Antibacterial and fungicidal activities of ethanol extracts of 38 species of plants

Galenic preparations are broadly used against microorganisms pathogenic to humans, thought their poteintial in this aspect is not studied completely. In our in vitro experiment we studied the influence of alcohol tinctures from 38 species of plants on 15 species of bacteria and one species of fungus. Zones of growth inhibition of colonies measuring over 8 mm were observed during the use of ethanol extracts of Maclura pomifera against eight species of microorganisms (Escherichia сoli, Proteus mirabilis, Serratia marcescens, Yersinia enterocolitica, Salmonella typhimurium, Rhodococcus equi, Campylobacter jejuni and Corynebacterium xerosis), Ginkgo biloba – against eight species (Enterococcus faecalis, S. marcescens, Y. enterocolitica, Klebsiella pneumoniae, Listeria іnnocua, L. monocytogenes, Р. аeruginosa and C. jejuni), Genista tinctoria – against seven species (E. coli, Enterobacter aerogenes, Proteus mirabilis, K. pneumoniae, S. typhimurium, Р. аeruginosa and Rh. equi), Phellodendron amurense – against seven species (E. faecalis, S. marcescens, S. typhimurium, Rh. equi, C. jejunі, C. xerosis and Candida albicans), Berberis vulgaris – against seven species (P. mirabilis, S. marcescens, K. pneumoniae, S. typhimurium, C. jejuni, Р. аeruginosa and C. xerosis), Vitex negundo – against six species (E. faecalis, E. coli, P. mirabilis, K. pneumoniae, S. typhimurium and Rh. equi), Koelreuteria paniculata – against six species (E. faecalis, P. mirabilis, S. marcescens, S. typhimurium, C. jejunі and E. coli), Magnolia kobus – against six species (E. faecalis, E. coli, P. mirabilis, S. marcescens, S. typhimurium, C. jejunі and C. xerosis), Liriodendron tulipifera – against six species (K. pneumoniae, Listeria іnnocua, Р. аeruginosa, C. jejuni, Rh. equi and C. albicans), Clematis flammula – against six species (E. faecalis, P. mirabilis, L. monocytogenes, Р. аeruginosa, C. jejuni and C. xerosis), Wisteria sinensis – against five species (E. coli, S. typhimurium, L. monocytogenes, Rh. equi and C. albicans), Chimonanthus praecox – against five species (E. faecalis, S. marcescens, L. monocytogenes, C. jejuni and Rh. equi), Colchicum autumnale – against five species (S. marcescens, K. pneumoniae, L. ivanovi, L. monocytogenes and Р. аeruginosa). As a result of the study, these plants were found to be the most promising for further study of in vivo antibacterial activity. In the search of antibacterial and antifungal activities, the following plants were observed to be less promising: Ailanthus altissima, Aristolochia manshuriensis, Artemisia absinthium, Callicarpa bodinieri, Campsis radicans, Catalpa duclouxii, Celastrus scandens, Dictamnus alba, Eucommia ulmoides, Geranium sanguineum, Laburnum anagyroides, Nepeta racemosa, Parthenocissus tricuspidata, Polygonatum multiflorum, Prunus dulcis, P. laurocerasus, Ptelea trifoliata, Pteridium aquilinum, Quercus castaneifolia, Q. petraea iberica, Salvia officinalis, Securigera varia, Styphnolobium japonicum, Tamarix elongata and Vitex agnus-castus.


Introduction
During recent years there have been reports from all over the globe about resistance to medicine of disease-causing bacteria of human and animals. Emergence of resistance to antibiotics in bacteria is a global problem (Lopes et al., 2018;Zhang et al., 2019). Antibiotic-resistance is now one of the most serious threats to the health of people (Steinberg et al., 2017;Tumen et al., 2018;Khan et al., 2019). Our civilization is approaching the period when antibiotics will be unable to control the courses of common infections, and small traumas could once more lead to people dying (Islam et al., 2019). Antibiotics have allowed humans to live longer and be healthier. Resistance to preparations for treatment of a common intestinal bacterium Klebsiella pneumonia (carbapenems) occurs more and more frequently. K. pneumonia can cause various nosocomial infections (pneumonia, infections of blood, infections among newborns, etc.). Resistance to fluoroquinolones used for treatment of urinary tract infections caused by E. coli has also become widely distributed. In the 1980s, when these preparations were first used, resistance to them was practically absent. Currently, in many countries this treatment is ineffective for over 50% of patients. Cases of no effect on gonorrhea treatment with reserve antibiotics -cephalosporins of the third generation -have been confirmed in many countries of the EU, Australia, Canada, South Africa and Japan. Salević et al. (2019) report that probability of death of humans infected with MRSA (methicillin-resistant Staphylococcus aureus) is 64% higher compared with people with medically-non-resistant form of this infection. Resistance to antibiotics among microorganisms also leads to increase in costs for medical services due to the longer period of stay in hospitals. Therefore, there is a necessity of developing alternative antimicrobial preparations for treatment of infectious diseases. Rates of development of new antimicrobial preparations should exceed the rates of development of resistance among microorganisms to currently in-use antibiotics.
In this article we continue to study antibacterial preparations in plant extracts due to spread of antibiotic poly-resistant bacterial strains which are hard to treat (Zazharskyi et al., 2019;Palchykov et al., 2020). Plants produce various secondary metabolites with different biological activity. For galenic preparations of some species of plants we have already found anti-parasitic and antimicrobial activities (Boyko & Brygadyrenko, 2016a;Palchykov et al., 2019;Zazharskyi et al., 2019b).
The objective of this article was determining antibacterial effects of 38 ethanol extracts on 16 species of microorganisms. Up to now, these species of plants have remained poorly studied with respect to antimicrobial activity and can have significant potential in contemporary human and veterinary medicine.

Material and methods
Leaves and shoots of 38 species of plants were collected in the territory of the Botanical Garden of Oles Honchar Dnipo National University (Khromykh et al., 2018;Boyko & Brygadyrenko, 2019), dried at room temperature, fragmented, weighed and kept in 70% ethyl alcohol for 10 days, and then filtered. We took 10 grams of dry fragmented plants per 100 g of 70% ethyl alcohol. Then, 0.1 mL of this filtered alcohol extract was transferred onto one paper disk of 6 mm diameter. The disks were dried in sterile conditions in the temperature of 10 ºС in a microbiological safety cabinet HR1200-IIA2-D (China).
Antibacterial activity of plant tinctures was determined using the disk diffusion method in agar. Out of the daily culture of ethanol strains of microorganisms we prepared a weighed amount according to the standard of opacity of bacterial suspension equaling 0.5 units of density according to McFarland (McF) 1.5 х 10 8 CFU (colony-forming units), which we determined using a densitometer (Densimeter II, Table 1).
The obtained weighed amount was transferred into Muller-Hinton agar (Himedia) with subsequent cultivation in a ТСО-80/1 thermostat for 24 h at the temperature of 37 ºС. On top of the inoculations, we put disks (n = 8) saturated with corresponding ethanol tinctures of 38 species of plants (Table 2).  As a positive control we used disks with 15 µg of azithromycin -broadspectrum macrolide (Valle et al., 2015). Disck with 15.0 µg amphotericinin were also used as a second control against Candida albicans. Twenty four hours later the growth of the culture was measured using a zone scale for reading the sizes of growth inhibition zones of microorganisms (Antibiotic Zone Scale-C, model РW297, India) and software TpsDig2 (2016, F. James Rohlf). The data in the tables are presented as x ± SD (standart deviation).

Results
Growth of separate strains of microorganisms of Enterococcaceae, Enterobacteriaceae, Morganellaceae and Yersiniaceae families was arrested by ethanol extracts of the species of plants we studied (Table 3, 4). We observed inhibition of growth of E. faecalis by Ginkgo biloba (18.4 mm, hereinafter the average radius of growth inhibition zone is given in mm). Slightly lower than Ginkgo biloba, but still high antibacterial effects were exhibited by Pteridium aquilinum (14.5), Polygonatum multiflorum (12.4), Clematis flammula (10.6), Magnolia kobus (10.4), Prunus laurocerasus (10.2), Vitex negundo (10.2). Moderate inhibition of growth of the colonies of E. faecalis was seen under the influence of Dictamnus albus (9.8), Koelreuteria paniculata (8.4), Chimonanthus praecox (8.2), Celastrus scandens (6.7) and Callicarpa bodinieri (6.2). Bacteria of E. faecalis were resistant to the influence of alcohol extracts of the rest of the species of plants we studied.
Interesting results were obtained for use of ethanol extracts against microorganisms of the Listeriaceae family (Table 5). While L. monocytogenes was highly susceptible to 8 plants (Ptelea trifoliata

3). Ethanol extract of
Ginkgo biloba exceeded the other tested plants regarding the width of growth inhibition zone of P. aeruginosa by 1.3-2.1 times. We determined tolerance of the tested strains of L. monocytogenes and P. aeruginosa strains to the action of azithromycin.

Discussion
Substances produced by Embryophyta as secondary metabolites were found to be biologically quite active compounds against microorganisms pathogenic for humans and agricultural animals. Some of the plants we studied may become the basis for the development of new pharmaceutical preparations (Zazharskyi et al., 2019c). Kavitha & Nelson (2016) consider that chloroform extract of leaves of Aristolochia manshuriensis will become the alternative for the treatment of threat of pathogenic organisms. The authors found twenty bioactive constituents and functional groups associated with ethanol, carbonic acid, alkanes, aldehides, aroma acids present in chloroform extract of leaves of A. manshuriensis. The studied extract inhibited Vibrio harveyi, V. vulnificus and Serratia marcescens. Hydroethanol extract from Celastrus scandens L., against the background of high antioxidant activity (2,2-diphenyl-1-picrylhydrazyl, chelation of metals, capabilities to restore three-valent iron in plasm, superoxide radical and nitrogen oxide), good anti-inflammatory activity, displayed low antibacterial and antifungal properties (Kumar & Sharma, 2018).
Artemisia absinthium L. are perennial plants with ubiquitous distribution in deserts and dry places of Eurasia, usually growing on slopes of hills, sides of the roads and fields. It is native to Europe, North Asia and North Africa. The plant can contain toxic substances (thujon for example) responsible for side effects. Absinthe is used in phytotherapy due to its tonic, spasmolytic, antipyretic and anthelmintic properties. Obistioiu & Chiurciu (2014) and Al-Ghamdi (2020) report fungicidal effect of A. absinthium.
Fungicidal activity of essential oil from Chimonanthus praecox L. was observed towards eight phytopathogenic fungi, the inhibiting power measuring 8-32 µg/mL (Gui & Qin, 2014). Adami & Naderi (2015) think that the most important compound in the plant Colchicum autumnale L. is colchicine alkaloid, though its antimicrobial activity is studied poorly.
Ethanolic and petroleum extracts of Pteridium aquilinum (L.) exhibit antibacterial properties (Kardong & Saikia, 2013) against four species of tested bacteria (B. subtilis, S. aureus, P. vulgaris and E. coli), producing inhibition zones ranging 16-20 mm. Bacteria of P. aeruginosa were resistant to extracts of this species (Kardong & Saikia, 2013). However, extracts prepared in methanol, chloroform and distilled water showed no inhibiting activity against all the tested organisms. The observed difference in antibacterial activity while using various methods of extraction may be explained by incomplete transition of active substances into solution in the temperature of environment and loss of active components during boiling (Kardong & Saikia, 2013).
Antimicrobial peptides arrest the growth of bacteria, fungi, plant pathogens and even viruses. They have a powerful pharmaceutical effect. Bark of Eucommia ulmoides Oliv. is used in traditional Chinese medicine. Peptide present in E. ulmoides had in vitro inhibiting effect (Liu & Han, 2007) on Candida аlbicans (MIC = 156 µg/mL). Liu & Han (2007) consider that this plant can be used as a new antibiotic of plant origin for pre-vention of candidosis. Screening of bioactive secondary metabolites demonstrated that roots of E. ulmoides contain 7 compounds, one of them being gliotoxin. Its activity was close to the activity of gentamicin antibiotic, and stronger that the activity of nystatin antifungal preparation (Zhang & An, 2019).
Extracts of Genista tinctoria contributed to proliferation of probiotic strains and increased the number of bacterial colonies of Bifidobacterium animalis subsp. lactis, B. longum and Lactobacillus casei (Skenderidis & Giavasis, 2019). Prebiotic effect correlates with the concentration of polysaccharides and polyphenols of G. tinctoria, the content of which can increase the stress-tolerance of B. lactis and B. longum in a modelled gastrointestinal environment. Skenderidis & Giavasis (2019) consider that encapsulated extracts from G. tinctoria could be used as prebiotic supplements for food products for stimulation of growth and increase in vitality of probiotic strains of Bifidobacterium and Lactobacillus.
Oil extract from Geranium sanguineum L. exhibited antibacterial effects against one standard strain of S. aureus ATCC 433000 and seventy clinical strains of S. aureus, including strains with multi-drug resistance (Bigos & Sienkiewicz, 2012). Wafa & Ouarda (2017) report high antimicrobial activities of methanol extracts of G. sanguineum against S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853, B. subtilis ATCC 6633 and C. аlbicans ATCC1024, while having moderate anti-inflammatory effect.
Plant extract of Vitex negundo L. was the most effective (Padder & Ganaie, 2015) both against Streptococcus mutans (MIC -minimum inhibitory concentration = 0.37 µg/mL) and P. aeruginosa (MIC = 0.75 µg/mL). Deogade et al. (2016) determined antibacterial activity of ethanol extract from leaves of V. negundo towards bacteria Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae, producing maximum inhibition zone of S. aureus (15 mm at the concentration of 80-100 mg/mL) and minimum ones of E. coli and K. pneumoniae (12 and 11 mm at the concentration of 100 mg/mL, respectively). The notable inhibiting activity of extract of this plant was due to its high content of phenols and flavonoids (Prashith & Raghavendra, 2014). Extracts and secondary metabolites of V. negundo, especially from the roots and leaves, have useful pharmacological properties: anti-inflammatory, anti-tumour, antioxidant and antimicrobial (Tan et al., 2017;Khan et al., 2019). Use of V. negundo may be promising for treatment of skin infections caused by Staphylococcus aureus (Triveni & Gaddad, 2016). Synthesis of nanoparticles of silver through self-restoration of silver nitrate by extracts of leaves of V. negundo is one of the new methods applied in the development of technologies for creation of nanoparticles (Bhavani & Geetha, 2013). Silver nanoparticles (56 nm) exhibited antimicrobial activity against E. coli and K. pneumonia. Essential oil from seeds of V. negundo (Ai, 2014) had significant antifungal impact on Candida albicans (MIC = 4.0 µg/mL). Moreover, this extract had hepatoprotectory properties, which could be associated with its antioxidant activity, and also protective effect against heightened level of lipids (Sharma & Suri, 2016).
Alcohol extracts from the pith of Liriodendron tulipifera L. showed antimicrobial activity towards S. aureus, Mycobacterium smegmatis, Candida albicans and Aspergillus niger. Hufford & Robertson (1975) attribute it to alkaloid fraction of dehydro glaucine and liriodenine as active components. Mechanisms of antimicrobial activity of extract from Magnolia kobus DC. on S. aureus were studied using light microscopy, transmission electronic microscopy and scanning electron microscopy. After 48 h of exposure to the extract, many cells of S. aureus completely decomposed (Hu & Ge, 2011). Methylene chloride extract from fruits of Maclura pomifera (Raf.) Schneid exerted strong in vitro antimicrobial and anti-Leishmania activities. Fractioning of this extract based on the activity led to production of isoflavons (osajin and pomiferin) as active compounds which demonstrated high activity against Cryptococcus neoformans, Staphylococcus aureus and Leishmania donovani (Dharmaratne & Nanayakkara, 2013). Lectin from M. pomifera in a specific way agglutinated bacterial suspensions of various strains of highly-pathogenic bacteria of Salmonella genus (Allen, 1985).
Methanol extracts of leaves and bark of Clematis flammula L. exhibited a broad spectrum of antibacterial activity due to fraction of ethylacetate (Khan & Omoloso, 2001). Non-filtered extracts of young shoots of the closely related plant Clematis vitalba L. were highly-active against pathogenic yeasts and yeast-like microorganisms (MIC = 1.4-12.3 µg/mL). After fractioning with petroleum alcohol, ethylacetate and methanol, antifungal activity was observed only in methanol fractions (Buzzini & Pieroni, 2003). Thebo (2014) surveyed the extract of the shell of Prunus dulcis (Mill.) in its biomedical aspects: antifungal activity of extract of almond shell was observed against the clinically isolated pathogenic fungus Tinea capitis using the strip method. The antioxidant potential of non-filtered extract of the coating of the fruits was also assessed using DPPH (2,2diphenyl-1-picrylhydrazyl) and the system of scaveranging of radicals. The total antioxidant activity ranged 94.4-95.5%; total content of phenols accounted for 4.46 mg/g in the extract of almond shell. This had a great therapeutic potential after 20 days of therapy against T. capitis-caused infection of the skin on the head. The survey has proven the clinical efficiency of Prunus dulcis for treating dermatological diseases.
Antimicrobial activity of essential oil from Dictamnus dasycarpus Turcz. was tested against nine microorganisms using methods of disk diffusions and broth microdilutions. The essential oil displayed bactericidal activity towards S. aureus ATCC 25923 and methicillin-resistant strain of S. aureus (Lei & Liao, 2007).
Methanol extracts of Parthenocissus tricuspidata (Siebold & Zucc.) Planch. demonstrated in vitro anti-malaria activity against Plasmodium falciparum, and also а schizonticidal activity towards P. berghei in blood of mice in the conditions of use of the doses causing no noticeable toxicity: these extracts elevated the share of oxidized hemoglobin in erythrocytes and inhibited synthesis of protein (Park & Moon, 2008).
The antimicrobial impact of the remaining species of plants is covered much less thoroughly in the literature. Thus, according to our results, ethanol extracts inhibit growth of colonies of many species of microorga-nisms of the Yersiniaceae, Enterobacteriaceae, Morganellaceae, Enterococcaceae, Listeriaceae, Pseudomonadaceae, Campylobacteraceae, Corynebacteriaceae, Nocardiaceae families and fungi of the Saccharomycetacea family. A somewhat disturbing find was that the strains of P. mirabilis, K. рneumoniae, S. marcescens, L. monocytogenes and C. jejuni, which we studied, were absolutely resistant to azithromycin (growth inhibition zone equaled 0.0 mm), and that C. albicans showed a low susceptibility to amphotericin (growth inhibition zone was 2.4 mm).