Biljana Nikolić

Protective effect of linalool, myrcene and eucalyptol against t-butyl hydroperoxide induced genotoxicity in bacteria and cultured human cells

We studied the protective effect of monoterpenes myrcene, eucalyptol and linalool against t-butyl hydroperoxide (t-BOOH) induced genotoxicity in reverse mutation assay with Escherichia coli WP2 IC185 strain and its oxyR mutant IC202, and with the comet assay in human hepatoma HepG2 and human B lymphoid NC-NC cells. The monoterpenes were tested in concentration ranges 0.05-1.5 mg/plate and 0.01-1.0 microg/ml in bacteria and mammalian cells, respectively. Suppression of t-BOOH induced mutagenesis was detected only in IC202 strain, and correlated with the observed inhibition of lipid peroxidation by the three monoterpenes. Linalool and myrcene strongly suppressed t-BOOH induced mutagenesis. Eucalyptol, in addition to moderate suppression of t-BOOH induced mutagenesis, suppressed also spontaneous mutagenesis. In NC-NC cells linalool and myrcene reduced t-BOOH induced DNA damage by about 50% at 0.01 microg/ml, while eucalyptol was less efficient (about 50% reduction at 1.0 microg/ml). In HepG2 cells linalool and eucalyptol reduced DNA damage by 30% and 40%, respectively, while myrcene was ineffective. The repair of t-BOOH induced DNA damage, studied in HepG2 cells, was not affected by monoterpenes. The results indicate that linalool, eucalyptol and myrcene have substantial protective effect against oxidant induced genotoxicity, which is predominately mediated by their radical scavenging activity.

Modulation of genotoxicity and DNA repair by plant monoterpenes camphor, eucalyptol and thujone in Escherichia coli and mammalian cells

The aim of this work was to examine the antigenotoxic potential of plant monoterpenes: camphor, eucalyptol and thujone in prokaryotic and eukaryotic cells and to elucidate their effect on DNA repair. We compared the effect of monoterpenes on spontaneous, UV- and 4NQO-induced mutagenesis in Escherichia coli K12 repair proficient, and MMR and NER deficient strains. Positive controls tannic acid and vanillin were included in bacterial tests. We also examined protective effect of monoterpenes against 4NQO-induced genotoxicity in Vero cell line by alkaline comet assay. The results obtained in repair proficient strain indicated antimutagenic potential of monoterpenes against UV- and 4NQO-induced mutagenesis, which was diminished with NER deficiency. Camphor and eucalyptol maintained UV-induced SOS response longer than in controls, while thujone decreased SOS response and reduced general protein synthesis and the growth rate. The three monoterpenes increased spontaneous and UV-induced recombination in recA730 and camphor additionally in recA(+) cells. Incubation of 4NQO-pretreated Vero cells with monoterpenes resulted in significant reduction of tail moment. However, higher concentrations of monoterpenes induced DNA strand breaks. Obtained results indicate that by making a small amount of DNA lesions camphor, eucalyptol and thujone can stimulate error-free DNA repair processes and act as bioantimutagens.

Composition of n-alkanes in natural populations of Pinus nigra from Serbia - chemotaxonomic implications.

This is the first report on the composition and variability of the needle‐wax n‐alkanes in natural populations of Pinus nigra in Serbia. Samples of 195 trees from seven populations belonging to several infraspecific taxa (ssp. nigra, var. gocensis, ssp. pallasiana, and var. banatica) were analyzed. In general, the size of the n‐alkanes ranged from C16 to C33, with the exception of ssp. nigra, for which it ranged from C18 to C33. The most abundant were C23‐, C25‐, C27‐, and C29‐alkanes. The needle waxes of Populations I–III and V were characterized by a higher content of C23‐, C25‐, and C27‐alkanes and a lower content of C24‐, C26‐, C28‐, and C30‐alkanes, compared to the other populations, and the trees of these populations could be assigned to ssp. nigra. The samples of Population VI were characterized by higher amounts of C22‐, C24‐, C30‐, and C32‐alkanes and lower amounts of C25‐ and C27‐alkanes, and the trees could be considered as ssp. pallasiana. The samples of Population VII, consisting of trees belonging to var. banatica, were richer in C29‐, C31‐, and C33‐alkanes. The wax compositions of Populations IV and V, both composed of trees previously determined as P. nigra var. gocensis, showed a tendency of splitting. Indeed, the alkane composition of Population IV was closer to that of ssp. pallasiana pines, while that of Population V was more similar to that of ssp. nigra pines. From the results presented here, it is obvious that in the central part of the Balkan Peninsula, significant diversification and differentiation of the populations of black pine exists, and these populations could be defined as different intraspecific taxa. Our results also indicate the validity of n‐alkanes as chemotaxonomic characters within this aggregate.

Chemotaxonomic Significance of the Terpene Composition in Natural Populations of Pinus nigra J.F.Arnold from Serbia

The essential‐oil variability in seven native populations belonging to different infraspecific taxa of Pinus nigra (ssp. nigra, var. gocensis, ssp. pallasiana, and var. banatica) growing wild in Serbia was analyzed. In the needles of 195 trees from seven populations, 58 essential‐oil components were identified. The major components were α‐pinene (43.6%) and germacrene D (29.8%), comprising together 73.4% of the total oil composition. Based on the average chemical profile of the main terpene components (with contents >5%), the studied populations were found to be the most similar to populations from central Italy and Greece (ssp. nigra). Cluster analysis showed the division of the populations into three principal groups: the first group consisted of Populations I, II, III, IV, and V (considered as ssp. nigra group), the second of Population VI (ssp. pallasiana group), and the third of Population VII, which had the most distinct oil composition (ssp. banatica group). The taxonomic implications of the essential‐oil profiles of the investigated taxa of this very complex species are discussed.

Comparative study of genotoxic, antigenotoxic and cytotoxic activities of monoterpenes camphor, eucalyptol and thujone in bacteria and mammalian cells

Genotoxic/antigenotoxic, mutagenic/antimutagenic and cytotoxic effects of monoterpenes camphor, eucalyptol and thujone were determined in bacteria and mammalian cells using alkaline comet assay, Escherichia coli K12 reversion test and MTT assay, respectively. When applied in low doses (up to 200 μM in bacterial assay and 50 μM in comet assay) monoterpenes protected repair proficient E. coli and Vero cells against UV-induced mutagenesis and 4NQO-induced DNA strand breaks, respectively. Antimutagenic response was not detected in nucleotide excision repair (NER) deficient bacteria. When monoterpenes were applied in higher doses, a weak mutagenic effect was found in mismatch repair (MMR) and NER deficient E. coli strains, while induction of DNA strand breaks was evident in human fetal lung fibroblasts MRC-5, colorectal carcinoma HT-29 and HCT 116 cells, as well as in Vero cells. Moreover, the involvement of NER, MMR and RecBCD pathways in repair of DNA lesions induced by monoterpenes was demonstrated in E. coli. Camphor, eucalyptol and thujone were cytotoxic to MRC-5, HT-29 and HCT 116 cells. The most susceptible cell line was HCT 116, with IC50 values of 4.5 mM for camphor, 4 mM for eucalyptol and 1 mM for thujone. Observed effects of monoterpenes are consistent with hormesis response, characterized by a low dose beneficial effect and a high dose adverse effect of a stressor agent, and provide a basis for further study of both chemopreventive and chemotherapeutic potential of camphor, eucalyptol and thujone.

Terpene Chemodiversity of Relict Conifers Picea omorika, Pinus heldreichii, and Pinus peuce, Endemic to Balkan

Terpenes are often used as ecological and chemotaxonomic markers of plant species, as well as for estimation of geographic variability. Essential oils of relic and Balkan endemic/subendemic conifers, Picea omorika, Pinus heldreichii, and P. peuce, in central part of Balkan Peninsula (Serbia and Montenegro), on the level of terpene classes and common terpene compounds were investigated. In finding terpene combinations, which could show the best diversity between species and their natural populations, several statistical methods were applied. Apart from the content of different terpene classes (P. omorika has the most abundant O‐containing monoterpenes and sesquiterpenes; P. heldreichii and P. peuce have the largest abundance of sesquiterpene and monoterpene hydrocarbons, resp.), the species are clearly separated according to terpene profile with 22 common compounds. But, divergences in their populations were established only in combination of several compounds (specific for each species), and they were found to be the results of geomorphologic, climatic, and genetic factors. We found similarities between investigated species and some taxa from literature with respect to terpene composition, possibly due to hybridization and phylogenetic relations. Obtained results are also important regarding to chemotaxonomy, biogeography, phylogeny, and evolution of these taxa.

Chemodiversity of Nonacosan-10-ol and n-Alkanes in the Needle Wax of Pinus heldreichii

This is the first report of individual variability and population diversity of the contents of nonacosan‐10‐ol and n‐alkanes in the needle cuticular waxes of Bosnian pines originated from Montenegro, regarded as Pinus heldreichii var. leucodermis, and from Serbia, regarded as P. heldreichii var. pančići. The amount of nonacosan‐10‐ol varied individually from 27.4 to 73.2% (55.5% in average), but differences between the four investigated populations were not statistically confirmed. The size of the n‐alkanes ranged from C18 to C33. The most abundant n‐alkanes were C23, C27, and C25 (12.2, 11.2, and 10.8% in average, resp.). The carbon preference index (CPI) of the n‐alkanes ranged from 0.8 to 3.1 (1.6 in average), while the average chain length (ACL) ranged from 20.9 to 26.5 (24.4 in average). Long‐chain and mid‐chain n‐alkanes prevailed (49.6 and 37.9% in average, resp.). It was also found that the populations of P. heldreichii var. leucodermis had predominantly a narrower range of n‐alkanes (C18C31) than the trees of the variety pančići (C18C33). Differences between the varieties were also significant for most of the other characteristics of the n‐alkane pattern (e.g., most abundant n‐alkanes, CPI, ACL, and relative proportion of short‐, mid‐, and long‐chain n‐alkanes). The principle component and cluster analyses of eleven n‐alkanes confirmed the significant diversity of these two varieties.

Chemotaxonomic implications of the n-alkane composition and the nonacosan-10-ol content in Picea omorika, Pinus heldreichii, and Pinus peuce.

The n‐alkane composition and the nonacosan‐10‐ol content in the needle cuticular waxes of Serbian spruce (Picea omorika), Bosnian pine (Pinus heldreichii), and Macedonian pine (Pinus peuce) were compared. The amount of nonacosan‐10‐ol in the needle waxes of P. omorika was higher than those in P. heldreichii and P. peuce. The range of n‐alkanes was also wider in P. omorika (C18–C35) than in P. heldreichii and P. peuce (C18–C33). The dominant n‐alkanes were C29 in the needle waxes of P. omorika, C23, C27, and C25 in those of P. heldreichii, and C29, C25, C27, and C23 in those of P. peuce. The waxes of P. omorika contained higher amounts of n‐alkanes C29, C31, and C33, while those of P. heldreichii and P. peuce had higher contents of n‐alkanes C21, C22, C23, C24, and C26. The principal component analysis of the contents of nine n‐alkanes showed a clear separation of the Serbian spruce populations from those of the two investigated pine species, which partially overlapped. The separation of the species was due to high contents of the n‐alkanes C29 and C31 (P. omorika), C19, C20, C21, C22, C23, and C24 (P. heldreichii), and C28 (P. peuce). Cluster analysis also showed a clear separation between the P. omorika populations on one side and the P. heldreichii and P. peuce populations on the other side. The n‐alkane and terpene compositions are discussed in the light of their usefulness in chemotaxonomy as well as with regard to the biogeography and phylogeny of these rare and endemic conifers.

Population Variability of Nonacosan-10-ol and n-Alkanes in Needle Cuticular Waxes of Macedonian Pine (Pinus peuceGriseb.)

This is the first report on population variability of nonacosan‐10‐ol and n‐alkanes in needle epicuticular waxes of Macedonian pine (Pinus peuce Griseb.) Hexane extracts of needle samples, originating from two natural populations in Montenegro (Zeletin and Sjekirica) and from one population in Serbia (Mokra Gora) were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The amount of nonacosan‐10‐ol varied individually from 41.3 to 72.31% (average 55.9%), with the Sjekirica population being statistically divergent (64.4% on average). The results showed n‐alkanes in epicuticular waxes ranging from C18 to C33. The most abundant alkanes were C29, C25, C27, and C23 (15.5, 11.1, 10.6, and 10.5% on average, resp.). The carbon preference index of Pinus peuce ranged from 1.0 to 4.3 (1.9 on average). Average chain length ranged from 18.4 to 27.7 (average 25.7). A high level of inidividual quantitative variation in all of these hydrocarbon parameters was also detected. These results were compared with published data on other species from the Pinus genus.

Essential Oil Variability in Natural Populations of Picea omorika, a Rare European Conifer

This study is the first report on the composition and variability of essential oil in the relic, endemic, and vulnerable tree species Serbian spruce, Picea omorika, in its natural populations. In the needles of 108 trees of four natural populations, 49 components of essential oils were identified. The main compounds were bornyl acetate (29.2%), camphene (18.7%), and α‐pinene (12.9%). Fourteen additional components had the contents of up to 0.5%: α‐cadinol (6.1%), limonene (5.8%), santene (3.5%), (E)‐hex‐2‐enal (2.9%), T‐cadinol (2.9%), δ‐cadinene (2.3%), tricyclene (2.1%), myrcene (1.6%), β‐pinene (1.2%), borneol (0.9%), germacrene D (0.9%), α‐muurolene (0.6%), and two unidentified compounds. Population IV from Mileševka Canyon had a much higher content of bornyl acetate (42.9%). Populations I–III from Mt. Tara were more abundant in sesquiterpenes (up to 18.2%). The content of bornyl acetate, the multi‐variation analyses according to seven selected components, especially the cluster analysis and genetic analysis of α‐cadinol, which suggested the monogenic type of heredity, showed a clear differentiation of the two geographic areas, the similarity of populations I–III from the area of Mt. Tara, and the separation of the population IV from Mileševka Canyon.