Planta Med. 57(1991) 347
Chemical, Physiological, and Toxicological Aspects of the Essential Oil of Some Species of the Genus Bystropogon* Dimitrios Economou1'2 and Adolf Na hrstedt2'3 1
Institut für Pharmazeutische Biologie und Phytochemie der Westfhlischen Wilhelms-Universität, Hittorfstr. 56, D-4400 Münster, Federal Republic of Germany 2 Part of the dissertation thesis of D. Economou (29) Address for correspondence
Abstract The composition of the essential oils of Bystropogon plumosus, B. origanfolius var. palmensis,
B. wildpretii, B. maderensis, and B. canariensis var. smithianus were studied by GLC and GLC/MS. The first four species provide relatively similar oils characterized by monoterpene ketones as the main constituents while the last species is quite different with main constituents consisting of mono- and sesquiterpene hydrocarbons. In B. plumosus the ontogenetic variation of the monoterpene ketones was studied for a period of four years. Dur-
ing this time the behaviour of puiegone was inversely correlated with that of menthone and isomenthone. Pulegone was the most active compound of the monoterpene ketones in antimicrobial tests using three species of bacteria and fungi, respectively.
Key words
with var. origanifolius, var. ferrensis (Ceb. et On.) I. LaSerna and var. palmensis Bornm., 3. B. odoratissimus Bolle, and 4. B. wildpretii I. La-Serna) and the section Canariense I. La-Serna (5. B. canariensis L'Her. with var. canariensis and var. smithianus Christ, 6. B. punctatus L'Her., and 7. B. maderensis Webb) (2). The authors of (2) and (3) regret that phytochemical data which may help to clarify the systematic position of these species are rare;
nevertheless they state that all members of the group
Plumosus smell like menthol whereas those of Canariense do not (3).
Bystropogon species are traditionally used
in South America to inhibit the germination of stored potatoes and to protect them against predatory insects (4). The dried leaves of B. plumosus are used in the Canary Islands as a remedy against colds in native folk medicine ("poleo"-tea) (5). Several batches of leaves of B. plumosus have been analyzed for their essential oils and these were found to contain more than 80 % monoterpene ketones with
pulegone, menthone, and iso-menthone as main con-
Essential oil, monoterpene ketones, pulegone, menthone, isomenthone, ontogenetic varia-
stituents (5). A search for monoterpene glycosides in B. plumosus failed but succeeded in the isolation of the new
tion, antimicrobial activity, toxicity, Bystropogon,
hemiterpene glucoside 2S-methyl-1 -yl-f3-n-glucoside (6).
Lamiaceae.
In the present study the Canarian species B. origanfoliusvar. palmensis(B in Table 1) and B. wildpretii
(C) of section Plumosus and B. maderensis (0) and B. Introduction
canariensis var. smithianus (E) of section Canariense were investigated with regard to their essential oil composition.
The genus Bystropogon (mci. Mintho-
The data obtained in (5) of a comparable batch of B.
stachys Bunge) includes evergreen shrubs and belongs to
plumosus (section Plumosus) are presented as A in Table 1 for comparison. The oil of B. plumosus and its main constituents were tested for their activities towards some bacteria and some fungi; the ontogenetic variation of the oil and of its main constituents was investigated and is discussed.
the plant family Lamiaceae (Stachyoideae Saturejeae:
Thyminae). Centers of distribution are the Canary Islands, Madeira, and South America (Chile, Argentina). According to Engler and Prantl (1) the genus is subdivided into the two sections Bystropogon Benth. (Canarian-Maderian species) and Minthostachys Benth. (South American species: B. rnol-
us Kunth., B. spicatus Benth., B. tomentosus Benth., B. canus Benth,, B. glabrescens Benth.), Based mainly on mor-
Materials and Methods Plant material
phological and anatomical data the section Bystropogon
B. plumosus and B. canariense var. smith ianus
was recently further divided (2) into the section Plumosus I. La-Serna (1. B. plumosus L'Her., 2. B. origanfolias L'Her.
were obtained from Dr. Buttler (Univ. Bochum); B. maderensis was collected from Madeira. All three species were cultivated in the Ex-
* Dedicated
perimental Garden of this Institute; vouchers are deposited under PBMS 2, 3, and 4, respectively. The essential oils B and C were dis-
to Professor Ernst Reinhard on the occasion of his
65th birthday on August 21, 1991.
tilled from dried leaves of B. origan(folius var. palmensis and B. wildpretii obtained through the courtesy of Dr. Ehmke (Techn.
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Received: March 13, 1991
348 Planta Med. 57(1991)
Diinitrios Economou and Adolf Nahrstedt
Univ. Braunschweig) from La Palma; the material was identified according to La Serna Ramos (2) by a local authority. All living plantlets used for this study were propagated vegetatively from one individuum.
B. pluinosus specimens used for studies of on-
togenetic variation were kept in pots and were stored in the greenhouse at 18°C from Nov. ito April 31, and subsequently outside. During the summer periods 1984—1986 the mean temperature was generally lower than 18°C varying between 9.9°C (Oct.) and 17.7°C (July, Aug.). Mean daily averages of air temperature in Tenerife at an altitude of ca. 600 mare 11.7 to 15.1 °C in the winter period (Nov. to April) and 14.6 to 19.2°C in the summer period (33).
220 °C, 4°/mm, 5 mm isothermally at 220°C; FID. Peaks were identified by comparison with the results obtained in (5) and by addition
of standards.
GLC/MS system
Hewlett-Packard HP 5986; ionization energy 70eV; DB-WAX capillary column 60m x 0.25mm id.; helium 1.5 mllmin, split 1 :20; 60—220°C, 4°/mm, 5mm isothermally at 220°C; FID. Peaks were identified using the MS data collection of Dragoco, Holzminden, FRG.
Tests of antimicrobial activity Agar diffusion test system; the samples were
Steam distillation
applied onto paper disks (diameter 6mm, Oxoid, Wesel FRG) which
were placed on the agar plates inoculated with the microorganisms. The diameter of the zone of inhibition was taken as a measure of activity. E. coli, S. aureus, and P. aeruginosa were
Carlo Erba GC 6000; DB-WAX capillary column,
mixed with a Columbia Agar (Merck, Darmstadt FRG) and incubated with the soaked paper disks for 24 hat 37°C. C. albicans and A. niger were incubated at 37°C, M. mucedo at ambient temperature for 48 h on a Sabourand-Glucose 2 % Agar (Merck, Darmstadt FRG). The microorganisms stem from the collection of the Institut für Pharmakologie und Toxikologie, TU Braunschweig. The essential oil used for testing contained as main constituents pulegone
30m x 0.25 mm id.; split ratio 1:35, helium 1.15 mllmin; 60—
(55%), isomenthone (14%), menthone (8.4%), piperitenone
GLC system
Table 1 Composition of the essential oils obtained from B. plumosus (A), B. origanifoliusvar. palmensis (B), B. wildpretii (C), B. maderensis(D), and B. canariensis var. smith/anus (E). Peak areas of the GLC signals were not corrected. The constituents of E were additionally analyzed by GLC/MS. The data of A were taken from (5).
yield/bOg
A(5)
B
B. plumosus
B. origanifolius var. palmensis
C B. wildpretii
D B. maderensis
E
B. canariensis var.
smith/anus
1.17 ml (fr. w.)
1.32 ml (dm)
1.50 ml (dm)
0.75 ml (fr. w.)
0.1 ml (fr. w.)
0.34 0.32 0.06 0.23 0.64
0.23 0.25
0.08
0.28 0.32
1.97 0.29
(dmorfr.w.) Monoterpene hydrocarbons
a-pinene
-pinene camphene sabinene myrcene a-phellandrene limonene f-phellandrene/1,8-cineole c/s-/3-ocimene
trans-fl-ocimene a-terpinene r-terpinene terpinolene p-cymene Sesquiterpene hydrocarbons
Monoterpene alcohols
—
2.50 0.06 0.13
0.10
—
0.23 0.59 —
0.75 —
0.18 0.90
0.09 0.01
0.14 0.30 0.01 2.20 0.05 0.07 0.42
—
0.10 —
—
0.32
0.10 22.15
0.19 0.06 1.58
—
0.10
—
0.07
—
6.30
—
—
—
—
1.68
—
—
—
—
3.32
0.03
—
0.01
80%) of which either pulegone (A, D) or menthone (B, C) are the main constituents. Sample E clearly differs from A to D in
that the yield is low (0.lml/lOOg fresh weight) with the main constituents being the monoterpene hydrocarbons (38.7%) of which ca. 22% was sabinene, and the sesquiterpene hydrocarbons (28.8%) of which more than 21% was germacrene D; the monoterpene ketone fraction, however, accounts for only 2.3%.
Thus, all investigated oils of species of the section Plumosus (A, B, C) represent a chemotype that is rich in essential oil, high in monoterpene ketones, and low in monoterpene hydrocarbons. This result confirms the ob-
— —
93.4
92.4
0.96 1.05
81.60 —
0.25 — —
4.60
93.1
—
—
0.52 —
0.67 0.25 0.67 0.19 — — —
0.52 0.16 —
1.17
82.3
servation (3) that members of the section Plumosus smell peppermint-like. The situation in section Canariense differs in that D belongs to the former type but E is characterized by poor oil production with low concentration of monoterpene ketones contrasting with a high amount of mono- and
sesquiterpene hydrocarbons. With respect to these biochemical parameters, section Plumosus seems to be
quite homogeneous whereas section Canariense combines different chemotypes; this view, however, based on some individuals of 3 out of 6 and 2 out of 4 species and varieties, respectively, needs further investigation together with the
question how far chemical polymorphism may occur, which is well known for essential oils obtained from Lamiaceae (7). In addition, the examination of hybrids ob-
served in the section Canariense (2) may contribute to taxonomic questions. The essential oils obtained from South American species are also high in pulegone, menthone, and isomenthone [see (5) for references, (30)1 and thus correspond with the section Plumosus of the Canary Islands and Madeira.
As expected for essential oil producing plants, the amount of oil as well as its composition vary during ontogenesis. This was shown for B. plumosus oil and its main constituents (Table 2). The yield of oil varied between
1.0 and 2.4% of fresh material from 1984 to 1987. The highest values (1.5 to 2.4%) were obtained during the winter period (November—April) when the plants were stored at a constant 18°C in the greenhouse (thus not under
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Monoterpene esters
Planta Med. 57(1991)
Dimitrios Economou and Adolf Nahrstedt
Table 2 Variation of yield of essential oil and its main components from B. plumosus during August 1983 to September 1987.
time of
yield of oil [%l
harvesting
1983
1984
1985
isomenthone
1.60 1.20 1.20 1.50
23.0
22.1
35.3
40.9 50.0 43.4
13.9 18.7 16.5
21.5
01.30 04.02 04.25 07.02 08.06 09.13 10.03 10.18 11.01 12.07
1.50 1.55
9.4 2.4 3.4
26.0
2.00 1.20 1.30 1.40 1.80 2.00 2.00 2.40
49.0 63.0 72.3 40.0 52.5 55.2 45.3 48.6 51.0 49.2
15.6
27.8
11.5
16.0 14.0
1.70 2.00 2.00 1.98 1.70 1.10 1.15 1.40 1.70 1.70
73.7 58.0 61.0 69.4 72.3 68.0 39.0 44.0 30.7 46.2
0.2 9.1 8.0 5.6 3.3 6.6 19.1 10.9 13.7 8.8
1.80 1.90 2.00 2.00 1.00 1.05 1.00 2.00 2.00
57.2 61.4 66.0 51.0 35.8
4.7 3.3 1.3 9.5 11.0 5.9
02.01
01.08 02.05 03.04 05.06 06.18 08.01
09.20 11.06 12.22 1987
menthone
08.08 09.12 10.15 12.15
03.04 03.15 04.18 06.06 06.20 07.19 09.27 10.18 12.06 1986
% of oil;
pulegone
01.30 03.26 06.11
09.02
7.5 11.0
The antimicrobial activity of the essential oil obtained from B. pluinosus and its main constituents was tested using some bacteria and fungi for comparison with some well known antibiotics in the agar diffusion test
23.3 20.8
9.5
following the suggestions of Jannsen et al. (8). The results in Table 3 demonstrate a weak activity of the oil towards bacteria with Staphylococcus aureus as the most sensitive and
16.8 23.2
Pseudomonas aeruginosa as the most resistant species, as expected (13). The oil shows a more pronounced activity against fungi, especially Mucor inucedo. Of the three main
18.5 15.1
12.4 7.2 3.4 6.6
constituents, pulegone, menthone, and isomenthone, pulegone is the only compound that acts on fungi and that also exhibits the most pronounced antibacterial activity. These results indicate that pulegone is mainly responsible for antimicrobial properties of the oil. Similar observations were obtained by Kellner and Kober who investigated the antiseptic properties of some essential oils (9) and their components (10) in the vapor phase on 8 species of bacteria and found that pulegone and menthone act as weak antiseptics. Göckeritz et al. (11) observed a lower fungistatic and a higher bacteriostatic concentration for both monoterpenes. Knobloch et al. (12) found that pulegone and menthone had a similar activity towards bacteria; the same group showed that pulegone and menthone only moderately influence membrane-associated basic biochemical processes whereas thymol or carvacrol are strongly inhibitory (13). Iii this context it is of interest that the insecticidal activity of Bystropogon essential oils is also mainly connected with pulegone (14).
17.3 14.9
24.3 13.9
8.8 6.0 3.0 10.9
51.4
9.1
38.0 20.7
14.0 16.5
21.0 6.6 8.4 20.0 32.0
33.9 55.4 54.9 57.0
9.2 6.5 5.8 8.8
27.2 9.4 8.8 7.7
40.4
2.10 2.10 1.20 1.15
19.1
15.6 13.7 12.3
natural conditions; see Materials and Methods). From these results the recommended time for harvesting the leaves is thus just before the onset of flowering in contrast to several
other Lamiacean plants in which harvesting is recommended during blooming or the late flowering period (31). After onset of flowering in late April, the yield decreased from ca. 2 % to 1—1.4% until the end of flowering in late
Escherichia Staphylococcus Pseudomonas co/i aureus aeruginosa
Mucor mucedo
2 8
5 13
28 49
22
18
36
0 0 0 0
0 0 0 0
34
9
12
34
12 21
2 4
10l
12
21
4
10il
6
4
4
0 0
isomenthone 5fi1 10/LI
6
14
4
0 0
50/Lg
20
22
10/Lg ketoconazol 10/Lg nystatin 20/Lg
16
31
7 0
pulegone menthone
tetracyclin ampicillin
5il
5l
(19, 2 1—25). Previously this oil was used medicinally as an
Aspergi/lus niger
4 14
5tl l0jil
Essential oils with high levels of pulegone are regarded as toxic (18, 19, 20). Several reports exist in the literature dealing with severe intoxications, sometimes ending with death, after ingestion of comparatively high doses of pennyroyal oil (from Mentha pulegium, sometimes Hedeoma pulegioides) that contains pulegone up to 80%
Candida albicans
2 10
ess.oil
to April. Without exception, isomenthone is higher than menthone. Interestingly, the amounts of both ketones vary inversely to those of pulegone in that they are usually low when pulegone is high and vice versa. This fits with the biogenetic scheme postulated for peppermint plants by several authors where pulegone is the precursor of menthone and isomenthone (15—17).
20.5
8.4
September. The content of pulegone varies between 22 and 72 % of the oil with high levels generally during November
Table 3 Antibacterial and antifungal activity of the essential oil obtained from B. plumosus (composition see Materials and Methods) and its main constituents (pulegone, menthone, isomenthone) in the microdiffusion test system. The values indicate the diameter of the inhibition zone in mm.
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350
Chemical. Physiological, and ToxicologicalAspects of the Essential Oilof Some Species of the Genus Bystropogon Planta Med. 57(1991) 351
the effects (25).
References 1 Engler, A., Prantl, K. (1897) Die natürlichen Pflanzenfamilien, IV.
Teil, 3. Aht. a, p. 314. Engelmann, Leipzig. La Serna Ramos, I. (1984) Phanerog. Monogr. 18. Cramer, Vaduz. La Serna Ramos, 1., Wildpret de la Torre, W. (1976) Vieraea 6, 77. Alliaga, T. J. (1984) Diss. Thesis, Univ. Gieflen, FRG. Nahrstedt, A., Economou, D., Hammerschmidt, F.-J. (1985) Planta Med. 51, 247. 6 Nahrstedt, A., Economou, D., Wray, V. (1990) J. Nat. Prod. 53, 1387. Hegnauer. R. (1966) Chemotaxonomie der Pflanzen, Vol. 4, p. 292, Birkhauser, Basel. 2
Jannsen, A. M., Scheffer, J. J. C., Baerheim Svendsen, A. (1987) Planta Med. 53, 395. 10
It seems obvious that essential oils are toxic
to different extents at high doses. Recently, the oils of Mentha rotund(folia and M. longifolia were shown to possess LD50 values in rats of 641 and 437mg/kg, respectively (26). On the other hand, intoxication was never observed
when such oils were used properly at a dosage recommended for medicinal usage. 5 g of dry herb of B. pluinosus
with a content of 1.5 % essential oil containing 70% pulegone provides a potential dose of pulegone of approximately 50mg (corresponding to ca. 0.8 mg/kg man); this calculation may be still too high as a normal tea preparation with hot water will never extract the entire essential oil. Unfortunately no information on the acceptable daily intake (ADI) dose of pulegone is available. For carvone, the ADI dose is estimated up to 1.25 mg/kg in man (27); the provisional limits for pulegone in beverages and foods are 100 and 25mg/kg, respectively (32). Accordingly, oils rich in
pulegone of some Bystropogon spec. are without doubt non-toxic in medicinal usage as antiseptics in traditional therapy. Acknowledgements Thanks are due to Dr. H. Buttler, Institut für spezielle Botanik (Univ. Bochum), for providing living plant mate-
rial and for several hints regarding the systematics of the genus Bystropogon, to Dr. A. Ehmke (Institut für Pharmazeutische Blobgie, TU Braunschweig) for collecting plant material on La Palma,
and to L. Kruger and coworkers (this Institute) for growing the plants. We are mostly indebted to Dr. F.-J. Hammerschmidt, Dragoco (Holzminden) for running the GLC/MS, and to Dr. med. V. Franke, formerly Institut für Pharmakologie und Toxikologie (TU
Braunschweig) for much help during the performance of the antimicrobial tests. Dr. G. Matthiaschk (BGA, Berlin) provided information on ADI values arid limits in foodstuffs. The linguistic help of Dr. V. Wray is very much acknowledged.
12 13
14 15 17
Kellner, W., Kober, W. (1954) Arzneimittelforsch. 4, 319. Keilner, W., Kober, W. (1955) Arzneimittelforsch. 5, 224. Giickeritz, D., Weuffen, W., Höppe, H. (1974) Pharmazie 29, 339. Knobloch, K., Pauli, A., Ibert, B., Weigand, H., Weis, N. (1989) J. Ess. Oil. Res. 1, 119. Knobloch, K., Weigand, H., Weis, N., Schwarm, H.-N., Vigenschow, H. (1986) in: Progr. in Ess. Oil Res. (Brunke, E.-J., ed.), p. 429, de Gruyter, Berlin. Vogler, U. (1985) Diplomarbeit, Univ. Kiel, FRG. Hefendehl, F. W. (1970) Phytochemistry 6, 823. Hefendehl, F. W., Murray, M. J. (1976) Lboydia 39, 39. Croteau, R. (1988) 1ST Atlas of Science 1. 182.
Steinegger, E., Hansel, R. (1988) Lehrbuch der Pharmakognosie und Phytopharmazie, p. 701, Springer, Berlin. 19 Nelson, S.D., Gordon, W. P. (1983) J. Nat. Prod. 46, 71. 20 Lewis, E. J., Friedrich, E. C. (1990) Planta Med. 56, 224. 21 Allen, W. T. (1887) Lancet 1022. 22 Jones, C. 0. (1913) Brit. Med. J. 745. 23 ValIance, W. B. (1955) Lancet 850. 24 Early, D. F. (1961) Lancet 580. 25 Gordon, W. P., Forte, A. J., McMurtry, R. J., Gal, J., Nelson, S. D. (1982) Toxicol. Appl. Pharmacol. 65, 413. 26 Perez-Raya, M. D., Utrilla, M. P., Navarro, M. C., Jimenez, J. (1990) Phytother. Res. 4, 232. 27 Toxicological evaluation of some flavouring substances and non-nutritive sweetening agents. FAO Nutrition Meetings Rep. Ser. No. 44A. WHO/Food Add./68.33 (1967). 28 European Pharmacopoeia, Vol. III, (1975), p. 68. 29 Economou, D. (1989) Diss. Thesis, Techn. Univ. Braunschweig, FRG. Baerheim Svendsen, A., Scheffer, J. J. C., Looman, A. (1987) Flavour Fragance J. 2, 45. Guenther, E. (1949) The Essential Oils, Vol. III, p. 396 if. Van Nostrand Comp., New York. 32 Flavouring substances and natural sources of flavourings. Council of Europe. 3"' edition; Strasbourg, 1981. x Climatological summaries of Santa Cruz de Tenerife, sea level 617 m. Deutscher Wetterdienst — Zentralamt, D-6050 Offenbach.
°
'
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abortifaciens and to induce menstruation at a dose of 1—3 minims (23) (1 minim = ca. 0.065 g). Later on it was abused for these purposes at doses of a tablespoonful (21) and a dose of 24 g (25) caused convulsions, uraemia, liver and lung damage; unfortunately most of the reports cited above do not give exactly the amount of pennyroyal oil administered but it seems likely that one or more doses of ca. 15 ml (about 250 mg/kg) are undoubtedly toxic (24). Cellular necrosis and acute lung and liver damage were also observed at doses of 400 mg/kg and higher given intraperitoneally to mice (25). The authors conclude that the isopropylidene grouping of pulegone and its derivatives is responsible for
Chemical, Physiological, and Toxicological Aspects of the Essential Oil of Some Species of the Genus Bystropogon* Dimitrios Economou1'2 and Adolf Na hrstedt2'3 1
Institut für Pharmazeutische Biologie und Phytochemie der Westfhlischen Wilhelms-Universität, Hittorfstr. 56, D-4400 Münster, Federal Republic of Germany 2 Part of the dissertation thesis of D. Economou (29) Address for correspondence
Abstract The composition of the essential oils of Bystropogon plumosus, B. origanfolius var. palmensis,
B. wildpretii, B. maderensis, and B. canariensis var. smithianus were studied by GLC and GLC/MS. The first four species provide relatively similar oils characterized by monoterpene ketones as the main constituents while the last species is quite different with main constituents consisting of mono- and sesquiterpene hydrocarbons. In B. plumosus the ontogenetic variation of the monoterpene ketones was studied for a period of four years. Dur-
ing this time the behaviour of puiegone was inversely correlated with that of menthone and isomenthone. Pulegone was the most active compound of the monoterpene ketones in antimicrobial tests using three species of bacteria and fungi, respectively.
Key words
with var. origanifolius, var. ferrensis (Ceb. et On.) I. LaSerna and var. palmensis Bornm., 3. B. odoratissimus Bolle, and 4. B. wildpretii I. La-Serna) and the section Canariense I. La-Serna (5. B. canariensis L'Her. with var. canariensis and var. smithianus Christ, 6. B. punctatus L'Her., and 7. B. maderensis Webb) (2). The authors of (2) and (3) regret that phytochemical data which may help to clarify the systematic position of these species are rare;
nevertheless they state that all members of the group
Plumosus smell like menthol whereas those of Canariense do not (3).
Bystropogon species are traditionally used
in South America to inhibit the germination of stored potatoes and to protect them against predatory insects (4). The dried leaves of B. plumosus are used in the Canary Islands as a remedy against colds in native folk medicine ("poleo"-tea) (5). Several batches of leaves of B. plumosus have been analyzed for their essential oils and these were found to contain more than 80 % monoterpene ketones with
pulegone, menthone, and iso-menthone as main con-
Essential oil, monoterpene ketones, pulegone, menthone, isomenthone, ontogenetic varia-
stituents (5). A search for monoterpene glycosides in B. plumosus failed but succeeded in the isolation of the new
tion, antimicrobial activity, toxicity, Bystropogon,
hemiterpene glucoside 2S-methyl-1 -yl-f3-n-glucoside (6).
Lamiaceae.
In the present study the Canarian species B. origanfoliusvar. palmensis(B in Table 1) and B. wildpretii
(C) of section Plumosus and B. maderensis (0) and B. Introduction
canariensis var. smithianus (E) of section Canariense were investigated with regard to their essential oil composition.
The genus Bystropogon (mci. Mintho-
The data obtained in (5) of a comparable batch of B.
stachys Bunge) includes evergreen shrubs and belongs to
plumosus (section Plumosus) are presented as A in Table 1 for comparison. The oil of B. plumosus and its main constituents were tested for their activities towards some bacteria and some fungi; the ontogenetic variation of the oil and of its main constituents was investigated and is discussed.
the plant family Lamiaceae (Stachyoideae Saturejeae:
Thyminae). Centers of distribution are the Canary Islands, Madeira, and South America (Chile, Argentina). According to Engler and Prantl (1) the genus is subdivided into the two sections Bystropogon Benth. (Canarian-Maderian species) and Minthostachys Benth. (South American species: B. rnol-
us Kunth., B. spicatus Benth., B. tomentosus Benth., B. canus Benth,, B. glabrescens Benth.), Based mainly on mor-
Materials and Methods Plant material
phological and anatomical data the section Bystropogon
B. plumosus and B. canariense var. smith ianus
was recently further divided (2) into the section Plumosus I. La-Serna (1. B. plumosus L'Her., 2. B. origanfolias L'Her.
were obtained from Dr. Buttler (Univ. Bochum); B. maderensis was collected from Madeira. All three species were cultivated in the Ex-
* Dedicated
perimental Garden of this Institute; vouchers are deposited under PBMS 2, 3, and 4, respectively. The essential oils B and C were dis-
to Professor Ernst Reinhard on the occasion of his
65th birthday on August 21, 1991.
tilled from dried leaves of B. origan(folius var. palmensis and B. wildpretii obtained through the courtesy of Dr. Ehmke (Techn.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Received: March 13, 1991
348 Planta Med. 57(1991)
Diinitrios Economou and Adolf Nahrstedt
Univ. Braunschweig) from La Palma; the material was identified according to La Serna Ramos (2) by a local authority. All living plantlets used for this study were propagated vegetatively from one individuum.
B. pluinosus specimens used for studies of on-
togenetic variation were kept in pots and were stored in the greenhouse at 18°C from Nov. ito April 31, and subsequently outside. During the summer periods 1984—1986 the mean temperature was generally lower than 18°C varying between 9.9°C (Oct.) and 17.7°C (July, Aug.). Mean daily averages of air temperature in Tenerife at an altitude of ca. 600 mare 11.7 to 15.1 °C in the winter period (Nov. to April) and 14.6 to 19.2°C in the summer period (33).
220 °C, 4°/mm, 5 mm isothermally at 220°C; FID. Peaks were identified by comparison with the results obtained in (5) and by addition
of standards.
GLC/MS system
Hewlett-Packard HP 5986; ionization energy 70eV; DB-WAX capillary column 60m x 0.25mm id.; helium 1.5 mllmin, split 1 :20; 60—220°C, 4°/mm, 5mm isothermally at 220°C; FID. Peaks were identified using the MS data collection of Dragoco, Holzminden, FRG.
Tests of antimicrobial activity Agar diffusion test system; the samples were
Steam distillation
applied onto paper disks (diameter 6mm, Oxoid, Wesel FRG) which
were placed on the agar plates inoculated with the microorganisms. The diameter of the zone of inhibition was taken as a measure of activity. E. coli, S. aureus, and P. aeruginosa were
Carlo Erba GC 6000; DB-WAX capillary column,
mixed with a Columbia Agar (Merck, Darmstadt FRG) and incubated with the soaked paper disks for 24 hat 37°C. C. albicans and A. niger were incubated at 37°C, M. mucedo at ambient temperature for 48 h on a Sabourand-Glucose 2 % Agar (Merck, Darmstadt FRG). The microorganisms stem from the collection of the Institut für Pharmakologie und Toxikologie, TU Braunschweig. The essential oil used for testing contained as main constituents pulegone
30m x 0.25 mm id.; split ratio 1:35, helium 1.15 mllmin; 60—
(55%), isomenthone (14%), menthone (8.4%), piperitenone
GLC system
Table 1 Composition of the essential oils obtained from B. plumosus (A), B. origanifoliusvar. palmensis (B), B. wildpretii (C), B. maderensis(D), and B. canariensis var. smith/anus (E). Peak areas of the GLC signals were not corrected. The constituents of E were additionally analyzed by GLC/MS. The data of A were taken from (5).
yield/bOg
A(5)
B
B. plumosus
B. origanifolius var. palmensis
C B. wildpretii
D B. maderensis
E
B. canariensis var.
smith/anus
1.17 ml (fr. w.)
1.32 ml (dm)
1.50 ml (dm)
0.75 ml (fr. w.)
0.1 ml (fr. w.)
0.34 0.32 0.06 0.23 0.64
0.23 0.25
0.08
0.28 0.32
1.97 0.29
(dmorfr.w.) Monoterpene hydrocarbons
a-pinene
-pinene camphene sabinene myrcene a-phellandrene limonene f-phellandrene/1,8-cineole c/s-/3-ocimene
trans-fl-ocimene a-terpinene r-terpinene terpinolene p-cymene Sesquiterpene hydrocarbons
Monoterpene alcohols
—
2.50 0.06 0.13
0.10
—
0.23 0.59 —
0.75 —
0.18 0.90
0.09 0.01
0.14 0.30 0.01 2.20 0.05 0.07 0.42
—
0.10 —
—
0.32
0.10 22.15
0.19 0.06 1.58
—
0.10
—
0.07
—
6.30
—
—
—
—
1.68
—
—
—
—
3.32
0.03
—
0.01
80%) of which either pulegone (A, D) or menthone (B, C) are the main constituents. Sample E clearly differs from A to D in
that the yield is low (0.lml/lOOg fresh weight) with the main constituents being the monoterpene hydrocarbons (38.7%) of which ca. 22% was sabinene, and the sesquiterpene hydrocarbons (28.8%) of which more than 21% was germacrene D; the monoterpene ketone fraction, however, accounts for only 2.3%.
Thus, all investigated oils of species of the section Plumosus (A, B, C) represent a chemotype that is rich in essential oil, high in monoterpene ketones, and low in monoterpene hydrocarbons. This result confirms the ob-
— —
93.4
92.4
0.96 1.05
81.60 —
0.25 — —
4.60
93.1
—
—
0.52 —
0.67 0.25 0.67 0.19 — — —
0.52 0.16 —
1.17
82.3
servation (3) that members of the section Plumosus smell peppermint-like. The situation in section Canariense differs in that D belongs to the former type but E is characterized by poor oil production with low concentration of monoterpene ketones contrasting with a high amount of mono- and
sesquiterpene hydrocarbons. With respect to these biochemical parameters, section Plumosus seems to be
quite homogeneous whereas section Canariense combines different chemotypes; this view, however, based on some individuals of 3 out of 6 and 2 out of 4 species and varieties, respectively, needs further investigation together with the
question how far chemical polymorphism may occur, which is well known for essential oils obtained from Lamiaceae (7). In addition, the examination of hybrids ob-
served in the section Canariense (2) may contribute to taxonomic questions. The essential oils obtained from South American species are also high in pulegone, menthone, and isomenthone [see (5) for references, (30)1 and thus correspond with the section Plumosus of the Canary Islands and Madeira.
As expected for essential oil producing plants, the amount of oil as well as its composition vary during ontogenesis. This was shown for B. plumosus oil and its main constituents (Table 2). The yield of oil varied between
1.0 and 2.4% of fresh material from 1984 to 1987. The highest values (1.5 to 2.4%) were obtained during the winter period (November—April) when the plants were stored at a constant 18°C in the greenhouse (thus not under
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Monoterpene esters
Planta Med. 57(1991)
Dimitrios Economou and Adolf Nahrstedt
Table 2 Variation of yield of essential oil and its main components from B. plumosus during August 1983 to September 1987.
time of
yield of oil [%l
harvesting
1983
1984
1985
isomenthone
1.60 1.20 1.20 1.50
23.0
22.1
35.3
40.9 50.0 43.4
13.9 18.7 16.5
21.5
01.30 04.02 04.25 07.02 08.06 09.13 10.03 10.18 11.01 12.07
1.50 1.55
9.4 2.4 3.4
26.0
2.00 1.20 1.30 1.40 1.80 2.00 2.00 2.40
49.0 63.0 72.3 40.0 52.5 55.2 45.3 48.6 51.0 49.2
15.6
27.8
11.5
16.0 14.0
1.70 2.00 2.00 1.98 1.70 1.10 1.15 1.40 1.70 1.70
73.7 58.0 61.0 69.4 72.3 68.0 39.0 44.0 30.7 46.2
0.2 9.1 8.0 5.6 3.3 6.6 19.1 10.9 13.7 8.8
1.80 1.90 2.00 2.00 1.00 1.05 1.00 2.00 2.00
57.2 61.4 66.0 51.0 35.8
4.7 3.3 1.3 9.5 11.0 5.9
02.01
01.08 02.05 03.04 05.06 06.18 08.01
09.20 11.06 12.22 1987
menthone
08.08 09.12 10.15 12.15
03.04 03.15 04.18 06.06 06.20 07.19 09.27 10.18 12.06 1986
% of oil;
pulegone
01.30 03.26 06.11
09.02
7.5 11.0
The antimicrobial activity of the essential oil obtained from B. pluinosus and its main constituents was tested using some bacteria and fungi for comparison with some well known antibiotics in the agar diffusion test
23.3 20.8
9.5
following the suggestions of Jannsen et al. (8). The results in Table 3 demonstrate a weak activity of the oil towards bacteria with Staphylococcus aureus as the most sensitive and
16.8 23.2
Pseudomonas aeruginosa as the most resistant species, as expected (13). The oil shows a more pronounced activity against fungi, especially Mucor inucedo. Of the three main
18.5 15.1
12.4 7.2 3.4 6.6
constituents, pulegone, menthone, and isomenthone, pulegone is the only compound that acts on fungi and that also exhibits the most pronounced antibacterial activity. These results indicate that pulegone is mainly responsible for antimicrobial properties of the oil. Similar observations were obtained by Kellner and Kober who investigated the antiseptic properties of some essential oils (9) and their components (10) in the vapor phase on 8 species of bacteria and found that pulegone and menthone act as weak antiseptics. Göckeritz et al. (11) observed a lower fungistatic and a higher bacteriostatic concentration for both monoterpenes. Knobloch et al. (12) found that pulegone and menthone had a similar activity towards bacteria; the same group showed that pulegone and menthone only moderately influence membrane-associated basic biochemical processes whereas thymol or carvacrol are strongly inhibitory (13). Iii this context it is of interest that the insecticidal activity of Bystropogon essential oils is also mainly connected with pulegone (14).
17.3 14.9
24.3 13.9
8.8 6.0 3.0 10.9
51.4
9.1
38.0 20.7
14.0 16.5
21.0 6.6 8.4 20.0 32.0
33.9 55.4 54.9 57.0
9.2 6.5 5.8 8.8
27.2 9.4 8.8 7.7
40.4
2.10 2.10 1.20 1.15
19.1
15.6 13.7 12.3
natural conditions; see Materials and Methods). From these results the recommended time for harvesting the leaves is thus just before the onset of flowering in contrast to several
other Lamiacean plants in which harvesting is recommended during blooming or the late flowering period (31). After onset of flowering in late April, the yield decreased from ca. 2 % to 1—1.4% until the end of flowering in late
Escherichia Staphylococcus Pseudomonas co/i aureus aeruginosa
Mucor mucedo
2 8
5 13
28 49
22
18
36
0 0 0 0
0 0 0 0
34
9
12
34
12 21
2 4
10l
12
21
4
10il
6
4
4
0 0
isomenthone 5fi1 10/LI
6
14
4
0 0
50/Lg
20
22
10/Lg ketoconazol 10/Lg nystatin 20/Lg
16
31
7 0
pulegone menthone
tetracyclin ampicillin
5il
5l
(19, 2 1—25). Previously this oil was used medicinally as an
Aspergi/lus niger
4 14
5tl l0jil
Essential oils with high levels of pulegone are regarded as toxic (18, 19, 20). Several reports exist in the literature dealing with severe intoxications, sometimes ending with death, after ingestion of comparatively high doses of pennyroyal oil (from Mentha pulegium, sometimes Hedeoma pulegioides) that contains pulegone up to 80%
Candida albicans
2 10
ess.oil
to April. Without exception, isomenthone is higher than menthone. Interestingly, the amounts of both ketones vary inversely to those of pulegone in that they are usually low when pulegone is high and vice versa. This fits with the biogenetic scheme postulated for peppermint plants by several authors where pulegone is the precursor of menthone and isomenthone (15—17).
20.5
8.4
September. The content of pulegone varies between 22 and 72 % of the oil with high levels generally during November
Table 3 Antibacterial and antifungal activity of the essential oil obtained from B. plumosus (composition see Materials and Methods) and its main constituents (pulegone, menthone, isomenthone) in the microdiffusion test system. The values indicate the diameter of the inhibition zone in mm.
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350
Chemical. Physiological, and ToxicologicalAspects of the Essential Oilof Some Species of the Genus Bystropogon Planta Med. 57(1991) 351
the effects (25).
References 1 Engler, A., Prantl, K. (1897) Die natürlichen Pflanzenfamilien, IV.
Teil, 3. Aht. a, p. 314. Engelmann, Leipzig. La Serna Ramos, I. (1984) Phanerog. Monogr. 18. Cramer, Vaduz. La Serna Ramos, 1., Wildpret de la Torre, W. (1976) Vieraea 6, 77. Alliaga, T. J. (1984) Diss. Thesis, Univ. Gieflen, FRG. Nahrstedt, A., Economou, D., Hammerschmidt, F.-J. (1985) Planta Med. 51, 247. 6 Nahrstedt, A., Economou, D., Wray, V. (1990) J. Nat. Prod. 53, 1387. Hegnauer. R. (1966) Chemotaxonomie der Pflanzen, Vol. 4, p. 292, Birkhauser, Basel. 2
Jannsen, A. M., Scheffer, J. J. C., Baerheim Svendsen, A. (1987) Planta Med. 53, 395. 10
It seems obvious that essential oils are toxic
to different extents at high doses. Recently, the oils of Mentha rotund(folia and M. longifolia were shown to possess LD50 values in rats of 641 and 437mg/kg, respectively (26). On the other hand, intoxication was never observed
when such oils were used properly at a dosage recommended for medicinal usage. 5 g of dry herb of B. pluinosus
with a content of 1.5 % essential oil containing 70% pulegone provides a potential dose of pulegone of approximately 50mg (corresponding to ca. 0.8 mg/kg man); this calculation may be still too high as a normal tea preparation with hot water will never extract the entire essential oil. Unfortunately no information on the acceptable daily intake (ADI) dose of pulegone is available. For carvone, the ADI dose is estimated up to 1.25 mg/kg in man (27); the provisional limits for pulegone in beverages and foods are 100 and 25mg/kg, respectively (32). Accordingly, oils rich in
pulegone of some Bystropogon spec. are without doubt non-toxic in medicinal usage as antiseptics in traditional therapy. Acknowledgements Thanks are due to Dr. H. Buttler, Institut für spezielle Botanik (Univ. Bochum), for providing living plant mate-
rial and for several hints regarding the systematics of the genus Bystropogon, to Dr. A. Ehmke (Institut für Pharmazeutische Blobgie, TU Braunschweig) for collecting plant material on La Palma,
and to L. Kruger and coworkers (this Institute) for growing the plants. We are mostly indebted to Dr. F.-J. Hammerschmidt, Dragoco (Holzminden) for running the GLC/MS, and to Dr. med. V. Franke, formerly Institut für Pharmakologie und Toxikologie (TU
Braunschweig) for much help during the performance of the antimicrobial tests. Dr. G. Matthiaschk (BGA, Berlin) provided information on ADI values arid limits in foodstuffs. The linguistic help of Dr. V. Wray is very much acknowledged.
12 13
14 15 17
Kellner, W., Kober, W. (1954) Arzneimittelforsch. 4, 319. Keilner, W., Kober, W. (1955) Arzneimittelforsch. 5, 224. Giickeritz, D., Weuffen, W., Höppe, H. (1974) Pharmazie 29, 339. Knobloch, K., Pauli, A., Ibert, B., Weigand, H., Weis, N. (1989) J. Ess. Oil. Res. 1, 119. Knobloch, K., Weigand, H., Weis, N., Schwarm, H.-N., Vigenschow, H. (1986) in: Progr. in Ess. Oil Res. (Brunke, E.-J., ed.), p. 429, de Gruyter, Berlin. Vogler, U. (1985) Diplomarbeit, Univ. Kiel, FRG. Hefendehl, F. W. (1970) Phytochemistry 6, 823. Hefendehl, F. W., Murray, M. J. (1976) Lboydia 39, 39. Croteau, R. (1988) 1ST Atlas of Science 1. 182.
Steinegger, E., Hansel, R. (1988) Lehrbuch der Pharmakognosie und Phytopharmazie, p. 701, Springer, Berlin. 19 Nelson, S.D., Gordon, W. P. (1983) J. Nat. Prod. 46, 71. 20 Lewis, E. J., Friedrich, E. C. (1990) Planta Med. 56, 224. 21 Allen, W. T. (1887) Lancet 1022. 22 Jones, C. 0. (1913) Brit. Med. J. 745. 23 ValIance, W. B. (1955) Lancet 850. 24 Early, D. F. (1961) Lancet 580. 25 Gordon, W. P., Forte, A. J., McMurtry, R. J., Gal, J., Nelson, S. D. (1982) Toxicol. Appl. Pharmacol. 65, 413. 26 Perez-Raya, M. D., Utrilla, M. P., Navarro, M. C., Jimenez, J. (1990) Phytother. Res. 4, 232. 27 Toxicological evaluation of some flavouring substances and non-nutritive sweetening agents. FAO Nutrition Meetings Rep. Ser. No. 44A. WHO/Food Add./68.33 (1967). 28 European Pharmacopoeia, Vol. III, (1975), p. 68. 29 Economou, D. (1989) Diss. Thesis, Techn. Univ. Braunschweig, FRG. Baerheim Svendsen, A., Scheffer, J. J. C., Looman, A. (1987) Flavour Fragance J. 2, 45. Guenther, E. (1949) The Essential Oils, Vol. III, p. 396 if. Van Nostrand Comp., New York. 32 Flavouring substances and natural sources of flavourings. Council of Europe. 3"' edition; Strasbourg, 1981. x Climatological summaries of Santa Cruz de Tenerife, sea level 617 m. Deutscher Wetterdienst — Zentralamt, D-6050 Offenbach.
°
'
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abortifaciens and to induce menstruation at a dose of 1—3 minims (23) (1 minim = ca. 0.065 g). Later on it was abused for these purposes at doses of a tablespoonful (21) and a dose of 24 g (25) caused convulsions, uraemia, liver and lung damage; unfortunately most of the reports cited above do not give exactly the amount of pennyroyal oil administered but it seems likely that one or more doses of ca. 15 ml (about 250 mg/kg) are undoubtedly toxic (24). Cellular necrosis and acute lung and liver damage were also observed at doses of 400 mg/kg and higher given intraperitoneally to mice (25). The authors conclude that the isopropylidene grouping of pulegone and its derivatives is responsible for
