Influence of plant origin natural α-pinene with different enantiomeric composition on bacteria, yeasts and fungi

Fitoterapia 127 (2018) 20–24

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Influence of plant origin natural α-pinene with different enantiomeric composition on bacteria, yeasts and fungi

T

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Kristina Ložienėa,d, , Jurgita Švedienėa, Algimantas Paškevičiusa,c, Vita Raudonienėa, Oksana Sytarb, Anatoliy Kosyanb a

Nature Research Centre, Institute of Botany, Vilnius, Lithuania SRL "Physiological bases of plant productivity", Educational and Scientific Center “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine c Laboratory of Microbiology of the Centre of Laboratory Medicine, Vilnius University Hospital Santaros Clinics, Vilnius, Lithuania d Department of Pathology, Forensic Medicine and Pharmacology, Institute of Biomedical Sciences, Faculty of Medicine, Vilnius University, Vilnius, Lithuania b

A R T I C LE I N FO

A B S T R A C T

Keywords: Juniperus communis α-Pinene Enantiomers Bacteria Yeasts Fungi

Although the nature-identical chemical compounds are cheaper, they not always repeat biological activity of plant origin natural chemical compounds, often react allergies and resistance of microorganisms. The aim of this study was to investigate effects of Juniperus communis origin α-pinene with different enantiomeric composition on bacteria, yeasts and fungi. Results showed that different enantiomeric composition of α-pinene have different activities on microorganisms: essential oil with (1S)-(−) ≈ (1R)-(+) enantiomeric composition of α-pinene influenced on some microorganisms stronger than essential oil with (1S)-(−) < (1R)-(+) enantiomeric composition of α-pinene; the pure natural α-pinene with enantiomeric composition S < R more strongly inhibited growth of investigated bacteria and Candida yeasts, α-pinene with enantiomeric composition S ≈ R – growth of Trichophyton and Aspergillus. (1S)-(−) and (1R)-(+) enantiomeric forms of α-pinene can have also different synergistic effects with other compounds of essential oil. The results of study showed that the same amount of αpinene with different enantiomeric composition can have diverse antimicrobial potential due different specific interactions with other chemical compounds of essential oil. Therefore, it is very important to determine and present the enantiomeric composition of those plant origin compounds, which are characterized by enantiomerisation, during the course of research of biological activities of natural plant products (essential oils and other) and their isolated compounds.

1. Introduction Although the nature-identical chemical compounds are cheaper, they not always repeat the characteristics and biological activity of natural chemical compounds, more often react an allergies and the resistance of microorganisms [1]. The increasing attention of scientists is drawn to the fact that the biologically active natural products and the compounds of plant origin have fewer side effects than their synthetic analogues [2,3]. The secondary plant metabolites essential oils are the biologically active natural products with strong antimicrobial properties against different human pathogenic microorganisms [4]. Recent work shows that essential oils can act as prooxidants affecting inner eukaryotic cell membranes and organelles such as mitochondria. Depending on the type and the concentration, they exhibit cytotoxic effects on living cells but are non-genotoxic usually [5]. It is assumed that the antimicrobial activity of essential oil may depend not only on its

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Corresponding author at: Nature Research Centre, Institute of Botany, Vilnius, Lithuania. E-mail address: [email protected] (K. Ložienė).

https://doi.org/10.1016/j.fitote.2018.04.013 Received 23 February 2018; Received in revised form 14 April 2018; Accepted 20 April 2018 Available online 22 April 2018 0367-326X/ © 2018 Elsevier B.V. All rights reserved.

major constituents, but also on the interactions between the major and the minor constituents, resulting in additive or synergistic antimicrobial effects of several compounds [6–8]. Nowadays is discussed about the use of Juniperus sp. essential oils for human needs in the traditional medicine and pharmaceutical industry which based on the presence specific biological active compounds [9]. An antimicrobial activity of juniper essential oils against microorganisms is very different and depends on their chemical composition [10–12]. The predominant compound of essential oil of common juniper (Juniperus communis) commonly is α-pinene [13–16]. This bicyclic monoterpene must amount 20% to 50% in J. communis essential oil (Iuniperi aetheroleum) according to requirements of European Pharmacopoeia [17]. The experiments with synthetical standards of (−) and (+) enantiomers of α-pinene showed their different biological activity [18,19]; the different enantiomers of α-pinene shown the selective anti-inflammatory and anti-catabolic effects on human

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below in Results and discussion). According to literature data, second quantitative components of the low-temperature fraction of essential oils of J. communis, growing wild in Lithuania, are sabinene and/or myrcene [15,25]. The difference of the boiling point temperature between α-pinene and these two components is about 9–11 °C. To obtain a pure α-pinene fraction was used a distillation unit consisting from 25 ml Favorsky flask, dephlegmator 25 cm high with vacuum thermal isolation, Liebig's refrigerator, an alonge and receiving flask. The distillation rate was 1 drop in 5 s. The 10 ml of essential oil of J. communis was dried over anhydrous sodium sulphate, filtered and putted in the reaction flask. The collection of pinene fraction was done when solution was boiled at 155.5–156.5 °C under extraction, with a refractive index of light close to 1.4664. The theoretical yield of reaction is 41.5%. The relatively low yield of pinene fraction due to the large interfractional losses was necessary to ensure a higher purity of the final product. The components of essential oil of J. communis can be divided into two fractions: the low-boiling fraction – 156–170 °C and the high-boiling fraction – 232–304 °C. The difference between the boiling point of α-pinene and the boiling point of the highboiling components is from 76 to 148 °C. Therefore, during distillation, a high degree of purification of α-pinene from other components can be achieved.

chondrocytes: (+)-α-pinene being the most promising for further studies to determine its potential value as an anti-osteoarthritic drug [20]. J. communis growing in different locations can accumulate the essential oils with different ratios of (+) and (−) enantiomers of α-pinene [16,21–24]. The different enantiomeric composition of this monoterpene can be the reason of different bioactivity directly and/or indirectly through the synergistic/antagonistic effect with other compounds of essential oil. The aim of this study was to investigate the effects of J. communis origin α-pinene with different enantiomeric compositions on bacteria, yeasts and fungi. 2. Materials and methods 2.1. Plant material Previous investigations showed that cones of J. communis growing wild in Lithuania can synthesize the monoterpene α-pinene with the different ratio of (+) and (−) enantiomers [16,24] and are the good subject for isolation of natural α-pinenes with different enantiomeric ratios. The cones with (1S)-(−) ≈ (1R)-(+) and (1S)-(−) < (1R)-(+) enantiomeric ratios of α-pinene were collected separately in August in Lithuania and dried at room temperature. The growing sites (coordinates) of each individual J. communis with their enantiomeric ratio of α-pinene were known from the previous above mentioned works.

2.4. Microorganisms The bacteria Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, fungi, Aspergillus fumigatus SC 6359, A. flavus CBS 120264, Trichophyton rubrum ATCC28188, T. mentagrophytes SC 91427, and yeasts Candida albicans CBS 2730, C. parapsilosis CBS 8836 were used in this study. The strains of bacteria were maintained on nutrient agar (Liofilchem, Italy) and the strains of fungi and yeast – on Sabouraud dextrose agar (Liofilchem, Italy) slants and stored at 4 °C. The inoculum of each strain to be tested was prepared with fresh cultures by suspending the microorganisms in sterile water with 0.05% Tween 80. The turbidity of the inoculum was adjusted by spectrophotometer according to the conidial size of the species (0.09 to 0.3 optical densities at 530 nm) [26].

2.2. Isolation and analysis of essential oil The essential oils with (1S)-(−) < (1R)-(+) and (1S)-(−) ≈ (1R)(+) enantiomeric ratios of α-pinene (no.1 and no.2, respectively) were isolated from J. communis dried cones separately by hydrodistillation in Clevenger type apparatus during two hours. 1% essential oils solutions were prepared in mixture of diethyl ether and n-pentane (1,1) for further investigations. The analysis of α-pinene and its enantiomers were carried out by a FOCUS GC (Thermo Scientific) gas chromatograph with a flame ionisation detector (FID). Data were processed with CHROMCARD S/W. The silica capillary column TR-5 (30 m, i.d. 0.25 mm, film thickness 0.25 μm) was used for the analysis of the monoterpene αpinene with the following GC parameters (Fig. 1): carrier gas helium flow rate 1.6 ml/min; temperature programme from 40 °C to 250 °C increasing at 4 °C/min; detector temperature 260 °C; split injector was heated at 250 °C. The identification of α-pinene and β-pinene was carried out by the comparison of the retention time (RT) of its GC peaks in FID chromatograms with RT of α-pinene and β-pinene analytical standards (Sigma-Aldrich). The percentage amounts of α-pinene were recalculated according to the areas of FID chromatographic peaks assuming that all constituents of the essential oil comprise 100%. α-Pinene enantiomers were separated on HP−Chiral−20B column (30 m length, 0.249 mm id, 0.25 μm film thickness) with helium as carrier gas (Fig. 1). The following GC parameters were used for the analysis of the α-pinene enantiomers: helium flow rate of 1.6 ml/min; temperature program from 85 to 160 °C increasing at 5 °C/min; detector temperature 260 °C; split injector was heated at 250 °C. The identification of (1R)-(+)-α-pinene and (1S)-(−)-α-pinene was carried out by the comparison of the retention time (RT) of its GC peak in FID chromatograms with RT of (1R)-(+)-α-pinene and (1S)-(−)-α-pinene analytical terpene standards (Sigma-Aldrich; purity (GC area %) ≥98.5% and ≥ 99.0%, respectively) under the same GC parameters and column. The percentage amounts of α-pinene enantiomers were recalculated according to the areas of FID chromatographic peaks assuming that monoterpene α-pinene fraction is 100%.

2.5. Antimicrobial properties of essential oils of Juniperus communis The minimum inhibitory concentration (MIC) and the minimum bactericidal/fungicidal concentration (MBC/MFC) of J. communis essential oils and natural origin α-pinene with different enantiomeric compositions were determined using a broth microdilution method. For the antimicrobial activity testing J. communis essential oils and pure natural α-pinene with different enantiomeric compositions were dissolved in 96% (v/v) ethanol. Final concentrations of J. communis essential oil no.1 and no.2 were 10% (w/v), (1R)-(+)-α-pinene, (1S)(−)-α-pinene and (1R)-(+)-α-pinene standard – 50% (w/v). The 96well plates were prepared by dispensing into each well 100 μl RPMI 1640 medium with L-glutamine without sodium bicarbonate (Sigma Aldrich, Germany). A 100 μl prepared samples of pinenes were added into the first wells. Then 100 μl from their serial dilutions was transferred into eighth consecutive wells. Thereafter, each well was inoculated with 100 μl of suspension containing 106 CFU/ml−1 of the culture and incubated at 30 °C for 48 h. A suspension of microorganisms in the medium was used as positive control. A 50% or 10% (v/v) ethanol was used as a negative control for the influence of the solvents. After incubation, the growth of microorganisms was indicated by the presence of the turbidity and a ‚pellet‘on the well bottom. MICs were determined presumptively as the first well, in ascending order, which did not produce a pellet. To confirm MIC and establish MFC, 10 μl of broth was removed from each well and inoculated on Sabouraud dextrose agar for yeasts and fungi, nutrient agar – bacteria. No visible colony growth after subsequent 24–48 h incubation was accepted as MFC. All tests were performed in duplicate [27].

2.3. Isolation of pinenes from essential oils α-Pinene is component of the low-temperature fraction of essential oils. This monoterpene was the main compound and amounted about 61% in both experimental samples of essential oils of J. communis (see 21

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A 140000

α-pinene (9.27 min)

Detector response

120000 100000 80000 60000 40000

β-pinene (10.79 min)

20000 0

8

8.5

9

9.5

10

10.5

11

11.5

12

Time (min) B

C

Detector response

30000

(1R)-(+)-α-pinene (9.90 min)

Detector response

35000

25000 20000 15000 (1S)-(-)-α-pinene (9.67 min)

10000 5000 0

9

9.5 10 Time (min)

10.5

20000 (1R)-(+)-α-pinene (9.95 min) 18000 (1S)-(-)-α-pinene (9.68 min) 16000 14000 12000 10000 8000 6000 4000 2000 0 9 9.5 10 10.5 Time (min)

Fig. 1. Capillary GC analysis of pinene isomers (A) and α-pinene enantiomers (B, C) in the studied Juniperus communis essential oils.

3. Results and discussion

(1R)-(+) was 1:7 in essential oil no.1, i. e. there strongly dominated (+)-α-pinene in this essential oil; while the near percentages of both αpinene enantiomers were characteristic of essential oil no.2: the percentage of (1R)-(+)-α-pinene was higher than percentage of (1S)(−)-α-pinene 1.4 time only (Table 1). Therefore, thereinafter the essential oils no.1 and no.2 will be named as S < R and S ≈ R, respectively. The previous studies of juniper essential oil samples, collected from different locations, showed that the ratio of α-pinene enantiomers varies greatly. It was reported that J. communis cones, collected from different populations in Finland, Lithuania and the northern part of Poland, accumulated more (1R)-(+)-α-pinene than (1S)-(−)-α-pinene [16,22,23]. (1S)-(−)-α-Pinene of 100% enantiomeric purity was detected only in 1.8% of J. communis trees growing in Lithuania [24]. The enantiomeric ratio of α-pinene can vary greatly depending on the origin of the material, place of cultivation and the part of the plant (cones or leaves) [21], however it remains very similar both in unripe and ripe cones, i. e. it not differed significantly in the cones during their ripening period [28]. Therefore the special interest to study juniper oils with presence of various ratios of enantiomers of this monoterpene.

3.1. Composition of isomeric and enantiomeric fractions of pinene in essential oils of Juniperus communis The percentage of α-pinene was very similar and amounted more than half of total content of essential oils in both investigated samples: 62.11% and 59.62% in the sample no.1 and no.2, respectively. Meanwhile, the percentages of other isomer were very low in both essential oils: the percentage of β-pinene was 52 and 24 times less in the sample no.1 and no.2, respectively, in comparison with α-pinene, and amounted only 2–4% of total pinene fraction (Table 1). α-Pinene was found as the main compound of essential oils of cones of J. communis growing in Poland [23], Lithuania [14,24], Italy [13], Algeria [28]. The enantiomeric composition of α-pinene fraction very differed in the investigated essential oils of J. communis: the ratio of (1S)-(−) and

Table 1 Isomeric composition of pinene and enantiomeric composition of α-pinene fraction in Juniperus communis investigated essential oils. Essential oil of Juniperus communis

No. 1 (S < R) No. 2 (S ≈ R)

Isomeric composition of pinene fraction (%)

Enantiomeric composition of α-pinene fraction (%)

α-Pinene

β-Pinene

(1S)-(−)

(1R)-(+)

98 96

2 4

13 41

87 59

3.2. Activity of essential oil of Juniperus communis and natural origin αpinene with different enantiomeric composition on different microorganisms The efficiency of α-pinene rich J. communis essential oils on investigated Candida yeasts and dermatophytes of genus Trichophyton was stronger than on bacteria and Aspergillus fungi (Table 2). The large scale of experiments in vitro also demonstrated that juniper oil with high 22

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Table 2 Minimum inhibitory concentrations (MICs) and minimum bactericidal/fungicidal concentrations (MBCs/MFCs) of Juniperus communis essential oil and isolated αpinene fraction with diffrent enantiomeric compositions against bacteria, yeasts, fungi, and dermatophytes. Microorganisms

Juniperus communis essential oil with different enantiomeric composition of α-pinene

Isolated pinene fraction with different enantiomeric composition of α-pinene

(1R)-(+)-α-pinene standard

(1S)-(−) < (1R)-(+) (essential oil No. 1)

(1S)-(−) ≈ (1R)-(+) (essential oil No. 2)

(1S)-(−) < (1R)-(+)

(1S)-(−) ≈ (1R)-(+)

MIC

MBC/MFC

MIC

MBC/MFC

MIC

MBC/MFC

MIC

MBC/MFC

MIC

MBC/MFC

0.63

2.5

0.63

2.5

0.2

0.2

0.8

3.13

0.2

3.13

0.16

0.63

0.04

0.63

0.01

0.78

0.2

3.13

0.01

3.13

0.01

0.04

0.01

0.04

0.01

0.2

0.2

0.2

0.0002

0.0007

0.04

2.5

0.04

2.5

0.01

0.2

0.05

3.13

0.0002

0.2

0.16

2.5

0.04

2.5

0.04

0.63

0.01

2.5

0.01

0.63

0.16

0.63

0.04

2.5

0.16

0.63

0.002

0.63

0.01

0.01

0.04

2.5

0.01

0.63

0.16

0.16

0.04

0.04

0.04

0.04

0.04

0.63

0.04

0.16

0.16

0.16

0.01

0.01

0.04

0.04

% Escherichia coli ATCC 25922 Staphyloccocus aureus ATCC 29213 Candida albicans CBS 2730 Candida parapsilosis CBS 8836 Aspergillus flavus CBS 120264 Aspergillus fumigatus SC 6359 Trichophyton mentagrophytes SC 91427 Trichophyton rubrum ATCC 28188

content of α-pinene, β-pinene not possessed or possessed low antimicrobial activity against food spoilage bacteria such as Escherichia coli, Staphylococcus aureus, and Bacillus cereus [29], but high antimicrobial activity against fungi [12,30]. Results showed that MICs of essential oil with S ≈ R and S < R enantiomeric composition of α-pinene were the same on E. coli, Candida yeasts, and T. rubrum (Table 2). The resistance of yeast Candida albicans and bacteria E. coli to both essential oils of juniper was the least and highest, respectively, in comparison with other microorganisms. The literature data suggest that (+) enantiomeric forms of both pinene isomers able to inhibit the phospholipase and the esterase activities of bacteria, and that (+)-α-pinene was highly toxic to C. albicans (killing 100% of inoculum within 60 min) [19]. The gram-negative bacteria E. coli was alike sensitive to both essential oils without reference to the enantiomeric composition of αpinene in them; however, four-times lower concentration of essential oil S ≈ R in comparison to essential oil S < R was needed that to cause a growth inhibition effect of S. aureus, i. e. the bactericidal effect of essential oil with S ≈ R on this gram-positive bacteria was stronger (Table 2). However, the results of our experimental work showed that antimicrobial (both fungistatic and fungicidal) activity of J. communis essential oil with α-pinene enantiomeric ratio S ≈ R was higher on Trichophyton mentagrophytes compared to the J. communis essential oil with α-pinene enantiomeric ratio S < R. It all goes to demonstrate that the essential oils with different enantiomeric ratio of α-pinene have differently effects on microorganisms. The experiment with the plant origin α-pinene showed that αpinene with the enantiomeric composition S < R more strongly inhibited the growth of investigated bacteria and Candida yeasts, αpinene with the enantiomeric composition S ≈ R – the growth of investigated fungi Trichophyton and Aspergillus (Table 2). Also α-pinene S ≈ R stronger fungicidally affected on all investigated species of dermatophytes: the fungicidal concentration of α-pinene S ≈ R was 4–16 time lower than α-pinene S < R. The results of our experimental work showed that (1S)-(−)-α-pinene analytical standard had no antimicrobial effect on any of the tested microorganism (therefore only the results of (1R)-(+)-α-pinene analytical standard are given in Table 2). The agar diffusion test also showed that only the positive enantiomers of α-pinene and β-pinene exhibited a bactericidal effect against

methicillin-resistant S. aureus (MRSA), pathogenic yeasts C. albicans and Cryptococcus neoformans and fungi Rhizopus oryzae [19]. Therefore, stronger fungicidal effect of α-pinene S ≈ R on cells of both Trichophyton rubrum and T. mentagrophytes in comparison with α-pinene S < R could be explained by synergetic influence of S enantiomer on R enantiomer. Minimum bactericidal concentration of (1R)-(+)-α-pinene analytical standard was less effective on E. coli and S. aureus (Table 2). α-Pinene S ≈ R bactericidal activity on these bacteria was like pure (1R)-(+)-α-pinene, i. e. the marked increase in S enantiomer and decrease in R enantiomer did not effect on bacteria; however, the low percentage of S enantiomer in α-pinene fraction acted synergistically: the bactericidal effect of α-pinene S < R was 4–16 time higher in comparison with α-pinene S ≈ R and analytical standard of (1R)(+)-α-pinene (Table 2). The minimum inhibitory concentration (excluding both dermatophytes, C. albicans and A. fumigatus) of pure natural α-pinene with enantiomeric composition S < R on investigated microorganisms was more effective than the activity of essential oil with S < R enantiomeric composition of this monoterpene, i. e. it demonstrate the significant antimicrobial impact of α-pinene with R enantiomeric form (Table 2). Meanwhile stronger effect of essential oil with S < R enantiomeric composition of α-pinene on mentioned exceptions (A. fumigatus, C. albicans and both dermatophytes) could demonstrate likely synergy of this enantiomeric form with other compounds of essential oil. The lower MIC and MFC of essential oil with S ≈ R enantiomeric composition of α-pinene required for inhibitory effect against microorganisms in comparison with S ≈ R α-pinene can demonstrate synergy of S enantiomeric form of α-pinene with other compounds of essential oil (Table 2). Although α-pinene is main and dominant compound, essential oils of juniper are composed of many chemical compounds (belonged to aldehydes, ketones, alcohols, esters, ethers, hydrocarbons): to 149 chemical compounds were identified in J. communis growing wild in Lithuania [15]. Though in some cases, the bioactivities of essential oils are closely related with the activity of the main components of the essential oils [31], however different chemical components of essential oil can interact to either reduce or increase antimicrobial efficacy [7]. For example, the using of checkerboard method showed that the combination of α-pinene and limonene demonstrated 23

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the synergistic anti-yeast activities, while the combination of α-pinene with linalool resulted in an additive effect [32].

[13]

4. Conclusion [14]

The same amount of α-pinene in essential oil, but different its enantiomeric composition, can have diverse antimicriobial potential due different specific interactions with other chemical compounds of essential oil. Therefore, it is very important to determine and present the enantiomeric composition of those compounds, which are characterized by enantiomerisation and form a significant part of essential oils, during the course of research of biological activities of natural plant products (essential oils and other) and their isolated compounds. The authors of manuscript not have any conflicts of interest.

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Declarations of interest

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