The Open Agriculture Journal




ISSN: 1874-3315 ― Volume 14, 2020
RESEARCH ARTICLE

Phytochemical Research and Antimicrobial Properties of Lipophylic Extracts of Some Species of Salix L. Genus from Ukraine



Natalia Borodina1, Ain Raal2, *, Volodimir Kovalyov1, Tetiana Osolodchenko3, Оleh Koshovyi1, Hoai Thi Nguyen4, Andrey Komissarenko1
1 Department of Pharmacognosy, The National University of Pharmacy, Kharkiv, Ukraine
2 Institute of Pharmacy, Faculty of Medicine, University of Tartu, Tartu, Estonia
3 Mechnikov Institute of Microbiology and Immunology of the National Academy of Medical Sciences of Ukraine, Kharkiv, Ukraine
4 Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, Hue City, Vietnam

Abstract

Background:

Willows are indispensable for the creation of anti-erosion plantations, preliminary soil-improving crops in the reclamation of disturbed lands, as well as for energy plantations designed to produce the biomass suitable for biofuels. In the process of care and thinning of these plantations, there are a huge amount of waste remains - young willow branches and leaves, which would be advisable to use in medical and pharmaceutical practice to create new supplements and medicines. They are known to possess antimicrobial, antifungal, and antiviral properties.

Objective:

The aim of the current paper is to determine the chemical composition of lipophylic extracts of some species of Salix L. genus and to study their antimicrobial properties.

Methods:

The lipophylic extracts from Salix cinerea L., S. incana Schrank, S. cаprea L., S. sachalinensis F. Schmidt, S. acutifolia L., S. fragilis L., S. caspica Pall., S. rosmarinifolia L. and S. myrsinifolia Salisb. fresh shoots were obtained using chloroform in the Soxhlet apparatus. The composition of volatile components and carboxylic acids was determined using quantitative content of chlorophylls and carotenoids was measured spectrophotometrically. The antibacterial activity was studied by the agar diffusion method.

Results:

The methods used allowed identifying 46 volatile compounds in the lipophylic extracts from the shoots of the plants of Salix L. genus, where eugenol and squalene dominated; as well as 42 organic acids, among which benzoic, methoxybenzoic, salicylic acids prevailed, and palmitic, linoleic and linolenic acids were found to prevail among fatty acids. All lipophylic extracts studied showed antimicrobial activity against Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Bacillus subtilis and Candida albicans.

Conclusion:

The results obtained indicate the possibility of the creation of a novel antimicrobial agent using the lipophylic extracts from the shoots of Salix L. genus rich in different biologically active substances.

Keywords: Antibacterial activity, Carotenoids, Clorophylls, Lipophylic extracts, Salix L. shoots, Volatile compounds.


Article Information


Identifiers and Pagination:

Year: 2020
Volume: 14
First Page: 136
Last Page: 144
Publisher Id: TOASJ-14-136
DOI: 10.2174/1874331502014010136

Article History:

Received Date: 08/04/2020
Revision Received Date: 22/06/2020
Acceptance Date: 23/06/2020
Print publication date: 18/08/2020
Collection year: 2020

© 2020 Borodina et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Institute of Pharmacy, Faculty of Medicine, University of Tartu, Nooruse 1, 50411 Tartu, Estonia; Tel: +372 7375288; Fax: +372 7375289; E-mail: ain.raal@ut.ee





1. INTRODUCTION

One of the prospective groups of plants, which, according to the literature data, are known to possess antimicrobial, antifungal, antiviral properties, are the plants from the Salicaceae family [1Kurkin VA. Salicaceae family plants are a promising source of new antimicrobial, anti-inflammatory and tonic drugs Paper presented at the Int scientific conf Search, development and implementation of new medicines and organizational forms of pharmaceutical activity, Tomsk 2000; 42-3.-5Ramos PAB, Moreirinha C, Silva S, et al. The health-promoting potential of Salix spp. Bark polar extracts: Key insights on phenolic composition and in vitro bioactivity and biocompatibility. Antioxidants 2019; 8(12): 609.
[http://dx.doi.org/10.3390/antiox8120609] [PMID: 31801290]
]. Willows (genus Salix L.) are indispensable for the creation of anti-erosion plantations, preliminary soil-improving crops in the reclamation of disturbed lands, as well as for energy plantations designed to produce the biomass suitable for biofuels [6Afonin AA. Methodological principles for the creation of sustainable highly productive willow stands (for example, autochthonous species Salix of the Bryansk forest) RIO of Bryansk State University; Bryansk 2005. (in Russian).-8Kuzovkina YA, Schulthess CP, Zheng D. Influence of soil chemical and physical characteristics on willow yield in Connecticut. Biomass Bioenergy 2018; 108: 297-306.
[http://dx.doi.org/10.1016/j.biombioe.2017.11.021]
].

The leader countries by area of land covered by energy crops in Europe are: Italy - 57 thousand hectares (the largest area in Europe), Poland - 13 thousand hectares, Sweden - 12 thousand hectares, Germany - 11 thousand hectares, Denmark - 10 thousand hectares, Finland - 8 thousand hectares. According to the estimates of the Bioenergy Association of Ukraine, the potential bioenergy 21 million tons, includes about a third - 7 million tons AD - energy crops on 2 million hectares of land. The total area of land involved in cultivation energy crops in Ukraine - about 4 000 hectares for August 2018. Most of them (approx. 3,000 ha) located on the territory of Volyn, Kyiv and Zhytomyr regions [6Afonin AA. Methodological principles for the creation of sustainable highly productive willow stands (for example, autochthonous species Salix of the Bryansk forest) RIO of Bryansk State University; Bryansk 2005. (in Russian)., 7Fuchilo YD, Sbitna MV, Fuchilo OY. Indigenous willow of Ukraine. Science Newsletter NLTU of Ukraine 2013; 23: 68-72. [in Ukrainian]., 9Gąsecka M, Siwulski M, Drzewiecka K, et al. Organic acid profile and phenolic and sugar content in Salix purpurea × viminalis L. cultivated with different spent mushroom substrate and copper additions Chemistry and Ecology 291;35(2): 191-203.]. In the process of care and thinning of these plantations, there are a huge amount of waste remains - young willow branches and leaves, which would be advisable to use in medical and pharmaceutical practice to create new supplements and medicines.

According to the literature data and our own phytochemical research on the willow genus plants, it was determined that they have a diverse chemical composition and contain various classes of natural substances [10Noleto-Dias C, Harflett C, Beale MH, Ward JL. Sulfated flavanones and dihydroflavonols from willow. Phytochem Lett 2020; 35: 88-93.
[http://dx.doi.org/10.1016/j.phytol.2019.11.008] [PMID: 32025274]
-14Gąsecka M, Mleczek M, Jutrzenka A, Goliński P, Stuper-Szablewska K. Phenolic compounds in leaves of Salix species and hybrids growing under different soil conditions. Chem Ecol 2017; 33(3): 196-212.
[http://dx.doi.org/10.1080/02757540.2017.1289186]
]. We have previously carried out a GC/MS research on the volatile compounds and carboxylic acids of the shoots of Salix caprea L., S. purpurea L., S. viminalis L., S. triandra L., S. cinerea L., S. fragilis L., S. rosmarinifolia L., S. myrsinifolia Salisb., S. acutifolia L., S. nigricans Smith., S. fragilis L., S. daphnoides Vill. of Ukrainian flora [15Borodina NV. The volatile components of Salix myrsinifolia salisb. shoots Collection of scientific works of PL Shupyk NMAPE employees 2016; 26: 303-8.-20Borodina NV, Kovalyov VV. Mineral composition of some species of the family Salicaceae Collection of scientific works of PL Shupyk NMAPE employees 2018; 29: 180-7.].

Lipophilic extracts of many plants have pronounced antibacterial properties [21Zazharskyi VV, Davydenko PO, Kulishenko OM, Borovik IV, Brygadyrenko VV. Antimicrobial activity of 50 plant extracts. Biosystems Diversity 2019; 27(2): 163-9.
[http://dx.doi.org/10.15421/011922]
]: such as eucalyptus and sage extracts are the main active components of such medicines as Chlorophyllipt and Salvin [22Kovalenko VN. Compendium 2014 " Medicines 2014; 2700., 23Koshovyi ON, Vovk GV, Akhmedov EYu, Komissarenko AN. The study of the chemical composition and pharmacological activity of Salvia officinalis leaves extracts getting by complex processing. Azerbaijan Pharmaceutical and Pharmacotherapy Journal 2015; 15(1): 30-4.], lipophilic extracts from Calendula officinalis [24Raal A, Orav A, Nesterovitsch J, Maidla K. Analysis of carotenoids, flavonoids and essential oil of Calendula officinalis cultivars growing in Estonia. Nat Prod Commun 2016; 11(8): 1157-60.
[http://dx.doi.org/10.1177/1934578X1601100831] [PMID: 30725580]
], Persicaria bistorta [25Klimczak U, Woźniak M, Tomczyk M, Granica S. Chemical composition of edible aerial parts of meadow bistort (Persicaria bistorta (L.) Samp.). Food Chem 2017; 230: 281-90.
[http://dx.doi.org/10.1016/j.foodchem.2017.02.128] [PMID: 28407912]
], Crataegus submollis Sarg [26Sydora NV, Kovaleva AM, Iakovenko VK, Ilyina TV, Krivoruchko EV. Phytochemical research of Crataegus submollis Sarg. leaves lipophilic complex and study of its antibacterial activity. Der Pharmacia Lettre 2016; 8(21): 19-23.], Genista tinctoria [27Vladimirova IN, Georgiyants VA. Lipophilic substances from Genista tinctoria. Chem Nat Compd 2013; 49(1): 91-2.
[http://dx.doi.org/10.1007/s10600-013-0514-3]
], Dahlia varieties [28Gontova T, Ilyinska N, Golembiovska O. A study of the component composition of phenolic compounds obtained from Dahlia varieties Ken"s Flame herb. Pharma Chem 2016; 8(18): 455-9.] and other spices [29Raal A, Relve P, Kõivupuu M. Modern beliefs regarding medicinal plants in Estonia. J Balt Stud 2018; 49(3): 387-403.
[http://dx.doi.org/10.1080/01629778.2018.1453851]
, 30Zdoryk OA, Khokhlova KO, Georgiyants VA, Vyshnevska LI. Investigation of physical and chemical stability of ointment with herbals. Int J Pharm Compd 2014; 18(3): 248-52.
[PMID: 25306773]
]. The main active ingredients of the lipophylic extracts are chlorophylls, carotenoids, the combination of unsaturated fatty acids, volatile compounds, and other biologically active substances that show various types of pharmacological activity [24Raal A, Orav A, Nesterovitsch J, Maidla K. Analysis of carotenoids, flavonoids and essential oil of Calendula officinalis cultivars growing in Estonia. Nat Prod Commun 2016; 11(8): 1157-60.
[http://dx.doi.org/10.1177/1934578X1601100831] [PMID: 30725580]
, 31Gligorić EI, Igić R, Suvajdžić LD, Teofilović BD, Grujić-Letić NN. Salix eleagnos Scop. " a novel source of antioxidant and anti-inflammatory compounds: Biochemical screening and in silico approaches. S Afr J Bot 2020; 128: 339-48.
[http://dx.doi.org/10.1016/j.sajb.2019.11.018]
-33Gonz?­lez-Alamilla EN, Gonzalez-Cortazar M, Valladares-Carranza B, et al. Chemical Constituents of Salix babylonica L. And their antibacterial activity against Gram-positive and Gram-negative animal bacteria. Molecules 2019; 24(16): 2992.
[http://dx.doi.org/10.3390/molecules24162992] [PMID: 31426583]
]. Thus, for the purpose of complex research, and further rational application of the plant raw material, we continue the research on the lipophylic extracts, obtained from the plant raw material of willow genus plants.

The aim of the study was to determine the chemical composition of lipophylic extracts of some species of Salix L. genus, as well as to study their antimicrobial properties.

2. MATERIALS AND METHODS

2.1. Plant Material

The plant material studied was collected from nine Salix genus species in June and July of 2015-2016 in Zakarpattia, Kharkiv and Kyiv regions (Table 1), the shoots were collected from at least ten different trees of same species, and a united sample was formed. The plant raw material samples were dried at room temperature for 10 days and were stored in well closed bags on the shelves at room temperature. All Salix species studied were identified by the taxonomic guide [34Kochno MA, Parkhomenko LI, Zarubenko AV. Dendroflora of Ukraine Wild and cultivated trees and bushes 2002., 35Dobrochaeva DN, Kotov MI, Prokudin YN, Barbarich AI. Key to Higher Plants of Ukraine 2nd ed. 1999; 546. (In Russian)] plus using the expert opinion of the botanist Horelov A.M. from the M.M. Gryshko National Botanical Garden of the National Academy of Sciences of Ukraine (Kyiv). The voucher specimen (No 98-107) is deposited in the herbarium of the Pharmacognosy Department, National University of Pharmacy, Kharkiv, Ukraine.

2.2. Extraction of Lipophylic Compounds

Lipophylic extracts were obtained from the 20.0 g of the crushed dried plant material (1-3 mm) of Salix cinerea L., S. incana Schrank, S. cаprea L., S. sachalinensis F. Schmidt, S. acutifolia L., S. fragilis L., S. caspica Pall., S. rosmarinifolia L. and S. myrsinifolia Salisb. by exhausting extraction of the crushed plant raw material under the same conditions using chloroform in the Soxhlet apparatus [26Sydora NV, Kovaleva AM, Iakovenko VK, Ilyina TV, Krivoruchko EV. Phytochemical research of Crataegus submollis Sarg. leaves lipophilic complex and study of its antibacterial activity. Der Pharmacia Lettre 2016; 8(21): 19-23., 36State Pharmacopoeia of Ukraine. (2004-2018) Amendment. 1-3: 1-4. Kharkiv (in Ukrainian)-38Harna SV, Vyetrov PP, Rusynov OI, Georgiyants VA. Optimization of technology for extraction of lipophilic complexes from medicinal plant raw materials. Message 1 Selection of extractant Zaporozhye medical journal 2010; 3: 92-4.]. The Soxhlet extractor (product identification number 8730/500ml) of borosilicate thermal glass SIMAX, a round-bottomed flask of 1000 ml capacity, produced in the Czech Republic, a one-place heating mantle LOIP LH-110 (LAB-KN-1000), produced in Russia, were used. The lipophylic extracts obtained were concentrated on the complete extragent removal and were used for further research. The quantitative content of the main biologically active compounds in the lipophylic extracts from the shoots of the plants of Salix L. genus was determined spectrophotometrically.

2.3. Determination of Sum of Lipophilic Extracts

The sum of lipophilic extracts was determined gravimetrically, according to the State Pharmacopoeia of Ukraine [36State Pharmacopoeia of Ukraine. (2004-2018) Amendment. 1-3: 1-4. Kharkiv (in Ukrainian), 38Harna SV, Vyetrov PP, Rusynov OI, Georgiyants VA. Optimization of technology for extraction of lipophilic complexes from medicinal plant raw materials. Message 1 Selection of extractant Zaporozhye medical journal 2010; 3: 92-4.].

Table 1
Studied shoots of different species of Salix genus collected in Ukraine.


2.4. Determination of Sum of Carotenoids and Chlorophylls

The quantitative estimation of the content of the sum of carotenoids (in terms of β-carotene at the wavelength 453 nm), and chlorophylls (in terms of chlorophyll A at the wavelength 670 nm) in the lipophylic extracts studied (in accurately weighed amounts of extracts of 0.05 g) was carried out spectrophotometrically on the Hitachi U3210 spectrophotometer by the generally accepted procedure [24Raal A, Orav A, Nesterovitsch J, Maidla K. Analysis of carotenoids, flavonoids and essential oil of Calendula officinalis cultivars growing in Estonia. Nat Prod Commun 2016; 11(8): 1157-60.
[http://dx.doi.org/10.1177/1934578X1601100831] [PMID: 30725580]
, 39Kuregyan AG. Spectrophotometry in the analysis of carotenoids. Basic research 2015; 2(23): 5166-72. In Russian, 40Vasilieva VI, Stoyanova OF, Shkutina IV, Karpov SI. Spectral methods of analysis. A practical guide, Lan, St Petersburg 2014. Russian]. The content of the sum of carotenoids (X, mg%) in terms of β-carotene and chlorophylls (X, mg%) in terms of chlorophyll A was calculated using the formula: X = (10xAxVx100)xE 1%1cm m, where: 10 - the content of chlorophyll A or β-carotene in 1 ml of a 1% solution, mg; А - optical density of the solution studied; V - volume of a measuring flask, ml; E1%1cm - extinction of chlorophyll A in chloroform at the wavelength 670 nm, which comprises 944,5, or E1%1cm - extinction of β-carotene in chloroform at the wavelength 453 nm, which comprises 2400; m - weight of a lipophylic extract sample, g [36State Pharmacopoeia of Ukraine. (2004-2018) Amendment. 1-3: 1-4. Kharkiv (in Ukrainian), 39Kuregyan AG. Spectrophotometry in the analysis of carotenoids. Basic research 2015; 2(23): 5166-72. In Russian, 40Vasilieva VI, Stoyanova OF, Shkutina IV, Karpov SI. Spectral methods of analysis. A practical guide, Lan, St Petersburg 2014. Russian].

The identification of carotenoid pigments in the extracts obtained was carried out by spectrophotometric measurement of extinction values of their 10% solutions in chloroform at the wavelengths 350-500 nm with an interval of 50 nm on the Hitachi U3210 spectrophotometer. The presence of three maxima in the absorption spectra of carotenoid pigments at 420, 450 and 470 nm was detected. According to the literature data, these maxima are characteristic of the absorption spectrum of β-carotene. The peaks in the range of 600-670 nm may be indicative of the presence of chlorophylls in the lipophylic extracts. Thus, the main absorption maxima of the lipophylic substances do not overlap, which allows the simultaneous determination of carotenoids and chlorophyll in the willow shoots extracts spectrophotometrically without preliminary separation [36State Pharmacopoeia of Ukraine. (2004-2018) Amendment. 1-3: 1-4. Kharkiv (in Ukrainian), 39Kuregyan AG. Spectrophotometry in the analysis of carotenoids. Basic research 2015; 2(23): 5166-72. In Russian, 40Vasilieva VI, Stoyanova OF, Shkutina IV, Karpov SI. Spectral methods of analysis. A practical guide, Lan, St Petersburg 2014. Russian].

2.5. Determination of Volatile Compounds

The GC/MS analysis of volatile compounds was carried out using the Agilent Technology 6890N chromatograph with the mass-spectrometric detector 5973N, with capillary column HP-5MS (silica, the column length was 30 m, inner diameter - 0,25 mm); carrier gas - helium (flow rate 1 ml/min). The sample volume was 0.1-0.5 µl with the flow split 1/50. The thermostat temperature was 50°C programmed for 4°C/min up to 220°C. The temperature of the detector and the vaporizer was 250°C [15Borodina NV. The volatile components of Salix myrsinifolia salisb. shoots Collection of scientific works of PL Shupyk NMAPE employees 2016; 26: 303-8., 16Borodina NV, Kovalyov VV, Koshovyi OM, Akhmedov EY. The chromatography-mass spectrometry study of Salix rosmarinifolia L. Azerbaijan Pharmaceutical and Pharmacotherapy Journal 2016; 16: 15-20., 41Il"ina TV, Kovaleva AM, Goryachaya OV, Vinogradov BA. Terpenoids and aromatic compounds from essential oils of Cruciata laevipes and C.glabra. Chem Nat Compd 2013; 48(6): 1106-8.
[http://dx.doi.org/10.1007/s10600-013-0482-7]
].

2.6. Determination of Organic Acids

Organic acids were also determined by the GC/MS method using the Agilent Technology 6890N chromatograph with the mass-spectrometric detector 5973N. The sample was injected in the column in the splitless mode, the rate of sample injection was 1.2 ml/min within 0.2 min. Detection was held under the following conditions: capillary chromatographic column INNOWAX, the length of which was 30 m, inner diameter - 0.25 mm; carrier gas - helium; carrier gas velocity - 1.2 ml/min; the temperature of the sample injection heater - 250°C, the temperature of the thermostat was programmed for 4°C /min from 50°C up to 250°C; the temperature of the detector - 250°C [16Borodina NV, Kovalyov VV, Koshovyi OM, Akhmedov EY. The chromatography-mass spectrometry study of Salix rosmarinifolia L. Azerbaijan Pharmaceutical and Pharmacotherapy Journal 2016; 16: 15-20., 17Borodina NV. The study of volatile components Salix caprea L. 4th European Conference on Biology and Medical Sciences 2015; 209-13., 42Krivoruchko E, Kanaan H, Samoilova V, Ilyina T, Koshovyi O. Carboxylic acids from brown algae Fucus vesiculosus and Padina pavonica. Ceska Slov Farm 2018; 66(6): 287-9.
[PMID: 29623711]
].

Identification of the volatile compounds and organic acids was carried out by comparing the mass-spectra of the compounds with the data of the mass-spectra libraries NIST05 and WILEY 2007 combined with the identification software AMDIS and NIST. The quantitative determination of the compounds was carried out, taking into account the internal standard, and was evaluated in mg/kg of the plant raw material [16Borodina NV, Kovalyov VV, Koshovyi OM, Akhmedov EY. The chromatography-mass spectrometry study of Salix rosmarinifolia L. Azerbaijan Pharmaceutical and Pharmacotherapy Journal 2016; 16: 15-20., 42Krivoruchko E, Kanaan H, Samoilova V, Ilyina T, Koshovyi O. Carboxylic acids from brown algae Fucus vesiculosus and Padina pavonica. Ceska Slov Farm 2018; 66(6): 287-9.
[PMID: 29623711]
].

2.7. Determination of Antibacterial Activity

The antibacterial activity of the extracts was studied by the agar diffusion method in the Mechnikov Institute of Microbiology and Immunology in the Microorganisms and Mediums Biochemistry Laboratory under the supervision of PhD (BiolSc) Osolodchenko T.P [23Koshovyi ON, Vovk GV, Akhmedov EYu, Komissarenko AN. The study of the chemical composition and pharmacological activity of Salvia officinalis leaves extracts getting by complex processing. Azerbaijan Pharmaceutical and Pharmacotherapy Journal 2015; 15(1): 30-4., 26Sydora NV, Kovaleva AM, Iakovenko VK, Ilyina TV, Krivoruchko EV. Phytochemical research of Crataegus submollis Sarg. leaves lipophilic complex and study of its antibacterial activity. Der Pharmacia Lettre 2016; 8(21): 19-23., 43Ilina T, Kashpur N, Granica S, et al. Phytochemical profiles and in vitro immunomodulatory activity of ethanolic extracts from Galium aparine L. Plants (Basel) 2019; 8(12): 541.
[http://dx.doi.org/10.3390/plants8120541] [PMID: 31775336]
, 44Koshovyi OM, Zagayko AL, Kolychev IO, Akhmedov EYu, Komissarenko AN. Phytochemical study of the dry extract from bilberry leaves. Azerbaijan Pharmaceutical and Pharmacotherapy Journal 2016; 16(1): 18-23.]. According to the WHO recommendations, the reference strains of Staphylococcus aureus АТСС 25923, Staphylococcus aureus 6538 АТСС, Escherichia coli АТСС 25922, Proteus vulgaris NCTC 4636, Pseudomonas aeruginosa АТСС 27853, Pseudomonas aeruginosa 9027 АТСС, Bacillus subtilis АТСС 6633 and Candida albicans 885/653 АТСС were used for the activity of substances assessment. The microbial suspension of microorganisms was prepared using the Densi-La-Meter device (PLIVALachema, Czech Republic; wavelength 540 nm). The suspension was prepared according to the instruction, which is added to the device, and an informational letter on the innovation in the healthcare system of Ukraine [45Boyko NN, Zaytsev AI, Osolodchenko TP. Vector algebra theory in analysis of properties of antibacterial medications. Annals of Mechnikov Institute 2014; 1: 20-6.]. The cultures were synchronized by low temperature (4°C). The microbial burden comprised 107 microbial cells per 1 ml of the medium, and was estimated by the McFarland standard. An 18-24 hours culture of microorganisms was taken for the research. The Mueller-Hinton Agar (Dagestan SIC "Culture Mediums") was used in the experiment. The Sabouraud agar was used for Candida albicans. The antibacterial activity of the samples studied was determined using the agar diffusion method, or the "wells" method on two layers of dense culture medium, placed in Petri dishes. The "starvation" media (agar-agar, water, salts) were used for the lower layer. The lower layer represents a pad of 10 mm in height, on which 3-6 thin-walled stainless steel cylinders 8 mm in diameter and 10 mm high are horizontally placed. The upper layer, which consists of an agar culture medium, melted and cooled to 40°C, where a relevant standard of a 24-hour test microorganism culture is placed, is poured around the cylinders. The upper layer was preliminarily mixed until the formation of a uniform mass. The cylinders were pulled out by sterile forceps, and the extract studied, taking into account its volume (0.3 ml), was placed in the slots formed.

The volume of the medium for the upper layer fluctuated between 14 and 16 ml. The dishes were dried for 30-40 min at room temperature, and then were placed into a thermostat for 18-24 hours. Diameters of the inhibition zones of the microorganisms were measured by a ruler with the measurement error ±0.1 mm.

2.8. Statistical Analysis

The mean and Standard Deviation (SD) of the sample were calculated according to the monograph "Statistical analysis of the results of a chemical experiment" of the State Pharmacopoeia of Ukraine, 2.2. The average sample μ was calculated as the arithmetic mean of all variants (n=5 of combined samples). At the same time, the spread of options around the average is characterized by the magnitude of the standard deviations. The uncertainty of this estimate is characterized by the value of the confidence interval, in which the true value μ is given with the given two-way probability P2. Under uncertainty, the confidence interval is understood, usually for the 95% significance level. Limit values of the confidence interval were calculated using Student's criterion. Quantitative data are presented as the mean ± SD [23Koshovyi ON, Vovk GV, Akhmedov EYu, Komissarenko AN. The study of the chemical composition and pharmacological activity of Salvia officinalis leaves extracts getting by complex processing. Azerbaijan Pharmaceutical and Pharmacotherapy Journal 2015; 15(1): 30-4.].

3. RESULTS

3.1. Total Content of Lipophylic Compounds, Chlorophylls and Carotenoids

The lipophylic extracts from the shoots of the plants of Salix L. genus were obtained. The lipophylic extracts obtained represent a salvelike mass of a dark-green colour with a characteristic pleasant aromatic odour. The lipophylic fractions are insoluble in water, soluble in chloroform, alcohol, hexane, vegetable oils. The percentage content of the extract in terms of the dried plant raw material comprised the highest yield - from Salix myrsinifolia Salisb. shoots - 15.71±0.10%, Salix acutifolia L. - 8.12±0.09% (Table 2).

3.2. Content of Volatile Compounds

Forty-six volatile compounds were identified in the lipophylic extracts from the shoots of willow genus plants by the GC/MS method (Table 3).

3.3. Contents of Organic Acids

Forty-two organic acids were detected, of which benzoic acid (from 1149.7 mg/kg to 3308.3 mg/kg), methoxybenzoic (from 7514.7 mg/kg to 14056.5 mg/kg), salicylic acid (from 1178.0 mg/kg to 5642.0 mg/kg) prevailed (Table 4).

3.4. Antimicrobial Activity

The results of the research on the antimicrobial properties of lipophylic extracts by the agar diffusion method are given in (Table 5).

Table 2
Quantitative content of the main biologically active substances in the lipophylic extracts from the shoots of the Salix genus plants.


Table 3
Volatile compounds of the lipophylic extracts from the shoots of the Salix L. genus plants.


Table 4
Organic acids of the lipophylic extracts from the shoots of the Salix L. genus plants.


Table 5
Results of the antimicrobial activity study of the lipophylic extracts from the shoots of the willow species.


The following criteria were used in the evaluation of the antibacterial activity: 1) absence of the microorganisms growth inhibition zone around the slots, as well as the inhibition zone up to 10 mm, show the absence of sensitivity of the microorganism towards the extract placed in the slot; 2) the growth inhibition zones of 10-15 mm in diameter indicate the low sensitivity of the culture towards the extract studied; 3) the growth inhibition zones of 15-25 mm in diameter are considered as the indicator of the sensitivity of the microorganism towards the extract studied; 4) the growth inhibition zones, the diameter of which exceeds 25 mm, indicate high sensitivity of the microorganism towards the extract studied.

The results of the experimental research show expressed antimicrobial activity of the lipophylic extracts towards the strains of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli, as well as the activity towards the yeast fungus Candida albicans.

4. DISCUSSION

4.1. Total Content of Lipophylic Compounds, Chlorophylls and Carotenoids

The quantitative content of lipophylic compounds in willow shoots differs depending on the chosen type of the plant raw material, but still is on the quite high level.

According to our data (Table 2), the content of the sum of chlorophylls in the shoots of the willow genus plants varies from 26.4 to 68.3 mg/%. The quantitative content of carotenoids in the lipophylic extracts from the shoots of the plants of Salix L. genus was also determined spectrophotometrically. The content of carotenoids ranges from 12.6 to 41.5 mg/%. Thus, the sum of chlorophylls in the willow species shoots during the vegetation period is higher compared to that of carotenoids, which confirms the high level of metabolism and anabolism in the plants.

Chlorophylls possess bactericidal and antioxidant activities, improve the state of blood vessels [46Leong HY, Show PL, Lim MH, Ooi CW, Ling TC. Natural red pigments from plants and their health benefits: A review. Food Rev Int 2017; 34(5): 463-82.
[http://dx.doi.org/10.1080/87559129.2017.1326935]
, 47Wang YT, Yang CH, Huang TY, Tai MH, Sie RH, Shaw JF. Cytotoxic effects of chlorophyllides in ethanol crude cxtracts from plant leaves. Evid Based Complement Alternat Med 2019; 20199494328
[http://dx.doi.org/10.1155/2019/9494328] [PMID: 31379971]
]. Carotenoids show a proven anticarcinogenic, immune modulating, antioxidant activity, suppress the photosensibilization processes and lower the risk of cardiovascular disorders, which contribute to the prospects of further research on the properties, obtained during the study of lipophylic extracts [48von Lintig J. Provitamin A metabolism and functions in mammalian biology. Am J Clin Nutr 2012; 96(5): 1234S-44S.
[http://dx.doi.org/10.3945/ajcn.112.034629] [PMID: 23053549]
-50Young AJ, Lowe GL. Carotenoids-Antioxidant Properties. Antioxidants 2018; 7(2): 28.
[http://dx.doi.org/10.3390/antiox7020028] [PMID: 29439455]
].

4.2. Content of Volatile Compounds

The dominating compounds among all the components are eugenol (4-allyl-2-methoxyphenol, the content of which comprised from 98.92 mg/kg to 259.72 mg/kg), and squalene (from 495.15 mg/kg up to 2153.98 mg/kg). Squalene should be noted as one of the dominating compounds in the lipophylic extracts, which belongs to the compounds of carotenoid nature. The results of experimental and clinical research show squalene has a positive impact on lipid metabolism. In medicine, squalene is added as a component of agents for the external therapy of inflammatory skin diseases. In this case, the property of squalene to deeply penetrate the skin, being able to carry elements of other compounds, is used; as a result, potent medicines can be applied on the skin pointwise, not being troubled of their adverse effects [31Gligorić EI, Igić R, Suvajdžić LD, Teofilović BD, Grujić-Letić NN. Salix eleagnos Scop. " a novel source of antioxidant and anti-inflammatory compounds: Biochemical screening and in silico approaches. S Afr J Bot 2020; 128: 339-48.
[http://dx.doi.org/10.1016/j.sajb.2019.11.018]
].

Aromatic components, such as benzyl alcohol, salicylic alcohol, pyrocathechol, etc., are also found in quite a large amount, which indicates the presence of antiseptic properties in the extracts. A large amount of aldehydes and ketones, which comprise over 10% of the total compounds, is also found.

Previously, cis-3-hexenyl acetate, cis-3-hexenol, and benzaldehyde were found from leaves of six willow varieties [51Peacock L, Lewis M, Powers S. Volatile compounds from Salix spp. varieties differing in susceptibility to three willow beetle species. J Chem Ecol 2001; 27(10): 1943-51.
[http://dx.doi.org/10.1023/A:1012278417424] [PMID: 11710603]
]. Karimi et al. [52Karimi I, Hayatgheybi H, Shamspur T, Kamalak A, Pooyanmehr M, Marandi Y. Chemical composition and effect of an essential oil of Salix aegyptiaca (musk willow) in hypercholesterolemic rabbit model. Revista Brasileira de Farmacognosia-Brazilian Journal of Pharmacognosy 2011; 21(3): 407-14.
[http://dx.doi.org/10.1590/S0102-695X2011005000030]
] showed that the main compound of essential oil of S. aegyptiana was 1,4-dimethoxybensene, also phenylethyl alcohol, carvone and citronellol are present among principal compounds. The phenylethyl alcohol was also found in the Salix species studied (41-1182 mg/kg).

4.3. Contents of Organic Acids

A considerable part of the extracts is represented by fatty acids - essential components of plant cells, which take part in lipid biosynthesis and almost all physiological processes, as well as biochemical reactions, which proceed in human and animal cells. Fatty acids show F-vitamin activity, reparative and many others, in addition, they provide pharmacological effects of a range of medicines. The biological value of lipophylic extracts depends on the composition of fatty acids. Linoleic (from 2488.0 mg/kg to 10796.9 mg/kg) and linolenic (from 2487.45 mg/kg to 10796.87 mg/kg) acids are found in quite large quantity in the extracts. Palmitic acid (from 10542.3 mg/kg to 18854.5 mg/kg) dominates among the saturated fatty acids. The high content of hydroxycinnamic acid derivatives and salicylic acid in the lipophylic extracts of willow shoots is of much interest. This might explain their high pharmacological activity to some extent. In our study [53Koshovyi O, Raal A, Kovaleva A, et al. The phytochemical and chemotaxonomic study of Salvia spp. growing in Ukraine. J Appl Biol Biotechnol 2020; 8(3): 29-36.], it was shown that Salix spp. branches contain 3.0-10.1 mg/kg of copper. According to Rohnert et al. [54Rohnert U, Koske D, Schneider W, Elstner EF. Inhibition by salix-extracts and phytodolor(R) of copper-catalyzed oxidative destructions. Zeitschrift fur Naturforschung Section C ". J Biosci 1998; 53(3-4): 233-40.
[PMID: 9687162]
], the oxidation of low-density lipoprotein by copper ions is strongly inhibited by different aqueous extracts of Salix spp. but linolenic acid has no significant effect on it.

The study of the chemical composition of lipophylic extracts from the shoots of willow genus plants indicates that they must possess a wide spectrum of antimicrobial activity. Thus, the experiment carried out significantly broadens the data on the chemical composition of the plant raw material of Salix L. genus plants and extracts on their basis.

4.4. Antimicrobial Activity

The results of the experimental research show expressed antimicrobial activity of the lipophylic extracts towards the strains of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli, as well as the activity towards the yeast fungus Candida albicans. As it was found by Gonzalez-Alamilla et al. [33Gonz?­lez-Alamilla EN, Gonzalez-Cortazar M, Valladares-Carranza B, et al. Chemical Constituents of Salix babylonica L. And their antibacterial activity against Gram-positive and Gram-negative animal bacteria. Molecules 2019; 24(16): 2992.
[http://dx.doi.org/10.3390/molecules24162992] [PMID: 31426583]
], the hydroalcoholic extract also showed an antibacterial effect against S. aureus (MIC 0.39 mg/ml) and less to E. coli. The water infusion of S. balylonica leaves showed just weak activity to E. coli and no activity against C. albicans [55Zazharskyi VV, Davydenko PO, Kulishenko OM, Borovik IV, Brygadyrenko VV. Antimicrobial activity of 50 plant extracts. Biosystem Diversity 2019; 27(2): 163-9.
[http://dx.doi.org/10.15421/011922]
].

Thus, it can be concluded that the lipophylic extracts are a prospective substance for the creation of a novel antimicrobial agent from willow shoots, and further require more detailed research on their antimicrobial activity.

CONCLUSION

1. A comparative study of the chemical composition of lipophylic extracts from the shoots of 9 willow species of Ukrainian flora - Salix cinerea L., S. incana Schrank, S. cаprea L., S. sachalinensis F. Schmidt, S. acutifolia L., S. fragilis L., S. caspica Pall., S. rosmarinifolia L. and S. myrsinifolia Salisb - was carried out. 46 volatile compounds and 42 carboxylic acids were detected and identified. The quantitative content of lipophylic compounds in the willow species plant raw material and chlorophylls and carotenoids in the willow shoots extracts was determined. The balanced natural complex of these biologically active compounds is responsible for the pharmacological effect of the obtained lipophylic extracts from the plant raw material studied.

2. For all the studied lipophylic extracts from willow shoots, the antimicrobial activity towards Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Bacillus subtilis and Candida albicans was established.

3. The results obtained indicate the possibility of the creation of a novel antimicrobial agent using the lipophylic extracts from willow shoots, which will allow the more effective application of the Salix L. genus plants raw material, as well as broadening the nomenclature of Ukrainian medicinal products.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No human or animals were used in this research.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

Not applicable.

FUNDING

The research was funded by the Ministry of Health Care of Ukraine at the expense of the State Budget in the framework # 2301020.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Declared none.

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