Polina D. Blagojević

Antimicrobial Plant Metabolites: Structural Diversity and Mechanism of Action

Microbial infectious diseases continue to be one of the leading causes of morbidity and mortality. It has been estimated that microbial species comprise about 60% of the Earths biomass. This, together with the fact that their genetic, metabolic and physiological diversity is extraordinary, makes them a major threat to the health and development of populations across the world. Widespread antibiotic resistance, the emergence of new pathogens in addition to the resurgence of old ones, and the lack of effective new therapeutics exacerbate the problems. Thus, the need to discover and develop new antimicrobial agents is critical to improve mankinds future health. Plant secondary metabolites (PSMs) offer particular promise in this sense. Plant Kingdom could be considered a rich source of the most diverse structures (e.g. there are more than 12,000 known alkaloids, more than 8,000 phenolic compounds and over 25,000 different terpenoids), many of which were proven to possess strong antimicrobial properties (e.g. thymol, eurabienol, etc.). In many instances, PSMs can be easily isolated from the plant matrix, either in pure state or in the form of mixtures of chemically related compounds. What is also important is that the development of bacterial resistance toward natural plant products (that are generally regarded as eco-friendly) has been thus far documented in a very limited number of cases (e.g. for reserpine). Having all of the mentioned advantages of PSMs as potential antimicrobials in mind, a major question arises: why is it that there are still no commercially available or commonly used antibiotics of plant origin? This review tries to give a critical answer to this question by considering potential mechanisms of antimicrobial action of PSMs (inhibition of cell wall or protein synthesis, inducing leakage from the cells by tampering with the function of the membranes, interfering with intermediary metabolisms or DNA/RNA synthesis/function), as well as their physical and chemical properties (e.g. hydrophilicity/lipophilicity, chemical stability). To address the possible synergistic/antagonistic effects between PSMs and with standard antibiotics, special attention has been given to the antimicrobial activity of PSM-mixtures (e.g. essential oils, plant extracts). Moreover, possible ways of overcoming some of PSMs molecular limitations in respect to their usage as potential antibiotics were also discussed (e.g. derivatization that would enable fine tuning of certain molecular characteristics).

Toxic essential oils. Part II: Chemical, toxicological, pharmacological and microbiological profiles of Artemisia annua L. volatiles

Botanical drugs based on Artemisia annua L. (Asteraceae) are important in the treatment of malaria. Alongside with artemisinin, this aromatic species produces high and variable amounts of other chemicals that have mostly unknown biological/pharmacological activities. Herein, we have studied the toxicological/pharmacological profile of volatile constituents of a Serbian population of A. annua. Fifty-eight components were identified, among them, artemisia ketone (35.7%), α-pinene (16.5%) and 1,8-cineole (5.5%) were the most abundant ones. Significant variability of A. annua volatile profile was confirmed by means of agglomerative hierarchical cluster analysis indicating the existence of several different A. annua chemotypes. In an attempt to connect the chemical profile of A. annua oil with its biological/toxicological effects, we have evaluated in vivo and/or in vitro toxicity (including hepato- and nephrotoxicity/protection), antinociceptive, antioxidant (DPPH, ABTS and superoxide radical scavenging activity assays), enzyme inhibiting (protein kinase A and α-amylase) and antimicrobial potential of A. annua oil and of its constituents. Our results revealed that the beneficial properties of A. annua botanical drugs are not limited only to their antimalarial properties. Taking into account its relatively low toxicity, the usage of A. annua volatiles (at least of the herein studied population) does not represent a health risk.

Comparative study of the leaf volatiles of Arctostaphylos uva-ursi (L.) Spreng. and Vaccinium vitis-idaea L. (Ericaceae).

The first GC and GC/MS analyses of the essential oils hydrodistilled from dry leaves of Arctostaphylos uva-ursi and Vaccinium vitis-idaea enabled the identification of 338 components in total (90.4 and 91.7% of the total GC peak areas, respectively). Terpenoids, fatty acids, fatty acid- and carotenoid derived compounds were predominant in the two samples. Both oils were characterized by high relative percentages of α-terpineol and linalool (4.7-17.0%). Compositional data on the volatiles of the presently analyzed and some other Ericaceae taxa (literature data) were mutually compared by means of multivariate statistical analyses (agglomerative hierarchical cluster analysis and principal component analysis). This was done in order to determine, based on the essential oil profiles, possible mutual relationships of the taxa within the family, especially that of species from the genera Arctostaphylos and Vaccinium. Results of the chemical and statistical analyses pointed to a strong relation between the genera Vaccinium and Arctostaphylos.

Toxic essential oils. Part III: Identification and biological activity of new allylmethoxyphenyl esters from a Chamomile species (Anthemis segetalis Ten.)

To determine the exact structure of previously tentatively identified minor essential-oil constituents of a Chamomile species (Antemis segetalis Ten. (Asteraceae)), we have synthesized a small combinatorial library of 54 regioisomeric allylmethoxyphenyl pentanoates and 2-pentenoates (49 completely new compounds). GC-MS in combination with 1D- and 2D-NMR analyses of the library compounds provided unambiguous data that led to a straightforward identification of the mentioned A. segetalis constituents as eugenyl angelate, 2-methylbutanoate and 3-methylbutanoate (0.21, 0.22, and 0.13 mg/100 g of fresh plant material, respectively). To assess the safety and potential beneficial pharmacological uses of these naturally occurring esters and several other library compounds (these were tested to provide relevant data for a SAR (structure-activity relationship) analysis), we have studied the effect of these compounds in several models of toxicity (acute toxicity against Artemia salina, cytotoxicity against two cell lines (fibroblast and melanoma)), as well as their acetylcholinesterase inhibitory and antibacterial activities. Anthemis segetalis constituents showed low to moderate activity in all tests. The obtained results suggest that the intake of these compounds in naturally available amounts, on their own, would probably not represent a risk to human health but the possible adverse interactions with the plant matrix should not be neglected.

A note on the volatile secondary metabolites of Foeniculum vulgare Mill. (Apiaceae)

An analysis (GC and GC/MS) of Foeniculum vulgare Mill. (fennel) root and schizocarp essential oils and diethyl ether extracts enabled the identification of 89 different components, representing 98.2-100% of the total samples. One fourth of the identified compounds (24 in total) are reported for the first time as fennel volatiles. The most abundant classes of constituents of all four analyzed samples were the phenylpropanoids (69.5-85.5%) and monoterpenoids (11.7-26.9%). The dominant volatile metabolites of the schizocarps were fenchone (13.3-18.8%) and (E)-anethole (66.1-69.0%). Contrary to that, terpinolene (6.2-6.5%) and dillapiole (71.4-77.5%) were the major volatiles of fennel roots. The most striking differences between the chemical composition of the analyzed oils and their corresponding extracts were a rather surprising presence of a significant relative amount of apiole in the root oil (9.3%; this compound was detected only in trace amounts in the root extract) and the high percentage of 10-nonacosanone in the schizocarp extract (5.8%; this ketone was absent from the schizocarp oil). Possible explanations of the mentioned differences were put forward. The AMS (average mass scan of the total ion chromatogram) profiles/fingerprints of the volatile oils and extracts of F. vulgare roots and fruits are also given in the paper.

New volatile sulfur-containing compounds from wild garlic (Allium ursinum L., Liliaceae)

In many countries, the leaves of Allium ursinum L. (Liliaceae) are a popular substitute for garlic and, for centuries, the herb has been taken internally to treat an array of medical conditions. Herein, we report the chemical composition of 12 different A. ursinum essential-oil samples (five populations; fresh/air-/oven-dried plant material; leaves/inflorescences). GC-MS/GC-FID analyses, quantitative structure-property relationship modeling (simple 0D/1D-descriptors) of retention indices and the synthesis of selected compounds, enabled the identification of >200 different constituents, mainly organo(poly)sulfides. Some of these were new compounds (allyl (methylthio)methyl, (methylthio)methyl (Z)/(E)-1-propenyl and allyl 1-(methylthio)propyl disulfides) or were previously not detected in samples of natural origin (heptyl methyl, methyl octyl, allyl hexyl (1), allyl octyl (2) and propyl (propylthio)methyl sulfides). A multivariate statistical analysis revealed the onset of significant changes in the plant material volatile profile during the drying process.

Plant volatiles providing additional evidences to the occurence of a wild-growing population of Calamintha vardarensis (Greuter et Burdet) Šilić outside of its natural habitat.

The essential‐oil profile of a Calamintha species, wild‐growing in the urban settings of the city of Niš (South Serbia) and botanically tentatively identified as C. vardarensis (endemic species native to FYR Macedonia and East Serbia), has been statistically (multivariate statistical analyses) compared with those of other Calamintha species, including two previously investigated C. vardarensis populations, as a means of corroboration of the surprising occurence of this Calamintha population outside of its natural distributional range. Agglomerative hierarchical clustering reveals a close link of C. vardarensis from Niš (with neo‐menthol (40.0%), menthone (21.8%), and pulegone (27.2%) as its major oil contributors) and C. vardarensis from FYR Macedonia.

Toxic essential oils. Part IV: The essential oil of Achillea falcata L. as a source of biologically/pharmacologically active trans-sabinyl esters

Herein we report on the comprehensive chemical analysis of the essential oils obtained from above- and underground parts of a previously unreported chemotype of Achillea falcata L. (Asteraceae) and, for the first time, on the biological/toxicological profile of its dominant/newly discovered volatile metabolites. Detailed spectral analyses, in combination with chemical synthesis and theoretical study, of selected constituents, enabled the identification of trans-sabinol and its esters - the formate, tiglate (new compounds), acetate, butanoate, isobutanoate, 2-methylbutanoate and 3-methylbutanoate - in both aerial and underground parts of A. falcata. Evaluation of acute toxicity in Artemia salina model, in vitro and in silico (molecular docking) evaluation of acetylcholinesterase inhibitory activity and in vivo (mice) evaluation of antinociceptive activity (hot plate, tail immersion and acetylcholine-induced abdominal writhing tests) of trans-sabinol and its esters suggested that they may interact with different targets in crustacean/mammalian organisms. Alongside moderate acute toxicity (LD50 (48 h) = 0.03-0.26 mmol/L), the tested compounds exert influence on both the peripheral and central nervous systems (in the hot plate test, trans-sabinyl tiglate, at 50 mg/kg, produced a 140% baseline increase 15 min after the treatment) and to moderately inhibit acetylcholinesterase (at the concentration of 20 µg/mL, these compounds caused a reduction of acetylcholinesterase activity up to 40%).

Commercial Carlinae radix herbal drug: Botanical identity, chemical composition and antimicrobial properties

Context: Carlinae radix is an herbal drug, commonly used by the locals in southeastern Serbia for the treatment of respiratory and urogenital diseases and, externally, for various skin conditions. There still seems to be no detailed studies correlating the chemical composition of this drug and its ethnopharmacological uses. Objective: Chemical composition, antimicrobial activity and mode of action of C. radix essential oil, isolated from commercial samples (confirmation of whose true biological identity was also the aim of this work) were analyzed. Antimicrobial potential of decoctions (extracts prepared by boiling plant material in a given solvent), used in ethnomedicine preferentially to the pure essential oil, was also investigated. Materials and methods: The essential oil obtained by hydrodistillation was screened for antimicrobial activity by disc diffusion and broth microdilution methods. Effects of the oil on the growth of Staphylococcus aureus cells were investigated using turbidimetric measurements and visualized using scanning electron microscopy. Analyses of the chemical composition of the oils were done using gas chromatography and gas chromatography/mass spectrometry. Results and discussion: Both the essential oil and the decocts exhibited a very high antimicrobial activity against all tested strains, with S. aureus as the most sensitive one [e.g., for the oil sample the values for minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were 0.02, 0.04 µL/mL, respectively]. Growth curves of S. aureus demonstrated a significant decrease in turbidity (for the MIC concentration this amounted to ca. 70%) showing a concentration-dependent lysis of the cells, confirmed by scanning electron microscopy. Chemical composition, anatomical and morphological features of the sample pointed to Carlina acanthifolia L. (Asteraceae) instead of Carlina acaulis L. (Asteraceae). Conclusion: The results showed significant antimicrobial effect of the essential oil and the decoctions and support the use of this plant in ethnomedicine for the treatment of various human infections, especially those caused by S. aureus. Adulteration of the drug would not cause significant differences in its biological activity, since chemical composition of the sample showed high similarity with those containing C. acaulis roots.

Volatiles of Curcuma manggaVal. & Zijp (Zingiberaceae) from Malaysia

Analysis by GC and GC/MS of the essential oil obtained from Malaysian Curcuma mangga Val. & Zijp (Zingiberaceae) rhizomes allowed the identification of 97 constituents, comprising 89.5% of the total oil composition. The major compounds were identified as myrcene (1; 46.5%) and β‐pinene (2; 14.6%). The chemical composition of this and additional 13 oils obtained from selected Curcuma L. taxa were compared using multivariate statistical analyses (agglomerative hierarchical cluster analysis and principal component analysis). The results of the statistical analyses of this particular data set pointed out that 1 could be potentially used as a valuable infrageneric chemotaxonomical marker for C. mangga. Moreover, it seems that C. mangga, C. xanthorrhiza Roxb., and C. longa L. are, with respect to the volatile secondary metabolites, closely related. In addition, comparison of the essential oil profiles revealed a potential influence of the environmental (geographical) factors, alongside with the genetic ones, on the production of volatile secondary metabolites in Curcuma taxa.