Salvia_110330

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EVALUATION OF THE ESSENTIAL OIL PRODUCTION AND COMPOSITION OF

SALVIA SPECIES BEYOND THEIR NATIVE AREA OF DISTRIBUTION

Mth, Imre1, Mth , kos2, Hohmann Judit3and Janicsk, Gbor1

1

Institute of Ecology and Botany, Hungarian Academy of Sciences, Vcrtt, Hungary2 University of West Hungary, Faculty of Agriculture and Food Science, Department ofBotany, Mosonmagyarvr, Hungary3 Szeged University, Faculty of Pharmacy, Department of Pharmacognosy, Szeged, Hungary

Abstract

Members of the Salvia genus, similarly to many representatives of the sub-families

Lamioideae and Nepetoideae can be grown under the continental conditions of Hungary,

beyond their native area of distribution.

Salvia tomentosa_Mill., S. officinalis L.,, S. fruticosa Mill., S. lavandulifolia_Vahl., S.

candelabrum Boiss., and S. ringens Sm. are essential oil containing species with essential oils

of similar characteristics. They belong to the predominantly monoterpenoid bearing oils.

Thujone content shows significant variations.

The investigations have demonstrated that since S. tomentosa Mill. has a similar essential oil

composition to S. officinalis L. with a favourably lower thujone content, it could be regarded

as a potential prospective substitute for essential oil production.

Key words: Salvia species, essential oil production, essential oil composition, native area

Introduction

The genus Salvia with its nearly 1000 species (Alizar, 1993) represents a huge and important

taxonomic unit of the tribe Menthae, Lamiaceae Subfam. Nepetoideae (Cantino, et al. 1992 ) .

Species of this genus, like Salvia officinalis L. possess significant pharmacologic as well as

economic values.

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Data from the special literature describe that Salvia has undergone marked species radiations

in three regions of the world: Central and South America (500 spp.), Central

Asia/Mediterranean (250 spp.) and Eastern Asia (90 spp.) (Walker et al., 2004).

In view of the native area of distribution ofSalvia species, the Central European country with

its continental climate, Hungary cannot be regarded as part of the native area for most of the

species. e.g. Salvia fruticosa Mill., is an endemic species of the Eastern Mediterranean basin,

and the native distribution ofSalvia officinalis L. is also more or less restricted to the Western

Part of the Balkan Peninsula (Karousous et al., 2000). Remarkably, however, some Salvia

species, among them also S. officinalis L. is, cultivated as a culinary, medicinal herb and/or as

an ornamental plant, in Hungary.

In a broader aspect, members of the Family Lamiaceae are numerous in Hungarian flora.

Among the some 2200 flowering species, 16.9 % belong to the group of Pontusian

Mediterranean - Submediterranean floristic elements. 73 species are members of the Family

Lamiaceae.

Aim of present investigations was to assess the chemical variability of selected species of the

Family Lamiaceae, with a special emphasis on Salvia officinalis L. and related taxa under

environmental conditions beyond the northern area of the species. Our work concentrates on

the investigations carried out by Hungarian researchers predominantly to the species growing

in Hungary and or prosperous economic plants (members of Section Salvia) that can be

introduced to Hungary.

Materials and Methods

Plant Materials

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The plant materials of the experiments were grown and collected in the experimental

populations of the Institute for Botany and Ecology of the Hungarian Academy of Sciences,

Vcrtt, Hungary. Seeds were obtained by botanic garden seed exchange. Transplant raising

including germination took place in a hot bed. Seedlings were transplanted into the open field,

in May. The overwintering of plants took place under botanic garden open field conditions.

The species, native in Hungary were grown from seeds collected in Hungary. Plant material

of chemical analyses was freshly harvested and dried at 40 oC. Voucher specimens of

experimental plants has been deposited in Herbarium of the Research Institute for Ecology an

Botany of the Hungarian Academy of Sciences, Vcrtt.

Gas chromatography

Essential oil analyses (10, 11)

The essential oil content of freshly harvested or ground air dry samples was determined by

steam distillation in a Clevenger apparatus, according to Pharm. Hung. VII. The amount if

essential oils were expressed as v/w percentage. Occasionally n-heptane additional phase was

applied, in cases of low essential oil content. Essential oil composition was determined by a

HP 5890 II. gas-chromatograph, equipped with am HP 5 capillary column (30m x 0.35 m x

0.25). The temperature program varied in the range 60 210 oC, at a velocity of 3 oC/min and

210250 oC, velocity 5 oC/min. Temperature of detector and injector: 250 oC, carrier gas: N2.

Finnigan GCQ mass detector (ion-trap) gaschromatograph (GC/MS). Conditions of GC/MS

measurements were identical, with the exception of He-carrier gas. Flow velocity: 31.9

ml/min. Ionization of positive ion was in the range: 40400 amu.

Identification of components was made on the basis of authentic samples using Kovats-index

calculation and the database of GC/MS equipment.

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Determination of Caffeic and rosmarinic acid

0.4 g ground samples were subjected to ultrasonic extraction using 60 % (70%) aqueous

methanol solutions in 3 different aliquots. Subsequently, a 25 ml strain solution was prepared

from the united fractions. 25 l portions of the solution were used for thin layer

chromatography, using toluol ethylacetate-formic acid (5:4:1) mixture mobile phase, in the

presence of authentic samples. Kieselgel 60 (Merck) layers were used in thin layer

chromatography. TLC/densitometric investigations were carried out by a Shimadzu CS-9301

PC (Japanese) equipment.

Evaluation took place under an UV lamp, densitometric measurements were made at 325 nm

according to a method elaborated and validated by authors.

Determination of ursolic and oleanolic acids

Ursolic and oleanolic acids were determined jointly by TLC/densitometry. For the

measurements 0.5 g air dry samples subjected to ultrasonic extraction in 80 % aqueous-

methanolic solution. 5 ml aliquots of strain solutions were prepared, out which 20 and 40 l

aqueous-methanolic (80%) strain solution. 40 l was applied to thin layer chromatography.

Triterpene carbon acids were separated from the other components by using a mobile phase.

Chloroform-methanol (19+1). This system did not make it possible to separate the two

triterpene acids, therefore these compounds were measured jointly. A mixture of sulphonic

acid and anis aldehyde were used as visualizing agent. Densitometric measurement took place

after a 5 min heating to 105 oC and 40 min delay at 536 nm, in the reflection mode.

Results and Discussion

Essential oil content of Salvia species in Hungary

Production biological investigations into the species of Family Lamiaceae have been going on

in the Institute of Ecology and Botany of the Hungarian Academy of Sciences, Vcrtt, for

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nearly three decades. Under experiment garden conditions, a Lamiaceae collection numbering

more than 150 species has rendered it possible to make observations on the establishment of

species not native to this floristic area and to obtain a comprehensive assessment of these

species. Earlier results of our investigations have been reported by (Mth et al., 1993, Mth,

1997, Mth and Csed, 2007, Mth et al., 2007)

It order to arrive at conclusions of taxonomic significance we analysed both the essential

production and composition of 6 Salvia species of the Section Salvia. Regarding essential oil

production the differences were significant. The highest essential oil yield was produced by S.

lavandulifolia Vahl. (0.89 ml/100 g fr.weight), followed by S. fruticosa Mill. (0.46 ml/100 g)

and S. tomentosa (0.36 ml/100 g).

In terms of essential oil composition, the pattern ofS. officinalis L. seemed to be similar to

that ofS. candelabrum Boiss., while in the case ofS. ringens Sm. a n-heptane co-phase had to

be deployed before farther analysis.

The greatest difference in essential oil composition could be observed in the - and -thujone

contents, with the following sequence of species: S. officinalis L., S. fruticosa Mill. (S.

triloba), S. ringens Sm., S. tomentosa Mill. The thujone content ofS. candelabrum Boiss. was

nearly negligible, whereas S. candelabrum Boiss. contained absolutely no thujone, which is in

accordance with observations by other authors. In the case of latter taxon, 1,8-cineole has

proved to be the main component.

Among the monoterpenes, the camphor content was highest in S. fruticosa Mill. As compared

to the S. tomentosa plants of Bulgarian origin (Mth I. et al., 1997, Dobos et al., 1997) the

relative modest total thujone content (10 %) plants growing in our experimental garden seems

to be higher. Based on our observations, the composition of S. tomentosa Mill. is relatively

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similar to that of S. officinalis L. which data seem to indicate that this species could be a

potential promising substitute forS. officinalis L..

The essential oil character ofS. lavandulifolia Vahl., due to the perfect lack of thujone and the

remarkably high 1,8-cineole content is perfectly different. Despite the significantly highest

essential oil producing capacity of this species, due to continental climatic conditions its

production can be regarded as insecure.

Table 1. Main essential oil components of members of the Section Salvia , in Hungary*

S.tomentosa

S.officinalis

S.fruticosa

S.lavandulifolia

S.candelabrum

S.ringens

ml/100 dry mass 0.39 0.25 0.46 0.89 0.25 +

Terpenes:

Monoterpenes

-thujene 0.2 0.2 0.2 04.4 0.2-pinene 1.6 1.1 1.2 2.7 6.5 2.5-pinene 7.2 2.3 1.7 6.8 7.4 2.9Myrcene 1.4 0.9 4.3 5.8 1.4 2.2

-terpinene 0.2 0.2 0.1 0.4p-cymol 0.2 0.2 0.1 0.4 1.1 0.4

Limonene 1.9 1.3 0.2 1.7

1,8-cineole 11.6 3.5 16.9 47.0 20.5 16.6

-terpinene 0.6 0.5 0.5 1.1 0.5terpinolene 0.4 0.4 0.6 0.5 0.5

-thujone 7.4 36.0 21.4 1.6 9.7-thujone 3.3 9.7 3.7 0.5 1.8Camphore 10.1 9.6 26.0 12.6 9.0 16.0

Borneole 9.3 8.4 1.0 2.2 4.4 2.0

Terpinene-4-ol 0.6 0.5 0.4 0.7 1.1

-terpineole 0.4 0.1 0.5 0.3 1.9 0.5bornyl-acetate 3.3 4.9 1.0 0.6 0.9 1.4

Sesquiterpenes

-caryophyllene 5.9 0.7 1.4 4.9 0.2 2.3-humulene 6.2 4.2 2.8 1.0 1.1 4.8-muurolene 0.3 0.3-kadinene 5.5 0.7seline (3,7), 11-diene

0.6 0.1 0.2

caryophyllenoixide 0.9 0.2 0.1 0.3

viridiflorol 17.1 4.6 5.6 4.5 0.8 13.5

humulene 1,2-epoxide

0.5 0.8 0.2 0.4

Sclarene 0.9 1.9 0.8 2.6

* According to Mth et al., 2007

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The essential oil production ofSalvia species can undergo significant differences according to

the origin of the plants (Mth I. et al., 1993), time of harvest and plant organ analysed. This

is demonstrated by Table 2. on the example ofS. officinalis L.(Mth I. et al., 1992). Similar

differences can be anticipated also in the case of other species.

Table 2 - Changes in the essential oil content ofSalvia officinalis L. during the vegetation

period (dry wt. %)

Leaves Generative organs

April 0.88May 0.62

June 0.81

July 1.04 1.59

August 1.31 0.70

September 1.03 0.63

October 0.85

November 0.83


Rosmarinic-, caffeic- and ursolic acid contents of various organs of 5 species (S. officinalis L.,

S. candelabrum Boiss., S. tomentosa Mill., S. lavandulifolia Vahl., S. fruticosa Mill.) studied,

with monthly sampling, in the course of the growth season showed a rather similar pattern for

all species. The sequence of species was: triterpene carbonic acids, followed by rosmarinic

acid, and finally with an order of magnitude lower values, the caffeic acid.

Table 3 - Average content of selected non-volatile components of members of the Section

Salvia, in the growth season (AprilSeptember)

Species Rosmarinic acid Caffeic acid Ursole/Oleonolic

Acid

S. offi cinalis 0.18 0.02 0.57

S. candelabrum 0.2 0.04 0.36S. tomentosa 0.18 0.02 0.44

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S. lavanduli foli a 0.30 0.03 0.40

S. fr uticosa 0.19 0.04 0.42


The occurrence of rosmarinic-, caffeic- and ursolic acids

As regards the variations in the production of rosmarinic-, caffeic- and ursolic acids of various

plant organs, Table 4 contains average values of samples taken in the course of the entire

growth season. The table contains also the values of standard deviation that in this case refer

to the extent of expected range of changes. It can be stated that the chemically more sensitive

rosmarinic- and caffeic acid content undergo more significant changes, though remarkably

only in the case of leaf samples. The term generative organs, in this case, spans over the

phases of budding, flowering and fruiting, so obviously data regarding these organs show a

substantial variability. As compared to rosmarinic acid, the presence of caffeic acid is several

order of magnitude lower, while the S.D. values seem to refer to no great differences

according to organs. The accumulation of ursolic/oleanolic acids is highest in the leaves. In

the course of the growth season the greatest variations, i.e. changes were observed in the case

of the stems.

Table 4 - Average non-volatile content of organs ofSalvia officinalis L., in Hungary, in the

growth season

Organs Rosmarinic acid Caffeic acid Ursolic-/Oleanolic acidX + S.D. CV (%) X + S.D. CV (%) X + S.D. CV (%)

Vegetative 0.31 44.2 0.030 78.5 0.34 8.0

Leaves 0.18 93.3 0.038 0.56 31.5

Stems 0.30 44.3 0.018 48.9 0.22 57.3

Generative

organs

0.32 90.0 0.026 23.6 0.11 30.8


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Changes in the rate of oleanolic and ursolic acids were also studied with several other species

of the Family Lamiaceae (Janicsk et al., 2006). Table 5 comprises the summarized

(cumulate) values of 76 taxa. Representatives of the genus Salvia, subfamily Nepetoideae are

figured separately. S. officinalis L., S. lavandulifolia Vahl. and S. tomentosa Mill. are also

combined into a separate unit. As indicated by the table, the ursolic acid content was highest

in each instances. (It should be mentioned, that in the overall database only 4 samples

contained oleanolic acid in higher quantity). Species of the Lamioideae sub-family contained

substantially higher quantities than species of the sub-family. This seems to underline our

earlier observations of chemotaxonomic importance (Mth et al., 1993) according to which

at the sub-family level, differences can be detected in the rate of ursolic / oleanolic acids.

Remarkably, all three species of the Section Salvia had a higher than average content of

triterpene-carbonic acid content. While the average for genera was rather similar to the plant

individuals of the sub-family Nepetoideae, S. tomentosa Mill., S. officinalis L. and S.

lavandulifolia Vahl. produced outstanding values of both oleanolic and ursolic acid content.

Table 5 - Average Oleanolic and Ursolic acid content of taxonomic groups of the Family

Lamiaceae, in Hungary (dry wt. %)

Number of taxa Oleanolic acid Ursolic acid

Subfam.

Lamioideae

13 0.012 0.042

Subfam.

Nepetoideae

3 1.254 2.964

Genus Salvia 33 0.227 0.485

Species of

Section Salvia

3 1.254 2.964


The occurrence of miscellaneous components

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Phenylpropanoids, i.e. phenylethanoilds are special components of the Family Lamiaceae

Representatives of this group of compounds have been detected in species of the Sub-Family

Lamioideae (Calis et al., 1984, Calis et al., 1991, Mth and Csed, 2007).

In Hungary, martinozid has been isolated from the polar fraction ofS. officinalis L. extracts

for the first time (Hohmann, et al. 2003), though in the rather minute quantity of 0.0006 %.

Although this compound has been known for while, its first occurrence in Salvia officinalis L.

is significant due to the fact that it indicates its presence not only in the genus Salvia but also

in the Sub-Family Nepetoideae. (Mth and Csed, 2007).

Conclusions

Our investigations have demonstrated that members of the Salvia genus, similarly to many

representatives of the sub-family Lamioideae and Nepetoideae can be grown under the

continental conditions of Hungary. Hungarian grown species of the section Salvia possess

similar characteristics to those growing in their natural habitats.

Differences among species within the section Salvia are not explicit, which from the chemical

point of view also underlines the close relationship of these species. All of them are essential

oil containing species and their essential oils have similar in characteristics. They belong to

the predominantly monoterpenoid bearing oils. Among the constituents thujone content shows

significant variation. The relatively high interspecific variability of chemical components, e.g.

in Salvia officinalis L. calls attention to the fact that this is a factor to be considered in the

evaluation of the biological activity of species.

The investigations have demonstrated that since S. tomentosa has a similar essential oil

composition to S. officinalis. It also contains favourably less thujone, therefore, it could be

regarded as a potential prospective substitute.

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