enol at mle 112 (C,H 12 0+) was a cyclic process resulting from transfer of a y-hydrogen from the isopropyl side chain to the carbonyl, with simultaneous ,8-cleavage. Various fragmentation pathways could explain the species at ml e 69 (C.H 5 0+, perhaps by loss of C a H6 from mle Ill), mle 41 (Ca H 5 +, or C2 HO+ via ring cleavage), or mle 55 (C,H 7 +). Finally, the only possible facile cleavage of the menthofuran molecule can be explained by a retro-Diels-Alder type fragmentation to give two rather stable unsaturated fragments, the furane-like C7 HgO+ at mle 108, and CaH 6 • Oil composition. During three consecutive seasons, the composition of the oil showed little variation when analyzed at the same stages of plant ontogeny but did vary between stages (Tables 4 and 5). Can. Insl. Food Sci. Technol. J. Vol. 10. No.4, October 1977
Southern Alberta oil obtained at 20%bloom appeared to have the most acceptable profile from the standpoint of chemically evaluated oil quality. The oil at 5% bloom seemed to be an immature product, while the oil at 50% bloom had already reached a high content of menthofuran, and had reverted to a somewhat immature status. This indicated that secondary growth was corning up from the root shoots while the main portion of the mint field was in bloom. Therefore, in an attempt to develop a quality peppermint oil, the harvest time would have to be limited to a fairly narrow time period. Further into the growing season, after passing the stage of 50% bloom, the oil became more mature, and, eventually, entered a phase where the menthofuran started to decrease. However, even this oil could not be considered a quality oil for dentifrice
252
Table 5. Oil composition of peppermint plants grown in a solid stand in Southern Alberta, and harvested at different stages of plant ontogeny. Stage of ontogeny vegetative vegetative bud forming 5% bloom 20% bloom 50% bloom 75% bloom full bloom end of bloom a
Total menthol
L-Menthol
Menthyl ester
Total menthone
Menthofuran
Pulegone
36.1" 35.4 47.6 49.5 53.1 48.8 51.9 54.0 57.1
32.6 32.9 42.6 42.0 47.5 44.7 43.2 45.1 47.0
2.3 1.9 3.3 3.4 4.2 3. I 4.2 6.6 9.6
43.6 43.5 35.0 30.8 27.8 29.7 23.7 17.9 17.2
0.7 0.5 1.8 1.9 2.8 6.0 6.2 10.4 6.0
1.2 1.2 0.9 1.0 1.5 1.6 2.7 2.3 0.8
Results in percent.
The major and minor hydrocarbon constituents of peppermint oil are listed in Table 6. Three stages of development, 5% bloom, 75% bloom, and the end of bloom, are given to illustrate the changes during a season. The major terpene hydrocarbons were limonene and ,a-pinene, fol-
or chewing gum production, because it was a relatively harsh and overmatured product at this late date of harvest. By the same token, the best quality of oil from central Alberta would be an oil taken from a harvest between 50% and full bloom. Table 6. Hydrocarbon constituents of peppermint oils. Constituent
Retention time (Menthol = 1.00)'
Camphene ,B-Caryophyllene p-Cyrnene Limonene ,B-Myrcene a-Pinene ,B-Pinene Sabinene y- Terpinene
0.14 0.93 0.41 0.29 0.27 0.12 0.19 0.23 0.38
TOTALc Monoterpene hydrocarbons Sesquiterpene hydrocarbons (includes OLC peaks 36 & 37) Benzene related hydrocarbons
A
L
B
E
Southern I'
II
III
0.2 0.5 0.5 3.0 0.7 0.8 1.1 0.5 0.3
trace 0.6 0.6 2.7 0.8 0.7 1.6 0.4 0.4
trace 0.7 0.4 2.6 0.6 0.7 1.4 0.2 0.6
6.53
6.53
2.75 0.52
1.11 0.58
R T A Central
Northern
Commercial oil (United States)
II
III
III
0.3 1.8 0.5 1.6 0.6 0.7 0.9 0.8 0.7
0.3 1.7 0.5 1.5 0.7 0.5 0.7 0.8 0.7
0.3 1.7 0.3 2.0 0.7 0.6 0.9 0.8 0.6
trace 0.5 trace 0.8 trace 0.2 0.9 0.3 trace
0.2 2.3 0.4 1.6 0.7 0.5 0.8 0.8 0.6
6.16
5.67
5.32
6.04
2.13
5.90
1.42 0.43
3.95 0.45
3.70 0.50
3.79 0.33
0.45 trace
3.98 0.38
Northern
Commercial oil (United States)
'Conditions of OLC separation as on Figure 2. 'Harvest time I, 5% bloom; II, 75% bloom; III, end of bloom. Results in percent. 'Constituents below O. I% are not listed. Table 7. Alcohol and ester constituents of peppermint.oils. Constituent
Retention time (Menthol = 1.00)'
Isomenthol Linalool Menthol Menthyl acetate + neoisomenthyl acetate Neoisomenthol Neomenthol 3-0ctanol Sabinene hydrate
A
L
B
E
Southern II
III
R T A Central II
III
III
1.05 0.90 1.00
0.3' 0.3 42.0
0.3 0.3 43.2
0.3 0.4 47.0
0.9 0.5 36.3
0.5 0.3 40.3
0.6 0.5 44.2
0.4 0.9 58.9
0.7 0.5 42.8
0.95
3.4
4.2
9.6
3.4
3.7
3.8
1.9
6.6
0.95 0.91 0.59 0.74
0.9 2.6 0.3 0.7
0.9 2.5 0.4 0.7
1.3
1.3
3.3 0.4 0.7
3.0 0.6 0.7
1.2 3.4 0.7 0.7
1.2 3.3 0.6 0.7
trace 3. I 2.0 0.9
1.2 4.2 0.4 0.7
46.78 3.40
47.95 4.20
53.09 9.60
42.73 3.39
46.51 3.69
50.46 3.83
64.25 1.89
50.14 6.56
TOTAL' Monoterpene alcohols Esterified "Menthol"
'See Table 6 for explanation. 'Results in percent. 'Constituents below 0.1 % are not listed.
253
J.
Insl. Can. Sci. Technol. Alimenl. Vol. 10. No.4. October 1977
lowed by sesquiterpenes (,B-caryophyllene, and GLC peaks 36, and 37, Germacrene D). Altogether, eleven constituents above 0.1% were found. The total monoterpene hydrocarbon contents decreased from south to north when equal stages of plant development were compared. Thus, the north had only one third of the amount found in plants grown in the south. A similar trend was observed for benzene related constituents. The composition of alcohol and ester Constituents of peppermint oil is given in T~ble 7. The presence of all four menthol stereoisomers was Confirmed. The L-menthol content in southern Alberta (42.0-47.5%) was higher than that of central Alberta oils (32.3-44.2%). The northern oil obtained from a harvest, which, by plant morphology, would correspond to the end of bloom stage, was in fact a quite overmature oil. Generally, the shorter northern growing season decreased development stages to an even narrower time period than that experienced in central Alberta. The content of neomenthol was significant, and amounted to 2.5-3.4%. Neoisomenthol and isomenthol contents were low, especially in southern oils. The former had a maximum amount of 1.3%, while the latter never exceeded
0.3%. The content of total monoterpene alcohols increased with time during a season. Esterified menthol, like the free alcohols, also increased. This was particularly so in southern oils. The northern oil, though overmatured, did not accumulate the ester to more than 1.9%. The monoterpene ketone and oxide constituents are given in Table 8. During a given season, menthone was higher in central Alberta oil than in the southern Alberta oil. The overmatured southern oil had 15.1% of menthone at the end of bloom, while central oils never fell below 21.2%. Of interest was the northern oil, which contained only 11.9% of menthone at the end of bloom. In all of the oils, the isomenthone, at about 3%, was practically unchanged throughout the season, with'a small drop in content being experienced only at the end of the season. Piperitone and pulegone were low, except in the northern oil. The menthofuran contents of a high 6.3% in southern oils, 1.1-2.9% in central oils, and 0.3% in northern Alberta oils were theoretically expected (Burbott and Loomis, 1967). Finally, 1,8-cineol, generally near 5-6%, appeared not to vary significantly with the stages of plant ontogeny. The compositional data from Tables 6-8 were used to
Table 8. Monoterpene ketone and oxide constituents of peppermint oils. Constituent
Retention time (Menthol = 1.(0)'
Cineol-I,8 Isomenthone Menthone Menthofuran Piperitone Pulegone
0.34 0.84 0.77 0.74 1.29 1.11
TOTAL' Monoterpene ketones Monoterpene oxides
A
L
B
E
Southern II
III
5.9' 3.5 27.3 1.9 0.5 0.9
6.3 3.0 20.7 6.3 0.6 2.7
5.1 2.1 15.1 6.0 0.2 0.8
32.18 7.84
27.04 12.59
18.19 11.11
R T A Central
Northern
Commercial oil (United States)
II
III
III
5.2 3.0 29.5 1.1 1.1 1.1
5.7 3.4 26.0 1.2 1.2 0.8
5.1 2.8 21.2 2.9 0.9 0.9
4.2 4.4 11.9 0.3 6.6 2.0
5.2 3.3 19.4 2.0 0.6 0.9
34.77 6.31
31.44 6.86
25.77 8.02
24.79 4.52
24.15 7.19
'See Table 6 for explanation. 'Results in percent. 'Constituents below 0.1% are not listed. Table 9. Significant constituent ratios for Alberta peppermint oils. A
B
Terpenes' All constituents
Menthone Isomenthone
C Limonene 1,8-Cineol
5% bloom 75% bloom end of bloom
0.109 0.110 0.112
9.77 7.70 7.59
0.31 0.27 0.40
5% bloom 20% bloom 75% bloom end of bloom Commercial oil from United States
0.125 0.125 0.129 0.113
7.80 7.95 6.85 7.15
0.50 0.47 0.43 0.51
0.101
5.81
0.37
Ratio of Percentages D E Menthofuran NeomenthoJ Menthyl acetate "Menthone related constituents'"
"Menthone related constituents" "Menthol related constituents"
G
CENTRAL ALBERTA 0.032 0.88 0.038 0.92 0.107 0.86 SOUTHERN ALBERTA 0.059 0.77 0.092 0.62 0.209 0.60 0.34 0.260
15.12 14.54 16.20
0.75 0.62 0.51
18.92 19.90 20.36 18.66
0.67 0.58 0.59 0.38
0.75
12.99
0.45
0.081
a"Terpenes": Monoterpene hydrocarbons emerging prior to and including 1,8-cineol. '''Menthone related constituents": menthofuran + menthone + isomenthone. '''Menthol related constituents": neomenthol + menthol + menthyl acetate + isomenthol 1961). Can. Inst. Food Sci. Technol. J. Vol. 10, No.4. October 1977
F "Menthol related constitutents"" Neomenthol
+ neoisomenthol (all ratios according to Smith and Levi,
254
calculate ratios of percentages of various combinations of constituents, according to Smith and Levi (1961), in order to check the possibility of differentiating the oils from Alberta from those of the United States and other geographical origins. These ratios are presented in Table 9. Ratio A, the terpenes emerging in GLC analysis prior to and including 1,8-cineo1 over all constituents, was a reflection of a genuine natural and non-redistilled oil. Ratio B, menthone over isomenthone, depended on harvest time. This ratio, between 6.8-9.8 with an average of 7.8, was high when compared to oils from the United States which range from 2.7-6.6. Though ecological factors influenced this ratio, there was not a clear cut difference between ratios for central and southern Alberta. Ratio C, which is genetically controlled, and which varies from 0.2-0.7 for peppermint oils, averaged 0.33 for central and 0.48 for southern oils. An increase in ratio D, menthofuran over menthone related constituents, with plant maturation was a noteworthy trend. Ratio D was strongly influenced by the stage of plant ontogeny. Prior to blooming, the plant gave an oil with a lower menthofuran content than was found during or after bloom. The fact that climate co-determined ratio D was reflected by lower values for the oils from the cooler central Alberta, and higher values from the warmer southern Alberta. Other environmental factors, such as the level of available N in a range of 56 to 448 kg N/ha, solid stand growing, or cultural treatments in a strip row, had no influence on this ratio (Nelson et al., 1971a,b). Thus, the values of 0.09 for the U.S. Midwest and 0.09-1.13 for Washington and Oregon, like Alberta results, were undoubtedly influenced solely by climatic factors. Ratio E, neomenthol over menthyl acetate, was higher in central than in southern oils. In the latter, decrease of the ratio during plant maturation was evident. Climatic factors also influenced ratio F, menthol related constituents over neomenthol. It ranged between 18.7-20.4 for southern and 14.5-16.2 for central Alberta oils. None of these values coincided with those of the U.S. Midwest (12.6-13.8), or Oregon (13.0-13.8), but the ratios from Central Alberta were close to those of Washington (14.9-17.3). The last ratio, G, reflected the phenomenon of menthone reduction during plant maturation, and exposure of the plants to prolonged sunlight. The values were lower for southern than for central Alberta, with values at the end of bloom of 0.38 and 0.51, respectively. The fairly conclusive distinction of Alberta grown peppermint oils from those of the United States is illustrated on Figures 4 and 5. The oils grown in the U.S. Midwest, Oregon, or Washington were readily distinguished by plotting ratio E vs ratio D for oil constituents, as reported earlier by Smith and Levi (1961), or, more recently, by Nelson et al (1971a,b). In such a plot the central Alberta oils were also readily differentiated. However, the oils from an early harvest in southern Alberta generally coincided with plots for Oregon and partially overlapped with the U.S. Midwest, while the oils of more matured plants, or late harvests gave similar plots to oils from Washington. A better differentiation of Alberta oils was obtained when ratio F was plotted vs ratio G (Figure 5). Here the plots of oils from central Alberta were grouped in an area well separated from those for Oregon and the U.S. Midwest, and only the late harvest, mostly at the end of bloom, merged 255
.5....--------------------.4
P .3 Q
~ 0::
SOUTHERN ALBERTA
.2
.1
o
.2
.4
.6
1.2
RATIO «Eo
Fig. 4.
Distribution plot of ratio D (menthofuran/menthone related constituents) vs ratio E (neomenthol!menthyl acetate) showing the variation with the geographical origin of the oil.
u.•
o ~
0::
.4
.5
.6
.7
.8
RATIO "G" Fig. 5.
Distribution plot of ratio F (menthol related constituents/neomenthol) vs ratio G (menthone related constituents/menthol related constituents) showing the variation with the geographical origin of the oil.
with the plots of Washington oils. Southern Alberta oils at any time of harvest were sharply separated from all other oils. In conclusion, the data for Alberta indicate that optimum cultural treatments on a solid stand, including high rates ofN, and irrigation (in southern Alberta) can provide satisfactory yields of both oil and herb. An oil of optimum profile, with a quality comparable to that of imported oils, can be obtained. However, harvesting must be done over a fairly narrow time period. A complete determination of the feasibility of growing peppermint in Alberta would require additional examination of the cost of establishing J. Inst. Can. Sci. Technol. Aliment. Vol. 10, No.4, October 1977
and maintaining a mint stand, especially in the light of the extent of winter kill. Furthermore, a marketing study would be necessary, since each peppermint producing area must generally establish its own market, rather than expect to simply replace existing sources.
Acknowledgement This work was supported by a grant from the Alberta Agricultural Research Trust. The assistance of T. Leishman in field distillation of the oils is gratefully acknowledged. Gratitude is also expressed to Dr. R. E. Harris, CDA Research Station, Beaverlodge, and Dr. N. Marchak, at Two Hills, for the growing of mint on observation plots, to Dr. R. Gaudiel, Alberta Horticultural Research Center, Brooks, for his valuable suggestions on cultural treatments of mint fields, and to I. P. Callison & Sons, Inc., Intern., Chehalis, Washington, for some oil composition control data.
References BeIAfi-Relhy, K.. Iglewski. 5., Kerenyi. E. and Kolta, R. 1973. Untersuchung der Zusammensetzung von einheimischen und auslandischen atherischen 6Jen. II. Analvse eines ungaris~ chen Pfefferminzoles. Acta Chim (Budapest) 76: 167. • Burbon, A. J. and Loomis, W. D. 1967. Effect ofIight and temperature on monoterpenes in peppermint Plant Physiol. 42:20. Dorrell, D. G. 1972. CDA Research Branch, Morden, Manitoba. Franz, C. ~ra7;t~ E~:~. ~2~6~~d N nutrients on the formation of essential oils of Mentha piperita.
Can. Inst Food Sci. Technol. J. Vol. 10. No.4, October 1977
Gillen. D. G. and Scanlon, 1. T. 1972. The separation of menthol-menthone stereoisomers. J. Chromato~. Sci. 10:729. Gretskaya, R. L. 1973. Effectiveness of Nand P fertilizers for irrigated peppermint. Tr. Vses. Nauchno-Issled. Inst. Kul't. 6:75. Hamon, N. W. and Zuck, D. A 1972. Peppermint as a cash crop in Saskatchewan. Can. 1. Plant Sci. 52:837. Handa, K. L, Smith, D. M., Nigam, I. C. and Levi, L 1964. Essential oils and their constituents. XXIII. Chemotaxonomy of the genus Mentha. J. Pharm. Sci. 53: 1407. Hefendehl. F. W. and Ziegler, E. 1975. Analytik von PfefferminzOlen. Deu!. Lebensmittel Rundschau 71 :287. Kruepper. H.. Lossner, G. and Shroeder, H. 1968. Effect of irrigation on the yield and quality of peppermmt (Mentha piperita). Pharmazie 23: 192. Lawrence, B. M., Hogg, 1. W., and Terhune, S. J. 1972. Essential oils and their constituents. X. Some new trace constituents in the oil of Mentha piperiw,. L. Flavor Ind. 3:467. Moshonas, M. G. and Lund, E. 0.1970. The mass spectraofsesquiterpene hydrocarbons. Flavour Ind. 1:375-378. Nelson, C. E.. Early, R. E. and Mortensen, M. A. 1971a. Effects of growing method and rate and time of N fertilization on peppermint yield and oil composition. Ext. Servo Wash. Agr. Exptl. Sta.. College of Agri., Wash. State Univ. eire. 541. Nelson, C. E., Mortensen, M. A. and Early, R. E. 1971b. Evaporative cooling of peppermint by sprinkling., Ibid. eire. 539. Nigam, I. C. and Levi. L. 1964. Essential oils and their constituents. XX. Detection and estimation of menthofuran in Mentha arvensis and other mint species by coupled QL- and TLchromatography. J. Pharm. Sci. 53: 1008. Nigam, I. c., Sahasrabudhe, M. and Levi, L. 1963. Coupled GL-TL-chromatography. Simultaneous detennination of piperitone and piperitone oxide in essential oils. Can. J. Chern. 41:1535. Petrowitz. H. 1. 1960. Zurkieselgelschicht-Chromatographie der stereoisomeren Menthole. Agnew. Chern.. 72:921. Smith. D. M. and Levi, L. 1961. Treatment of compositional data for the characterization 01" essentialoils. Determination of geographical origins of peppermint oils by GLC analysis. J. Agr. Food Chem. 9:230. Thomas. A F. and WiJlhalm. B. 1966. The mass spectra of the menthols, carvomenthols and their acetates and related alcohols. J. Chem. Soc. (B), 219. Vlahov, R., Holub. M., Ognyanov, I. and Herout, V. 1967. Sesquiterpenic hydrocarbons from the essential oil of Mentha piperita of Bul~arian ori~in. Coil. Czech. Chern. Comm. 32:808. Watson, V. K. and St. John, J. L. 1~955. Pepperlllint oil. R~lation of maturity and curing of peppermint hay to yield and composition of oil. J. Agr. Food Chern. 3: 1033. Willhalm._B. and Thomas, A. F. 1965. The mass spectra of menthone, isomenthone. and carvomenthone. J. Chem. Soc. (B). 6478. Received January 3, 1977
256
S. Molnar Alberta Horticultural Research Center Brooks, Alta. TOJ OJO * Previous report: Sage oil. Salvia officinalis. L (Labiatae). Can. lost. Food Sci. Techno!' J.. 10:201-207 (1977) ... Recipient of Canadian Commonwealth Fellowship. Permanent address: Universiti Kebangsaan Malaysia, Kuala Lumpur. Malaysia.
Abstract
Introduction
parts of the flowering plants, when steam distilled, give the mint oil. This oil is one of the indispensable items in the food flavoring industry, with the major market being chewing gum and toothpaste production. The fresh or dried herb, itself, has very limited practical use for flavoring. Mentha arvensis, L. (synonymous to Mentha canadensis) is found among the native wild flora of the Prairies and Rocky Mountains, especially near banks of irrigation ditches, sloughs, lakes, and streams. Growing along with it are numerous species of this genus, as well as some mint garden 'escapees' such as citrata, pulegium, rotundifolia, spicata, or even piperita. According to Canadian Food and Drug Regulations (B.1O.019), peppermint oil must be obtained from the leaves and flowering tops of Mentha piperita, L., and/or Mentha arvensis, var. piperascens, Holmes. In addition, either oil must contain not less than 50% of free and combined menthol. The main commercial growing regions of mint are in the United States, mostly in the Midwest States of Michigan and Wisconsin, and in Oregon, Washington and Idaho. Their total area of peppermint harvested in 1976 is estimated at a record 29,000 ha, with an average oil yield of 61.5 kg/ha (USDA Statistical Reporting Service, June 1976). Mint is not grown commercially in Canada at present, although, preliminary research in Manitoba (Dorrell, 1972), and in Saskatchewan (Hamon and Zuck, 1972) indicated that good quality mint oil can be produced. The current domestic requirement of the oil is met by imports exceeding 45 tons per year (DBS, 1971-1975). . In this investigation the performance of black MItcham mint (Mentha piperita Huds., var. officinalis, forma rubescens Camus) grown in Southern, Central, and Northern Alberta (latitudes of 50° 33',53° 42', and 56°, respectively) was assessed during five seasons. Determinati.oOS were made of the effects of irrigation and harvest t1m.e upon the yield of oil and the amount of the major and mInor oil constituents.
Several species of mint are grown, but the most common for commercial use are peppermint (Mentha piperita, L.), and spearmint (Mentha spicata, L.). The above ground
(a) Plant material. In the spring of 1969 certified Mentha
This study was undertaken to indicate whether or not a satisfactory peppermint oil could be produced in the Alberta environment with its short growing season. The horticultural performance of the solid mint stand over three consecutive seasons was rated as good for both central Alberta and southern Alberta, where irrigation was also used. Satisfactory maximum yields, near 7100 kg/ha of the ~plant and 86 kg/ha of the oil (1.2% of dried herb), were obtained at the full bloom stage. TL- and G L-chromatography and mass spectral analysis were used to determine the major and minor oil constituents. The oil composition, which changed with time, had its most ideal profile at 20% bloom in southern Alberta, and at full bloom in central Alberta. At these stages the respective percentages for southern and central oils were: total menthol (53.1, 54.6), free menthol (47.5. 41.2), menthyl esters (4.2,3.8), menthone (27.8, 25.8), and menthofuran (2.8, 1.7). The central Alberta oils, at all stages of plant development, were lower in free menthol, menthyl esters, and, especially, in menthofuran. Alberta oils could be differentiated from oils of other geographical origins through compositional analysis. Finally, the quality of Alberta oils was assessed relative to a standard quality peppermint oil from the United States which is used by the domestic food industry.
Resume On a etudie la possibilite de produire de I'huile de menthe dans les regions d'Alberta ou la saison de croissance est courte. La performance horticole de la menthe en semis dense a ete bonne, au cours de trois saisons consecutives, dans les regions du centre et du sud d'Alberta ou I'on pratique I'irrigation. Des rendements maximums satisfaisants, 7100 kg/ha de plante, et 86 kg/ha d'huile (1.2% de I'herbe, base seche), ont ete obtenus au stade de pleine floraison. On a determine les constituants majeurs et mineurs de l'huile a I'aide de chromatographies en couche mince et gaz-liquide et de spectrometrie de masse. La composition de I'huile, qui a varie avec Ie temps, a eu son meilleur profil au stade de 20% de floraison dans Ie sud d'Alberta, et au stade de pleine floraison dans Ie centre d'AIberta. Aces stades, les compositions (%) des huiles ont ete respectivement pour les regions du sud et du centre comme suit: menthol total (53.1, 54.6), menthollibre (47.5,41.2), esters menthyliques (4.2, 3.8), menthone (27.8, 25.8), et menthofuran (2.8, 1.7). Les huiles du centre d'Alberta, a tous les stades de developpement de la plante, ont ete plus faibles en menthol libre, en esters menthyliques, et surtout, en menthofuran. II est possible, par la composition, de diffhencier les huiles d' Alberta des huiles en provenance d'ailleurs. On a enfin evalue la qualite des huiles d'Alberta par rapport a une huile de menthe standard des Etats-Unis qui est utilisee en industrie alimentaire au Canada.
247
Materials and Methods J. Inst. Can. Sci. Technol. Aliment. Vol.
10. No.4. October 1917:1 ::1
piperita L., var. black Mitcham roots were obtained from Oregon and were planted and propagated in a greenhouse. On the 15th of May, 1970, they were transplanted into field plots in rows 90 em apart with 30 cm between plants. Plots were at Beaverlodge in Northern, Two Hills in Central, and Brooks in Southern Alberta. Before planting, 335 kg/ha of 11-48-0 N:P:K were applied to the sandy loam (pH 7.9, low in organic matter and lime). In June 220 kgl ha of 34-0-0 were applied as a side dressing about 8-10 cm along both sides of the row, and slightly below the surface (Gretskaya, 1973; Franz, 1972; Nelson et al., 1971a,b). In Southern Alberta irrigation equivalent to 30-45 cm was applied four times per season using furrows placed 90 cm apart (Kruepper et al., 1968). Field care during the first season consisted of rototilling between the rows to remove stolons, roots, and weeds. Weeds within the rows were removed by hand. Harvesting was done at various stages of bloom in August and early September, with a total of seven to ten harvests about 10 days apart. Over the winter the plots were either covered by straw, or left unprotected. The following season the same N:P fertilizer as before was applied in early spring and incorporated by a shallow rototilling of the entire soil surface, while the N fertilizer was side banded in June at the same rate as before. The selective control of annual and perennial weeds within the established field was achieved by applying TREFLAN (a,a,a-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) at a rate of 0.55 to 0.85 kg/ha, and/or TERBACIL (3-tert-butyl-5-chloro-6-methyluracil) at a rate of 0.9 to 1.8 kg/ha. Both herbicides were incorporated in the soil by preemergence broadcast plus rototilling. In the third season the plants were allowed to completely fill in between the rows, and they had done so by the middle of August. Subtreatments with fertilizer and herbicides were continued for two years. The essential oil was obtained from plants which were cut close to the ground, and then partially air dried for two days prior to steam distillation (Watson and St. John, 1955). The resultant oil was separated from the water, and dried over anhydrous sodium sulfate. It was stored in sealed tubes at 4°C to await chemical analysis. (b) Chemicals. Pure compounds used as standards in TLchromatography, GL-co-chromatography, and infrared and mass spectral analysis were obtained from Aldrich Chern. Co. Inc., Milwaukee, Wisc., K & K Labs. Inc., Plainview, N.Y., and Eastman Org. Chern., Rochester, N.Y. Isomenthol and neoisomenthol (from isomenthone) and neomenthol (from menthone) were synthesized by us USing aluminum isopropoxide and applying the MeerweinPonndorf-Verley reduction procedure. Neomenthyl- and ~eoisomenthyl acetate were also prepared by us by refluxIn~ the appropriate alcohols in xylene with glacial acetic aCId, acetic anhydride and fused sodium acetate. A sample of standard quality essential oil from peppermint, representative of those supplied to the food industry, and one which meets the standards of the United States and other Pharmacopoeia, was obtained from H. G. Hotchkiss Essential Oil Co., Lyons, Wayne County, N.Y. (c) Chemical analysis. Preparative TL-chromatography Was done using silicic acid as adsorbent, with a solvent syst~m ofbenzene:ethyl acetate, 95:5 (v/v). The separation of oIl constituents into hydrocarbon, oxide, ester, carbonyl, Can. Ins!. Food Sci. Technol. J. Vol. 10. No.4, October 1977
and alcohol classes was based on the differences in th . polarity. The compounds within each band were recover~~ In ethyl acetate and further analyzed by G LC. GL-chromatography was performed on a Bendix M 2500 gas chromatograph equipped with 1.8 m X 3 mm I.D. glass columns packed with 15% EGS on Chromosorb P, AW, 100/120 mesh. Operating conditions were: injection temperature 200°C, N 2 carrier gas flow rate 30 mIl min, and temperature programming from 60-150°C at I ° I min. A flame ionization detector was used. A Varian Aerograph 1400 with a TC detector was used in separations for mass spectral determinations. Two GLC columns, designated 'fresh' (one which was freshly prepared), and 'aged' (one which had been in use for at least four months) were used in combination to achieve an effective separation of the major and minor oil constituents. G LC runs were usually preceded by separation of the oil constituents into classes by preparative TLC. As an alternative to a detailed analysis, GL-chromatogram computations were limited to total menthol (1.menthol + its stereoisomers + menthyl esters), L-menthol, menthyl acetate, total menthone (menthone + isomenthone), menthofuran, and pulegone.
Results and Discussion Generally, the conditions of the Alberta environment were sufficiently conducive to the growing of mint. For example, the soil in Southern Alberta was a sandy loam with available nutrients in kg/ha of N:27, P: 124 and K:750, and low sulfates at 3.8 ppm. Over the second to fifth years of growth the average precipitation (cm) and daily bright sunshine (hr) were: May 16-31 (l.8, 143), June (6.9, 312), July (2.3,350), August (3.0,328), and September 1-15 (1.2, 118). Similarly, in Central Alberta, precipitation and daily bright sunshine were: May 16-31 (1.6, 137), June (9.6, 260), July (7.6,280), August (6.6,282), and September 1-15 (1.8, 93). Daily temperature data are available from the Atmospheric Environment division of Environment Canada. The major drawback of the Alberta environment was the short growing season and the cold and long winters. Results of horticultural observation. Morphologically the mints grown in southern, central, and northern Alberta did not differ. Under the cultural treatments of strip row, and, later, solid stand growing, the over-all horticultural performance of the plant was rated as 4 (good) for southern and central Alberta, and 3 (medium) for northern Alberta. When compared to other special crops, this rating was lower than those given to parsley, sage, or spearmint, all rated as 5 (very good), and, except for northern Alberta, was better than anise, which was rated as 3. Winter kill of the irrigated southern Alberta stand was 30% with snow fencing or straw cover, while it was 70% for non-irrigated stands in central and northern Alberta which were not protected by straw cover or wind breaks. Mint hay and oil yielas. Dry matter and oil yields at each stage of plant ontogeny are given in Table 1. Yields for northern Alberta were low, and, therefore, are not listed. The solid stands gave hay yields which increased with time until the beginning of September. The lowest yields were obtained before bloom at the vegetative stage of the plant. The highest yields were found at full bloom, which occurred near the end of August, but was a week 248
Table I. Yields of herb and oil of peppermint grown in solid stands in Alberta and harvested at different stages of plant ontogeny. Southern Alberta
Central Alberta Yield (kg/ha) Stage of ontogeny Vegetative Bud forming Beginning of bloom Bloom, 5% flowerettes 20% bloom 50% bloom 75% bloom 80% bloom Full bloom End of bloom
Yield (kg/ha)
Oil yield (wt % of herb)
Oil yield (wt %of herb)
Date of harvest
dry herb'
oil'
dry
fresh
Date of harvest
dry herb"
oil'
dry
fresh
Aug. I
3,167
41.50
1.31
0.24
July 5 July 27
4,043 4,408
41.44
0.93 0.94
0.18 0.18
Aug. 5
3,920
47.10
1.20
0.24
Aug. I
4,377
56.90
1.03
0.21
Aug. 7
4,357
52.28
1.20
0.24
Aug. 15 Aug. 22
5,384 7,012
54.91 79.24
1.02 1.13
0.24 0.24
Aug. 29 Sept. 4
7,112 7,045
85.34 71.16
1.20 0.99
0.26 0.22
Aug. 9 Aug. 14 Aug. 20 Aug. 27 Aug. 29 Sept. 3 Sept. 12
5,883 5,951 6,776 6,913 7,007 7,134 6,832
65.86 68.43 81.98 84.34 86.88 87.75 71.05
1.12 1.15 1.21 1.22 1.24 1.23 1.04
0.20 0.22 0.27 0.26 0.27 0.27 0.23
'Samples of harvested fresh mint were dried at 70°C in an air circulating oven. The dry matter of the herb at various stages was: 18.2%, vegetative; 20.0%, bud forming; 20.1-20.9%, dunng bloom; 21.8-22.5%, full bloom. Results m thiS and followmg tables are an average of three consecutive growing seasons. 'Hay was cured by air drying for two days prior to the distillation. During curing, the moisture loss was 30% at early stages of plant ontogeny, and close to 50% at later stages.
earlier in central than in southern Alberta. The results obtained after irrigation and one application of 225 kg N/ha indicated that yields in southern Alberta were slightly higher than in Washington state with row cultivation, but were lower than with solid stand (Nelson et al., 197Ia). Yields in Alberta were greater than those in Manitoba (Dorrell, 1972). Hamon and Zuck (1972) found increases in the number of leaves and stolons, and greater height and bushiness as a result of fertilization and irrigation under Saskatchewan conditions. Average oil yields were found to depend on the stage of plant development. The highest yields, 87.7 and 85.3 kg/ha, respectively, were experienced at full bloom in southern and central Alberta. This high yield was only possible over a narrow span of time. Seven days earlier, at 75% bloom, the oil yield was less, while six days later, at the end of bloom, a dramatic drop was experienced. Under similar cultivation conditions, Washington yields were 98.0-117.6 kg/ha for early harvests, and 132.7 for late harvests timed between the 17th to 31st of August (Nelson et al., 1971a). In central Alberta higher oil yields per plant were obtained until 5% bloom. During subsequent stages of blooming, and especially after 50% bloom, the southern Alberta yields were higher. In Washington Nelson et al. (l97Ia) reported yields of 2.3% (July 13), 1.8% (August 3), 1.69% (August 17), and 1.55% (August 31). Their results for solid stand did not differ from those of strip row cultivation but higher yields of oil per plant were gained from the addition of only 112 kg N/ha, along with irrigation. Treatment at the level of 112 kg N/ha was not done in Alberta. However, results of a 140 kg N/ha application with irrigation on a mint strip row are available from Saskatchewan (Hamon and Zuck, 1972). Their yields of oil in kg/ha were 44.0 for early harvest and 70.2 for late harvest, with a dramatic drop to 33.6 at the end of bloom. Similar yields were reported for Manitoba (Dorrell, 1972), for which the average yield of oil on a dry matter basis was 0.94%, with a peak yield of 1.15% that was recorded in the 249
middle of August. Somewhat similar results were obtained in central Alberta when no fertilizer was applied. The yield of oil was only 33.6kg/ha, and was 0.95% on a dry matter basis for mint hay. These results confirmed the suggestion that higher oil yields on the Prairies can be expected only after fairly heavy application of N fertilizer. Thin layer chromatography. The TL-chromatogram of peppermint oil and some of its associated pure compounds is illustrated in Figure I. The Rf values and tentative identities of the spots, and the number of constituents found in each spot are given in Table 2. As seen from Figure I, the TLC method was able to Rf SOLVENT
VALUE
-+
,-
,
FRONT
functional group
.73__ .66~-- hydrocorbon .59 _-OCH 3
.
47
constituents
oil
,
n 10
o
- -OCCH3 ::;CO
.28
t-
-OH
.12
f-
START,OO t-
Fig. I.
spot number
TL-chromatogram of peppermint oil and its associated pure compounds. Adsorbent: 300 p. of MN-Kieselgel N (Macherey Nagel & Co.); spot detection: spraying 1% vanillin in cone. sulfuric acid and heating in an oven at 105° for 10 min. Volume of oil applied: I p.1 (a band of 50 /.II was applied in preparative runs). J. Inst. Can. Sci. Technol. Aliment. Vol. 10. No.4. October 1977
Table 2. Thin layer chromatography data for peppermint oils from Alberta. Spot Number
Rf Value
Tentative Identity
Identity Confirmed by Preparative TLGL Chromatography, MS and IR Analyses
Numberof Constituents in the Spot
I 2
0.13 0.20
? Menthol
2
3 4
0.26 0.29
Piperitone Neomenthol
5 6 7 8 9
0.33 0.35 0.38 0.46 0.53
10
0.67
1,8-Cineol Pulegone Isomenthone Menthone Menthyl Acetate Menthofuran
II
0.72
Minor constituents 38 & 39a Menthol (32), 3-0ctanol (16), Isomenthol (33), and minor or trace constituents 7,15,17-19,&25. Piperitone (4) Neomenthol (27), Neoisomenthol (31), and linalool (26) I ,8-Cineol (10) Pulegone (35) Isomenthone (24) M~nthone (22), and trace constituent (II) Menthyl acetate (30), and a minor constituent, neoisomenthyl acetate (29) Menthofuran (20), and trans-sabinene hydrate (21) a-thujene (I), a-pillene (2), camphene (3), {3-pinene (4), sabinene (5), {3-myrcene (7), limonene (8), {3-phellandrene (9), cisocimene (12), p-cymene (13), y- terpinene (14), {3-caryophyllene (28), and minor and trace constituents 23, 34, 36, 37 (Germacrene D), 41-46.
Terpene Hydrocarbons
9 I 3 I I I '2 2 2 22
aGLC chromatogram peak numbers (see Figure 2).
separate a great number of oil constituents, and, partIcularly, the menthone related constituents (Petrowitz, 1960) into menthol (Rf 0.20), neomenthol (0.29), isomenthone (0.38), menthone (0.46), menthyl acetate (0.53), and menthofuran (0.67). However, menthol was not well separated from isomenthol, nor was neomenthol from neoisomenthol, or menthyl acetate from neomenthyl acetate. Nevertheless, TLC combined with GLC separation was proven to be an effective method for analysis of most of the hydrocarbon, oxide, ester, alcohol, and carbonyl constituentsof the oil. Gas liquid chromatography. Both columns were used for each oil analysis (Figures 2 and 3). On a fresh column myrcene was resolved from limonene, and pulegone from sesquiterpenes. If a preliminary TLC separation was omitted, then 1,8-cineol was eluted along with peak 11, menthofuran with trans-sabinene hydrate, and menthyl acetate with neoisomenthol. On an aged column the chromatogram showed that 1,8-cineol was well resolved from peak 11, and menthofuran from sabinene hydrate, while pulegone appeared just after ,8-caryophyllene, and piperitone was eluted along with two sesquiterpenes. The separation of menthone and menthol stereoisomers was best on freshly prepared columns. The elution order was menthofuran, menthone, isomenthone, neomenthol, neoisomenthyl acetate, menthyl acetate, neoisomenthol (only the latter two have a trend to inverse on an aged column), menthol, isomenthol, pulegone, and piperitone. This elution sequence is different from that on Carbowax 20M, or sucrose diacetate hexaisobutyrate, SAIB (Handa et al., 1964). Gillen and Scanlon (1972) indicated that the limiting factor on a number ofliquid phases for the separation of menthone and menthol stereoisomers was the resolution of neoisomenthol either from neomenthol or menthol. In our analyses this was the case only on the aged column, while on the fresh column it was the Can. Inst. Food Sci. Techno!. J. Vol. 10, No.4. October 1977
resolution of neoisomenthol from its own acetate, and from menthyl acetate. In this study TL- and GL-chromatography had to be used in combination in order to get the best separation of peppermint oil constituents. This technique, as proven by Nigam et al. (1963) and Nigam and Levi (1964), offers advantages in chemical and taxonomical characterization of oils. However, recent analysis of peppermint oil by using liquid-liquid-chromatography for preliminary separation of hydrocarbons from oxygenated constituents, followed by preparative GLC using capillary columns (BelAfi-Rethy et al., 1973), appeared even more advantageous when an oil analysis that includes all trace constituents is required. Also superior was a combination of alkali extraction, fractional distillation, and subfractionation by alumina and silver nitrate-alumina column chromatography, which was then followed by capillary and preparative G LC (Lawrence et al., 1972). The latter authors characterized a total of 99 constituents in oil from the Wilamette Valley, Oregon. Nevertheless, a fast and accurate characterization of major and minor oil constituents, that reflects the oil's origin, quality, and degree of purification, can be achieved by a simple GLC separation (Hefendehl and Ziegler, 1975). Mass spectral analysis. Additional confirmation of the identities of peppermint oil constituents was provided by a combination of TL- and GL-chromatography, and mass spectral analysis. Data on some menthone-related oil constituents are provided in Table 3. The fragmentation patterns of most oil components were compatible with those of standard compounds, or with literature data (e.g., menthols and theIr acetates, Thomas and Willhalm, 1966; menthone and isomenthone, Willhalm and Thomas, 1965). Further discussion is restricted to four major oil constituents.
250
22
®
20,21
Alberta oil fresh column
32
10
8
26,27 24
36,37
11 18 1 '} 3
4 5 67
9
12
15
17
23 19
42 43
44 45
46
22
®
32
29
30 31
Commercial oil from United States 10 27
Fig. 2.
GL-chromatogram of peppermint oil on a freshly packed column. (a) Alberta oil. (b) Commercial oil from the United States. Peak designations: a-thujene (I), a-pinene (2), camphene (3), {3pinene (4), sabinene (5), {3-myrcene (7), limonene (8), {3-phellandrene (9), 1,8-cineol (10), trans-2-hexanal (II), cisocimene (\ 2), p-cymene (13), y-terpinene (14), aliphatic alcohols (\5-19, with 3-octanol at peak 16), menthofuran (20), trans-sabinene hydrate (21), menthone (22), pentadecane (23), isomenthone (24), a sesquiterpene (25), linalool (26), neomenthol (27), {3-caryophyllene (28), neoisomenthyl acetate (29), menthyl acetate (30), neoisomenthol (31), menthol (32), isomenthol (33), a sesquiterpene (34), pulegone (35), sesquiterpenes (35-39, with Germacrene D at peak 37), piperitone (40), and sesquiterpenes (42-46).
The principal fragment from menthol, C.H,O+ at ml e 71, arose from cleavage of the 3,4-bond of the molecular ion, followed by loss of C6 H 13 • Removal of OH and H from the parent ion to give various ions at ml e 138 was followed by further breakdown to cyclic species at ml e 95 (C 7 H" +), and mle 81 (C 6 H g ). The fragment at mle 82 (C 6 H lO +) arose 251
from cleavage of an mle 138 ion. Acetic acid was eliminated from menthyl acetate to give the ion at mle 138 (C IO H 18 +). This occurred either by 1,2-elimination, or by loss along with a H from the isopropyl side chain. Further fragmentation was similar to that of menthol. J. Insl. Can. Sci. Technol. Alimenl. Vol. 10. No.4, October 1971
Alberta oil
"
aged column
31 '0
J2
22
" 20 36,37,40
28
~
L Fig. 3.
~
l
GL-chromatogram of Alberta peppermint oil on the aged column. Peak identities as in Figure 2. Table 3. Mass spectral data for some peppermint oil constituents. GLC Parent Peak Numbera Peak [M.]b 20 22 24 30 32 35 40
150 154 154 198 156 152 152
Base Peak 108 112 112 95 71 81 82
Compound Identity
mle'
Menthofuran Menthone Isomenthone Menthyl Acetate Menthol Pulegone Piperitone
15079109779115180 6941551391547056 694155139154 III 8113812367698296 81 95 138 82 123 55 41 96 1526710982436942 110951371094115254
aGLC separation data as on Figure 2. bA parent peak in the range ofmle 204 - 212 was ascribed to sesquiterpenes. Some sesquiterpenes of peppermint oil were determined by Vlahov et al. (1967), while a number of their fragmentation patterns were described by Moshonas and Lund (1970). 'In decreasing order of relative abundance. Table 4. Oil composition of peppermint plants grown in a solid stand in Central Alberta, and harvested at different stages of plant ontogeny. Stage of ontogeny
Total menthol
L-Menthol
Menthyl ester
Total menthone
Menthofuran
Pulegone
39.6a 40.0 40.4 44.9 46.5 49.2 54.6 53.1
30.2 31.8 32.3 36.3 39.2 40.3 41.2 44.2
4.4 4.0 3.1 3.4 3.5 3.7 3.8 3.8
43.3 40.0 36.1 32.5 28.1 29.4 25.8 24.0
0.8 0.8 1.0 l.l l.l 1.2 1.7 2.9
0.7 0.7 0.8 1.1 0.9 0.8 l.l 0.9
vegetative vegetative bud forming 5% bloom 50% bloom 75% bloom full bloom end of bloom aResults in percent.
The main fragmentation of menthone to give the enol at mle 112 (C,H 12 0+) was a cyclic process resulting from transfer of a y-hydrogen from the isopropyl side chain to the carbonyl, with simultaneous ,8-cleavage. Various fragmentation pathways could explain the species at ml e 69 (C.H 5 0+, perhaps by loss of C a H6 from mle Ill), mle 41 (Ca H 5 +, or C2 HO+ via ring cleavage), or mle 55 (C,H 7 +). Finally, the only possible facile cleavage of the menthofuran molecule can be explained by a retro-Diels-Alder type fragmentation to give two rather stable unsaturated fragments, the furane-like C7 HgO+ at mle 108, and CaH 6 • Oil composition. During three consecutive seasons, the composition of the oil showed little variation when analyzed at the same stages of plant ontogeny but did vary between stages (Tables 4 and 5). Can. Insl. Food Sci. Technol. J. Vol. 10. No.4, October 1977
Southern Alberta oil obtained at 20%bloom appeared to have the most acceptable profile from the standpoint of chemically evaluated oil quality. The oil at 5% bloom seemed to be an immature product, while the oil at 50% bloom had already reached a high content of menthofuran, and had reverted to a somewhat immature status. This indicated that secondary growth was corning up from the root shoots while the main portion of the mint field was in bloom. Therefore, in an attempt to develop a quality peppermint oil, the harvest time would have to be limited to a fairly narrow time period. Further into the growing season, after passing the stage of 50% bloom, the oil became more mature, and, eventually, entered a phase where the menthofuran started to decrease. However, even this oil could not be considered a quality oil for dentifrice
252
Table 5. Oil composition of peppermint plants grown in a solid stand in Southern Alberta, and harvested at different stages of plant ontogeny. Stage of ontogeny vegetative vegetative bud forming 5% bloom 20% bloom 50% bloom 75% bloom full bloom end of bloom a
Total menthol
L-Menthol
Menthyl ester
Total menthone
Menthofuran
Pulegone
36.1" 35.4 47.6 49.5 53.1 48.8 51.9 54.0 57.1
32.6 32.9 42.6 42.0 47.5 44.7 43.2 45.1 47.0
2.3 1.9 3.3 3.4 4.2 3. I 4.2 6.6 9.6
43.6 43.5 35.0 30.8 27.8 29.7 23.7 17.9 17.2
0.7 0.5 1.8 1.9 2.8 6.0 6.2 10.4 6.0
1.2 1.2 0.9 1.0 1.5 1.6 2.7 2.3 0.8
Results in percent.
The major and minor hydrocarbon constituents of peppermint oil are listed in Table 6. Three stages of development, 5% bloom, 75% bloom, and the end of bloom, are given to illustrate the changes during a season. The major terpene hydrocarbons were limonene and ,a-pinene, fol-
or chewing gum production, because it was a relatively harsh and overmatured product at this late date of harvest. By the same token, the best quality of oil from central Alberta would be an oil taken from a harvest between 50% and full bloom. Table 6. Hydrocarbon constituents of peppermint oils. Constituent
Retention time (Menthol = 1.00)'
Camphene ,B-Caryophyllene p-Cyrnene Limonene ,B-Myrcene a-Pinene ,B-Pinene Sabinene y- Terpinene
0.14 0.93 0.41 0.29 0.27 0.12 0.19 0.23 0.38
TOTALc Monoterpene hydrocarbons Sesquiterpene hydrocarbons (includes OLC peaks 36 & 37) Benzene related hydrocarbons
A
L
B
E
Southern I'
II
III
0.2 0.5 0.5 3.0 0.7 0.8 1.1 0.5 0.3
trace 0.6 0.6 2.7 0.8 0.7 1.6 0.4 0.4
trace 0.7 0.4 2.6 0.6 0.7 1.4 0.2 0.6
6.53
6.53
2.75 0.52
1.11 0.58
R T A Central
Northern
Commercial oil (United States)
II
III
III
0.3 1.8 0.5 1.6 0.6 0.7 0.9 0.8 0.7
0.3 1.7 0.5 1.5 0.7 0.5 0.7 0.8 0.7
0.3 1.7 0.3 2.0 0.7 0.6 0.9 0.8 0.6
trace 0.5 trace 0.8 trace 0.2 0.9 0.3 trace
0.2 2.3 0.4 1.6 0.7 0.5 0.8 0.8 0.6
6.16
5.67
5.32
6.04
2.13
5.90
1.42 0.43
3.95 0.45
3.70 0.50
3.79 0.33
0.45 trace
3.98 0.38
Northern
Commercial oil (United States)
'Conditions of OLC separation as on Figure 2. 'Harvest time I, 5% bloom; II, 75% bloom; III, end of bloom. Results in percent. 'Constituents below O. I% are not listed. Table 7. Alcohol and ester constituents of peppermint.oils. Constituent
Retention time (Menthol = 1.00)'
Isomenthol Linalool Menthol Menthyl acetate + neoisomenthyl acetate Neoisomenthol Neomenthol 3-0ctanol Sabinene hydrate
A
L
B
E
Southern II
III
R T A Central II
III
III
1.05 0.90 1.00
0.3' 0.3 42.0
0.3 0.3 43.2
0.3 0.4 47.0
0.9 0.5 36.3
0.5 0.3 40.3
0.6 0.5 44.2
0.4 0.9 58.9
0.7 0.5 42.8
0.95
3.4
4.2
9.6
3.4
3.7
3.8
1.9
6.6
0.95 0.91 0.59 0.74
0.9 2.6 0.3 0.7
0.9 2.5 0.4 0.7
1.3
1.3
3.3 0.4 0.7
3.0 0.6 0.7
1.2 3.4 0.7 0.7
1.2 3.3 0.6 0.7
trace 3. I 2.0 0.9
1.2 4.2 0.4 0.7
46.78 3.40
47.95 4.20
53.09 9.60
42.73 3.39
46.51 3.69
50.46 3.83
64.25 1.89
50.14 6.56
TOTAL' Monoterpene alcohols Esterified "Menthol"
'See Table 6 for explanation. 'Results in percent. 'Constituents below 0.1 % are not listed.
253
J.
Insl. Can. Sci. Technol. Alimenl. Vol. 10. No.4. October 1977
lowed by sesquiterpenes (,B-caryophyllene, and GLC peaks 36, and 37, Germacrene D). Altogether, eleven constituents above 0.1% were found. The total monoterpene hydrocarbon contents decreased from south to north when equal stages of plant development were compared. Thus, the north had only one third of the amount found in plants grown in the south. A similar trend was observed for benzene related constituents. The composition of alcohol and ester Constituents of peppermint oil is given in T~ble 7. The presence of all four menthol stereoisomers was Confirmed. The L-menthol content in southern Alberta (42.0-47.5%) was higher than that of central Alberta oils (32.3-44.2%). The northern oil obtained from a harvest, which, by plant morphology, would correspond to the end of bloom stage, was in fact a quite overmature oil. Generally, the shorter northern growing season decreased development stages to an even narrower time period than that experienced in central Alberta. The content of neomenthol was significant, and amounted to 2.5-3.4%. Neoisomenthol and isomenthol contents were low, especially in southern oils. The former had a maximum amount of 1.3%, while the latter never exceeded
0.3%. The content of total monoterpene alcohols increased with time during a season. Esterified menthol, like the free alcohols, also increased. This was particularly so in southern oils. The northern oil, though overmatured, did not accumulate the ester to more than 1.9%. The monoterpene ketone and oxide constituents are given in Table 8. During a given season, menthone was higher in central Alberta oil than in the southern Alberta oil. The overmatured southern oil had 15.1% of menthone at the end of bloom, while central oils never fell below 21.2%. Of interest was the northern oil, which contained only 11.9% of menthone at the end of bloom. In all of the oils, the isomenthone, at about 3%, was practically unchanged throughout the season, with'a small drop in content being experienced only at the end of the season. Piperitone and pulegone were low, except in the northern oil. The menthofuran contents of a high 6.3% in southern oils, 1.1-2.9% in central oils, and 0.3% in northern Alberta oils were theoretically expected (Burbott and Loomis, 1967). Finally, 1,8-cineol, generally near 5-6%, appeared not to vary significantly with the stages of plant ontogeny. The compositional data from Tables 6-8 were used to
Table 8. Monoterpene ketone and oxide constituents of peppermint oils. Constituent
Retention time (Menthol = 1.(0)'
Cineol-I,8 Isomenthone Menthone Menthofuran Piperitone Pulegone
0.34 0.84 0.77 0.74 1.29 1.11
TOTAL' Monoterpene ketones Monoterpene oxides
A
L
B
E
Southern II
III
5.9' 3.5 27.3 1.9 0.5 0.9
6.3 3.0 20.7 6.3 0.6 2.7
5.1 2.1 15.1 6.0 0.2 0.8
32.18 7.84
27.04 12.59
18.19 11.11
R T A Central
Northern
Commercial oil (United States)
II
III
III
5.2 3.0 29.5 1.1 1.1 1.1
5.7 3.4 26.0 1.2 1.2 0.8
5.1 2.8 21.2 2.9 0.9 0.9
4.2 4.4 11.9 0.3 6.6 2.0
5.2 3.3 19.4 2.0 0.6 0.9
34.77 6.31
31.44 6.86
25.77 8.02
24.79 4.52
24.15 7.19
'See Table 6 for explanation. 'Results in percent. 'Constituents below 0.1% are not listed. Table 9. Significant constituent ratios for Alberta peppermint oils. A
B
Terpenes' All constituents
Menthone Isomenthone
C Limonene 1,8-Cineol
5% bloom 75% bloom end of bloom
0.109 0.110 0.112
9.77 7.70 7.59
0.31 0.27 0.40
5% bloom 20% bloom 75% bloom end of bloom Commercial oil from United States
0.125 0.125 0.129 0.113
7.80 7.95 6.85 7.15
0.50 0.47 0.43 0.51
0.101
5.81
0.37
Ratio of Percentages D E Menthofuran NeomenthoJ Menthyl acetate "Menthone related constituents'"
"Menthone related constituents" "Menthol related constituents"
G
CENTRAL ALBERTA 0.032 0.88 0.038 0.92 0.107 0.86 SOUTHERN ALBERTA 0.059 0.77 0.092 0.62 0.209 0.60 0.34 0.260
15.12 14.54 16.20
0.75 0.62 0.51
18.92 19.90 20.36 18.66
0.67 0.58 0.59 0.38
0.75
12.99
0.45
0.081
a"Terpenes": Monoterpene hydrocarbons emerging prior to and including 1,8-cineol. '''Menthone related constituents": menthofuran + menthone + isomenthone. '''Menthol related constituents": neomenthol + menthol + menthyl acetate + isomenthol 1961). Can. Inst. Food Sci. Technol. J. Vol. 10, No.4. October 1977
F "Menthol related constitutents"" Neomenthol
+ neoisomenthol (all ratios according to Smith and Levi,
254
calculate ratios of percentages of various combinations of constituents, according to Smith and Levi (1961), in order to check the possibility of differentiating the oils from Alberta from those of the United States and other geographical origins. These ratios are presented in Table 9. Ratio A, the terpenes emerging in GLC analysis prior to and including 1,8-cineo1 over all constituents, was a reflection of a genuine natural and non-redistilled oil. Ratio B, menthone over isomenthone, depended on harvest time. This ratio, between 6.8-9.8 with an average of 7.8, was high when compared to oils from the United States which range from 2.7-6.6. Though ecological factors influenced this ratio, there was not a clear cut difference between ratios for central and southern Alberta. Ratio C, which is genetically controlled, and which varies from 0.2-0.7 for peppermint oils, averaged 0.33 for central and 0.48 for southern oils. An increase in ratio D, menthofuran over menthone related constituents, with plant maturation was a noteworthy trend. Ratio D was strongly influenced by the stage of plant ontogeny. Prior to blooming, the plant gave an oil with a lower menthofuran content than was found during or after bloom. The fact that climate co-determined ratio D was reflected by lower values for the oils from the cooler central Alberta, and higher values from the warmer southern Alberta. Other environmental factors, such as the level of available N in a range of 56 to 448 kg N/ha, solid stand growing, or cultural treatments in a strip row, had no influence on this ratio (Nelson et al., 1971a,b). Thus, the values of 0.09 for the U.S. Midwest and 0.09-1.13 for Washington and Oregon, like Alberta results, were undoubtedly influenced solely by climatic factors. Ratio E, neomenthol over menthyl acetate, was higher in central than in southern oils. In the latter, decrease of the ratio during plant maturation was evident. Climatic factors also influenced ratio F, menthol related constituents over neomenthol. It ranged between 18.7-20.4 for southern and 14.5-16.2 for central Alberta oils. None of these values coincided with those of the U.S. Midwest (12.6-13.8), or Oregon (13.0-13.8), but the ratios from Central Alberta were close to those of Washington (14.9-17.3). The last ratio, G, reflected the phenomenon of menthone reduction during plant maturation, and exposure of the plants to prolonged sunlight. The values were lower for southern than for central Alberta, with values at the end of bloom of 0.38 and 0.51, respectively. The fairly conclusive distinction of Alberta grown peppermint oils from those of the United States is illustrated on Figures 4 and 5. The oils grown in the U.S. Midwest, Oregon, or Washington were readily distinguished by plotting ratio E vs ratio D for oil constituents, as reported earlier by Smith and Levi (1961), or, more recently, by Nelson et al (1971a,b). In such a plot the central Alberta oils were also readily differentiated. However, the oils from an early harvest in southern Alberta generally coincided with plots for Oregon and partially overlapped with the U.S. Midwest, while the oils of more matured plants, or late harvests gave similar plots to oils from Washington. A better differentiation of Alberta oils was obtained when ratio F was plotted vs ratio G (Figure 5). Here the plots of oils from central Alberta were grouped in an area well separated from those for Oregon and the U.S. Midwest, and only the late harvest, mostly at the end of bloom, merged 255
.5....--------------------.4
P .3 Q
~ 0::
SOUTHERN ALBERTA
.2
.1
o
.2
.4
.6
1.2
RATIO «Eo
Fig. 4.
Distribution plot of ratio D (menthofuran/menthone related constituents) vs ratio E (neomenthol!menthyl acetate) showing the variation with the geographical origin of the oil.
u.•
o ~
0::
.4
.5
.6
.7
.8
RATIO "G" Fig. 5.
Distribution plot of ratio F (menthol related constituents/neomenthol) vs ratio G (menthone related constituents/menthol related constituents) showing the variation with the geographical origin of the oil.
with the plots of Washington oils. Southern Alberta oils at any time of harvest were sharply separated from all other oils. In conclusion, the data for Alberta indicate that optimum cultural treatments on a solid stand, including high rates ofN, and irrigation (in southern Alberta) can provide satisfactory yields of both oil and herb. An oil of optimum profile, with a quality comparable to that of imported oils, can be obtained. However, harvesting must be done over a fairly narrow time period. A complete determination of the feasibility of growing peppermint in Alberta would require additional examination of the cost of establishing J. Inst. Can. Sci. Technol. Aliment. Vol. 10, No.4, October 1977
and maintaining a mint stand, especially in the light of the extent of winter kill. Furthermore, a marketing study would be necessary, since each peppermint producing area must generally establish its own market, rather than expect to simply replace existing sources.
Acknowledgement This work was supported by a grant from the Alberta Agricultural Research Trust. The assistance of T. Leishman in field distillation of the oils is gratefully acknowledged. Gratitude is also expressed to Dr. R. E. Harris, CDA Research Station, Beaverlodge, and Dr. N. Marchak, at Two Hills, for the growing of mint on observation plots, to Dr. R. Gaudiel, Alberta Horticultural Research Center, Brooks, for his valuable suggestions on cultural treatments of mint fields, and to I. P. Callison & Sons, Inc., Intern., Chehalis, Washington, for some oil composition control data.
References BeIAfi-Relhy, K.. Iglewski. 5., Kerenyi. E. and Kolta, R. 1973. Untersuchung der Zusammensetzung von einheimischen und auslandischen atherischen 6Jen. II. Analvse eines ungaris~ chen Pfefferminzoles. Acta Chim (Budapest) 76: 167. • Burbon, A. J. and Loomis, W. D. 1967. Effect ofIight and temperature on monoterpenes in peppermint Plant Physiol. 42:20. Dorrell, D. G. 1972. CDA Research Branch, Morden, Manitoba. Franz, C. ~ra7;t~ E~:~. ~2~6~~d N nutrients on the formation of essential oils of Mentha piperita.
Can. Inst Food Sci. Technol. J. Vol. 10. No.4, October 1977
Gillen. D. G. and Scanlon, 1. T. 1972. The separation of menthol-menthone stereoisomers. J. Chromato~. Sci. 10:729. Gretskaya, R. L. 1973. Effectiveness of Nand P fertilizers for irrigated peppermint. Tr. Vses. Nauchno-Issled. Inst. Kul't. 6:75. Hamon, N. W. and Zuck, D. A 1972. Peppermint as a cash crop in Saskatchewan. Can. 1. Plant Sci. 52:837. Handa, K. L, Smith, D. M., Nigam, I. C. and Levi, L 1964. Essential oils and their constituents. XXIII. Chemotaxonomy of the genus Mentha. J. Pharm. Sci. 53: 1407. Hefendehl. F. W. and Ziegler, E. 1975. Analytik von PfefferminzOlen. Deu!. Lebensmittel Rundschau 71 :287. Kruepper. H.. Lossner, G. and Shroeder, H. 1968. Effect of irrigation on the yield and quality of peppermmt (Mentha piperita). Pharmazie 23: 192. Lawrence, B. M., Hogg, 1. W., and Terhune, S. J. 1972. Essential oils and their constituents. X. Some new trace constituents in the oil of Mentha piperiw,. L. Flavor Ind. 3:467. Moshonas, M. G. and Lund, E. 0.1970. The mass spectraofsesquiterpene hydrocarbons. Flavour Ind. 1:375-378. Nelson, C. E.. Early, R. E. and Mortensen, M. A. 1971a. Effects of growing method and rate and time of N fertilization on peppermint yield and oil composition. Ext. Servo Wash. Agr. Exptl. Sta.. College of Agri., Wash. State Univ. eire. 541. Nelson, C. E., Mortensen, M. A. and Early, R. E. 1971b. Evaporative cooling of peppermint by sprinkling., Ibid. eire. 539. Nigam, I. C. and Levi. L. 1964. Essential oils and their constituents. XX. Detection and estimation of menthofuran in Mentha arvensis and other mint species by coupled QL- and TLchromatography. J. Pharm. Sci. 53: 1008. Nigam, I. c., Sahasrabudhe, M. and Levi, L. 1963. Coupled GL-TL-chromatography. Simultaneous detennination of piperitone and piperitone oxide in essential oils. Can. J. Chern. 41:1535. Petrowitz. H. 1. 1960. Zurkieselgelschicht-Chromatographie der stereoisomeren Menthole. Agnew. Chern.. 72:921. Smith. D. M. and Levi, L. 1961. Treatment of compositional data for the characterization 01" essentialoils. Determination of geographical origins of peppermint oils by GLC analysis. J. Agr. Food Chem. 9:230. Thomas. A F. and WiJlhalm. B. 1966. The mass spectra of the menthols, carvomenthols and their acetates and related alcohols. J. Chem. Soc. (B), 219. Vlahov, R., Holub. M., Ognyanov, I. and Herout, V. 1967. Sesquiterpenic hydrocarbons from the essential oil of Mentha piperita of Bul~arian ori~in. Coil. Czech. Chern. Comm. 32:808. Watson, V. K. and St. John, J. L. 1~955. Pepperlllint oil. R~lation of maturity and curing of peppermint hay to yield and composition of oil. J. Agr. Food Chern. 3: 1033. Willhalm._B. and Thomas, A. F. 1965. The mass spectra of menthone, isomenthone. and carvomenthone. J. Chem. Soc. (B). 6478. Received January 3, 1977
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