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Green synthesis of copper nanoparticles using carvacrol extract and investigation of its antifungal properties: Candida albicans and Candida tropicalis, an in vitro studyRazieNaghdiTalaee1Emailrazieh_talaee@yahoo.com
MansourGhaderpoori2✉Emailmghaderpoori@gmail.com
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MohsenMohammadi3MohammadYarahmadi4Emailmohamadi419@yahoo.comEmailyarahmadimo13@gmail.com
BahramKamarehie2EmailB.kamarehie@gmail.com
FaramrzAzimi2
FatemehArbabi1Emailfatemeh.arbaby1478@gmail.com
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Student Research CommitteeLorestan University of Medical SciencesKhorramabadIran2
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Environmental Health Research CenterLorestan University of Medical SciencesKhorramabadIran3Department of Pharmacognosy and Pharmaceutical Biotechnology, Faculty of PharmacyLorestan University of Medical ScienceKhorramabadIran
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Department of Medical Parasitology and MycologyLorestan University of Medical SciencesKhorramabadIranRazie Naghdi Talaee1, Mansour Ghaderpoori2, Mohsen Mohammadi3, Mohammad Yarahmadi4, Bahram Kamarehie2, Faramrz Azimi2, Fatemeh Arbabi1
1. Student Research Committee, Lorestan University of Medical Sciences, Khorramabad, Iran (razieh_talaee@yahoo.com, fatemeh.arbaby1478@gmail.com).
2. Environmental Health Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran (mghaderpoori@gmail.com, B.kamarehie@gmail.com)
3. Department of Pharmacognosy and Pharmaceutical Biotechnology, Faculty of Pharmacy, Lorestan University of Medical Science, Khorramabad, Iran (mohamadi419@yahoo.com)
4. Department of Medical Parasitology and Mycology, Lorestan University of Medical Sciences, Khorramabad, Iran (yarahmadimo13@gmail.com).
Corresponding author: Mansour Ghaderpoori (mghaderpoori@gmail.com),
Abstract
The aim of this study was to investigate the antifungal effects of the combination of carvacrol and biosynthesized CuO NPs with the aqueous extract of Satureja khuzestanica plant against Candida albicans and Candida tropicalis fungi. Four different modes were used to investigate the effect of fungi inactivation, aqueous extract of Khuzestan savory plant alone, synthesizing CuO NPs green, Carvacrol 98%, and composition of green CuO NPs and carvacrol 98%. In order to reduce environmental degradation effects, a simple and green approach for preparation and synthesis of nanoparticles was used. Different concentration of substances produced was applied to investigate the antifungal effects on fungal pathogens namely Candida albicans and Candida tropicalis. In this study, Time-Kill Curve Assay were determined for the experiments. Also, the studied concentrations were 1/2MIC, MIC, 2MIC, 4MIC, and 8MIC. According to the obtained results, CuO NPs were spherical in shape and almost uniform in size, with an average diameter of 70 nm. The lowest concentration of MIC and MFC was observed in combined disinfectants Carvacrol 98%+CuO NPs. In this combination, Candida tropicalis, the concentration of MIC and MFC was equal to 31 ± 25 and 52.08 ± 18.04 µg/mL, respectively. Satureja khuzestanica + CuO NPs and Carvacrol 98% was strength in Candida tropicalis in all the investigated concentrations, e.g. MIC, 2MIC, and 4MIC.
Keywords:
Candida albicans
Candida tropicalis
Satureja khuzestanica
Green synthesis
Carvacrol
Introduction
It is estimated that 20–25% of hospitalized patients get hospital infections, and the cost of these patients is 2.9 times the other hospital costs. The costs due to the increase in the duration of the patient's hospitalization, laboratory costs and diagnostic studies increase the costs caused by hospital infections. Hospital infections are responsible for 20,000 direct deaths and 60,000 indirect deaths annually 1. According to numerous scientific reports, the prevalence of fungal infections around the world, the prevalence of systemic fungal infections, especially candidiasis, in patients with immune system defects is increasing 2, 3.
Nosocomial fungal infections are one of the important causes of death of patients hospitalized in health care environments, especially populations with immune system defects 4. It seems that nosocomial fungal infections will increase in the coming decades due to underlying factors 5. One of the predisposing factors for the occurrence of such infections is the widespread use of invasive treatment methods, such as stem cell transplantation and organ transplantation, chemotherapy, the use of immunosuppressive drugs, etc., which lead to the weakening of the immune system 6, 7.
A
Candidiasis refers to a group of diseases caused by different species of Candida. These fungal agents cause disease in humans and animals 1, 8. Pathogens causing such infections often include Candida, Aspergillus, Mucor and Fusarium species 9, 10. Candida has a wide pathogenicity in human and animal hosts. This fungus is the cause of 88% of hospital fungal infections and the fourth cause of blood infections, among all the factors that cause infection 11, 12. Today, more than 150 Candida species have been identified, some of which are known to be pathogenic. 65% of Candida species do not have the ability to grow at 37℃, while the ability to grow at this temperature is necessary for the pathogenicity of the infectious agent 1.The significant antimicrobial activity of many inorganic metal oxide nanoparticles, such as ZnO, CuO2, MgO, TiO2, and SiO2, and the selective toxicity potential that they show towards biological systems make them a suitable option in treatment, diagnosis, surgical devices and antimicrobial agents. Nanoparticles (NPs) are synthesized using different physical and chemical methods. In comparison with these methods, physical or chemical, green synthesis is one of the best methods for producing NPs in recent years 13. Among the advantages of the green synthesis method, we can mention the following: cost-effective, simple, use of lower temperature, use of non-toxic materials, in addition, it is compatible with medical and food applications 3, 14. The green synthesis method is being developed and compatible with the environment 15. Recently, with the increase in environmental problems, a lot of attention has been paid to the discussion of green chemistry, and the effort to synthesize NPs through environmentally friendly methods has increased greatly 16, 17. Green chemistry is a research or innovation method that includes an applied part for the design, development and efficient production of products that have the ability to minimize hazardous substances for health 18. The goal of new green technology projects is to minimize the potential risks of nano applications for humans and the environment. NPs resulting from chemical methods that are used today have caused many concerns due to the use of dangerous and toxic chemicals and the resulting environmental damage. Nano biotechnology is one of the most promising fields of science and nano technology in the new era 19. In recent years, the use of plant extracts for the preparation of metal nanoparticles has been proposed as an easy and suitable alternative to chemical and physical methods. In this method, plant extract is used as a reducing agent and coating for NPs 3, 16, 20. Various plants have been used for the synthesis of nanoparticles using the green synthesis method. NPs are synthesized using all plant parts such as stem, flower, fruit, leaf and skin 21, 22.
There are many biological methods for the synthesis of metal nanoparticles from bacteria and fungi, which also require sterile conditions and a timer to maintain the temperature of the culture medium. In recent years, the method of biosynthesis of metal nanoparticles (especially copper and silver nanoparticles) using plant extracts has attracted more attention than other methods due to its simplicity, low cost, high efficiency, non-toxicity and compatibility with the environment. The use of plants as sustainable and available resources in the preparation of biocompatible nanoparticles has been the focus of many researchers in recent years, and the advantages of this method include biocompatibility, cheapness, non-toxicity, and the production of nanoparticles with high purity 3. Due to its electrical, optical, catalytic properties, copper NPs have wide applications in medicine, antifungal and antibacterial 23. Copper oxide nanoparticles have antibacterial and antifungal properties. Although silver-based antibacterial agents show high antimicrobial activity and low toxicity, they are not very effective against fungi 24–26. Copper oxide nanoparticles can be a suitable candidate in this field due to its lower price, good antifungal ability and good stability. Copper nanoparticles are toxic to many microorganisms such as Escherichia coli bacteria, Staphylococcus aureus, Pseudomonas aeruginosa and non-toxic to animal cells 27. Until now, various plants have been used for the synthesis of NPs using the green synthesis method. NPs are synthesized using all plant parts such as stem, flower, fruit, leaf and skin 23, 28. The aim of this study was to investigate the antifungal effects of the combination of carvacrol and biosynthesized CuO NPs with the aqueous extract of Satureja khuzestanica plant against Candida albicans and Candida tropicalis fungi.
Materials and methods
Required laboratory materials
The materials required for the synthesis of CuO NPs and experiments in the mycology tests were CuSO4.5H2O, Satureja khuzestanica plant, Sabouraud Dextrose Broth (SDB), Sabouraud Dextrose Agar (SDA), ethanol (98%), Carvacrol (98%, C10H14O), Standard strain of Candida albicans fungus, Standard strain of Candida tropicalis fungus, and standard solution 0.5 McFarland. All material chemicals were used in the tests without further purification. All reagents and solvents were used as received from commercial suppliers without further purification.
Extraction of the aqueous extract of the savory plant
In this study, Satureja khuzestanica plant was obtained from Khorraman pharmaceutical company in Khorramabad Iran. In the first step, deionized water was used for cleaning and washing. In this study, the dried leaves and stems of the plant were used. Then, the plant was dried with an electric mill and the resulting powder was used for extraction. 10 g of the powder prepared from this plant was mixed with 100 mL of distilled water and kept at 40 ℃ for 60 min. Then, it was placed at a temperature of 95°C for 30 min for the purpose of temperature shock and extraction of the extract from the plant. The plant extract was filtered through Whatman No. 1 qualitative filter paper and centrifuged at 5000 rpm for 5 min to remove large particles. In order to prevent thermal damage, the filtered extract was kept at 4°C for the preparation of nanoparticles. The obtained extract is shown in Fig. 1 16, 20, 29.
Fig. 1
The obtained extract from Satureja khuzestanica
Synthesis of CuO NPs using the aqueous extract of Satureja khuzestanica
At first, 75 mL of the extract prepared in the previous step was added to the solution of 1 mM (100 mL) of copper sulfate, which was stirred at 40°C. This mixture was kept in this state for 72 h. After this step, the prepared solution is used in a centrifuge to separate the particles from the liquid, 12000 rpm for 20 min. Centrifugation was repeated three times in order to eliminate the remaining sediment impurities until the supernatant became clear. After centrifugation, the synthesized nanoparticles were dried in an oven at a temperature of 60°C.
Analyzes used for biosynthesized copper oxide nanoparticles
The production of NPs is based on the change in absorption and its color, which is confirmed by comparing the optical spectrometry of nanoparticles and plant extracts. The reduction of copper ions depends on the surface plasmon resonance (SPR) of copper oxide nanoparticles. 300 mL of the sample was diluted with 3 mL of distilled water and UV-Vis spectroscopy analysis was measured using a spectrophotometer in the range of 700 − 200 nm. In this research; X-ray diffraction, XRD, spectrum has been used to extract the accuracy of the synthesis of CuO NPs, to find out the type of crystal network and the size of the synthesized nanoparticles, using XRD device with voltage 40 KV and current 40 mA. In order to investigate the size of synthesized NPs and the morphology of synthesized, scanning electron microscope, SEM, analysis was used. For this purpose, the sediment obtained from the NPs was centrifuged three times at a speed of 1200 rpm and the resulting sediment was photographed by a growth electron microscope in such a way that the nanoparticles were fixed on a copper grid covered with carbon and after drying with an infrared lamp, photography was done with an accuracy of 2.32°A. Fourier transform infrared, FT-IR, spectroscopy is one of the most widely used methods in the qualitative identification of different molecules, determination of the molecular structure of different species, especially organic species, and identification of functional groups in the structure of a species, which was investigated by this method of synthesized CuO NPs.
Fungi tested in the study
In this study, the standard strain of Candida tropicalis, ATCC 1314, was obtained from the mushroom collection center of Tehran University of Medical Sciences and the standard strain of Candida albicans, 1264PTCC, was obtained from Razi Research Center of Lorestan University of Medical Sciences.
McFarland standard
To standardize the concentration of the inoculum for the antifungal sensitivity test, the standard turbidity of barium sulfate (BaSO4), equivalent to the 0.5 McFarland standard, was used. To prepare a suitable fungal suspension in order to determine its drug sensitivity, the number of yeasts in the inoculation samples must have a correct and acceptable standard. The number of these yeasts for the antibiogram method is conventionally 1×106 to 5×106 cells per mL 30, 31.
Inoculation of fungal suspension into culture medium
Disinfectants used in the present study
In this study, after synthesizing green CuO NPs and preparing the required culture media, disinfection experiments were performed. Four different modes were used to investigate the effect of fungi inactivation: (i) Aqueous extract of Khuzestan savory plant alone, (ii) synthesizing CuO NPs green, (iii) Carvacrol 98%, and (iv) composition of green CuO NPs and carvacrol 98%.
Determination of MIC and MFC
MIC and MFC mean Minimum Inhibitory Concentration and Minimum Fungi Concentration, respectively. To evaluate the effects of antimicrobial agents, different methods can be used, but in all methods, the working principle is to measure the effect of certain concentrations of a disinfectant in inhibiting the growth or destroying the tested fungi. Therefore, at this stage, MIC and MFC were determined. MIC refers to the smallest amount of disinfectant that can significantly inhibit the growth of an organism after a certain incubation period (16 to 20 h depending on the type of microorganism), which microorganisms will grow again if the antimicrobial agent is removed from the environment. This concentration is the most accurate and valuable concentration. MFC refers to the lowest concentration of disinfectant that can reduce the fungal population by 99.9% after 24 h. In other words, reduce the initial population by a thousand times and this concentration is greater than or equal to the MIC concentration. If MIC and MFC is equal, that antimicrobial substance is called fungicidal. When MFC is higher, the antimicrobial substance is called Fungistatic. Usually, in medicinal and therapeutic topics, fungi static substances are desirable, but in the discussion of disinfection, fungicidal substances are desirable to completely destroy microorganisms 32, 33.
Methods of measuring and checking antimicrobial effects
To determine and characterize the antifungal effects of a disinfectant, there are various methods, such as dilution methods, penetration methods, and bioautographic methods [3]. In this study, the MIC value was determined using the tube dilution method in the form of microdilution 34. Antibacterial test is to measure the effect of antibiotics or other antimicrobial agents to prevent the growth of fungi in a laboratory environment. Disk diffusion or Kirby-Bauer was used to compare the effect of different disinfectants on the studied fungi. In this method, after isolating the fungi, some of the mushroom colony is removed and dissolved in the serum medium. Then, the medium was cultured with a swab, and after culture, the antibiogram disks were transferred to the culture medium. After placing the disks, the plate was closed and they were placed in an incubator at 37 for one day and night. After the set time, the plate was examined under the light and the area of non-growth was measured. In this study, the times of 20 min to 24 hours were determined for the experiments. Also, the studied concentrations were 1/2MIC, MIC, 2MIC, 4MIC, and 8MIC. In the end, the results were reported as positive and negative, that is, the environments on which the fungus grew were considered positive (+) and the environments that did not grow were considered negative (-)30. The work steps are shown in Fig. 2.
Fig. 2
Work steps
Determine of CuO NPs characterization
The green synthesis CuO NPs was characterized by Fourier Transform Infrared spectroscopy spectra (FT-IR, Spectrum Two model, PerkinElmer Company), X-Ray Diffraction (XRD, X’ Pert Pro model, Panalytical Company), and Scanning Electron Microscopy (SEM, SIGMA VP-500 model, ZEISS Company).
Data analysis method
In this research, SPSS software was used for descriptive analysis (mean ± SD) and analytical analysis (statistical tests). After collecting the data and entering them into SPSS, it was found that the quantitative variable (the diameters of the growth inhibition zone) does not follow the normal distribution, as a result, two-factor factorial design analysis was used to investigate the interaction effect of disinfectant and fungus. In the following, it was used the POST HOC test and the corrected Benferroni test to determine the significant difference between different groups.
Results and discussion
In recent years, due to the problems and dangers caused by the indiscriminate use of chemical poisons, there has been a great tendency to use the potential of medicinal plants in the control of pests and their poisons. In this study, four types of disinfectants were used to inactivate and destroy the studied fungi.
Characterization of as-synthesis green CuO NPs
The basis of the synthesis of NPs is the regeneration of their salt ions and, in fact, neutralization of electric charge. Due to the regeneration of Cu ions and the production of NPs, the color change of the solution from brown to green is a sign of the synthesis of CuO NPs. In past studies which have been conducted on the biosynthesis of metal NPs, including CuO, the color change of the reaction mixture has been introduced as the first sign of the successful synthesis of NPs 3, 29. In present work, due to the reduction of CuO and the production of NPs, a color change from brown to green was observed due to the interaction of plant extract and CuO solution. Reduction of CuO ions to CuO NPs was investigated by visible-ultraviolet spectrophotometer. The results show an absorption maximum in the region of 290 nm, which can belong to CuO NPs, in Fig. 2A. In Renuga et al study, where CuO NPs were synthesized using Brassica oleracea var for antifungal purposes, the recorded wavelength for copper oxide nanoparticles was reported as 220 nm 15. In some studies, higher wavelengths have also been reported for green CuO. In the study of Leila et al., the reported wavelength was 396 nm 35. Another test that can be used to confirm the production of NPs is XRD, which provides comprehensive information about the chemical composition and crystal structure of natural and industrial materials [36, 37]. Each crystal has its own unique pattern caused by XRD. Based on the XRD pattern that can be seen in Fig. 2B, six separate peaks at 2θ angles including 33.62, 37.12, 48.12, 56.34, 66.4, and 72.5 degrees indicate the structure of monoclinic CuO NPs were biosynthesized, which complied with JCPDS standard number 801268 and confirmed the formation of CuO NPs. Since the peaks are completely elongated and have a small width, it can be concluded that the sample is completely crystalline. Also, the absence of any additional peak in XRD pattern indicates the absence of any impurity phase in the formed NPs. The XRD of CuO NPs results obtained with different studies conducted with different plants are highly consistent 15, 20, 21, 35.
Fig. 2
The results obtained for CuO NPs. A. region of absorption maximum, B. XRD pattern, C. SEM, and D. FTIR pattern.
To investigate the shape and size of produced CuO NPs, SEM images were used, which is shown in Fig. 2C. According to the obtained images, CuO NPs were spherical in shape and almost uniform in size, with an average diameter of 70 nm. In 2023, the results of Raju et al study on the biosynthesis of copper oxide nanoparticles with green plants showed that the diameter of the obtained particles was reported to be 20 nm 38. Fourier transform infrared spectrometry, FT-IR, analysis was performed to identify different functional groups and biomolecules 39, 40 present in CuO NPs synthesized from Satureja khuzestanica plant extract. Based on Fig. 2D, five different peaks can be seen at wavelengths 617, 994, 1145, 1635, and 3420 cm− 1, which peaks 617 and 994 cm− 1 are related to the formation of CuO NPs. The peaks in the areas of 1145, 1635, and 3420 cm− 1 are respectively related to C-O stretching bonds, C = O in phenolic compounds of aqueous plant extract, O-H in alcohols, phenols and carboxylic acids, respectively. Since the organic compounds of the plant extract are placed around the NPs and cause their stability, therefore, the movements of these organic groups can be seen in the FT-IR spectrum. So it can be said that the metabolite of the aqueous extract of the Satureja khuzestanica is similar to the phenolic compounds in the structure. Synthesized NPs are present and act as stabilizers and reducers. The FT-IR of CuO NPs results obtained with different studies conducted with different plants are highly consistent 15, 20, 41. Examining the results of this study showed that the use of Satureja khuzestanica plant aqueous extract is a simple, fast, cheap, environmentally friendly method that can be performed in any type of laboratory for the synthesis of CuO NPs. In this method, chemical and toxic reagents are not used, it does not cause any pollution to the environment, and therefore it is superior to other chemical and physical methods of nanoparticle synthesis. The functional groups present in the flower extract of the aforementioned plant were responsible for the regeneration of copper metal ions into copper nanoparticles. In this method, CuO NPs with good stability were synthesized. Therefore, the biomolecules in the extract not only regenerated the copper ions, but also protected them against oxidizing agents after being prepared. NPs obtained from this method are very stable and renewable. Also, the antibacterial effect of metal nanoparticles is related to their small size and high surface to volume ratio. This feature allows nanoparticles to have more contact with the membrane of bacteria, as a result, their inhibitory effect on pathogens increases 21, 28.
Antifungal activity of studied disinfectants
Determination of MIC and MFC
Table 1 presents minimum inhibitory concentration and minimum fungi concentration of different disinfectants studied on fungi Candida tropicalis and Candida albicans. Based on Table 1, the concentration of MIC and MFC of Satureja khuzestanica plant (1000 µg/mL) was higher than that of other studied disinfectants. Therefore, that was the weakest disinfectant for the inactivation of fungi Candida tropicalis and Candida albicans. Among the medicinal plants, the essential extract of the species belonging to the genus Satureja khuzestanica plant are of great importance due to the presence of various active chemicals, such as terpenes and monoterpenes (carvacrol, thymol) with a small amount of hydrocarbons. But there is little information about the antifungal activity of different extracts of these plants [42]. The lowest concentration of MIC and MFC was observed in combined disinfectants Carvacrol 98%+CuO NPs. In this combination, Candida tropicalis, the concentration of MIC and MFC was equal to 31 ± 25 and 52.08 ± 18.04 µg/mL, respectively. Roudbary et al reported that MIC of alcoholic Crocus sativun extract for Candida dubliniesis and Candida albicans was 250 and 125 µg/mL, respectively. Also, MFC for Candida dubliniesis and Candida albicans was 500 and 125 µg/mL, respectively. Also, MIC of safranol extract for Candida dubliniesis and Candida albicans was 12.5 and 6.25 µg/mL, respectively [43]. The results of the present study show that the use of Carvacrol 98%+CuO NPs has more inhibitory effects than the other studied compounds because it has a lower MIC than the others. Based on the information in Table 1, it can be seen that Candida tropicalis is more sensitive to the antifungal effects of Carvacrol 98%+CuO NPs than Candida albicans. This sensitivity can be related to the different percentage of compounds and types of cell wall and membrane compounds of the fungus and virulence factors 30, 44. Garcia-Marin et al. (2022), assessed biosynthesized CuO NPs against Candida albicans. In this study, Candida albicans was exposed to CuO NPs to determine their inhibitory capacity. Based on the results of this study, MIC was reported as 35.5 µg/mL 45. Tajik-Ijdan et al. (2017), evaluated comparing the effects of alcoholic extract of ginseng with itraconazole against Candida albicans and Candida krusei. The lowest and highest MIC for C. Kruzei and C. albicans was 0.5 and 0.0625 35.5 µg/mL, respectively. Also, the lowest MIC and MFC for alcoholic extract was 64 mg/mL. The highest inhibition zone for C. albicans was 14 mm and 14 to 32 mm for alcoholic extract and Foritraconazole, respectively 46. Carvacrol is the main composition of essential oil obtained from thyme, it has antimicrobial properties. The mechanism of the antimicrobial effect of essential oils is due to their reaction with the cell membrane of microorganisms and changes in the permeability of compounds such as potassium and hydrogen. Carvacrol is an isomer of thymol and has a smell similar to thymol. This substance is an edible plant extract that is insoluble in water but soluble in alcohol and ether 47.
Disinfectants | Candida albicans | Candida tropicalis | ||||
|---|---|---|---|---|---|---|
MIC | MFC | MIC | MFC | |||
Satureja khuzestanica | 1000 | 1000 | 500 | 1000 | ||
Satureja khuzestanica + CuO NPs | 62 ± 5 | 83.33 ± 33.12 | 83.33 ± 33 | 83.33 ± 33 | ||
Carvacrol 98% | 52.08 ± 18 | 62 ± 5 | 41.66 ± 16 | 41.66 ± 16 | ||
Carvacrol 98%+CuO NPs | 31 ± 25 | 52.08 ± 18.04 | 20.9 ± 8 | 20.9 ± 9 | ||
Mean diameter of the growth inhibition zones
Antibacterial analysis, determining the diameter of the halo of non-growth or disk diffusion, is a method for measuring the antibacterial effects of formulations and is one of the most important and widely used tests in the field of microbiology. In the method, studied bacteria are cultivated in a plate and then the test discs are transferred to the plate and after one day the area where the bacteria did not grow is calculated. As a result, the larger the halo, the stronger the antibacterial effect. The test is complementary to MIC and MBC analysis. The method is suitable for comparing the antibacterial power with a known antibiotic. The importance of the analysis to determine the diameter of the halo of non-growth or disk diffusion has increased today due to the increase in the drug resistance of bacteria 20, 48. Table 2 shows the mean diameter of the growth inhibition zones of the fungi under study based on the results of disc diffusion test different disinfectants studied on Candida tropicalis and Candida albicans. Based on the data in Table 2, more mean diameter of the growth inhibition zone is seen in combination CuO NPs. It can be said that the combination of biosynthesized NPs and carvacrol has a synergistic effect and the combination of these two together increased their antifungal effect, compared to the use of each separately. Taheri et al. (2017), studied aqueous and alcoholic extract of the melissa officinalis effect on Candida albicans, Candida glabrata and Candida krusei. They reported the mean diameter of the non-growing zone related to the extracts and aqueous extracts on Candida Albicans was 17.83 and 5.75 mm, respectively. Also, the mean diameter of the non-growing zone related to aqueous extracts on Candida Glabrata and Candida Krusei were 15.5 and 13.66 mm respectively 49. Table 3 presents the results related to the type of performance of disinfectants on the standard strains of on Candida tropicalis and Candida albicans. Based on Kirby-Bauer standard, in none of the examined concentrations, MIC, 2MIC, and 4MIC, for Satureja khuzestanica extract, no effect was observed, which shows that this compound alone does not have an antifungal effect. The results of the study showed that the effect of the disinfectant of Carvacrol 98%+CuO NPs, Satureja khuzestanica + CuO NPs and Carvacrol 98% was strength in Candida tropicalis in all the investigated concentrations, e.g. MIC, 2MIC, and 4MIC. Based on the results presented in Table 3, Candida tropicalis was more sensitive to the studied disinfectants than Candida albicans. The difference in the antifungal activity of herbal extracts depends on their components. A compound may alone or synergistically with other compounds cause the antifungal activity of the essential herbal extract. Therefore, it seems that the significant antifungal effects of the essential oils studied in this research are influenced by the composition 31. But the aggravating effects among all the compounds is the main factor determining the high antifungal activity of the essential herbal extract. Antifungal activity of essential oils may be in damaging lipids, proteins, cell wall, cell membrane and organelles of fungi 5, 50.
Time-kill kinetics assay
Various factors have an effect on the effectiveness of disinfectants, including contact time, disinfectant concentration, pH, humidity, temperature, etc 51. In the meantime, concentration and disinfection time are two very important factors that have an opposite relationship. If the variables of pH, humidity, temperature, etc. are constant, the higher the concentration of a disinfectant, the more effective that substance is and the less time it takes to destroy and inactivate fungi 30, 48. Table 4 shows comparison of antifungal activity of studied disinfectants on Candida tropicalis and Candida albicans based on time-kill assay. Based on the data in Table 4, fungi have grown in all the studied samples for disinfection 1.
Disinfectants | Candida albicans | Candida tropicalis | |||||
|---|---|---|---|---|---|---|---|
MIC | 2MIC | 4MIC | MIC | 2MIC | 4MIC | ||
Satureja khuzestanica | 0 | 5 ± 0.5 | 6 ± 0.5 | 0 | 4.5 | 6 | |
Satureja khuzestanica + CuO NPs | 13 | 14.5 ± 5 | 16 ± 0.72 | 15 + 1 | 16 ± 0.22 | 18 | |
Carvacrol 98% | 13.3 ± 0.75 | 15 ± 0.8 | 17 ± 0.45 | 18 ± 0.5 | 18.57 ± 0.93 | 19 ± 0.4 | |
Carvacrol 98%+CuO NPs | 15 | 15.5 | 17.3 ± 0.18 | 20 ± 1 | 21 ± 0.7 | 22 ± 0.45 | |
Disinfectants | Candida albicans | Candida tropicalis | |||||
|---|---|---|---|---|---|---|---|
MIC | 2MIC | 4MIC | MIC | 2MIC | 4MIC | ||
Satureja khuzestanica | none | none | none | none | none | none | |
Satureja khuzestanica + CuO NPs | Medium | Medium | Medium | Strength | Strength | Strength | |
Carvacrol 98% | Medium | Strength | Strength | Strength | Strength | Strength | |
Carvacrol 98%+CuO NPs | Medium | Strength | Strength | Strength | Strength | Strength | |
Candida albicans | Candida tropicalis | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
20 min | 1h | 2h | 3h | 4h | 5h | 6h | 12h | 24h | 20 min | 1h | 2h | 3h | 4h | 5h | 6h | 12h | 24h | |||
Satureja khuzestanica | 1/2 MIC | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | |
MIC | + | + | + | + | + | + | - | - | - | + | + | + | + | + | + | + | + | + | ||
2MIC | + | + | + | + | - | - | - | - | - | + | + | + | + | + | - | - | - | - | ||
4MIC | + | + | + | - | - | - | - | - | - | + | + | + | - | - | - | - | - | - | ||
8MIC | + | + | - | - | - | - | - | - | - | + | + | - | - | - | - | - | - | - | ||
Satureja khuzestanica + CuO NPs | 1/2 MIC | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | |
MIC | + | + | + | + | + | + | + | + | + | + | + | + | + | - | - | - | - | - | ||
2MIC | + | + | - | - | - | - | - | - | - | + | + | - | - | - | - | - | - | - | ||
4MIC | + | + | - | - | - | - | - | - | - | + | + | - | - | - | - | - | - | - | ||
8MIC | + | + | - | - | - | - | - | - | - | + | + | - | - | - | - | - | - | - | ||
Carvacrol 98% | 1/2 MIC | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | - | - | - | |
MIC | + | + | + | + | + | + | + | + | + | + | + | + | + | - | - | - | - | - | ||
2MIC | + | + | - | - | - | - | - | - | - | + | + | - | - | - | - | - | - | - | ||
4MIC | + | - | - | - | - | - | - | - | - | + | - | - | - | - | - | - | - | - | ||
8MIC | + | - | - | - | - | - | - | - | - | + | - | - | - | - | - | - | - | - | ||
Carvacrol 98% + CuO NPs | 1/2 MIC | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | |
MIC | + | + | + | + | + | + | + | + | + | + | + | + | - | - | - | - | - | - | ||
2MIC | + | - | - | - | - | - | - | - | - | + | - | - | - | - | - | - | - | - | ||
4MIC | + | - | - | - | - | - | - | - | - | + | - | - | - | - | - | - | - | - | ||
8MIC | + | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | ||
+ observable colony on an agar plate - lack of colony growth on agar plate | ||||||||||||||||||||
Conclusion
The results of the present study showed that carvacrol extract and CuO NPs have antifungal effects on the studied species, Candida albicans and Candida tropicalis strains. Based on the results obtained in the present research, Carvacrol 98% + CuO NPs showed a higher inhibitory power on the sensitive Candida tropicalis, lower MIC and MFC strain compared to the fluconazole resistant Candida albicans strain. According to the concordance of the disc placement results and MIC regarding the antifungal effect of the studied essential oil on the growth of Candida albicans and Candida tropicalis strains, this essential oil can be further studied and investigated as an antifungal source in the control of candidiasis.
Acknowledgments
The authors of the work appreciate the Lorestan University of Medical Sciences (LUMS) for providing financial and laboratory facilities (design Code: 1813) and instrumental support to do this study.
A
Funding
This study was financially supported (IR.LUMS.REC.1400.084) by Lorestan University of Medical Sciences (Iran).
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Author Contribution
R.NT. and F.A. carried out the experiment. M.G, M. N, and B. K. wrote the manuscript with support from F.A and M.Y. M.G Editing, Investigation. R. NT, M. N, and B. K investigation, methodology. All author writing, review, editing, Supervision.
Competing interests
The authors declare no competing interests.
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Data Availability
All data generated or analyzed during the project are included in this published article.
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