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Essential Oils and ChromosomeNumbers From Italian AchilleaSpeciesMassimo Maffei a , Franco Chialva b & ArnaldoCodignola ca Dipartimento di Morfofisiologia, University ofTurin, Viale P.A. Mattioli, 25, I-10125, Turin, ItalyPhone: 11-658387b Research Laboratory, Martini & Rossi IVLAS S.p.A.,I-10020, Pessione, Turin, Italyc Dipartimento di Biologia Vegetale, University ofTurin, Viale P.A. Mattioli, 25, I-10125, Turin, ItalyPublished online: 28 Nov 2011.

To cite this article: Massimo Maffei , Franco Chialva & Arnaldo Codignola (1989)Essential Oils and Chromosome Numbers From Italian Achillea Species, Journal ofEssential Oil Research, 1:2, 57-64, DOI: 10.1080/10412905.1989.9697751

To link to this article: http://dx.doi.org/10.1080/10412905.1989.9697751

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RESEARCH REPORT J . Ess Oil Res., 2,57-64, (MarcWApril, 1989)

Essential Oils and Chromosome Numbers From Italian Achillea Species

Massimo Maffei* Dipartimento di Morfofisiologia, University of Turin,

Viale P.A. Mattioli, 25 Tel. 11-658387.1-10125 Turin, ltaly

Franco Chialva Research Laboratory, Martini G Rossi ZVLAS S.p.A.

Z-10020 Pessione, Turin, Ztal y

Amaldo Codignola Dipartimento di Biologia Vegetale, University of Turin,

Viale P.A. Mattioli, 25 1-10125 Turin, ltaly

Received: December 1988

ABSTRACT: Essential oils of five Achillea species, A. ptarmica, A. erba- rotta, A . moschata, A. nana and A. nobilis, growing spontaneously in the Northwest Italian Alps were analyzed by gas chromatography-mass spec- trometry. Several monoterpenes, sesquiterpenes and phenols were identified. Camphor and 1,8 cineole were the major constituents of A. erba-rotta, whereas germacrene D was particularly abundant in A. ptarmica and A. nobilis. A. moschata oil contained mainly terpinen-4-01 whereas trans- and cis-isoeugenol were present in good percentages in A. nana. The caryological analyses revealed a diploid (2n = 18) chromosome number for all plants. The absence in the essential oil of proazulene compounds was related to the dip- loid (2n = 18) chromosome number of the above mentioned species.

KEY WORD INDEX: Achillea species, Asteraceae, essential oils, chromo- some numbers.

INTRODUCTION: Some.Achillea species are listed in various Phar- macopeias. In addition to their pharmaceutical uses, other interesting and useful cosmetic and fragrance properties have been reported in the literature ( 172). The compounds principally used for these purposes are contained in the volatile oil. The presence or absence of several compounds and their concen- trations depend on genetic and environmental factors (3,4). From a genetic point of view, it was found that this genus makes a polyploid complex with a basic number x = 9 chromosomes (5). In previous works (6,7) we reported on the chromosome numbers of some Achillea species growing spontaneously at high elevations (from 1500 to 2580 m) in the Northwest Italian Alps. A diploid (2n = 18) chromosome number was found for many of them. The

*Senior author

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58 JOURNAL OF ESSENTIAL OIL RESEARCH

presence of some Achillea oil components (such as azulene and chamazulene) has been suggested to occur particularly in tetraploid (5,8-12), hexaploid and octoploid (5) types. These compounds are absent in the diploid types.

This communication, which is a part of a hybridization and selection project of Italian Achillea species, describes the results of an investigation on essential

Table 1. Chemicat composition (monoterpenes) of the oils distilled from some Achilles species.

% = area percentage

Monoterpenes

Hydrocarbons a-thujene a-pinene camphene P-pi nene sabinene a-phellandrene 6-3-carene a-terpinene 1 imonene P-phel landrene p-cymene r-terpinene

Alcoho 1 s 1 ina loo 1 terp i nen-4-01 pi nocarveo 1 a-terp i neo 1 borneol myrteno 1 carveol geraniol

Esters isoamyl-tiglate monoterpene acetate c hrysan t heny 1 acetate bornyl acetate geranyl acetate geranyl propionate

Aldehydes myrtena 1

Ketones a-thu jone P-thujone camphor pinocarvone j a smone

Ox ides 1.8-cineole

Achillea Achillea erba-rotta moschata

0.17 0.11 0.35 0.35 1.12 0.10 0.30 0.39 0.33 1.38 0.83 0.38

1.39 0.71 0.46 4.36 0.70 0.36 0.36 0.36

0.00 0.76 0.47 9.69 0.12 0.12

0.42

1.27 0.48 31.17 0.32 0.19

16.43

0.96 0.28 0.44 0.40 1.17 0.23 0.00 3.02 0.19 6.78 3.05 1.59

0.23 22.32 0.41 1.95 0.31 0.22 0.64 0.14

0.00 0.00 1.68 7.94 0.18 0.00

0.73

0.00 0.00 4.14 0.83 0.00

7.58

Achi 1 lea nana

%

0.00 0.00 0.00 0.00 2.36 0.00 0.00 0.00 0.26 0.00 0.00 0.19

0.84 7.22 1.63 1.02 0.37 0.14 0.19 0.15

1.14 0.19 0.00 0.14 1.21 2.60

1.90

0.00 0.00 0.11 0.28 0.26

3.76

Achi 1 lea nobilis

%

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.41 2.62 0.17 1.18 0.73 0.25 0.12 0.12

0.00 3.87 0.00 0.12 1.10 0.00

0.20

0.00 0.00 3.28 0.00 0.31

0.30

Ac h i 1 lea ptarmica

%

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.15 0.00 0.00 0.00

0.00 3.26 0.14 1.48 0.00 0.00 0.00 21.22

0.00 0.00 0.00 3.73 1.38 0.00

0.00

0.00 0.00 0.00 0.00 0.00

0.52

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JOURNAL OF ESSENTIAL OIL RESEARCH 59

oil constituents from some Achillea species: A. ptarmica, A . erba-rotta, A. moschata, A. nana and A. nobilis, growing spontaneously in the North West Italian Alps. It is also intended to determine if any correlation exists between chromosome numbers and oil composition in the above mentioned species, with particular reference to the presence of proazulene compounds.

EXPERIMENTAL: The species studied and the location and elevation where they were collected were as follows: 0 A. ptarmica L.: Val dAyas (Valle d’Aosta) 1550 m; 0 A. erba-rotta All.: Colle del Nivolet, Gran Paradiso National Park (Pied-

0 A. moschata Wulfen: Valle d’Ayas 2300 m; 0 A. nana L.: Colle del Nivolet 2450 m; 0 A. nobilis L.: Val d’Ayas 1300 m.

mont) 2580 m;

Table II. Chemical composition (sesquiterpenes and phenols) of the oils distilled from some Achi//ea species.

% = area percentage

Achillea Achillea erba-rotta moschata

Compound % - %

Sesquiterpenes

Hydrocarbons caryophyl lene unknown sesqui. HC germacrene 0 unknown sesqui. HC P-bi sabolene unknown sesqui. HC unknown sesqui. HC unknown sesqui. HC

Alcoho 1 s c i s-nero 1 i do 1 trans-nerolidol unkn. sesqui. alcohol unkn. sesqui. alcohol

Oxides caryophyl lene oxide

Pheno 1s

methyl isoeugenol cis-isoeugenol trans- i soeugenol thymol + carvacrol

0.13 0.20 0.38

0.87 0.12 0.00 0.00

1 .a9

0.68 0.35 0.00 1.30

1.77

0.12 1.23 0.31 0.86

0.00 2.43 0.11 0.35 8.57 0.13 0.00 0.00

1.07 0.42 0.00 5.74

0.86

0.00 0.36 1.31 0.65

Achi 1 lea nana %

0.14 1.29 0.97 0.24 0.00 0.27 0.00 0.00

1.96 2.26 0.00 2.13

0.17

0.27 16.44 21.60

6.55

Achi 1 lea nobilis

%

0.24 1.01

45.98 5.73 0.00 0.15 0.00 0.15

1.69 0.82 4.71 0.69

4.27

0.19 1.94 0.11 0.30

Achillea ptarrn i ca

%

0.45 0.01

32.32 3.34 5.04 0.69 1.43 1.24

0.00 0.00 0.00 1.70

0.52

0.78 0.44 0.00 0.00

unknown sesqui. HC = unidentified sesquiterpene hydrocarbon unkn. sesqui. alcohol = unidentified sesquiterpene alcohol

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60 JOURNAL OF ESSENTIAL OIL RESEARCH

A voucher specimen of all plants studied was deposited at Hortus Botanicus Universitatis Taurinensis (Turin, Italy). The species were classified using “Flora d’Italia” (13) and “Flora Europaea” (14).

Essential oil analyses-The plants, harvested in full bloom, were steam dis- tilled using a modified Clevenger apparatus. The essential oils were analyzed using a gas chromatograph with a flame ionization detector. Equipment de- scription, separation, operating conditions and temperature programming were as described previously (15). The compounds were identified using gas chromatography-mass spectrometry at the same conditions described in detail elsewhere (15).

Caryological analyses-chromosome numbers were determined using sev- eral root tips of plants harvested in the field or from seedlings germinated in petri dishes. The seeds from the latter were collected directly in the field. The root tips were pretreated with 1% colchicine solution (Merck) for 1 h. After fixation they were stained by means of the Feulgen reaction (hydrolysis 6 min. in 1N HC1 at 60°C). Several metaphase plates per species were observed and measurements were done on all photographs obtained. The chromosome for- mulas were estimated according to Levan, et al. (16)

Table 111. Percentage of monoterpenes, sesquiterpenes and phenols along with synthetic parameters of the oils

distilled from some Achillea species. % = area percentage

ComDound

Monotewenes

hydrocarbons alcohols esters aldehydes ketones oxides

SesquiterDenes

total

Pheno 1s

total

camphor/borneo 1

borneol comp. / geranyl comp.

Achi 1 lea Achi 1 lea erba-rotta moschata

w w

5.81 18.11 8.70 26.22

11.16 9.80 0.42 0.73

33.43 4.97 16.43 7.58

7.69 18.82

2.52 2.32

44.53 13.35

17.32 25.70

Achillea Achillea Achillea nand nobilis ptarmica % % % ---

2.81 0.00 0.15 11.56 5.60 26.10 5.28 5.09 5.11 1.90 0.20 0.00 0.65 3.59 0.00 3.76 0.30 0.52

9.43 66.44 46.74

44.86 2.54 1.22

0.30 4.49 0.00

0.13 0.70 0.17

borneol comp./geranyl comp. = ratio between borneol complex (borneol + bornyl acetate) and geranyl complex (geraniol + geranyl acetate + geranyl propionate).

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JOURNAL OF ESSENTIAL OIL RESEARCH 61

RESULTS: Essential oil analyses-The GC/MS analyses performed on the essential oil of the plants under discussion have individualized 50 compo- nents. The bulk of these components was composed of monoterpenes (see Table I) followed by some sesquiterpenes and phenols (see Table 11). Cam- phor occurred in all samples, with the exception ofA. ptarmica, and reached the highest values in A. erba-rotta. 1,8 Cineole was present in A. er/,tr-rotta and A. moschata, in much higher amounts than in A. nobilis. The highest percentages of bornyl acetate and alpha-terpineol were present in A. erba- rotta and A. moschata while the percentage of terpinen-4-01 was particularly high inA. moschata.

A. nana showed good percentages of methyl-isoeugenol and trans-isoeugenol. Germacrene D was particularly abundant in A. nobilis and in A. ptarmica. The latter possessed the highest percentage of geraniol.

Considering the total monoterpene and sesquiterpene composition (see Table 111) different percentages of monoterpenes were observed among oil samples. In particular: A. ptarmica and A. moschata had the highest percent- age of monoterpene alcohols and, with the exception of A. ptarmica, mono- terpene hydrocarbons. Monoterpene ketones, oxides and esters were more abundant in A. erba-rotta. A. nobilis and A. ptarmica were particularly rich in sesquiterpenes, whereas A. nana possessed the highest percentage of phenols. From Table I11 one can see that A. erba-rotta had the highest camphor/ borneol ratio, while A. moschata had the highest borneoYgerany1 ratio.

I I Table IV. Chromosome number of some Achilles

species along with bibliographic references

TAXA Chromosome Number References

Achillea erba-rotta 2n=18 5, 6, 23 2n-18 Achi 1 lea moschata 2n=18 6, 23 2n=18 Achi 1 lea nana 2n=18 5, 6 2n=18 Achillea nobilis 2n=18 5, 24 2n=18 Achillea ptarmica 2n=18 25. 26 2n=18

-

I I

Caryological analyses-After the assessment of the chromosomes coming from the metaphase plates it was found out that all plants had a diploid (2n = 18) chromosome number, revealing a basic number x= 9 chromosomes. Table IV gives the 2n chromosome number along with the bibliographic references. Figure 1 shows the chromosomes of the Achillea species ordered in a caryotypic pattern. From a first observation it can be seen that the chromo- somes are composed mainly of “M, m, sm” and “st” types, showing the ab- sence of “t” and “T” types. The chromosome length ranged from 1.4 to 2.8 un.

Further caryotypical analyses are under way to better define the chromosome formulas.

DISCUSSION: High elevations and dramatic changes in environmental con- ditions may operate a selective pressure on plants. The action of the latter can

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62 JOURNAL OF ESSENTIAL OIL RESEARCH

Figure 1. Chromosomes of some Achillea species ordered in a caryotypical pattern. A = A. ptarmica (x 3434); B = A. erba-rotta (x 2769); C = A. mos- chata (x 2769); D = A. nana (x 2658); E = A. nobilis (x 2658).

find its expression on the physiology, anatomy and caryology of the exposed plants. With regard to aromatic plants, it is well known how environmental conditions can affect the yield and composition of the essential oils produced (17-20), while a different caryological situation may dictate drastic differences in the oil quality (21,22). The genus Achillea has been demonstrated to show high levels of biochemical polymorphism, mainly connected to the synthesis of proazulene compounds (5).

The results of our research clearly indicate that Alpine Achillea species pos- sessing a diploid (2n = 18) chromosome number completely lack proazulene compounds. A survey of the literature indicates the presence of the latter proazulene compounds in tetraploid (5,8-12), hexaploid and octaploid (5) Achillea types. It appears that diploid (2n = 36, 54, 72) azulene-producing Achillea types originate from arid zones showing a xerophytic nature, whereas diploid populations of a mesophytic character, which can be found in cold and humid localities, possess a diploid (2n = 18) chromosome number and are nonazulenic types (5). This last observation corresponds to the environmental conditions typical of the areas from where our plants came. The chromosome counts for the species under study are in agreement with the most recent data reported in literature (5-7,23-26).

It is also interesting to note that diploid Achillea species are only present at high elevations, whereas the polyploid ones belong rather to the plains (27). This observation suggests that a selective pressure made by elevation and environment is needed for the proazulene biosynthetic pathways to be acti-

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JOURNAL OF ESSENTIAL OIL RESEARCH 63

vated. It is perhaps more speculative to propose that this pressure of elevation and environment also could have an effect on the biosynthesis of other oil components of the analyzed plants, even though sesquiterpenes seem to be more abundant in plants growing at lower elevations (i.e. A. ptarmica and A. nobilis).

The biosynthesis of camphor in Salvia officinalis, Tanacetum vulgare and Rosmarinus officinalis has been shown to involve the dehydrogenation of borneol which in turn is the result of the conversion of geranyl pyrophosphate (28). The camphor/borneol and the borneougeranyl ratios might be used as parameters that could indicate the efficiency of the synthesis of camphor and borneol respectively. In A. erba-rotta the occurrence of high percentages of camphor was positively correlated to the highest values of the camphor/ borneol and borneoYgerany1 ratios.

In conclusion, our data, gives a more detailed description of the oil chemical characteristics of some Alpine Achillea species. In addition, the correlation existing between diploid (2n= 18) chromosome number and the absence of proazulene compounds has been confirmed. Furthermore, the caryological homogeneity (such as chromosome number, length and formulas) present among plants allows the formation of interspecific hybrids. Thus it is possible to use these Achillea species for the breeding and selection of new and useful cultivars.

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