EVALUATION OF THE ESSENTIAL OIL PRODUCTION AND COMPOSITION OF SALVIA SPECIES BEYOND THEIR NATIVE AREA OF DISTRIBUTION Mth,

Imre1, Mth , kos2, Hohmann Judit3 and Janicsk, Gbor1

1 2

Institute of Ecology and Botany, Hungarian Academy of Sciences, Vcrtt, Hungary University of West Hungary, Faculty of Agriculture and Food Science, Department of Botany, Mosonmagyarvr, Hungary 3 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.

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 of Salvia 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 of Salvia 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

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 210 250 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: 40 400 amu. Identification of components was made on the basis of authentic samples using Kovats-index calculation and the database of GC/MS equipment.

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 % aqueousmethanolic 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

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 of S. officinalis L. seemed to be similar to that of S. candelabrum Boiss., while in the case of S. 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 of S. 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

similar to that of S. officinalis L. which data seem to indicate that this species could be a potential promising substitute for S. officinalis L.. The essential oil character of S. 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 0.39 S. officinalis 0.25 S. fruticosa 0.46 S. lavandulifolia 0.89 S. candelabrum 0.25 S. ringens +

ml/100 dry mass Terpenes: Monoterpenes -thujene -pinene -pinene Myrcene -terpinene p-cymol Limonene 1,8-cineole -terpinene terpinolene -thujone -thujone Camphore Borneole Terpinene-4-ol -terpineole bornyl-acetate Sesquiterpenes -caryophyllene -humulene -muurolene -kadinene seline (3,7), 11diene caryophyllenoixide viridiflorol humulene 1,2epoxide Sclarene
*

0.2 1.6 7.2 1.4 0.2 0.2 1.9 11.6 0.6 0.4 7.4 3.3 10.1 9.3 0.6 0.4 3.3 5.9 6.2 0.3 5.5 0.6 0.9 17.1 0.5 0.9

0.2 1.1 2.3 0.9 0.2 0.2 1.3 3.5 0.5 0.4 36.0 9.7 9.6 8.4 0.5 0.1 4.9 0.7 4.2

0.2 1.2 1.7 4.3 0.1 0.1 0.2 16.9 0.5 0.6 21.4 3.7 26.0 1.0 0.4 0.5 1.0 1.4 2.8

04.4 2.7 6.8 5.8 0.4 0.4 47.0 1.1 0.5

0.2 6.5 7.4 1.4 1.1 20.5

2.5 2.9 2.2 0.4 1.7 16.6 0.5 0.5 9.7 1.8 16.0 2.0 0.5 1.4 2.3 4.8 0.3 0.7

12.6 2.2 0.7 0.3 0.6 4.9 1.0

1.6 0.5 9.0 4.4 1.1 1.9 0.9 0.2 1.1

0.1 0.2 4.6 0.8 1.9 0.1 5.6 0.2 0.8 0.3 4.5

0.2

0.8 0.4

13.5

2.6

According to Mth et al., 2007

The essential oil production of Salvia 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 of S. 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 of Salvia officinalis L. during the vegetation period (dry wt. %) Leaves 0.88 0.62 0.81 1.04 1.31 1.03 0.85 0.83 Generative organs

April May June July August September October November

*

1.59 0.70 0.63

According to Mth et al., 2007

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 (April September)

Species S. officinalis S. candelabrum S. tomentosa

Rosmarinic acid 0.18 0.2 0.18

Caffeic acid 0.02 0.04 0.02

Ursole/Oleonolic Acid 0.57 0.36 0.44

S. lavandulifolia S. fruticosa
*

0.30 0.19

0.03 0.04

0.40 0.42

According to Mth et al., 2007

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 of Salvia officinalis L., in Hungary, in the growth season

Organs

Rosmarinic acid X + S.D. CV (%) 0.31 44.2 Vegetative 0.18 93.3 Leaves 0.30 44.3 Stems 0.32 90.0 Generative organs
*

Caffeic acid X + S.D. 0.030 0.038 0.018 0.026

CV (%) 78.5 48.9 23.6

Ursolic-/Oleanolic acid X + S.D. CV (%) 0.34 8.0 0.56 31.5 0.22 57.3 0.11 30.8

According to Mth et al., 2007

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. %)

Subfam. Lamioideae Subfam. Nepetoideae Genus Salvia Species of Section Salvia

*

Number of taxa 13 3 33 3

Oleanolic acid 0.012 1.254 0.227 1.254

Ursolic acid 0.042 2.964 0.485 2.964

According to Mth et al., 2007

The occurrence of miscellaneous components

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 of S. 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.

References Alizar, G. 1993. Catalogue Synonymique des Salvia du Monde. (Lamiaceae) VI. Biocosme Msogen, Nice, 10 (3-4):33-117. Calis, I., Lahloub, M.E., Rogenmoser, E., Sticher, O. 1984. 7-Isomartynoside, A Phenylpropanoid Glycosides from Galeopsis pubescens. Phytochemistry, 23, 2313-2315. Calis, I., Basaran, A.A., Saracoglu, I., Sticher, O., Redli, P. 1991. Phlinosides A., B. and C. three phenylpropanoid glycosides from Phlomis linearis. Phytochemistry, 30, 3073-3075. Cantino, P.D., Harley, R.M., Wagstaff, S.J, 1992. Genera of Labiatae: Status and classification. In Harley, R.M., Reynholds, T., (eds.) Advances in Labiate Science, Royal Botanic Gardens, Kew, pp. 511-522. Dobos, ., Nagy, G., Genova, E.M., Mth, I., Miklssy, V.V. and Janicsk, G. 1997. Comparative analysis of Salvia officinalis and Salvia tomentosa essential oils. In: Franz, Ch., Mth, . and Buchbauer, G. eds. Essential oils: Basic and Applied Research. Proceedings of the 27th International Symposium on Essential Oils. Allured Publishing Corp, Vienna, 241-243. Hohmann, J., Rdei, D., Mth, I., Blunden, G. 2003. Phenylpropanoid glycosides and diterpenoids from Salvia officinalis. Biochem. Syst. Ecol. 31: 427-429. Janicsk, G., Veres, K., Kakay, A.Z. and Mth, I. 2006: Study of the oleanolic and ursolic acid contents of some species of the Lamiaceae. Biochemical Systematics and Ecology, 34, 392-396. Karousou, R., Hanlidou, E. and Kokkini, St. 2000. The sage plants in Greece: Distribution and infraspecific variation. In: Kintzios, Sp.E. ed. SAGE. The genus Salvia. Harwood Academic Publishers, Amsterdam, pp. 27 -46.

Mth, ., Lemberkovics, ., Mth, I. Jr., Mth, I. and Ngyen, H. 1992. Production biology of Mediterranean Lamiaceae species in the temperate belt. Acta Horticulturae, 344, 121122. (1992) Mth, I. Jr., Olh, L., Mth, ., Miklssy, V.V., Bernth, J., Blunden, G., Patel, A.V. and Mth, I. 1992. Changes in the essential oil production of Salvia officinalis under climatic conditions of the temperate belt. Planta Med., 58, Supplement, A 680 Mth, I. Jr., Miklssy, V.V., Mth, ., Bernth, J., Olh, L., Blunden, G., Patel, A.V. 1993. Essential oil content as chemotaxonomic marker for the genus Salvia with reference to its variation in Salvia officinalis L. Acta Horticulturae, 330, 123-131. Mth, I., Nagy, G., Dobos, ., Miklssy, V.V., Janicsk, G. 1997. Comparative studies on the essential oils of some species of Sect. Salvia. In: Franz, Ch., Mth, . and Buchbauer, G. eds. Essential oils: Basic and Applied Research. Proceedings of the 27th International Symposium on Essential Oils. Allured Publishing Corp, Vienna, pp. 241-243. Mth, I. 1997. Some aspects of recent researches on Lamiaceae species in Hungary. Archiv za Farmaciju, 47: 395-404. Mth, I. and Csed, K. 2007. Chemical differences and similarities in the family Lamiaceae. Revista de Medicina si Farmacie 53: 1-14. Mth, I., Hohmann, J., Janicsk, G., Nagy , G., Rdei, D. 2007. Chemical diversity of the biological active ingredients of Salvia officinalis and some closely related speces (in Hungarian) Acta Pharm. Hung. 77: 37-45. Walker, J.B., Syst, W., Kenneth, J.S., Treutlein, J. and Wink, M. 2004. Salvia (Lamiaceae) is not monophyletic: implications for the systematics, radiation, and ecological specializations of Salvia and tribe Menthae. American Journal of Botany 91:1115-1125.