https://orcid.org/0000-0002-8078-8664
Figures
Abstract
Background
The diversity found in the chemical compounds of a single species in different regions results in different biologic characteristics which can be considered as a strong source for identifying new chemotypes. Hymenocrater incanus Bunge and Dracocephalum kotschyi Boiss. are exclusive species of the Lamiaceae family which grow in the western and central habitats of Iran. This study was designed and carried out to determine the yield, identify the chemical compounds, and evaluate the antimicrobial characteristics of the essential oil (EO) of these two species in Iran for the first time.
Methods
The flowering twigs of the species D. kotschyi and H. incanus were collected from the villages of Totmach and Kamu in Isfahan province respectively, in May 2019. The EO of these plants was extracted and separated using the water distillation method, utilizing the Clevenger device. The EO compounds were analyzed using a gas chromatograph coupled with a mass spectrometer (GC-MS). The evaluation of antimicrobial characteristics was carried out by determining the growth inhibition zone implementing the Agar method, the minimum inhibition concentration (MIC), and the minimum bactericidal/fungicidal concentration (MFC/MBC) utilizing liquid dilution culture.
Results
The results indicated that the highest yield belonged to the EO of D. kotschyi at %2.6 (w/w). In this EO there were citral (%25.44), neral (%20.87), α-pinene (%14.48), trans-geranic acid methyl ester (%9.74), and D-limonene (%6.87). Moreover, H. incanus had the dominant compounds (-)-Spathulenol (%12.61), caryophyllene (%10.00), linolenic acid (%8.54), 1,8-cineole (%5.95), palmitic acid (%5.35), and α-cadinol (%5.17). The largest diameter of growth inhibition zone belonged to the H. incanus EO against the Gram-positive bacteria S. pyogenes (~17.67mm). The strongest inhibition activities in the form of growth inhibition diameter exhibited by the D. kotschyi EO were against the Gram-negative bacteria S. paratyphi-A serotype (~ 12 mm), K. pneumoniae, and Sh. dysenteriae (~ 11 mm) which was significant compared to the Gram-positive rifampin (~ 8 mm).
Citation: Ghavam M (2022) A GC-MC analysis of chemical compounds and identification of the antibacterial characteristics of the essential oil of two species exclusive to Iranian habitats: New chemotypes. PLoS ONE 17(10): e0273987. https://doi.org/10.1371/journal.pone.0273987
Editor: Guadalupe Virginia Nevárez-Moorillón, Universidad Autonoma de Chihuahua, MEXICO
Received: May 27, 2022; Accepted: August 19, 2022; Published: October 6, 2022
Copyright: © 2022 Mansureh Ghavam. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the article and its Supporting Information files.
Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
List of abbreviations: EOs, Essential oils; GC-MS, Gas chromatography–mass spectrometry; μg, Microgram; mL, Miligram; DMSO, dimethyl-sulfoxide; MFC, Minimum Fungicidal Concentration; MBC, Minimum Bactericidal Concentration; MIC, Minimal inhibitory concentrations; RI, Retention Index
1. Introduction
Due to the excessive prescription of antibiotics and the increase in antibiotic resistance in bacteria the search for an alternative has become vital. Therefore, extensive research has been carried out about the potential use of bactericidal compounds in the EO of plants to control and treat illnesses [1, 2]. The use of Eos in pharmaceutical and food industries, alternative medicine, and herbal treatments are based on their antimicrobial effects [3, 4]. It has been proven that many Eos possess antimicrobial effects which makes them an environmentally friendly alternative to eliminate harmful bacteria and microbes [5, 6]. Researchers have proven that sometimes natural EOs have a better performance than artificial medicine. EOs have various mechanisms to fight against microorganisms such as preventing the building of cell walls and nucleic acids, inhibiting protein synthesis, and changing the cell membrane performance [7, 8]. The hydrophobe characteristic of the EOs allows them to penetrate the lipids in the cell membrane and increase its penetrability, disrupting all vital activities related to the cell membrane, the drainage of ions, vital compounds, and eventually apoptosis [9].
Nowadays, research on medicinal plants has been prioritized to facilitate the combination of various compounds to reach diverse biologic effects [10, 11]. Previous research has shown that the EO of many plants in the Lamiaceae family contains compounds of strong antimicrobial effects [12–15]. Of this family, the Hymenocrater type has 12 species in the world among which, 9 are bushes with beautiful colorful which grow in Iran. These plants grow in mountainous areas and 5 of these species are exclusive to Iran, with limited dispersal [16–18]. The species of this type are called “Arvaneh” flower or “Sheikh Ali” medicine in Iran and are used for culinary and medical purposes. These are used traditionally in the treatment of respiratory and heart diseases, skin allergies, scars, and as a nerve tonic, anti-inflammatory, air freshener, and mosquito repellant [19, 20]. In previous studies, various biologic activities have been reported for different species of Hymenocrater such as anti-oxidant [21, 22], anti-microbial [21, 23], anti-microbial [24], larvicidal [25], anti-diabetes, anti-Alzheimer’s, anti-obesity, and skin-repairing [26] effects. The Hymenocrater incanus Bunge species is an exclusive one in Iran which can be found in the west, south, and parts of central Iran [16]. The dominant compounds in the EO of this species are recorded as β-caryophyllene, 1,8-cineole, α-pinene, and β–pinene [27]. On the biologic activities of the H. incanus EO, there have only been reports of anti-microbial effects on some micro-organisms [28].
Dracocephalum is another type from the Lamiaceae family which has 186 different species throughout the world and 11 in Iran [17]. Five of these species are native to Iran and scattered in the north, west, east, and center of Iran [16]. The species of this type are called "Badarshaboo" in Farsi [29] Dracocephalum is a source of terpenoids such as Luteolin, Apigenin, Oleic acid, Ursolic acid, Neral, and Geraniol [30]. The EO of the plants of this type possess anti-oxidant, anti-bacterial, anti-bacterial, and antiseptic activities; they are also used to treat stomachache and bloating [31]. Dracocephalum kotschyi Boiss. is a species exclusive to the north, west, and center of Iran [32]. This plant is used for treating rheumatic pain, muscle building, wound healing, reducing blood fat, toning the immune system, and is regarded to have anticancer and anti-microbial effects in traditional medicine. The aroma and taste of the EO of these plants make them useful in many industries such as cosmetics, perfume manufacturing, drinks, ice-cream making, confectionery, food industries, etc. [33, 34]. The EO of this species is traditionally used as an anti-spasm agent [35]. Ethnobotanic monitoring reports in Iran indicate that this plant is known as “Semsa’” in Lorestan province and its leaves are traditionally used in cooking meat and fish, processing dairy products [41] (Ashrafi et al., 2017), and used as an antipyretic and sedative as well [36]. In Zanjan province, this plant is known as “Akhbash” and its leaves are infused to treat rheumatism [37]. Studies have shown that the EO of this species has pain-killing [38], antinociceptive [33], anti-spasm [39], anti-toxoplasma [40], anti-microbial [12] effects. The most important reported compounds of the EO of this species are monoterpenes such as α-pinene, limonene, geraniol, neral, and citral [29, 34, 39, 41–46] (7;; 5;. Plant EOs are affected by various factors such as genetics, evolution, environmental conditions, geographic changes, physiologic factors, and harvest time [44, 47]. Thus, plants that vary genetically and climatically and grow in different habitats can be considered strong sources to identify new chemotypes [28]. The difference in the chemical compounds in the EO of a species in various locations results in different anti-microbial activities [48].
Considering the traditional usage and the reported anti-microbial activities of the species of Hymenocrater and Dracocephalum types, and regarding the variety in the chemical compounds reported for D. kotschyi and H. incanus; it seems important to identify the chemotypes of these two species in various areas. Therefore, the present study was designed and carried out to identify the chemical compounds and evaluate and compare the antimicrobial activity of the EO of these two species for the first time in the natural habitats of Isfahan province in Iran.
2. Materials and methods
2.1. Species collection and identification
Initially, the natural habitats for D. kotschyi and H. incanus species were identified to be Totmach village (N 33˚ 41ʹ15” and E 51˚ 37ʹ 11” and 2077 m elevation) and Kamu village (N 33˚ 39ʹ42ʺ and E 51˚ 15ʹ 45ʺ and 2942 m elevation) in Isfahan province, Iran. Then, the flowering twigs of the species under study were collected randomly from various bases (70 bases in each spot) in May 2019. The specimens were moved to the laboratory after harvest and were spread on flat clean surfaces to dry. These species were identified and verified by a botanist and are recorded and kept under codes 1016 and 1017 in the herbarium of the Faculty of Natural Resources and Geoscience of the University of Kashan.
2.2. EO extraction and yield
Firstly, 100 g of the dry plant material of each harvest spot was ground to 1 cm pieces by an electric grinder. The plant materials were put into a 2-liter flask along with distilled water. The flask was coupled to a Clevenger device and after 2 hours the EO was extracted by the water distillation method. The EO gathered in the output pipes was separated using n-pentane. After that, the EO was completely separated from the water and purified using sodium sulphate. The extracted essence was poured into a dark and closed container and kept in a freezer (4°C). To determine the yield of the EO, the humidity percentage was calculated. This stage was repeated three times for the plant materials of each spot and the yield was reported as mean ± standard deviation [49].
2.3. Determining the chemical compounds of the EO
To determine the compounds of the EO a gas chromatograph connected to a mass spectrometer (GC-MS) model Agilent N-5973 made in the US, with an HP-5MS (%5 methyl phenyl siloxane, 30 m length, internal diameter 0.25 mm, absorbing material thickness 0.25 μm) was utilized. Regarding open temperature programming; first, the temperature reached 60°C in 5 mins. After that, the temperature was increased gradually with a 3°C/min rate until it reached 246°C. Helium was used as the carrier gas with a flow velocity of 1.5 mL/min. The ionization voltage was 70 eVs. The volume of the injected specimen was 1 μl with a 1.50 split and a detector temperature of 250°C. To calculate the retention index (RI) for the peaks, a mixture of aliphatic hydrocarbons (C8-C21) was injected into the GC system as per the analytical conditions mentioned above. The calculation and identification of the EO compounds were carried out using the linear retention indices and comparing them to the indices in references [50], and with the help of the mass spectrums of standard compounds and the data available in e-libraries.
2.4. Evaluating the anti-microbial activities of the EO
2.4.1. Supply and culture of microbial variants.
To evaluate the antimicrobial activity of the Eos, 12 standard microbial strains were obtained from the Iranian Research Organization for Science and Technology. These variants included four Gram-positive bacteria- Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 29737), Staphylococcus epidermidis (CIP 81.55), and Streptococcus pyogenes (ATCC 19615)- five Gram-negative bacteria—Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 10031), Pseudomonas aeruginosa (ATCC 27853), Salmonella paratyphi-A serotype (ATCC 5702), and Shigella dysenteriae (PTCC 1188)-, two fungal variants- Aspergillus brasiliensis (ATCC 16404), and Aspergillus niger (ATCC 9029)-, and a yeast—candida Albicans (ATCC 16404). The bacterial variants were cultured in an Agar nutrient medium, whereas the fungi and yeast variants were cultured in the Sabouraud Dextrose Agar medium.
2.4.2. Determining the growth inhibition zone by the Agar diffusion method.
Various EOs were dissolved in Dimethyl sulfoxide (DMSO) to the concentration of 300 μg/mL. Plates of Mueller-Hinton Agar growth medium were supplied for the bacteria and Sabouraud Dextrose Agar for the fungi and the yeast. Microbial suspensions were prepared from the 24 h culture of various microbial variants with 0.5 McFarland turbidity, and 100 μl of each was cultured in similar growth media conditions. Utilizing Pasteur Pipettes, wells were made in the growth media with uniform distance and a 6 mm diameter. 10 μl EO was added to each well. To determine the sensitivity of the variants, the antibiotics rifampin and gentamicin (for the bacteria) and nystatin (for the fungi and the yeast) were used as a positive control and DMSO as a negative control. The plates impregnated with bacterial variants were incubated for 24 h in 37°C, plates impregnated with fungi variants for 72 h in 30°C and plates impregnated with the yeast for 48 h in 30°C in an incubator. Measuring the growth inhibition zone diameter was carried out using an antibiogram (in mm) [51] (Cockerill et al., n.d.). The tests were repeated for each EO sample and each variant, three times, and the growth inhibition zone diameter was reported in mean ± standard deviation.
2.4.3. Determining the Minimum Inhibitory Concentration (MIC), and the Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) with dilution in liquid growth media.
A specific amount of every EO sample was weighed and the primary stock was prepared with a 4000 μg concentration. Then, concentrations of 1000, 2000, 500, 250, 125, 62.5 μg/mL were prepared from the primary concentration. A 96-well sterile microplate was utilized, and 95 μl of the broth growth medium, 5 μl of microbial suspension with 0.5McFarland, and 100 μl of each EO concentration were added to each microplate well. As a negative control, the growth medium was used instead of EOs, and as a positive control, antibiotic powders of gentamicin and rifampin (for the bacteria) and nystatin antibiotic powder (for the fungi and the yeast) were used instead of the EOs. The plates impregnated with bacterial variants were incubated at 37°C for 24 h, and the ones impregnated with yeast and fungi at 30°C for 48 and 72 h in an incubator. MIC was determined with regards to the turbidity and the change in the color in each of the microplate wells.
The agar dilution assay was used to determine the MICs for the fungal strains based on the protocol introduced by of [52]. The suitable amounts of EO at different concentrations (2000, 1000, 500, 250, 125, 62.5, 31.25 and 15.63 μg/mL) were added to SDA containing 50% (v/v) Tween 20. Nystatin powder was used as the positive control, and the negative control was the plate with SDA containing 50% (v/v) Tween 20 without any EO. The culture media were spot inoculated with 4 ml of spores (104 spores /mL). The inoculated plates were incubated at 30°C for 72 h, the test was performed in triplicate for each essential oil, and the minimal concentration of the essential oil that inhibited the growth of the fungi was reported as the MIC.
After a 24 h heating period, 5 μl of each microplate well with no growth was injected into an agar nutrient medium to be incubated at 37°C for 24 h. Each concentration that did not exhibit growth had killed the microbe variant and was designated as the MFC/MBC [51] (Cockerill et al., n.d.). The tests were repeated three times for each EO and each variant.
3. Results and discussion
3.1. EO yield
The D. kotschyi EO (pale yellow) with a %2.6 (w/w) yield was significantly different from the H. incanus EO (dark yellow) with a %0.1 (w/w) yield. In previous studies, the highest yield from D. kotschyi was at %1.7 from Khansar area [43], %0.6 from Lorestan province [41], %0.45 from Zanjan [29], %0.24 from Fereydounshahr [42], and %0.2 from Daran [12]. Moreover, the yield of H. incanus was reported to be %0.6 in the Abadeh region [27] and %0.02 in the Daran area [28]. These quantities for medicinal herbs vary according to habitat and region because the metabolic activity of the plants fluctuates under the influence of environmental factors [53].
3.2. EO compounds
The results of the analyses on the EOs revealed the existence of 54 different chemical compounds in the EO of the two species under study (Table 1) which the GC-MS chromatogram depicts in Figs 1 and 2. 36 of these compounds belonged to the H. incanus EO which is in accordance to the results of [27] with 35 compounds, and contradicts the results of [12] with 25. 21 various compounds were detected in the D. kotschyi EO which is in agreement with the findings of [43] with20 compounds, [12] with 21 compounds, and [42] with 23. Some previous studies reported the D. kotschyi EO compounds to be more than 40 which is in contrast with our findings [39, 41, 44].
Oxygenated monoterpenes constituted %58.62 of the D. kotschyi EO compounds which agrees with the findings of [28, 43, 44]. The EO of H. incanus contained %31.13 sesquiterpenes hydrocarbons as the dominant group of chemical compounds. [28] found nonterpenoids (others) as the dominant compounds of this EO at %48.98 which contradicts our findings. The diversity of phytochemical compounds in various chemotypes is impacted by factors such as environmental, soil, and seasonal conditions [54].
The results of variance analysis indicated that a significant difference existed among the means of the amounts of various components from the EOs under study (P≤0.01). The dominant and main compounds in the EO of D. kotschyi were citral (%25.44), neral (%20.87), α-pinene (%14.48), trans-geranic acid methyl ester (%9.74), and D-limonene (%6.87). In some previous studies, citral at %12.08 [41] and %19.63–12.49 [55] was reported to be the primary compound of D. kotschyi which agrees with our findings. Moreover, previous studies reported neral as the third main compound in the EO of this species at %20.9 in Mazandaran province, %11.58 in Ardebil province, and %11.25 in Isfahan province [28, 44, 56]. The α-pinene compound in the EO of this species was reported by [28, 41] to be the dominant compound at %10.43 and %13.66 respectively, which contrasts our present findings. Trans-Geranic acid methyl ester has never been reported in previous studies on this plant, which makes our study the first to report the presence of these compounds in this EO; this could be the result of chemotypical variations. Some previous studies reported D-limonene as the second dominant compound for this EO at %19.96 [43], and %46.79 [29]. The reason for the difference between our findings and those of previous studies can be due to ecologic conditions, environmental conditions, the growth stage of the plant, harvest time, EO extraction methods, or even storage conditions [57].
The dominant compounds of the H. incanus EO were (-)-Spathulenol (%12.61), caryophyllene (%10.00), linolenic acid (%8.54), 1,8-cineole (%5.95), palmitic acid (%5.35), and α-cadinol (%5.17). (-)-spathulenol has been reported as the stereoisomer spathulenol in previous studies at %2.7 [27]. Thus, the existence and dominance of this compound in this EO in our study is a specific characteristic that has not been reported before. In previous reports on the compounds of the EO of this species, caryophyllene was mentioned as the dominant compound (%17.6), and as a peripheral compound (%3.68) [27, 28]. The existence of acidic compounds such as oleic acid (%23.53), and palmitic acid (%28.23) as the second and third dominant compounds of this EO was previously reported [28]. The compound 1,8-cineole was reported as the first compound at %16.9 by [27], and as a peripheral compound at %0.22 by [28] which contradicts the findings of the present study. Similarly, [58] reported the fifth dominant compound of this EO to be α-cadinol at %7.66. The difference in type and quantity of the chemical compounds in the EO indicates the unique properties of the species in the area under study which can result from a difference in the environmental conditions of the habitat and imply the existence of a chemotype of this species [28, 59].
3.3. Antimicrobial activity
The ANOVA results showed that there is a significant difference between the diameter of the growth inhibition zone for the EOs under study and the Gram-positive antibiotics against the microbial variants (P≤0.01) (Table 2). The biggest growth inhibition zone diameter belonged to the H. incanus EO against the Gram-positive bacteria S. pyogenes (~17.67 mm) which can be called an acceptable antibacterial activity compared to the antibiotics rifampin (~21 mm) and gentamicin (~ 32 mm). These bacteria are responsible for the most common bacterial pharyngitis [60], scarlet fever, and impetigo. The present study is the first report of the inhibitory power of the H. incanus EO against S. pyogenes and can introduce a potential natural tool against this bacteria. The biological activity of the Eos depends on their chemical compounds [61]. Due to the hydrophobic nature of the effective compounds in Eos, they cannot penetrate and access the active points inside Gram-negative bacteria. Therefore, Gram-positive bacteria usually show more vulnerability to these Eos [62]. The dominance of sesquiterpenes such as (-)-spathulenol, caryophyllene, and α-cadinol, the monoterpene 1,8-cineole, and acidic compounds like linolenic acid and palmitic acid seems to be the main reason for this antibacterial activity. [63] have also attributed the antimicrobial activity of the H. incanus EO to the dominance of oxygenated sesquiterpenes. The antibacterial activity of spathulenol has been proven [64]. Similarly, [65] has reported that the antibacterial activity of the H. longiflorus was due to the marginal or rare existence of antibacterial compounds, specifically 1,8-cineole. The antimicrobial activity of caryophyllene against Gram-positive bacteria has been proven [66]. [67] have attributed the strong antibacterial activity of the Calendula officinalis L. to the impressive amounts of α-cadinol. The antimicrobial activity of the acidic compounds against many microorganisms has been proven [28, 68]. Moreover, the synergy resulting from the diversity among the main and peripheral EO compounds in their antimicrobial activities must be considered [28]. Therefore, compounds with lower percentages such as α-pinene, β–pinene, linalool, cis-β-farnesene, α-humulene, δ-cadinene, and phytol can be considered as other effective factors in the antibacterial activity. In many studies, the activity against S. pyogenes is attributed to the presence of α-pinene and linalool in the EOs [15]. Strong antibacterial activity is reported for phytol [69]. The antibacterial and anti-biofilm effects of α-humulene extracted from plants have been proven [70]. The inhibitory activity of β–pinene against some Streptococcus variants has been recorded.
The strongest inhibitory activity of the D. kotschyi EO was against the Gram-negative bacteria S. paratyphi-A serotype with the growth inhibition zone diameter of ~12 mm, comparative to rifampin (~8 mm) and gentamicin (~18 mm). Similarly, [41] reported a growth inhibition zone diameter of 30 mm for the D. kotschyi EO. Salmonellas cause typhoid fever, gastroenteritis, and sepsis in humans and can be contracted by direct contact, or food and water contaminated by human or animal excrement [71]. The difference in the antibacterial activity of the EO of a species from various habitats results from the effect of the geographical location of the plant on the type and percentage of its phytochemical compounds [1]. This strong activity seems to be mainly due to the dominance of oxygenated monoterpenes such as citral and neral, hydrocarbon monoterpenes like α-pinene and D-limonene, and acidic compound trans-geranic acid methyl ester. The molecular structure of terpenes makes them hydrophobe which enables them to precipitate on the lipophilic plasma membrane of microorganisms. This increases the permeability of the membrane which in turn causes the loss of vital electrolytes [72]. The antibacterial effects of citral and neral are proven [73]. The inhibitory effect of α-pinene has been reported against some salmonella variants [74]. Moreover, minor and peripheral compounds such as trans-β-ocimene, linalool, β–myrcene, geraniol, germacrene D, and geranyl acetate could be effective in this activity. The inhibitory effect of myrcene [74], linalool [75]. and EOs containing sabinene and trans-β-ocimene [76] against some Salmonella variants has been recorded.
Another strong inhibitory activity of the D. kotschyi EO was producing a growth inhibition zone diameter of about 11 mm against Gram-negative bacteria K. pneumoniae and Sh. dysenteriae which was extremely significant and impressive compared to the positive controls rifampin (~8 mm) and gentamicin (~17 mm). In previous studies the growth inhibition zone diameter of this EO against K. pneumoniae was reported to be about 10 mm by [52], 18 mm by [41], and 10 mm by [46]. Furthermore, the growth inhibition zone diameter of this EO against Sh. dysenteriae has been reported to be about (9.50±0.50mm) [58]. Klebsiella pneumoniae is an opportunistic bacterium in clinical settings and could cause nosocomial infections such as ureteral, respiratory, and blood flow infections, especially in ICU patients [77]. There have been reports of inhibitory activities against K. pneumoniae for geraniol [78], myrcene, and α-pinene [74], linalool [79], and Limonene [80]. Shigella is one of the most important causes of intestine infections and diarrhea [81]. Dysentery caused by Shigella is usually serious and can lead to death if not treated on time [82]. The effect of some compounds such as geraniol and α-pinene against some Shigella sp. has been proven [83, 84].
The D. kotschyi EO had a growth inhibition zone diameter of about 8 mm against the yeast C. albicans which was weaker compared to nystatin (~33 mm) which contradicts the results reached by [58] for this EO against C. albicans (16.33±0.58). C. albicans is an opportunistic fungal pathogen that dwells in the gut and urethra of %70 of humans, and %75 of women suffer from a Candida infection at least once in their lives. This yeast will turn into an opportunistic pathogen in immunodeficient patients, people with impaired immune systems, or even healthy people [85, 86] and cause reproductory, skin, mouth, and blood infections [6, 87]. [88] have reported strong activity for linalool and citral against C. albicans. (da Silva et al., 2008) [89, 90] (Leite et al., 2014) have shown significant in vitro activity for citral against C. albicans. A growth inhibition zone diameter of (~11 mm) against C. albicans was recorded for a pure compound of α-pinene. The inhibitory effect of the EOs containing germacrene D against this yeast has been reported [91] (Mevy et al., 2007). The anti-Candida effects of Limonene [80], linalool [92], and geraniol [93, 94] have been proven.
On the other hand, the findings of the minimum inhibitory or killing concentration using the dilution method in liquid growth media indicated that the value for MBC and MIC for D. kotschyi ad H. incanus EOs against all microorganisms under study fluctuated between 1000 and 4000 μg/mL which implies a weak performance compared to the positive control antibiotics (Table 3). The least value for minimum inhibitory concentration belongs to the D. kotschyi EO against S. pyogenes at 250 μg/mL which was quite weak compared to the positive controls rifampin and gentamicin (MIC = 0.975 μg/mL).
There has not been any report of the effect of this EO against S. pyogenes which makes our report the first to do so. It seems that the dominance of sesquiterpenes in the D. kotschyi EO has a vital role in this inhibitory activity compared to the H. incanus EO. Similarly, [58] deemed the dominance of sesquiterpenes as the main factor in the inhibitory activity of Rosa damascene Mill EO against this bacteria. The strong antimicrobial activity of sesquiterpenes is proven [95].
4. Conclusion
The results show that the D. kotschyi and H. incanus EOs had significant differences in yield, chemical compounds, and antimicrobial characteristics. The dominance of oxygenated sesquiterpene compounds in the D. kotschyi EO such as citral and neral and the acidic compound trans-geranic acid methyl ester for the first time caused antibacterial effects which were stronger than rifampin against Gram-negative bacteria S. paratyphi-A serotyp, K. pneumoniae, and Sh. dysenteriae. In the H. incanus EO, the dominance of hydrocarbon sesquiterpenes such as (-)-Spathulenol and caryophyllene, and acidic compounds like linolenic acid and palmitic acid are the unique attributes linked to the conditions of the growth media, causing the largest diameter of growth inhibition zone against Gram-positive bacteria, S. pyogenes. Therefore, the present study confirms the effects of habitat on the synthesis of chemical compounds in the EO of these two species as new chemotypes which can be a potential natural antibiotic against some infectious diseases. Nevertheless, more research for clinical applications is needed in the future.
References
- 1. Azizian Shermeh O., Taherizadeh M., Valizadeh M., & Zaboli A. (2018). Investigation of antioxidant and antimicrobial activities and phytochemical Compounds of essential oil and different extracts of Cymbopogon olivieri (Boiss.) Bor. from Sistan and Baluchestan province. Journal of Sabzevar University of Medical Sciences, 25(2), 279–292.
- 2. Panáček A., Kvitek L., Prucek R., Kolář M., Večeřová R., Pizúrová N., et al. (2006). Silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity. The Journal of Physical Chemistry B, 110(33), 16248–16253. pmid:16913750
- 3. Nascimento G. G. F., Locatelli J., Freitas P. C., & Silva G. L. (2000). Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazilian Journal of Microbiology, 31, 247–256.
- 4. Sadeghi E., Dargahi A., Mohammadi A., Asadi F., & Sahraee S. (2015). Antimicrobial effect of essential oils: a systematic review. Food Hygiene, 5(2 (18)), 1–26.
- 5. Herro E., & Jacob S. E. (2010). Mentha piperita (peppermint). Dermatitis, 21(6), 327–329. pmid:21144345
- 6. Heydari M., & Bagheri M. (2019). The Antimicrobial Effects of Hydro-Extract of Mentha Piperita Lamiaceae Essential Oil Nanoemulsion on Gram-negative Bacteria of Escherichia coli: A Laboratory Study. Journal of Rafsanjan University of Medical Sciences, 18(6), 515–528.
- 7. Conlon B. P., Nakayasu E. S., Fleck L. E., LaFleur M. D., Isabella V. M., Coleman K., et al. (2013). Activated ClpP kills persisters and eradicates a chronic biofilm infection. Nature, 503(7476), 365–370. pmid:24226776
- 8. Rahman M. A., & Bari M. A. (2013). Antibacterial activity of cell suspension cultures of Castor (Ricinus communis L. cv. Roktima). European Journal of Medicinal Plants, 65–77.
- 9. Smith-Palmer A., Stewart J., & Fyfe L. (2001). The potential application of plant essential oils as natural food preservatives in soft cheese. Food Microbiology, 18(4), 463–470.
- 10. Ranasinghe L., Jayawardena B., & Abeywickrama K. (2002). Fungicidal activity of essential oils of Cinnamomum zeylanicum (L.) and Syzygium aromaticum (L.) Merr et LM Perry against crown rot and anthracnose pathogens isolated from banana. Letters in Applied Microbiology, 35(3), 208–211.
- 11. Soleimani N., & Ebraze N. (2016). Evaluate anti-bacterial effects of cinnamomun verum and ferula gummosa essential oil on some pathogen gram positive and negative bacteria. New Cellular and Molecular Biotechnology Journal, 6(23), 87–94.
- 12. Ataee M., Hosseini H., Noori N., & Khanjari A. (2013). Effect of Zataria multiflora boiss. essential oil on growth curve and shigatoxin 2 production of enterohemorrhagic escherichia coli O157: H7. Journal of Medicinal Plants, 12(48), 62–71.
- 13. Ghavam M., Manca M. L., Manconi M., & Bacchetta G. (2020). Chemical composition and antimicrobial activity of essential oils obtained from leaves and flowers of Salvia hydrangea DC. ex Benth. Scientific Reports, 10(1), 1–10.
- 14. Mamadalieva N. Z., Youssef F. S., Ashour M. L., Akramov D. K., Sasmakov S. A., Ramazonov N. S., et al. (2021). A comparative study on chemical composition and antimicrobial activity of essential oils from three Phlomis species from Uzbekistan. Natural Product Research, 35(4), 696–701. pmid:30919659
- 15. Tabatabaei Yazdi F., Alizadeh Behbahani B., Heidari Sureshjani M., & Mortazavi S. A. (2014). The in vitro study of antimicrobial effect of Teucrium polium extract on infectious microorganisms. Avicenna Journal of Clinical Medicine, 21(1), 16–24.
- 16.
Jamzad Z. (2012). Flora of Iran: Lamiaceae. Tehran: Research Institute of Forests and Rangelands, 213–219.
- 17.
Mozaffarian V. (1996). A dictionary of Iranian plant names, Farhang Moaser. Tehran: Iran, 542–544.
- 18. Serpooshan F., Jamzad Z., Nejadsattari T., & Mehregan I. (2018). Molecular phylogenetics of Hymenocrater and allies (Lamiaceae): new insights from nrITS, plastid trnL intron and trnL‐F intergenic spacer DNA sequences. Nordic Journal of Botany, 36(1_2), njb–01600.
- 19. Al-Anee R. S. A., Sulaiman G. M., Al-Sammarrae K. W., Napolitano G., Bagnati R., Lania L., et al. (2015). Chemical characterization, antioxidant and cytotoxic activities of the methanolic extract of Hymenocrater longiflorus grown in Iraq. Zeitschrift Für Naturforschung C, 70(9–10), 227–235. pmid:26479342
- 20. Morteza-Semnani Katayoun, Ahadi H., & Hashemi Z. (2016). The genus Hymenocrater: a comprehensive review. Pharmaceutical Biology, 54(12), 3156–3163. pmid:27363483
- 21. Ahmadi F., Sadeghi S., Modarresi M., Abiri R., & Mikaeli A. (2010). Chemical composition, in vitro anti-microbial, antifungal and antioxidant activities of the essential oil and methanolic extract of Hymenocrater longiflorus Benth., of Iran. Food and Chemical Toxicology, 48(5), 1137–1144. pmid:20132856
- 22. Emrani S., Zhiani R., & Dolatabadi S. (2015). Identification of Chemical Compositions and Protective Effects of Essential Oil of Arvaneh (Hymenocrater platystegius) on Oxidative Stress Induced by H2O2 in PC12 Cells. The Neuroscience Journal of Shefaye Khatam, 3(3), 27–36.
- 23. Morteza-Semnani K, Saeedi M., & Akbarzadeh M. (2012). Chemical composition and antimicrobial activity of the essential oil of Hymenocrater calycinus (Boiss.) Benth. Journal of Essential Oil Bearing Plants, 15(5), 708–714.
- 24. Masoudi S., Rustaiyan A., Mohebat R., & Mosslemin M. H. (2012). Composition of the essential oils and antibacterial activities of Hymenocrater yazdianus, Stachys obtusicrena and Nepeta asterotricha three Labiatae herbs growing wild in Iran. Natural Product Communications, 7(1), 1934578X1200700138. pmid:22428263
- 25. Taran M., Karimi N., Abdi J., Sohailikhah Z., & Asadi N. (2013). Larvicidal effects of essential oil and methanolic extract of Hymenocarter longiflorus (Lamiaceae) against Echinococcus granulosus. Journal of Essential Oil Bearing Plants, 16(1), 85–91.
- 26. Bahadori S., Bahadori M. B., Zengin G., Maggi F., Dinparast L., & Aktumsek A. (2017). Chemical composition profile of the essential oil from Hymenocrater bituminous and its health functionality. International Journal of Food Properties, 20(sup1), S972–S980.
- 27. Mirza M., Ahmadi L., & Tayebi M. (2001). Volatile constituents of Hymenocrater incanus Bunge, an Iranian endemic species. Flavour and Fragrance Journal, 16(4), 239–240.
- 28. Ghavam M., Afzali A., & Manca M. L. (2021). Chemotype of damask rose with oleic acid (9 octadecenoic acid) and its antimicrobial effectiveness. Scientific Reports, 11(1), 1–7.
- 29. Khodaei M., Amanzadeh Y., Faramarzi M. A., & Hamedani M. P. (2018). Chemical analysis and anti-bacterial effect of essential oils from three different species of Dracocephalum in Iran. American Journal of Essential Oils and Natural Products, 6(3), 31–34.
- 30. Zeng Q., Jin H., Qin J., Fu J., Hu X., Liu J., et al. (2010). Chemical constituents of plants from the genus Dracocephalum. Chemistry & Biodiversity, 7(8), 1911–1929. pmid:20730957
- 31. Dastmalchi K., Dorman H. J. D., Koşar M., & Hiltunen R. (2007). Chemical composition and in vitro antioxidant evaluation of a water-soluble Moldavian balm (Dracocephalum moldavica L.) extract. LWT-Food Science and Technology, 40(2), 239–248.
- 32. Rechinger K.-H., & Druk A. (1982). Flora Iranica, Labiatae. Akademische Druck Verlagsantalt. Graze, 150–151.
- 33. Golshani S., Karamkhani F., Monsef-Esfehani H. R., & Abdollahi M. (2004). Antinociceptive effects of the essential oil of Dracocephalum kotschyi in the mouse writhing test. J Pharm Pharm Sci, 7(1), 76–79. pmid:15144738
- 34.
Saeidnia S., Gohari A. R., Ito M., Kiuchi F., & Honda G. (2005). Bioactive constituents from Dracocephalum subcapitatum (O. Kuntze) lipsky. Zeitschrift Für Naturforschung C, 60(1–2), 22–24. https://doi.org/10.1515/znc-2005-1-204 pmid:15787238
- 35. Omidbaigi R. (2005). Production and processing of medicinal plants. Beh-Nashr: Mashhad, 210–225.
- 36. Mehrnia M., & Hosseini Z. (2020). Ethnobotanical study of native medicinal plants of Aleshtar region (Lorestan).
- 37. Saadatpour M., Barani H., Abedi Sarvestani A., & Forouzeh M. R. (2017). Ethnobotanical study of Sojasrood medicinal plants (Zanjan Province). Journal of Medicinal Herbs, 8(3), 185–193.
- 38. Amirghofran Z., Azadbakht M., & Karimi M. H. (2000). Evaluation of the immunomodulatory effects of five herbal plants. Journal of Ethnopharmacology, 72(1–2), 167–172. pmid:10967468
- 39. Sadraei H., Asghari G., & Kasiri F. (2015). Comparison of antispasmodic effects of Dracocephalum kotschyi essential oil, limonene and α-terpineol. Research in Pharmaceutical Sciences, 10(2), 109. pmid:26487887
- 40. Khamesipour F., Razavi S. M., Hejazi S. H., & Ghanadian S. M. (2021). In vitro and in vivo Anti‐Toxoplasma activity of Dracocephalum kotschyi essential oil. Food Science & Nutrition, 9(1), 522–531. pmid:33473313
- 41. Ashrafi B., Ramak P., Ezatpour B., & Talei G. R. (2017). Investigation on chemical composition, antimicrobial, antioxidant, and cytotoxic properties of essential oil from Dracocephalum kotschyi Boiss. African Journal of Traditional, Complementary and Alternative Medicines, 14(3), 209–217.
- 42. Fallah S., Mouguee S., Rostaei M., Adavi Z., Lorigooini Z., & Shahbazi E. (2020). Productivity and essential oil quality of Dracocephalum kotschyi under organic and chemical fertilization conditions. Journal of Cleaner Production, 255, 120189.
- 43. Jalaei Z., Fattahi M., & Aramideh S. (2015). Allelopathic and insecticidal activities of essential oil of Dracocephalum kotschyi Boiss. from Iran: A new chemotype with highest limonene-10-al and limonene. Industrial Crops and Products, 73, 109–117.
- 44. Mirzania Foroogh, & Sonboli A. (2021). Chemical diversity of essential oil composition from five populations of Dracocephalum Kotschyi Boiss.
- 45. Moridi Farimani M., Mirzania F., Sonboli A., & Moghaddam F. M. (2017). Chemical composition and antibacterial activity of Dracocephalum kotschyi essential oil obtained by microwave extraction and hydrodistillation. International Journal of Food Properties, 20(sup1), 306–315.
- 46. Shakib P., Taherikalani M., & Ramazanzadeh R. (2018). Chemical composition, genotoxicity and antimicrobial activities of Dracocephalum kotschyi Boiss against OXA-48 producing Klebsiella pneumoniae Isolated from major hospitals of Kurdistan Province, Iran. Microbiology Research Journal International, 1–8.
- 47. Mirzania Foroogh, Sarrafi Y., & Farimani M. M. (2018). Comparison of chemical composition, antifungal antibacterial activities of two populations of Salvia macilenta Boiss. Essential oil. Rec. Nat. Prod, 12, 385–390.
- 48. Popović-Djordjević J., Cengiz M., Ozer M. S., & Sarikurkcu C. (2019). Calamintha incana: essential oil composition and biological activity. Industrial Crops and Products, 128, 162–166.
- 49. Azarnivand H., Ghavam Arabani M., Sefidkon F., & Tavili A. (2010). The effect of ecological characteristics on quality and quantity of the essential oils of Achillea millefolium L. subsp. Millefolium. Iranian Journal of Medicinal and Aromatic Plants Research, 25(4), 556–571.
- 50.
Adams R. P. (2007). Identification of essential oil components by gas chromatography/mass spectrometry (Vol. 456). Allured publishing corporation Carol Stream, IL.
- 51. Cockerill F. R., Wilkler M. A., Alder J., Dudley M. N., Eliopoulos G. M., Ferraro M. J., et al. Clinical and Laboratory Standards Institute (CLSI), 2012. Performance Standards for Antimicrobial Susceptibility Testing: Sixteenth Informational Supplement. M100–S20, 30(1).
- 52. Ghavam M (2021) Relationships of irrigation water and soil physical and chemical characteristics with yield, chemical composition and antimicrobial activity of Damask rose essential oil. PLoS ONE 16(4): e0249363. pmid:33861759
- 53. Yousefi M., Nazeri V., & Mirza M. (2015). Effects of environmental conditions on the quantity and quality of Salvia leriifolia Benth. essential oil. Iranian Journal of Medicinal and Aromatic Plants, 30(6).
- 54. Petropoulos S., Fernandes Â., Barros L., Ciric A., Sokovic M., & Ferreira I. C. F. R. (2018). Antimicrobial and antioxidant properties of various Greek garlic genotypes. Food Chemistry, 245, 7–12. pmid:29287429
- 55. Asghari G., & Keyhanfard N. (2014). Seasonal variation of mono-and sesquiterpenoid components in the essential oil of Dracocephalum kotschyi Boiss. Research Journal of Pharmacognosy, 1(4), 41–47.
- 56. Ebadollahi A. (2020). Terpene Rich Essential Oil of Dracocephalum kotschyi Boiss as Efficient Alternative to Synthetic Chemicals in Management of Callosobruchus maculatus Fabricius. Journal of Applied Sciences and Environmental Management, 24(3), 501–506.
- 57. Dadkhah A., Fatemi F., & Dini S. (2017). The protective effects of Iranian golpar (Heracleum persicum) essential oil in liver damages induced by CCl4 in wistar rats. Journal of Medicinal Plants, 16(61), 110–122.
- 58. Ghavam M., Afzali A., Manconi M., Bacchetta G., & Manca M. L. (2021). Variability in chemical composition and antimicrobial activity of essential oil of Rosa× damascena Herrm. from mountainous regions of Iran. Chemical and Biological Technologies in Agriculture, 8(1), 1–16.
- 59. Ramak P., Karimian V., & Siahmansour R. (2020). Comparison of the nutrients and chemical composition of Allium jesdianum Boiss & Buhse in the habitats and field. Iranian Journal of Horticultural Science, 51(1), 19–31.
- 60. Fox J. W., Marcon M. J., & Bonsu B. K. (2006). Diagnosis of streptococcal pharyngitis by detection of Streptococcus pyogenes in posterior pharyngeal versus oral cavity specimens. Journal of Clinical Microbiology, 44(7), 2593–2594. pmid:16825390
- 61. Asri Y., Sadeh Hoseinabad Ghaeini F., Vaziri A., & Akbarzadeh M. (2017). Evaluation of essential oil composition in three population of Hymenocrater calycinus (Boiss.) Benth. in different regions of Mazandaran province. Eco-Phytochemical Journal of Medicinal Plants, 4(4), 15–23.
- 62. Eghbal H., Mohammadi A., Mohammad Nejad Khiavi N., Ahmadi Sabegh M., & Jahani N. (2021). Comparison of the Antibacterial Properties of Essential Oils of Malva Sylvestris and Salvia Officinalis on Common Bacteria of Oral Infection with Chlorhexidine Mouthwash. Journal of Mashhad Dental School, 45(3), 217–229.
- 63. Ghavam M. In vitro biological potential of the essential oil of some aromatic species used in Iranian traditional medicine. Inflammopharmacol 30, 855–874 (2022). pmid:35322321
- 64. Taherkhani M., Masoudi S., Fatah Elahi R., Baradari T., & Rustayian A. (2012). Identify the compounds in the essential oils of two plants from the Apiaceae family, Reichenb Torilis leptophylla and Boiss Thecocarpus meifolius, and study their antibacterial properties. Iran. J. Chem. Chem. Eng, 31, 65–70.
- 65. Abu-Mejdad N. M. (2014). Antifungal activity of some plant extracts against two yeast isolates in vitro. Reasercch Journal of Pharmaceutical, Biological and Chemical Sciences, 5(2), 1992–1998.
- 66. Yoo H.-J., & Jwa S.-K. (2018). Inhibitory effects of β-caryophyllene on Streptococcus mutans biofilm. Archives of Oral Biology, 88, 42–46. pmid:29407750
- 67. Moghtader M., Salari H., Mozafari H., & Farahmand A. (2016). Evaluation the qualitative and quantitative essential oil of Calendula officinalis and its antibacterial effects. Cellular and Molecular Researches (Iranian Journal of Biology), 29(3), 331–339.
- 68. Mattana P., Rosa P., Poli J., Richards N., Daboit T., Scroferneker M. L., et al. (2014). Lipid profile and antimicrobial activity of microbial oils from 16 oleaginous yeasts isolated from artisanal cheese. Revista Brasileira de Biociências, 12(2), 121.
- 69. Prabha S. P., Karthik C., & Chandrika S. H. (2019). Phytol–A biosurfactant from the aquatic weed Hydrilla verticillata. Biocatalysis and Agricultural Biotechnology, 17, 736–742.
- 70. Jang H.-I., Rhee K.-J., & Eom Y.-B. (2020). Antibacterial and antibiofilm effects of α-humulene against Bacteroides fragilis. Canadian Journal of Microbiology, 66(6), 389–399. pmid:32073898
- 71. Tsen H.-Y., Lin J.-S., & Hsih H.-Y. (2002). Pulsed field gel electrophoresis for animal Salmonella enterica serovar Typhimurium isolates in Taiwan. Veterinary Microbiology, 87(1), 73–80. pmid:12079748
- 72. Bakkali F., Averbeck S., Averbeck D., & Idaomar M. (2008). Biological effects of essential oils–A review. Food and Chemical Toxicology, 46(2), 446–475. pmid:17996351
- 73. Saddiq A. A., & Khayyat S. A. (2010). Chemical and antimicrobial studies of monoterpene: Citral. Pesticide Biochemistry and Physiology, 98(1), 89–93.
- 74. Hmamouchi M., Hamamouchi J., Zouhdi M., & Bessiere J. M. (2001). Chemical and antimicrobial properties of essential oils of five Moroccan Pinaceae. Journal of Essential Oil Research, 13(4), 298–302.
- 75. Varia R. D., Patel J. H., Modi F. D., Vihol P. D., & Bhavsar S. K. (2020). In vitro and In vivo Antibacterial and Anti-inflammatory Properties of Linalool. Int. J. Curr. Microbiol. Appl. Sci, 9, 1481–1489.
- 76. Pirbalouti A. G., Hossayni I., & Shirmardi H.-A. (2013). Essential oil variation, antioxidant and antibacterial activity of mountain fennel (Zaravschanica membranacea (Boiss.) M. Pimen.). Industrial Crops and Products, 50, 443–448.
- 77. Paczosa M. K., & Mecsas J. (2016). Klebsiella pneumoniae: going on the offense with a strong defense. Microbiology and Molecular Biology Reviews, 80(3), 629–661. pmid:27307579
- 78. Ilić B. S., Kocić B. D., Ćirić V. M., Cvetković O. G., & Miladinović D. L. (2014). An in vitro synergistic interaction of combinations of Thymus glabrescens essential oil and its main constituents with chloramphenicol. The Scientific World Journal, 2014.
- 79. Yang S.-K., Yusoff K., Ajat M., Wee C.-Y., Yap P.-S.-X., Lim S.-H.-E., et al. (2021). Combinatorial Antimicrobial Efficacy and Mechanism of Linalool Against Clinically Relevant Klebsiella pneumoniae. Frontiers in Microbiology, 12, 382. pmid:33815320
- 80. Oumzil H., Ghoulami S., Rhajaoui M., Ilidrissi A., Fkih‐Tetouani S., Faid M., et al. (2002). Antibacterial and antifungal activity of essential oils of Mentha suaveolens. Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives, 16(8), 727–731. pmid:12458474
- 81. Olad G., Tavalaee M., Ebrahimi F., Salimian J., Nazarean S., & Rahimi A. A. (2011). Shigella dysentery stxA mutant (R170L-A231D-G234E) gene design and optimization of recombinant protein expression and purification. Journal of Shahrekord Uuniversity of Medical Sciences, 13.
- 82. Madanchi H., Honari H., Safaei S., & Sayadmanesh A. (2012). Fusion of cholera toxin B subunit (ctxB) with Shigella dysenteriae type I toxin B subunit (stxB), cloning and expression that in E. coli. Journal of Fasa University of Medical Sciences, 2(3), 135–141.
- 83. Bilia A. R., Santomauro F., Sacco C., Bergonzi M. C., & Donato R. (2014). Essential oil of Artemisia annua L.: an extraordinary component with numerous antimicrobial properties. Evidence-Based Complementary and Alternative Medicine, 2014. pmid:24799936
- 84. Mirza Z. R. M. H., Hasan T., Seidel V., & Yu J. (2018). Geraniol as a novel antivirulence agent against bacillary dysentery-causing Shigella sonnei. Virulence, 9(1), 450–455. pmid:29256316
- 85. Kabir M. A., Hussain M. A., & Ahmad Z. (2012). Candida albicans: a model organism for studying fungal pathogens. International Scholarly Research Notices, 2012.
- 86. Mirabadi M., Azadegam Ghomi H., & Didehdar M. (2018). Evaluation of antifungal effects of garlic essential oil (Alliumsstadium) in Candida albicans, Candida glabrata and Candida tropicalis in vitro. Applied Biology, 7(28), 11–19.
- 87. Shokri H., & Amri Saroukolaei S. (2016). Evaluation of Vacuolar Aspartic Proteinase (VAP) Gene Expression in Candida albicans Strains Isolated from Systemic Candidiasis Patients. Avicenna Journal of Clinical Medicine, 23(2), 103–109.
- 88. Zore G. B., Thakre A. D., Jadhav S., & Karuppayil S. M. (2011). Terpenoids inhibit Candida albicans growth by affecting membrane integrity and arrest of cell cycle. Phytomedicine, 18(13), 1181–1190. pmid:21596542
- 89. da Silva C. de B., Guterres S. S., Weisheimer V., & Schapoval E. E. S. (2008). Antifungal activity of the lemongrass oil and citral against Candida spp. Braz J Infect Dis, 12(1), 14.
- 90. Leite M. C. A., Bezerra A. P. de B., Sousa J. P. de, Guerra F. Q. S., & Lima E. de O. (2014). Evaluation of antifungal activity and mechanism of action of citral against Candida albicans. Evidence-Based Complementary and Alternative Medicine, 2014. pmid:25250053
- 91. Mevy J. P., Bessiere J. M., Dherbomez M., Millogo J., & Viano J. (2007). Chemical composition and some biological activities of the volatile oils of a chemotype of Lippia chevalieri Moldenke. Food Chemistry, 101(2), 682–685.
- 92. Kunduhoglu B. (2017). Anti-yeast activity of cinnamaldehyde, eugenol and linalool. World J Res Rev, 5(5), 32–34.
- 93. Kim E., & Park I.-K. (2012). Fumigant antifungal activity of Myrtaceae essential oils and constituents from Leptospermum petersonii against three Aspergillus species. Molecules, 17(9), 10459–10469. pmid:22945026
- 94. Sharma Y., Khan L. A., & Manzoor N. (2016). Anti-Candida activity of geraniol involves disruption of cell membrane integrity and function. Journal de Mycologie Medicale, 26(3), 244–254. pmid:27554866
- 95. Buckle J. (2015). Basic plant taxonomy, basic essential oil chemistry, extraction, biosynthesis, and analysis. Clinical Aromatherapy, 37–72.