Antibacterial and fungicidal effect of ethanol extracts from Juniperus sabina , Chamaecyparis lawsoniana , Pseudotsuga menziesii and Cephalotaxus harringtonia

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Introduction
Over recent years, due to spread of antibiotic-polyresistant strains of bacteria which are poorly susceptible to treatment, more and more often reports emerge, describing the potential of the search for efficient antibacterial substances in ethanol plant extracts (Boyko et al., 2016;Zazharskyi et al., 2019a, b;Palchykov et al., 2019Palchykov et al., , 2020. Potentially, a subject of significant interest in this aspect is various groups of gymnosperms used for the greening of settlements. Cephalotaxus harringtonii (Forbes) K. Kokh known as Japanese plum-yaw is an evergreen coniferous shrub or small tree from the Taxaceae family. Scioheliophyte, eutrophic, mesohyte. Quite winter-hardy (second category), quite drought tolerant (second category) species. Needs protection against wind over winter. The plants grow in deep rich soils. C. harringtonii can be used in group plantations in parks. The species is native to Japan, but sometimes is used in Western couuntires. Several forms are grown for this purpose. In Japan, C. harringtonii grows from Kyuschu in the south to Hokkaido in the north. Particularly, the species is found in Hondo in Chiba Prefecture on Kiyosumi Mountain located in Awa district of Awa province. It is also common in Nagasaki and Hiroshima Prefectures. The nana variety occurs in the eastern part of Honshu, and also Hokkaido, especially on coastal rocks and highland areas (Tripp, 1995). In Europe, C. harringtonii has been cultivated since 1829. Many gardeners are familiar with this species named after Charles Harrington, the fourth duke Harrington, one of the first who grew the plant in a European garden in Elvaston. Omacetaxine, a substance obtained from the leaves of the plant is a new preparation aginst leukemia.
Pseudotsuga menziesii (Mirbel) Franco -Douglas fir is a tall coniferous tree from the Pinaceae family, which relates to the first and the second layers of forests. Also known as Douglas fir, Douglas pine, Oregon pine, and Columbian pine. Evergeen, phytoncidal, decorative fruit plant. Heliophyte, xeromesophyte, neutrophil, mesotrophic-eutrophic. Prefers acidic and neutral soils. Winter-hardy (second category), drought-tolerant (first category) plant. Grows better in well-drained, neutral loams. For greening it is used in parks, garden squares, embankments, more rarely in the streets. It is native to the western part of North America. Morphologically, P. menziesii is flexible, has many forms. P. menziesii var. menziesii forms a deeper root system. P. menziesii var. glauca manifests more flexibility, occurring in relic forests of British Columbia (Canada); in semi-arid vermouth steppe in the greater part of its range, forms the deepest roots. P. menziesii contains poriolflavanone (one of the flavonoids) produced in response to Poria weirii (Barton) infection. Different groups of Indians used bark, resin and needles of this plant for treating various diseases.
Chamaecyparis lawsoniana (A. Murray) Parl. is a tree of the second layer, belonging to the Curpessaceae family. Evergreen, phytoncidal, quite winter-hardy (second category) and drought-hardy (second category) plant. Mesophyte, but badly widstands air dryness, heliophyte, mesotrophic. The plant is not demanding in soil fertility requirements, but prefers light (light-loamy and sabulous) conditions. Ch. lawsoniana grows better in places sufficiently protected from wind and with good exposure to sunlight. It grows well in urban conditions; its blue-grey and blue forms are the most common. The species is characteristic for Oregon and North-West California. It grows at the height of 1,500 m above the sea level in the valleys of the Klamath Mountains, along streams. The species is very sensitive to infection by Phytophthora lateralis Tucker & Milbrath.
Juniperus sabina L. or savin juniper, savin juniper of the Cupressaceae family is a dioecious, humifuse shrub 1.0-1.5 m high. It rapidly grows in width and forms dense thickets. More rarely, small trees 4 m in height with curved trunks are seen. The prostrate or shrub-like tree form occurs in many cities of Europe. Evergreen, phytoncidal, ground cover, soil protective, winter-hardy (first category) and drought-hardy (first category) species. Heliophyte, mesophyte, xeromesophyte. It is widely used for decorating rocky hills, slopes, in singular or group plantations on lawns or forest edges. Smoke and gas-resistant. Resilient in urban conditions, can be used for greening streets, except in industrial areas. Light-loving, undemanding in soil requirements, has soil-protecting abilities. Forests and groves of J. sabina are common in the steppe zone, on rocky mountain slopes and sandy dunes; sometimes the plant extends from the lower to the higher mountain belt (1,000-2,300 m above the sea level), where it forms thickets. The range includes Asia Minor, the Caucasus, Russia (Urals, Siberia and Primorje), South-East Asia, South and Central Europe. J. sabina grows in very rare relic communities, and has a limited distribution, being at the verge of extinction (García-Cervigón et al., 2018;Lambevska-Hristova & Bancheva, 2019). Shoots contain essential oil and are poisonous Batsatsashvili et al., 2017). Shoots and juniper berries contain glycosides, saponins, flavonids, tannins and up to 17% sabinol, which causes severe poisoning and miscarriages in cattle. Toxic properties of juniper sabina limit its application in therapeutic use. Most often, in folk medicine it is used as an external preparation. Ointment is rubed into the roots of hair in treatment of alopecia, applied to body areas affected by scabies, tincture is used against warts. Powder is sprinkled over pus ulcer. Essention from fresh branches with leaves is used in homeopathic treatment of diseases of the kidneys and the bladder, stranguria, gout, painful menstruations and impaired monthly cycle (Gubanov et al., 1976). The oils of this plant were dominated by α-pinene, sabinene, and cedrol with moderate amounts of limonene, terpinen-4-ol, and elemol (Adams et al., 1998). Alongside some well-known compounds from acidic fraction of the extract of n-Hexane from leaves of J. sabina,  isolated two new lignans, derivative of naphthalene called junaphtoic acid and 3-O-demethylyatein. While studying phylogeographic peculiarities of J. sabina, Guo et al. (2010) presumed absence of regional interpopulational differentiation.
These species of plants remain poorly studied in relation to antimicrobial activity and can have a significant potential in human and veterinary medicine and. The objective of the article was determining the antibacterial effect of ethanol extracts of J. sabina, Ch. lawsoniana, P. menziesii and C. harringtonia on some species of microorganisms of the families Enterobacteriaceae, Staphylococcaceae, Yersiniaceae, Bacillaceae, Listeriaceae, Corynebacteriaceae, Campylobacteraceae, Nocardiaceae, Pseudomonadaceae and Saccharomycetaceae in vitro.

Materials and methods
Leaves and shoots of four species of coniferous plants were collected in the territory of Botanical Garden of Oles Honchar Dnipro National University (Khromykh et al., 2018;, dried at room temperature, cut, weighed and kept 24 h in 70% ethyl alcohol, filtrated. Antibacterial activity of the plant tinctures were identified using disk-diffusion in agar. Out of the 24 h culture of the ethanol strains of microorganisms of the family, a weighed amount was prepared according to the standard of turbidity of bacterial suspension equaling 0.5 units of density according to McFarland (McF) 1.5 × 10 8 CFU (colony-forming units), which was determined using a densitometer (Densimeter II). The obtained weighed amount was re-inoculated to Muller-Hinton agar (Himedia) followed by cultivation in a thermostat TSO-80/1 over 24 h at the temperature of 37 °С. On top of the re-inoculations, the disks saturated with tinctures of the extracted ethanol tinctures of the four species of plants were placed (Table 1).

Table 1
Used part of the four species of plants and the most important information on their antibacterial activity Cephalotaxus harringtonii (Forbes) K. Koch leaves Watanabe & Fukao (2009) As a positive control we used disks with 15.0 µg of azithromycinmacrolid antibiotic of broad spectrum. Discs with 15.0 µg amphotericinin were also used as a second control against Candida albicans. After 24 h growth of the culture was assessed using a multi-angle ruler for measuring growth inhibition zones in microorganisms (Antibiotic Zone Scale-C, model РW297, India) and the program TpsDig2 (2016, F. James Rohlf). The data in tables are presented as x ± SD (standard deviation).

Results
We observed inhibition of growth of separate strains of microorganisms exposed to ethanol extracts of the studied species of plants (Table 2). Extract from J. sabina moderately inhibited growth of E. coli (4.5 and 4.8 mm, hereafter average radius of the growth inhibition zone is indicated in millimeters), E. faecalis (4.3), poorly inhibited growth of S. typhimurium (3.4), S. adobraco (2.7), P. vulgaris (2.8), P. mirabilis (2.1), Y. enterocolitica (2.5), S. marcescens (1.2), K. pneumoniae (2.5). Ethanol extract of Ch. lawsoniana had more notable antibacterial action towards E. coli (8.8 and 9.3) and P. vulgaris (5.1), compared with J. sabina. Extract from P. menziesii was more efficient against P. mirabilis (10.5) compared with the three other plant preparations and the control. Ethanol extract of C. harringtonia had high inhibiting effect against strain of E. coli 055 (10.7), and also P. vulgaris (8.3) and S. marcescens (10.4). During the study on the effect of the preparations of plants against bacteria of the Staphylococcaceae family, we determined an intense antibacterial effect towards them demonstrated by ethanol extracts from C. harringtonia and J. sabina. We found significant inhi-biting effect of the extracts from J. sabina, Ch. lawsoniana and C. harringtonia on the microorganisms of the Bacillaceae family: B. cereus -14.2, 1.6 and 5.7, B. subtilis -8.4, 10.6 and 0.0 mm, respectively. We determined high sensitivity of L. ivanovi and L. monocytogenes to C. harringtonia (9.3 and 8.1), L. ivanovi to C. harringtonia (9.3), L. іnnocua to J. sabina and Ch. lawsoniana (12.4 and 10.6, respectively). Against C. xerosis, we observed growth inhibition zone of 6.4 and 6.8 mm produced by alcohol extracts of Ch. lawsoniana and P. menziesii and 10.6 and 9.7 mm by J. sabina and Ch. lawsoniana (9.3 mm during impact of azithromycin in the control). Bacteria of C. jejuni were highly susceptible only to ethanol extract of C. harringtonia (11.4). High antibacterial effect of the extracts from J. sabina, Ch. lawsoniana and P. menziesii was exerted against Rhodococcus equi (27.5, 14.3 and 8.7) during moderate exposure to ethanol extract of C. harringtonia (4.4). Bacteria of P. аeruginosa were highly sensitive to ethanol extract from J. sabina (10.5), moderately sensitive to Ch. lawsoniana (6.4 mm). We should note antibiotic resistance of the studied strains of L. monocytogenes, C. jejuni, P. vulgaris, P. mirabilis, P. аeruginosa, K. pneumoniae and S. marcescens to azithromycin. Extract from C. harringtonia exerted high inhibiting action towards C. albicans (9.8). Compared to amphotericin, higher growth inhibition zones were also produced by ethanol extracts from Ch. lawsoniana and P. menziesii (3.7 and 3.4 mm, respectively).

Discussion
Research has been conducted on species of gymnosperm plants, which apart from antibacterial and antifungicidial activity are also charac-terized by cytostatic properties against different types of the tumour tissues. Shokrzadeh et al. (2009), Janar et al. (2012 and Huyan et al. (2016) noted moderate inhibiting activity of diterpenoids of J. sabina towards five species of human tumour cells. Zhao et al. (2015) determined anti-inflammatory effect of shoots and leaves of J. sabina due to flavone glycoside isoscutellarein 7-O-β-D-rhamnopyranosyl -(1→3)-α-L-xylopyranoside. Analgesic and anti-inflammatory effects of general flavanoids from leaves of J. sabina have been proven on rodents (Zhao et al., 2018). Abdel-Kader et al. (2019) determined the hepatoprotective activity of J. sabina, which was assessed by biochemical parameters of blood serum of rats, such as aspartate-aminotransferase (AST), alanine aminotrasferase (ALT), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP) and total bilirubin. Khani et al. (2017) and Elisoveckaya & Brindza (2018) determined that essential oil from J. sabina has insecticidal, antifeedant, deterrent and repellent properties. Results of the studies by Živić et al. (2019) and Semerdjieva et al. (2019) allow one to state that ethanol extract of J. communis has a high antioxidant activity due to the content of total phenol compounds and total amount of flavonoids. 10.5 ± 0.98 0 ± 0 0 ± 0 0 ± 0 0 ± 0 P. аeruginosa ATCC 2799 0 ± 0 6.4 ± 0.78 0 ± 0 0 ± 0 0 ± 0 Klebsiella pneumoniae АТСС 13883 2.5 ± 0.21 1.7 ± 0.12 0 ± 0 0 ± 0 0 ± 0 Yersinia enterocolitica АТСС 9610 2.5 ± 0.22 0 ± 0 0 ± 0 0 ± 0 12.8 ± 1.27 Serratia marcescens АТСС 8100 1.2 ± 0.23 0 ± 0 2.8 ± 0.23 10.4 ± 0.94 0 ± 0 Candida albicans АТСС 2091 2.2 ± 0.41 3.7 ± 0.34 3.4 ± 0.31 9.8 ± 0.92 0 ± 0 * 2.4 ± 0.21 ** Note: * -discs with 15.0 μg of azithromycin were used for all bacteria as positive control; ** -discs with 15.0 μg amphotericinin were used as positive control for Candida albicans (Valle et al., 2015). Palá-Paúl et al. (2015) researched Ch. lawsoniana. They showed that the essential oils from young shoots and leaves of Ch. lawsoniana exert high antibacterial and antifungicidal activity towards Candida albicans, Bacillus subtilis, Staphylococcus aureus and Micrococcus luteus: essential oil was more effective against gram-positive rather than gram-negative bacteria. Recently, interest has developed to the use of essential oils from shoots of Ch. lawsoniana in commercial products, such as preparations against pests and cosmetics (Duringer et al., 2015). To assess the relative toxicological risk from these oils for fresh-water and marine organisms, acute aquatic toxicity of these oils was assessed using the planktonic crustacean Daphnia magna Straus, rainbow trout Oncorhynchus mykiss (Walbaum) and microalga Raphidocelis subcapitata (Korshikov) Nygaard, Komárek, J. Kristiansen & O. M. Skulberg. For the oils from leaves of Ch. lawsoniana, toxicity against D. magna or O. mykiss was not observed even in 5.0 mg/L (the highest tested concentration and the threshold of solubility). Research on toxicity against R. subcapitata using density of cells of algae showed that the value of EC 50 over 72 and 96 h equaled 1.7 mg/L, and the concentration without observed effect (NOEC) was 0.63 mg/L. Forty-eight h EC 50 for D. magna equaled 1.9 mg/L; value of NOEC for density of the cells of algae equaled 1.25 (72 h) and 0.63 mg/L (96 h). The emergence of antibiotic-resistant bacteria has caused difficulties in the treatment of infectious diseases (Kim et al., 2015). Methicillin-resistant Staphylococcus aureus is one of the commonest recognized antibiotic-resistant bacteria. New antibiotics for the treatment of MRSA in humans are urgently needed. Raw materials obtained from natural sources can be used for the development of new antibiotics, for example Chamaecyparis obtusa (Siebold & Zucc.) Endl., which is traditionally used for treating asthma. Kim et al. (2015) studied antibacterial activity of essential oil from leaves of Ch. obtusa against MRSA. Growth of MRSA and production of acid during metabolism of glucose was inhibited in Staphylococcus by the concentration of 0.1 mg/mL of Ch. obtusa. Smith et al. (2007) showed that within the framework of a project aimed at characterizing the antibacterial or immune-modulating activity of compounds of immature conifer cones against multi-drug resistance (MDR) strains of S. aureus, eight compounds were extracted from Ch. lawsoniana. Active compounds are mostly diterpenes with minimum inhibitory concentrations equaling 4-128 µg/mL against strains of S. aureus which are resistant to methicillin (EMRSA). Out of Ch. lawsoniana, diterpene ferruginol, pisiferol and its epimer 5-epipisiferol, formazan oxide, transcommunic acid and torulosal, sesquiterpenic oplopanonil acetate and germacrane 4β-hydroxigermacra-1(10)-5-dien were obtained. Some of these compounds also exerted modulating activity in potentiation of antibiotic activity against efflux strains, and ferruginol used in sub-inhibiting concentration caused 80-fold increase in the activity of oxacillin against strain of EMRSA-15. Yang et al. (2007) determined that essential oil from leaves of Ch. obtusa has relatively strong antibacterial activity against gram-positive bacteria and some fungi. Massicotte et al. (1992) determined that tubers on the roots of P. menziesii are present as ectomycorrhizal formations. Spectroscopy showed that the crystals found in the zone of loose hyphae of fungi perhaps contain calcium oxalate. Bacteria locate either in the hyphae of fungi in the bark of roots, or as colonies on the surface. These results allow us to assume that the roots of plants can be a valuable resource, because they contain a large amount of anti-fungi and allelopathc compounds (Massicotte et al., 1992). Palá-Paúl et al. (2015) report new data on antibacterial and antifungal activity of essential oil from Ch. lawsoniana. Leaves of juniper are practically safe for aquatic organisms (Duringer et al., 2015). Cedar oil did not exhibit toxicity against Oncorhynchus mykiss (Walbaum) or Selenastrum capricornutum Printz even in 0.5 mg/L (the highest tested concentration and threshold of solubility). Kim (2015) determined formation of biofilm of MRSA using a scanning microscopy and staining with с safranin. Chamaecyparis obtusa (Siebold & Zucc.) Endl. Inhibited the formation of biofilm of MRSA in concentrations of over 0.1 mg/mL. These data indicate that Ch. obtusa has antibacterial effect against MRSA, which could be associated with such components of the plant tissues as sabinene (19.1%), α-terpinyl acetate (17.0%), bornyl acetate (10.5%), limonene (8.5%), elemol (7.5%), myrcene (5.9%), γterpinene (4,0%) and hibaene (3.0%). Lee (2009) and Morikawa et al. (2012)  Watanabe & Fukao (2009) determined that C. harringtonia exerted a broad anti-tumour activity in rodents and anti-leukemia effect in people. They discovered that the substances from the plants are metabolized to the acidic product HHT-acid (2'-hydroxy-2'-(α-acetic acid)-6'hydroxy-6'-methylheptanoil cephalotaxine) during in vitro incubation with human or mice blood plasma. Concentration of HHT which inhibited by 50% growth of human leukemia cells HL-60 was 20 ng/mL, whereas for HHT-acid it equaled 14,500 ng/mL (acidic form was more than 700 times less cytotoxic than HHT). LD 50 for HHT equaled 6.7 mg/kg, at the same time HHT-acid exhibited no notable toxic effects even in the doses of 280 mg/kg.
Extract from immature fruits of C. harringtonia is most efficient in inhibiting the growth of Bacillus cereus and Leuconostoc mesenteroides (Tsenkovskii) van Tieghem, its antibacterial activity was tested against 22 species of gram-positive, 7 species of gram-negative bacteria and 13 species of fungi. Extract from C. harringtonia exerted antibacterial activity towards gram-positive bacteria with minimum inhibiting concentrations (MIC) of 25-200 µg/mL in agar broth and 5-40 µg/mL in liquid broth, antimicrobial effect was higher in acidic and alkaline media than in neutral conditions (Watanabe & Fukao, 2009).
From leaves of C. harringtonia f. fastigiata, Morita et al. (2000Morita et al. ( , 2010 a new alkaloid cephastigiamide A was extracted and its structure was studied using two-dimensional NMR spectroscopy and chemical degradation. Harringtonin, desoxyharringtonin and homodesoxyharringtonin demonstrated notable antiplasmatic action towards Plasmodium falciparum Welch, but not towards Leishmania major Yakimoff et Schokhor. Six new alkaloids (cephalezomines A-F) were isolated together with ten already known alkaloids from leaves of C. harringtonia var. nana; cephalezomines A-F displayed potent cytotoxicity against tumour cells (Morita et al., 2010(Morita et al., , 2012. Takano et al. (2010) and Dwivedi et al. (2015) focused on infections associated with biomaterials. Microbial adhesion on implants caused formation of biofilm, which ultimately led to damage to the implants. Therefore, for effective treatment against biofilms of S. aureus, which are responsible for most infections associated with biomaterials, these authors propose using silver nanoparticles and extracts from leaves of Pseudotsuga menziesii.
The antibacterial in vitro effect which we observed from J. sabina exceeded a 8 mm wide zone of inhibition of growth of 7 species of bacteria (S. aureus, B subtilis, B. cereus, L. іnnocua, C. xerosis, Rh. equi and P. аeruginosa), Ch. lawsoniana had the same effect against 5 species (E. coli, B. subtilis, L. іnnocua and Rh. equi), P. menziesii -only against 2 species (Rh. equi and P. mirabilis) and C. harringtoniaagainst 10 species of microorganisms (E. coli, S. aureus, S. epidermidis, L. ivanovi, L. monocytogenes, C. xerosis, C. jejuni, P. vulgaris, S. marcescens and C. albicans). Further detailed study of individual chemical substances present in those plants should be undertaken in both in vitro and in vivo experiments.

Conclusion
None of the four species of gymnosperms produced notable (growth inhibition zone of over 8 mm) effect towards E. faecalis, E. aerogenеs, S. typhimurium, S. adobraco, K. pneumoniae and Y. enterocolitica. Negative effect (growth inhibition zone of over 8 mm) on E. coli was exerted by alcohol extracts of Ch. lawsoniana and C. harringtonia, extracts of J. sabina and C. harringtonia took effect on S. aureus, only C. harringtonia affected S. epidermidis, against B. subtilis -J. sabina and Ch. lawsoniana were effective, against B. cereus -only J. sabina, against L. ivanovi -only C. harringtonia, against L. іnnocua -J. sabina and Ch. lawsoniana, against L. monocytogenes -only C. harringtonia, against C. xerosis -J. sabina and C. harringtonia, against C. jejunionly C. harringtonia, against Rh. equi -J. sabina, Ch. lawsoniana and P. menziesii, against P. vulgaris -only C. harringtonia, against P. mirabilis -only P. menziesii, against P. аeruginosa -only J. sabina, against S. marcescens -only C. harringtonia, and against C. albicans only alcohol extract of C. harringtonia had negative effect. We consider that it is practical to recommend ethanol extracts from J. sabina, Ch. lawsoniana, P. menziesii and C. harringtonia, or individual compounds present in them, to be used in further studies on treatment against polyresistant strains of the abovementioned microorganisms.