Genus Abies Mill. (Pinaceae) as the source of plant antimicrobials: A Review Rod Abies Mill. (Pinaceae) kao izvor biljnih antimikrobnih supstanci: Pregled

Antimicrobial resistance is one of the major global health problems and it’s related to the enormous number of human deaths. The occurrence and severity of infections caused by microbial pathogens illustrate the need for the identification and characterization of novel antimicrobial agents of natural origin. This review discusses a well-known coniferous genus Abies Mill. in terms of antimicrobial potential. Data regarding the antibacterial, antifungal, and antiviral properties of Abies species were collected and summarized in this review. It was found that 13 different Abies species are recognized as potential sources of antimicrobial compounds. The most investigated species was A. spectabilis (syn. A. webbiana ), followed by A. alba , A. cilicica , A. sibirica , A. nordmanniana , A. numidica , A. koreana , A. balsamea , A. holophylla , and A. concolor . Individual studies on A. firma , A. beshanzuensis , and A. cephalonica were also taken into account. The largest number of analyzed results were related to the antibacterial activity of Abies -derived products, but studies on antifungal, and particularly antiviral capacity were also noted. The most investigated products were essential oils and extracts. The broadest antimicrobial activity was observed for A. cilicica . This study noted that some endemic and endangered Abies species were being used for antimicrobial purposes. In that term, the rationalization of the sampling practices and the im - plementation of the conservation activities are of great importance. This review represents a comprehensive overview of the current knowledge on the antimicrobial potential of the genus Abies .


INTRODUCTION -Uvod
Antimicrobial resistance (AMR) is one of the most challenging problems of global health in the 21 st century (Hernando-Amado et al., 2019). According to O'Neill (2016), AMR is related to 750,000 annual human deaths worldwide, and by the year 2050, that number could increase up to 10 million. As humanity, we are indeed faced with the lack of new antimicrobials, the rise of AMR, the toxicity of synthetic antimicrobial drugs, and their many potential side effects, as well as the economic burden that is unavoidable in the constant race with microbes to develop efficient and affordable medicines. In the abovementioned issues, plants could represent a potential solution (Chassagne et al., 2021), since they produce secondary metabolites, well-known for their bioactive capacity.

Materijal i metode
This review discusses the genus Abies in terms of antimicrobial potential. Despite the fact that there are individual published results regarding the antimicrobial activity of particular Abies species, to the best of our knowledge there is no systematic overview that debates the antibacterial, antifungal, and antiviral properties of the genus Abies in a comprehensive manner. Available data regarding the antimicrobial potential of Abies species were extracted from scientific databases Web of Science, Scopus, and PubMed, by using the search terms such as: "Abies", "antimicrobial activity", "antimicrobial resistance", "phytochemical composition", and "plant antimicrobials".

Rezultati i diskusija
This review revealed that 13 Abies species were recognized in terms of antimicrobial potential. The total number of published studies for each species that is taken into consideration is presented in Graph 1. Details regarding the antimicrobial potential of particular species are presented below. Species are listed in accordance with their phylogenetic similarity (Xiang et al., 2018).

Abies alba Mill.
Abies alba, known as the European Fir or Silver Fir is native to the mountains of Europe, but successfully cultivated in North America. It occurs at altitudes of 300 to 1,700 m, on mountains with rainfall over 1,000 mm per year (Simonetti & Simonetti, 1990;Farjon, 2017). Bağci and Diğrak (1996) reported a modest antibacterial activity of the essential oil (EO) made from A. alba twigs. Broznić et al. (2018) Garzoli et al. (2021), who detected the most abundant components of the liquid phase of A. alba EO: α-pinene, β-pinene, limonene, and γ-terpinene. This EO exhibited antibacterial activity against E. coli, Pseudomonas fluorescens, Acinetobacter bohemicus, Kocuria marina, and Bacillus cereus. Values of the minimum inhibitory concentration and the minimum bactericidal concentration (MIC and MBC) were 51.28 mg/ml for E. coli, P. fluorescens, and K. marina, while lower MIC and MBC values were noted for A. bohemicus and B. cereus (12.82 mg/ml and 25.64 mg/ml, respectively). The MBC/MIC ratio defined the A. alba EO as bactericidal against all tested bacterial strains.

Abies cilicica (Antoine et Kotschy) Carrière
Abies cilicica (Cilician Fir) occurs in the mountains adjacent to the northeastern Mediterranean coast of Turkey, Syria, and Lebanon (Gardner & Knees, 2013). This species is dominant in the Abieti-Cedrion phytocoenosis, a type of forest that occurs between 800 and 2,100 m elevation. The annual precipitation is typically 1,000-1,500 mm, mostly falling in winter (Boydak, 2007). Bağci and Diğrak (1996) investigated the antimicrobial activity of essential oils made from twigs of A. cilicica subsp. cilicica and A. cilicica subsp. isaurica against series of microorganisms: E. coli, Bacillus megaterium, B. cereus, B. subtilis, B. brevis, P. aeruginosa, Listeria monocytogenes, K. pneumoniae, Enterobacter aerogenes, S. aureus, Saccharomyces cerevisiae, and C. albicans. This study recorded a very high antimicrobial effect of the tested EOs, with stronger antifungal potential, and low activity against the Gram-negative pathogen E. coli. The main components of the A. cilicica subsp. cilicica EO are δ-3-carene, α-pinene, longipinene, β-caryophyllene, α-humulene, and germacrene D (Bağci & Diğrak, 1996). Kizil et al. (2002) proved the antimicrobial potential of the resins obtained from the roots and stems of A. cilicica on a panel of microorganisms: S. aureus, Streptococcus pyogenes, Bacillus thurigiensis, B. brevis, B. subtilis, B. megatherium, B. cereus, P. aeruginosa, E. coli, and C. albicans. Results suggested that increasing the resin concentration (from 40 to 80 µg per disk) led to the formation of broader inhibition zones. Later investigation by Dayisoylu et al. (2009) tested the antimicrobial properties of the EO from the resin of the cones of A. cilicica subsp. cilicica and included a wide list of microbial species: Corynebacterium xerosis, B. brevis, B. megatherium, B. cereus, Mycobacterium smegmatis, P. aeruginosa, S. aureus, K. pneumoniae, E. faecalis, Micrococcus luteus, E. coli, Kluyveromyces fragilis, Rhodotorula rubra, and S. cerevisiae. Interestingly, all strains except for E. coli were successfully inhibited by this EO. The values of minimum inhibitory concentration (MIC) of EO for inhibited bacteria were in the range of 0.50-3.50 µg/ ml and for yeasts 0.50-1.75 µg/ml. The EO from the resin of the investigated plant contains limonene, β-pinene, α-pinene, and myrcene, with limonene being the most effective in terms of antibacterial potential, and myrcene as an antifungal agent. The aqueous and ethanolic extracts of resin obtained from cones of A. cilicica subsp. isaurica were investigated in terms of antimicrobial potential by Yavaşer et al. (2015). Activity is proven only for the ethanolic extract against S. aureus (11 mm), B. cereus (17 mm), and M. luteus (18 mm). However, there were no inhibition zones in the case of E. faecalis, E. coli, and L. monocytogenes. The resins are rich in terpenes and extracts may contain terpenoids, steroids, tannins, glycosides, anthraquinones, saponins, flavonoids, alkaloids, etc. The antibacterial activity of the ethereal extract made from A. cilicica is recorded by Erylmaz et al. (2016), against S. aureus (including methicillin-resistant strain, MRSA), B. subtilis, E. coli, P. aeruginosa, and K. pneumoniae, while the same substance did not show antifungal properties against C. albicans. The crude extracts from the leaf and flowering cones of A. cilicica subsp. cilicica were active against E. faecalis, Proteus vulgaris, K. pneumoniae, C. albicans, and Aspergillus niger. The growth of bacteria and fungi isolates was inhibited by methanolic, ethanolic, and acetonic extracts to different degrees, according to the tested organisms, plant fraction, and examined solvent (Saleh & Al-Mariri, 2016).

Abies nordmanniana (Steven) Spach
Abies nordmanniana (Nordmann Fir or Caucasian fir) is indigenous to the mountains south and east of the Black Sea, in Turkey, Georgia, and the Russian Caucasus. Typically, it occurs at altitudes of 900 to 2,200 m on mountains characterized by precipitation of over 1,000 mm (Tarkhnishvili et al., 2011). In the study of Bağci and Diğrak (1996), the essential oils of two subspecies were investigated A. nordmanniana subsp. nordmanniana and A. nordmanniana subsp. bornmiielleriana, and proven for their high antimicrobial activity against Bacillus megaterium, B. brevis, B. subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella pneumoniae, and Listeria monocytogenes. Antimicrobial activity of the methanolic and ethanolic extracts of leaves, cones, twigs, and stem barks of A. nordmanniana subsp. equi-trojani were detected against Mycobacterium smegmatis, B. subtilis, Salmonella typhimurium, Sarcina lutea, E. coli, S. aureus, Candida utilis, and S. cerevisiae in the research of Sakar et al. (1998). Observed effects are in relation to di-ORIGINAL SCIENTIFIC PAPER Genus Abies Mill. (Pinaceae) as the source of plant antimicrobials: A Review terpenoids, flavonoids, and tannin precursors. Erylmaz et al. (2016) noted relatively narrow antibacterial activity of A. nordmanniana ethereal extract, but interestingly, inhibition was observed against Gram-negative E. coli and P. aeruginosa, known for their extended antimicrobial resistance. The same study debates the antimicrobial activity of species refer as Abies equi-trojani, but according to the Euro+Med PlantBase, this is the synonym of A. nordmanniana subsp. equi-trojani. The ethereal extract showed antibacterial activity against B. subtilis and P. aeruginosa.

Abies numidica de Lannoy ex Carrière
Abies numidica is an endemic Algerian plant, growing in a high-altitude Mediterranean climate at 1,800-2,004 m with an annual precipitation of 1,500 to 2,000 mm (Yahi et al., 2011). Tlili Ait Kaki et al. (2012) found that pure essential oil of A. numidica was not active against investigated microorganisms, while particular dilution performed activity against MRSA, E. coli, K. pneumoniae, P. aeruginosa, Acinetobacter sp., and S. epidermidis. Inhibition zones were detected for all the investigated microorganisms at the dilution of 1/1000 (using dimethyl sulfoxide). The essential oil of A. numidica used in the investigation of Ramdani et al. (2014) was very active against B. cereus, E. coli, and E. faecalis. Modest activity was noted for S. aureus, S. epidermidis, and M. luteus, while low antibacterial activity was described for K. pneumoniae. Antifungal effects were very strong against S. cerevisiae. Unlike essential oils, Mostefa et al. (2016) investigated the phytochemical composition and antimicrobial potential of A. numidica hydro-methanolic cones extract. Chromatography revealed several new chemical compounds for this species, mainly abietane diterpenes. Investigated extract, as well as the individual compounds, performed antibacterial activity against B. subtilis (MIC=62.50 µg/ ml), E. faecalis, S. aureus, M. luteus, and Listeria innocua (MIC≤250 µg/ml).

Abies sibirica Ledeb.
Abies sibirica or Siberian Fir is native to the taiga ecosystems in Siberia, Turkestan, Xinjiang, Mongolia, and Heilongjiang. The species inhabits a cold boreal climate at elevations of 1,900-2,400 m (Katsuki et al., 2011) where average annual precipitation exceeds 600 mm (Bazhina, 2014). As one of the dominant tree species in European Russia, as well as of the Siberian taiga, it has been used in conventional and traditional medicine since ancient times (Makarova et al., 2013). Abies sibirica is recognized as a source of agents with anti-inflammatory, antimicrobial, wound healing, regenerating, and antifungal properties (Ayupova et al., 2014). Noreikaitė et al. (2017) showed that EO from A. sibirica exhibits antifungal activity against C. albicans. Truchan et al. (2019) detected the mild antibacterial effects of essential oil of this plant species against P. aeruginosa and MRSA strain, with clear inhibition zones of 9.40±0.1 mm, and 9.40±0.25 mm, respectively. The antiviral potential of polyprenols from A. sibirica is proven against the Influenza virus Safatov et al., 2000;Safatov et al., 2005). Furthermore, Sokolova et al. (2018) investigated compounds like borneol and camphor from this plant in order to design novel inhibitors for the Vaccinia virus.

Abies koreana E.H.Wilson
Abies koreana or Korean Fir is endemic species, native to the higher mountains of South Korea and occurs in habitats at 1,000 to 1,900 m of altitude, characterized by high rainfall, with cool and humid summers and heavy winter snowfall (Kim et al., 2011). Bağci and Diğrak (1996) observed the extensive antimicrobial potential of the essential oil of this plant species against different microorganisms, including Gram-positive and Gram-negative bacteria, as well as fungi. Furthermore, the antibacterial activity of A. koreana EO against several bacterial strains is reported by Jeong et al. (2007). The results obtained from the disk diffusion method indicated that tested EO exhibits a variable degree of antibacterial activity on different tested strains, with Staphylococcus epidermidis being the most susceptible strain, followed by MSSA and MRSA, Staphylococcus haemolyticus, S. simulans, and S. flexneri. Gram-negative strains displayed variable degrees of susceptibility, with the maximum activity observed against E. coli and P. aeruginosa. Oh et al. (2007) identified 47 compounds from the A. koreana essential oils, with limonene being the most abundant, followed by bornyl acetate, α-pinene, camphene, β-himachalene, β-myrcene, γ-selinene, γ-gurjunene, β-eudesmene, β-pinene, and other minor constituents. The same investigation confirmed the inhibitory activity of this EO against E. coli, S. epidermidis, and C. albicans. S. epidermidis was more sensitive in comparison to E. coli, while investigated EO led to extensive inhibition of C. albicans with the inhibition zones of 34.0±2.83 mm.

Abies balsamea (L.) Mill.
Abies balsamea, known as the Balsam Fir, is species native to most of eastern and central Canada and the northeastern USA (Farjon, 2013a). This species is shade tolerant and grow in cool climates, with a mean annual temperature of 4 °C, with consistent moisture at their roots (Walters & Reich, 2000). Pichette et al. (2006) investigated the chemical profile and antimicrobial acti-vity of A. balsamea essential oil. The analysis revealed β-pinene to be the main component, followed by δ-3carene, α-pinene, and bornyl acetate. Tested EO showed antibacterial activity against S. aureus (MIC value was determined at 56 µg/ml), while individual compounds were effective against E. coli as well. The antibacterial potential of the A. balsamea needle extract was investigated by Vandal et al. (2015), while Coté et al. (2016) studied the antibacterial effects of A. balsamea oleoresin. The latter substance was effective against S. aureus, including the MRSA strain. According to this research, oleoresin is mainly composed of monoterpenes, sesquiterpenes, and diterpenes. Resin acids, isopimaric and levopimaric acids detected in the sample are also related to the antibacterial properties of the whole oleoresin.

Abies concolor (Gordon) Lindl. ex Hildebr.
Abies concolor, commonly known as the White Fir, is species native to the mountains of western North America, with typical occurrence at elevations between 900 and 3,400 m (Farjon, 2013b). The essential oil of this species is tested by Bağci and Diğrak (1996) against various microorganisms, but antimicrobial activity was not detected. Nevertheless, the antibacterial properties of the seed and cone EO of A. concolor were observed in the study of Wajs-Bonikowska et al. (2017) against S. aureus, Enterococcus faecium, E. faecalis, E. coli, and K. pneumoniae. Obtained results showed that seed EO was more efficient in comparison to cone EO. This investigation also debates the chemical profile of the investigated EOs. As the main constituent was noted β-pinene, followed by limonene, camphene, β-phellandrene, and α-pinene.

Abies spectabilis (D.Don) Mirb. /syn. Abies webbiana (Wall ex D.Don) Lindl./
Abies spectabilis (East Himalayan Fir) is the dominant tree in the forests of the central and western Himalayas, especially from 3,000 m to 4,050 m, with occasional occurrences on ridges below this height (Thomas, 2019). The study performed by Vishnoi et al. (2007) investigated the antimicrobial activity of A. spectabilis methanolic extract in the range of 625 µg/ml to 5 mg/ ml, and obtained significant results against several bacterial and fungal species: S. aureus, S. epidermidis, M. luteus, E. coli, Salmonella typhi, Vibrio cholerae, Shigella dysenteriae, A. niger, and C. albicans. The inhibition zones were larger with the increased concentration of the extract. Constituents that are probably related to the observed antibacterial activity are abiesin, methyl betuloside, and betuloside identified in the leaves. Ambre et al. (2019) investigated compounds present in A. spectabilis chloroform leaf extract that are responsible for inhibitory ac-tivity against S. aureus. Authors identified seven metabolites, namely betuloside, 2,7-dihydroxy-4'-methoxyisoflavanone, genistein 7-Obeta-D-glucoside, β-sitosterol, abietane, coniferol, and 1-(3,4-dihydroxyphenyl)-1-decene-3,5-dione-Pos. Previous studies also recorded the antimicrobial potential of this species. Donovan et al. (2009) noted that genistein 7-O-beta-D-glucoside-isoflavone possesses antiviral activity; Ododo et al. (2016) presented an inhibitory effect of β-sitosterol against S. aureus and E. coli; González (2015) reported the antibacterial activity of diterpene abietane against S. aureus, B. subtilis, P. aeruginosa, and E. coli; Makwana et al. (2015) noted activity of monolignol coniferol on E. coli. These results are in accordance with the investigation of Timothy et al. (2021) who tested the ethanolic extract of A. spectabilis on various microorganisms: S. aureus, Streptococcus mutans, E. faecalis, and C. albicans, which exhibited very good antimicrobial properties. Furthermore, a recent study by Gautam et al. (2022) on ethanolic extract made from A. spectabilis leaves showed a wide antimicrobial range of activity, with fungal species being more susceptible to the tested compound in comparison to the bacteria. The authors stated that high phenolic content could be in relation to the described properties.

Abies holophylla Maxim.
Abies holophylla or Manchurian Fir is native to a mountainous region of northern Korea (Katsuki et al., 2013). This species has pronounced heat tolerance, and it is exceptionally winter hardy, capable of withstanding temperatures up to -34 o C. Manchurian fir grows in the mountains, but also at lower elevations and in valleys where it is exposed to hot summer temperatures (Meyer, 2010). The investigation of Lee and Hong (2009) Lee et al. (2014) confirmed the antibacterial activity of EO against K. pneumoniae, Haemophillus influenzae, S. pyogenes, Streptococcus pneumoniae, and Neisseria meningitidis. As the major constituents α-pinene, camphene, β-pinene, 3-carene, limonene, bornyl acetate, borneol, β-caryophyllene, α-caryophyllene, caryophyllene oxide, and α-bisabolol were identified.
This review showed that the most investigated products in terms of antimicrobial potential were essential oils and extracts. Additionally, the antimicrobial activity of resin, honeydew honey, and some individual chemical compounds was also investigated. Studied products for each species are presented in Figure 1. The representation in Figure 1 refers to those Abies species for which more than one study on antimicrobial activity was available.
Overall analyses revealed that different products derived from Abies species have proven inhibitory activity against 44 Gram-positive and Gram-negative species of bacteria, including some multidrug-resistant strains. Furthermore, inhibitory effects were detected against eight species of fungi and two viruses ( Table 1).
The broadest antimicrobial activity was noted in A. cilicica, with proven inhibitory action against 18 bacteria and five fungi. Very high antimicrobial properties were observed in A. spectabilis, A. alba, A. koreana, A. numidica, and A. holophylla (Graph 2).

Pseudomonas fluorescens
Since there are endemic species in the genus Abies (Xiang et al., 2018), investigations of bioactive potential should consider that fact, mostly because of the sampling behavior, but also in terms of correct understanding of generated results. This review noticed that some endemic Abies species were investigated in terms of antimicrobial potential: A. koreana (Korea Peninsula), A. cephalonica (Greece), A. firma (Japan), A. numidica (Algeria), and A. beshanzuensis (China). Furthermore, the conservation status of the reviewed species varies from least concern to critically endangered. Thus, the near-threatened Abies species that are likely to become endangered in the near future are A. holophylla, A. cilicica, and A. spectabilis; A. koreana holds endangered status, and it is characterized by a higher risk of extinction in the wild; while A. numidica and A. beshanzuensis are considered critically endangered species with the highest risk of extinction in the wild. Due to the presented issues, bioprospecting of novel antimicrobial agents from mentioned species should be rationalized, and due to the already recognized antimicrobial potential, conservational practices and in vitro elicitation of desirable chemical compounds should be implemented.