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mljgoums 2023, 17(3): 45-54 |
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Maryam F, Vahid P, Amirahmadi A, Salimi F. Perovskia abrotanoides Kar. as a Promising Source of Antimicrobial Compounds against Foodborne Pathogens. mljgoums 2023; 17 (3) :45-54
URL: http://mlj.goums.ac.ir/article-1-1366-en.html
URL: http://mlj.goums.ac.ir/article-1-1366-en.html
1- Department of Plant sciences, School of Biology, Damghan University, Damghan, Iran
2- Department of Plant sciences, School of Biology, Damghan University, Damghan, Iran ,poozesh@du.ac.ir
3- Department of Molecular & Cellular, School of Biology, Damghan University, Damghan, Iran
2- Department of Plant sciences, School of Biology, Damghan University, Damghan, Iran ,
3- Department of Molecular & Cellular, School of Biology, Damghan University, Damghan, Iran
Keywords: Listeria monocytogenes [MeSH], Yersinia enterocolitica [MeSH], Salmonella enterica [MeSH], Antioxidant [MeSH]
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.PNG)
Figure 1- Antioxidant activity of the extracts derived from P. abrotanoides collected from habitat 1 (2019) and habitat 2 (2018) based on the TLC-DPPH assay. M, E, D, and H indicate methanol, ethyl acetate, dichloromethane, and hexane extracts, respectively.
.PNG)
Figure 2- Antioxidant activities of extracts of P. abrotanoides (400 µg/ml) from different habitats based on the DPPH test. Antioxidant activity of P. abrotanoides extracts [400 (black column), 200 (white column), 100 (gray column), and 50 µg/ml (crosshatched column) concentrations]
DISCUSSION
Microbial food contamination is an important aspect of both pharmaceutical and food industries (28). P. abrotanoides is a promising medicinal plant with antimicrobial properties. It is locally known as hoosh, visk, brazambal, domou, and gevereh, which grows in mountainous areas of Iran, including the Semnan, Golestan, Isfahan, Khorasan, and Mazandran provinces (18, 29). The composition of P. abrotanoides essential oil and its bioactivities such as antifungal, antibacterial, anti-helminthic, anti-nociceptive, antiprotozoal, insecticidal, wound healing, and antioxidant effects have been demonstrated previously. Research has shown the presence of phenolic, flavonoid, and terpenoid components such as thymol, menthol, carvacrol, γ-terpinene 4-ol, and p-cymene in the essential oil of Perovskia. These compounds can be responsible for the favorable antioxidant and anti-microbial activities of this plant. Hozoorbakhsh et al. reported the inhibitory effects of P. abrotanoides essential oil against Mycobacterium tuberculosis (8, 15-18). Another study also showed that the essential oils of P. abrotanoides exert antimicrobial activity against Microsporum gypseum, Candida albicans, Aspergillus fumigatus, and Salmonella typhi (30).
In a study by Mahboubi and Kazempour, essential oil from leaves of P. abrotanoides (15.2 mm on B. cereus) showed higher antimicrobial activity compared with the fixed oil from the stem (8.34 mm on S. aureus) and leaves (11.2 mm on S. aureus). The study also reported the antimicrobial activity of the essential oil against C. albicans and Gram-positive bacteria. In the mentioned study, Aspergillus niger and Gram-negative bacteria were the least susceptible tested microorganisms (17).
Phytochemical studies have indicated the presence of monoterpenes and sesquiterpenes such as α-cadinol, 1,8‐cineole (eucalyptol), myrcene, pinene, camphor, caryophyllene, humulene, camphene, and bisabolol in high concentration (31). It has been shown that n-hexane and ethyl acetate extracts of P. abrotanoides aerial parts have considerable inhibitory effects on the growth of Leishmania donovani and Trypanosoma brucei rhodesiense (8). In another study, the ethanol extract of P. abrotanoides exhibited moderate to high levels of antibacterial activity against C. albicans, S. aureus, Staphylococcus epidermidis, Bacillus cereus, and Enterococcus faecalis with MIC values of 58, 45, 53, 63, and 85 mg/ml, respectively (32).
In the present study, the highest antimicrobial activity against Y. enterocolitica was related to the ethyl acetate (MIC:200, MBC:800) and methanol (MIC:400, MBC:1600) extracts of P. abrotanoides collected from habitat 1 in 2019. It seems that these antimicrobial compounds were semi-polar and polar. Since the MBC/MIC value of these extracts was lower than the other extracts, it can be concluded that they may have bactericidal activity (33). In a similar study, MIC values below 100 μg/ml, from 100 to 500 μg/ml, from 500 to 1000 μg/ml, and over 1000 μg/ml indicated good, moderate, weak, and no antimicrobial activity, respectively (31).
The best inhibitory effects on L. monocytogenes were caused by the dichloromethane (MIC: 50, MBC:400) and methanol (MIC:100, MBC:800) extracts of P. abrotanoides collected from habitat 1 in 2019. These extract act as bacteriostatic antimicrobial compounds (MBC/MIC>4). According to the results, P. abrotanoides collected from habitat 1 in 2019 had more antibacterial potential compared with P. abrotanoides collected from habitat 2 and 3 in 2018 and 2019, or even habitat 1 in 2018. Aoyagi et al. showed that methanol extract of P. abrotanoides had various MIC values (ranging from 78-250 μg/ml) against various pathogens (31). The presence of several known abietane diterpenoids and 11‐O‐ and 12‐O‐acetylcarnosic acids in the methanol extract of P. abrotanoides has been confirmed (34).
It seems that hexane is not a suitable solvent for extracting compounds with antioxidant activity. In our study, hexane extracts (except for hexane extract of plants collected from habitat 3 in 2019) had weaker antioxidant activity. Overall, extracts of P. abrotanoides collected in 2019 had a significantly higher antioxidant potential compared with those from plants collected in 2018 (except for the hexane and dichloromethane extracts of P. abrotanoides collected from habitat 2 and 3 in 2018, respectively). It seems that both polar and non-polar compounds in P. abrotanoides extracts possess antioxidant activity. However, it is suggested that the number or concentration of polar compounds is more than non-polar compounds since the methanol and ethyl acetate extracts showed a significant DPPH scavenging activity. Similar to our findings, Ghafourian and Mazandarani study reported that P. abrotanoides extract had good antioxidant activity, especially in the DPPH method (32). In our study, the extracts of P. abrotanoides collected from habitat 3 in 2018 and 2019 showed a greater antioxidant activity than those obtained from plants collected from the other habitats.
Many studies have been carried out on the relationship of plant chemical contents with biological activity and environmental variables in natural and cultivated plant species (35-37). This valuable information has been used to determine the medicinal significance and economic importance of plant products (38). Today, it is known that exposure of plants to environmental stress such as salinity may increase the production of reactive oxygen species, which can lead to cell damage (39, 40). Salt-tolerant plants generally have a better defense mechanism against oxidative stress through antioxidant compounds, which can scavenge reactive oxygen species (41, 42). In the present study, plants from habitat 3 had the highest anti-salinity and antioxidant activity (43), indicating a possible direct relationship between antioxidant capacity and salt tolerance.
CONCLUSION
Our results confirm the antimicrobial and antioxidant activities of various extracts of P. abrotanoides grown in three different habitats. It can be concluded that both environmental and genetic factors can affect the quantity and quality of medicinal plants.
ACKNOWLEDGEMENTS
The authors would like to thank Damghan University for supporting this resarch.
DECLARATIONS
FUNDING
The authors received financial support from Damghan University, Iran.
Ethics approvals and consent to participate
Not applicable since the study did not involve human or animal samples.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest regarding the publication of this article.
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INTRODUCTION
Food contamination with foodborne pathogens cause nutrient deterioration. It also adversely affects the quality and safety of foods by producing poisonous and foul-smelling compounds. According to a report by the Centers for Disease Control and Prevention, bacterially contaminated food causes significant mortality (3,000 deaths) and morbidity (48 million illnesses) in the United States annually (1, 2). Listeria monocytogenes, Salmonella enterica, and Yersinia enterocolitica are responsible for listeriosis, salmonellosis, and yersiniosis, respectively. They are major foodborne diseases that cause considerable public health concerns in the world. (3). L. monocytogenes causes a mortality rate of up to 30%. Major clinical manifestations of L. monocytogenes-related infections are gastroenteritis, meningitis, and septicemia. The persistence of L. monocytogenes in food processing for a long time, even at low temperatures, and its ability to generate biofilms or integrate into previously developed biofilms by other species, make it very elusive to control (4).
Food contamination with Salmonella can occur during the manufacturing and processing steps of livestock feed and food. This bacterium is also resistant to the harsh conditions of food matrices. This facilitates the transmission of this high-infective foodborne pathogen to human beings and enhances the number of outbreaks (5). Y. enterocolitica is considered the main species in the genus associated with yersiniosis. Associated symptoms include diarrhea, fever, and abdominal pain (6).
In order to guarantee food safety from foodborne-related illnesses, compounds with inhibitory effects on their growth are critically needed (4). Plants are promising sources of bioactive compounds such as antimicrobials and antioxidants. Herbal antioxidants improve food quality as natural antimicrobial compounds. Natural antioxidants in plants can scavenge free radicals before triggering oxidative chain reactions in the cell membrane or lipid-containing portions (11, 12).
Perovskia is a small genus of medicinal plants from the Lamiaceae family, a potential source of phenolic acids (7). Its aromatic shrubs grow wild in arid habitats of central Asia, including Afghanistan, Pakistan, Turkmenistan, and Iran, particularly in the northern, eastern, and central parts of Iran (7). There are three species of this plant in Iran including, P.abrotanoides Kar., P. atriplicifolia Benth., and P. artemisoides Boiss.. P. abrotanoides, locally known as brazambal, is an aromatic herb that has been used to treat rheumatic pains and leishmaniasis in Iranian traditional medicine (8). Jaafari and coworkers reported the anti-leishmaniasis activity of phenolic and terpenic constituents of P. abrotanoides stems and leaves (9).
Previous phytochemical investigations on the genus Perovskia indicate that this plant contains flavonoids, phenolics, and anthocyanins with leishmanicidal, anti-plasmodial, cytotoxic, anti-inflammatory, anti-diabetic, anti-lipidemic, and anthelmintic activities (8, 15-19). It also has structurally unique diterpenoids possessing abietane and rearranged icetexane scaffolds. The aerial parts and roots of P. abrotanoides contain isoprenoids and tanshinones, respectively (20, 21).
In this study, we aimed to determine the antimicrobial activity of extract from P. abrotanoides plants collected from three different parts of Iran and two different periods against P. abrotanoides, L. monocytogenes, Salmonella spp, and Y. enterocolitica. Given the great diversity of climate in Iran, plants grown under different conditions may have different antioxidant capacities (13, 14). Hence, we investigated the antioxidant activity of P. abrotanoides collected from different habitats.
MATERIALS AND METHODS
Chemical materials including hexane, dichloromethane, ethylacetate, methanol, dimethyl sulfoxide (DMSO), phosphate buffer saline (PBS), agar, thin-layer chromatography (TLC) plates, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Merck Co., Germany. Culture media including nutrient broth, brain heart infusion, tryptic soy broth, Mueller-Hinton agar, and tryptic soy agar were purchased from Ibresco, Iran. Gram-negative and Gram-positive bacterial species were obtained from the Iranian Biological Resource Center (Tehran, Iran).
The medicinal plant P. abrotanoides Kar. was collected from the mountain regions of eastern Alborz (with considerably different climates from Semnan and Mazandaran provinces) (Table 1). The flowering branches were properly rinsed with distilled water, shade-dried, and grounded into fine powder. The extraction of plant material was done according to a previous study with minor modifications (22). Briefly, 5 g of powdered branches were added into organic solvents (100 ml) with a different polarity index (PI), including hexane (PI=0), dichloromethane (PI=3.1), ethylacetate (PI=4.4), and methanol (PI=5.1). The mixture was heated at 40 oC on a hotplate with continuous stirring for 2 hours. The resulting solution was filtered using a Whatman filter paper and then air-dried to obtain the extracts.
The antimicrobial potential of the extracts (400 µg/ml) was evaluated using the agar well diffusion test. Antibiotic discs (6 mm diameter) and diluted DMSO were used as the positive and negative controls, respectively. Bioactive compounds were integrated into the medium, and culture plates were incubated at 37 °C for 24 hours. Inhibitory properties were assessed by measuring the diameter of inhibition zone (mm) around the wells and antibiotic disk. The test was repeated three times, and the results were reported as mean ± standard deviation (SD) after three repeats (23).
The minimal inhibitory concentration (MIC) of the extracts was determined according to the Clinical and Laboratory Standards Institute (CLSI), protocol M7-A6 (2012) (24). The lowest concentration of each extract that inhibited bacterial growth was determined as MIC. The wells without extracts and bacterial inoculum were considered as growth and sterility controls, respectively.
To determine the minimal bactericidal concentration (MBC), 10 µl of the sample from each well of MIC microtiter plate were directly transferred to Mueller-Hinton agar or tryptic soy agar plates and incubated for 24 hours at 37 oC. The MBC was determined as the lowest concentration of the extract in which no detectable growth was observed (25).
The antioxidant activity of extracts was evaluated qualitatively by the TLC-DPPH method. For this purpose, the crude extracts were developed on TLC silica gel 60 F254 plates (Mecherey-Nagel) in dichloromethane and methanol (93:7 v/v) mixture as solvent systems. Then, to recognize the radical scavenging activity of the extracts, the plates were sprayed with DPPH reagent solution (0.05% DPPH/methanol). The plates were incubated at room temperature in the dark for 30 minutes. The formation of yellow spots against a purple background, following spraying DPPH occurred for bonds containing compounds with antioxidant activity (26).
The radical scavenging activity of the extracts (800 µg/ml) was assessed according to the DPPH method in a 96-well microtitre plate as described previously (27). The antioxidant activity of the extracts was further evaluated at 400, 200, 100, and 50 µg/ml. Triplicate measurements were made and the ability to scavenge the DPPH radical was calculated by the following formula, where Ablank is the absorbance of the control and Asample is the absorbance of the sample:
Antioxidant activity%= (Ablank- Asample/ Ablank) × 100.
Data were expressed as means ± SD and analyzed using one-way ANOVA. All analyzes were carried out in SPSS (version 18) and at a statistical significance of 0.05.
RESULTS
Crude extracts of P. abrotanoides were obtained using solvent extraction. According to the weight of the obtained extracts, hexane and ethyl acetate had a higher capacity to extract compounds compared with dichloromethane and methanol (Table 2).
The crude extracts of P. abrotanoides showed significant inhibitory effects on L. monocytogenes. In this regard, extracts of samples 3-2018 and 3-2019 showed the highest and lowest anti-Listeria activities, respectively (Table 3). The extracts showed a lower antimicrobial activity against Y. enterocolitica than L. monocytogenes. In most cases, significant antimicrobial activity was related to the ethyl acetate or methanolic extracts. The ethyl acetate extract collected from habitats 1 and 2 in 2019 showed the highest antimicrobial activity against Y. enterocolitica (Table 3). Finally, the extracts of P. abrotanoides showed minimal antimicrobial activity against S. enterica (Table 3).
MIC and MBC values of the extracts with considerable antimicrobial activity in the agar well diffusion test were determined via broth microdilution and culturing methods. The lowest MIC (200 µg/ml) and MBC (400 µg/ml) values against Y. enterocolitica belonged to the ethyl acetate extract of 1-2019 (Table 4). The lowest MIC (50 µg/ml) and MBC (400 µg/ml) values on L. monocytogenes belonged to the dichloromethane extract of 1-2019 (Table 4).
All investigated extracts showed antioxidant properties in the TLC-DPPH assay (Figure 1). Results of the one-way ANOVA indicated that the antioxidant activity of the extracts derived from plants collected in 2019 was higher than that of those collected in 2018 (except for the dichloromethane and hexane extracts of 3-2018 and 2-2019, respectively). In most cases, the methanol and ethyl acetate extracts showed greater antioxidant activity compared with the hexane and dichloromethane extracts (Figure 2a).
The antioxidant activity of the extracts increased in a dose-dependent manner. The most radical scavenging activity was related to the ethyl acetate extract (400 µg/ml) of 3-2019. Also, the weakest DPPH scavenging activity was related to the ethyl acetate (50 µg/ml) and methanol (100 µg/ml) extracts of plants collected from 3-2019. According to the results, the antioxidant activity of methanol and dichloromethane extracts of P. abrotanoides collected from habitat 3 in 2018 showed negligible differences in various concentrations (Figure 2b).
Table 1- The geographical location of P. abrotanoides habitats
Food contamination with foodborne pathogens cause nutrient deterioration. It also adversely affects the quality and safety of foods by producing poisonous and foul-smelling compounds. According to a report by the Centers for Disease Control and Prevention, bacterially contaminated food causes significant mortality (3,000 deaths) and morbidity (48 million illnesses) in the United States annually (1, 2). Listeria monocytogenes, Salmonella enterica, and Yersinia enterocolitica are responsible for listeriosis, salmonellosis, and yersiniosis, respectively. They are major foodborne diseases that cause considerable public health concerns in the world. (3). L. monocytogenes causes a mortality rate of up to 30%. Major clinical manifestations of L. monocytogenes-related infections are gastroenteritis, meningitis, and septicemia. The persistence of L. monocytogenes in food processing for a long time, even at low temperatures, and its ability to generate biofilms or integrate into previously developed biofilms by other species, make it very elusive to control (4).
Food contamination with Salmonella can occur during the manufacturing and processing steps of livestock feed and food. This bacterium is also resistant to the harsh conditions of food matrices. This facilitates the transmission of this high-infective foodborne pathogen to human beings and enhances the number of outbreaks (5). Y. enterocolitica is considered the main species in the genus associated with yersiniosis. Associated symptoms include diarrhea, fever, and abdominal pain (6).
In order to guarantee food safety from foodborne-related illnesses, compounds with inhibitory effects on their growth are critically needed (4). Plants are promising sources of bioactive compounds such as antimicrobials and antioxidants. Herbal antioxidants improve food quality as natural antimicrobial compounds. Natural antioxidants in plants can scavenge free radicals before triggering oxidative chain reactions in the cell membrane or lipid-containing portions (11, 12).
Perovskia is a small genus of medicinal plants from the Lamiaceae family, a potential source of phenolic acids (7). Its aromatic shrubs grow wild in arid habitats of central Asia, including Afghanistan, Pakistan, Turkmenistan, and Iran, particularly in the northern, eastern, and central parts of Iran (7). There are three species of this plant in Iran including, P.abrotanoides Kar., P. atriplicifolia Benth., and P. artemisoides Boiss.. P. abrotanoides, locally known as brazambal, is an aromatic herb that has been used to treat rheumatic pains and leishmaniasis in Iranian traditional medicine (8). Jaafari and coworkers reported the anti-leishmaniasis activity of phenolic and terpenic constituents of P. abrotanoides stems and leaves (9).
Previous phytochemical investigations on the genus Perovskia indicate that this plant contains flavonoids, phenolics, and anthocyanins with leishmanicidal, anti-plasmodial, cytotoxic, anti-inflammatory, anti-diabetic, anti-lipidemic, and anthelmintic activities (8, 15-19). It also has structurally unique diterpenoids possessing abietane and rearranged icetexane scaffolds. The aerial parts and roots of P. abrotanoides contain isoprenoids and tanshinones, respectively (20, 21).
In this study, we aimed to determine the antimicrobial activity of extract from P. abrotanoides plants collected from three different parts of Iran and two different periods against P. abrotanoides, L. monocytogenes, Salmonella spp, and Y. enterocolitica. Given the great diversity of climate in Iran, plants grown under different conditions may have different antioxidant capacities (13, 14). Hence, we investigated the antioxidant activity of P. abrotanoides collected from different habitats.
MATERIALS AND METHODS
Chemical materials including hexane, dichloromethane, ethylacetate, methanol, dimethyl sulfoxide (DMSO), phosphate buffer saline (PBS), agar, thin-layer chromatography (TLC) plates, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Merck Co., Germany. Culture media including nutrient broth, brain heart infusion, tryptic soy broth, Mueller-Hinton agar, and tryptic soy agar were purchased from Ibresco, Iran. Gram-negative and Gram-positive bacterial species were obtained from the Iranian Biological Resource Center (Tehran, Iran).
The medicinal plant P. abrotanoides Kar. was collected from the mountain regions of eastern Alborz (with considerably different climates from Semnan and Mazandaran provinces) (Table 1). The flowering branches were properly rinsed with distilled water, shade-dried, and grounded into fine powder. The extraction of plant material was done according to a previous study with minor modifications (22). Briefly, 5 g of powdered branches were added into organic solvents (100 ml) with a different polarity index (PI), including hexane (PI=0), dichloromethane (PI=3.1), ethylacetate (PI=4.4), and methanol (PI=5.1). The mixture was heated at 40 oC on a hotplate with continuous stirring for 2 hours. The resulting solution was filtered using a Whatman filter paper and then air-dried to obtain the extracts.
The antimicrobial potential of the extracts (400 µg/ml) was evaluated using the agar well diffusion test. Antibiotic discs (6 mm diameter) and diluted DMSO were used as the positive and negative controls, respectively. Bioactive compounds were integrated into the medium, and culture plates were incubated at 37 °C for 24 hours. Inhibitory properties were assessed by measuring the diameter of inhibition zone (mm) around the wells and antibiotic disk. The test was repeated three times, and the results were reported as mean ± standard deviation (SD) after three repeats (23).
The minimal inhibitory concentration (MIC) of the extracts was determined according to the Clinical and Laboratory Standards Institute (CLSI), protocol M7-A6 (2012) (24). The lowest concentration of each extract that inhibited bacterial growth was determined as MIC. The wells without extracts and bacterial inoculum were considered as growth and sterility controls, respectively.
To determine the minimal bactericidal concentration (MBC), 10 µl of the sample from each well of MIC microtiter plate were directly transferred to Mueller-Hinton agar or tryptic soy agar plates and incubated for 24 hours at 37 oC. The MBC was determined as the lowest concentration of the extract in which no detectable growth was observed (25).
The antioxidant activity of extracts was evaluated qualitatively by the TLC-DPPH method. For this purpose, the crude extracts were developed on TLC silica gel 60 F254 plates (Mecherey-Nagel) in dichloromethane and methanol (93:7 v/v) mixture as solvent systems. Then, to recognize the radical scavenging activity of the extracts, the plates were sprayed with DPPH reagent solution (0.05% DPPH/methanol). The plates were incubated at room temperature in the dark for 30 minutes. The formation of yellow spots against a purple background, following spraying DPPH occurred for bonds containing compounds with antioxidant activity (26).
The radical scavenging activity of the extracts (800 µg/ml) was assessed according to the DPPH method in a 96-well microtitre plate as described previously (27). The antioxidant activity of the extracts was further evaluated at 400, 200, 100, and 50 µg/ml. Triplicate measurements were made and the ability to scavenge the DPPH radical was calculated by the following formula, where Ablank is the absorbance of the control and Asample is the absorbance of the sample:
Antioxidant activity%= (Ablank- Asample/ Ablank) × 100.
Data were expressed as means ± SD and analyzed using one-way ANOVA. All analyzes were carried out in SPSS (version 18) and at a statistical significance of 0.05.
RESULTS
Crude extracts of P. abrotanoides were obtained using solvent extraction. According to the weight of the obtained extracts, hexane and ethyl acetate had a higher capacity to extract compounds compared with dichloromethane and methanol (Table 2).
The crude extracts of P. abrotanoides showed significant inhibitory effects on L. monocytogenes. In this regard, extracts of samples 3-2018 and 3-2019 showed the highest and lowest anti-Listeria activities, respectively (Table 3). The extracts showed a lower antimicrobial activity against Y. enterocolitica than L. monocytogenes. In most cases, significant antimicrobial activity was related to the ethyl acetate or methanolic extracts. The ethyl acetate extract collected from habitats 1 and 2 in 2019 showed the highest antimicrobial activity against Y. enterocolitica (Table 3). Finally, the extracts of P. abrotanoides showed minimal antimicrobial activity against S. enterica (Table 3).
MIC and MBC values of the extracts with considerable antimicrobial activity in the agar well diffusion test were determined via broth microdilution and culturing methods. The lowest MIC (200 µg/ml) and MBC (400 µg/ml) values against Y. enterocolitica belonged to the ethyl acetate extract of 1-2019 (Table 4). The lowest MIC (50 µg/ml) and MBC (400 µg/ml) values on L. monocytogenes belonged to the dichloromethane extract of 1-2019 (Table 4).
All investigated extracts showed antioxidant properties in the TLC-DPPH assay (Figure 1). Results of the one-way ANOVA indicated that the antioxidant activity of the extracts derived from plants collected in 2019 was higher than that of those collected in 2018 (except for the dichloromethane and hexane extracts of 3-2018 and 2-2019, respectively). In most cases, the methanol and ethyl acetate extracts showed greater antioxidant activity compared with the hexane and dichloromethane extracts (Figure 2a).
The antioxidant activity of the extracts increased in a dose-dependent manner. The most radical scavenging activity was related to the ethyl acetate extract (400 µg/ml) of 3-2019. Also, the weakest DPPH scavenging activity was related to the ethyl acetate (50 µg/ml) and methanol (100 µg/ml) extracts of plants collected from 3-2019. According to the results, the antioxidant activity of methanol and dichloromethane extracts of P. abrotanoides collected from habitat 3 in 2018 showed negligible differences in various concentrations (Figure 2b).
Table 1- The geographical location of P. abrotanoides habitats
| Habitat | Province | Height above sea level | Longitude | latitude | Collection year | Sample name |
| 1 | Semnan | 1504m | N 36°16´22.46˝ | E 54°5´3.24˝ | 2018 | 1-2018 |
| 2019 | 1-2019 | |||||
| 2 | Mazandaran | 1672m | N 36°14´44.48˝ | E 53°43´47.3˝ | 2018 | 2-2018 |
| 2019 | 2-2019 | |||||
| 3 | Semnan | 1285m | N 36°21´42.47˝ | E 54°53´19.5˝ | 2018 | 3-2018 |
| 2019 | 3-2019 |
Table 2- The dry weight of crude extracts of P. abrotanoides
| Habitat | 1 | 2 | 3 | |||
| Year | 2018 | 2019 | 2018 | 2019 | 2018 | 2019 |
| Dry weight (g) of P. abrotanoides | 2.5 | 4.8 | 2.5 | 6 | 5.8 | 6 |
| Solvent | ||||||
| Hexane | 0.59 | 0.89 | 0.77 | 1.16 | 1.11 | 0.93 |
| Dichloromethane | 0.2 | 0.37 | 0.3 | 1.05 | 0.3 | 0.86 |
| Ethyl acetate | 1.17 | 0.89 | 0.76 | 1.2 | 1.32 | 0.9 |
| Methanol | 0.21 | 2.16 | 0.21 | 0.59 | 0.98 | 1.37 |
Table 3- Antimicrobial activity of the P. abrotanoides extracts against the tested pathogens.
| Solvent | Dose | Habitat 1 | Significance | Habitat 2 | Sig. value |
Habitat 3 | Significance | |||||||||||
| Year | Year | Year | ||||||||||||||||
| 2018 | 2019 | 2018 | 2019 | 2018 | 2019 | |||||||||||||
| L. monocytogenes | ||||||||||||||||||
| Hexane | 400 μg/ml | 11±0.1 | 12±0.3 |
NS | 19±0.6 | 12±0.5 | * | 20±0.2 | 0±0 | * | ||||||||
| Dichloromethane | 11±0.2 | 20±0.4 | * | 12±0.2 | 16±0.3 | * | 21±0.7 | 0±0 | * | |||||||||
| Ethyl acetate | 11±0.2 | 11±0.3 | NS | 20±1 | 20±0.8 | NS | 16±0.5 | 0±0 | * | |||||||||
| Methanol | 13±0.5 | 16±0.7 | * | 15±0.06 | 15±0.1 | NS | 20±0.4 | 0±0 | * | |||||||||
| Y. enterocolitica | ||||||||||||||||||
| Hexane | 400 μg/ml | 0±0 | 0±0 | NS | 0±0 | 0±0 | NS | 0±0 | 0±0 | NS | ||||||||
| Dichloromethane | 0±0 | 12±0.3 | * | 0±0 | 11±0.5 | * | 0±0 | 0±0 | NS | |||||||||
| Ethyl acetate | 12±0.45 | 16±0.7 | * | 12±0.6 | 16±0.4 | * | 13±0.4 | 0±0 | * | |||||||||
| Methanol | 12±0.5 | 12±0.8 | NS | 9±0.2 | 13±0.3 | NS | 11±0.1 | 0±0 | * | |||||||||
| S.enterica | ||||||||||||||||||
| Hexane | 400 μg/ml | 12±0.3 | 0±0 | NS | 0±0 | 0±0 | NS | 0±0 | 0±0 | NS | ||||||||
| Dichloromethanee | 12±0.4 | 0±0 | * | 0±0 | 0±0 | * | 0±0 | 0±0 | NS | |||||||||
| Ethyl acetate | 0±0 | 0±0 | * | 0±0 | 0±0 | * | 0±0 | 0±0 | * | |||||||||
| Methanol | 0±0 | 0±0 | NS | 0±0 | 11±0.3 | NS | 0±0 | 9±0.1 | * | |||||||||
| Positive controls | L. monocytogenes | Y. enterocolitica | S. enterica | |||||||||||||||
| Trimethoprim | 5 μg/disk | 40±2 | 17±0.3 | 27±0.5 | ||||||||||||||
| Trimethoprim + sulfamethoxazole |
1.25 mg 23.75 mg/disk |
44±0.7 | 23±0.9 | 25±0.6 | ||||||||||||||
| Gentamicin | 10μg/disk | 34±0.6 | 25±0.5 | 30±0.8 | ||||||||||||||
| Penicillin | 10μg/disk | 0±0 | 0±0 | 0±0 | ||||||||||||||
Figure 1- Antioxidant activity of the extracts derived from P. abrotanoides collected from habitat 1 (2019) and habitat 2 (2018) based on the TLC-DPPH assay. M, E, D, and H indicate methanol, ethyl acetate, dichloromethane, and hexane extracts, respectively.
Figure 2- Antioxidant activities of extracts of P. abrotanoides (400 µg/ml) from different habitats based on the DPPH test. Antioxidant activity of P. abrotanoides extracts [400 (black column), 200 (white column), 100 (gray column), and 50 µg/ml (crosshatched column) concentrations]
DISCUSSION
Microbial food contamination is an important aspect of both pharmaceutical and food industries (28). P. abrotanoides is a promising medicinal plant with antimicrobial properties. It is locally known as hoosh, visk, brazambal, domou, and gevereh, which grows in mountainous areas of Iran, including the Semnan, Golestan, Isfahan, Khorasan, and Mazandran provinces (18, 29). The composition of P. abrotanoides essential oil and its bioactivities such as antifungal, antibacterial, anti-helminthic, anti-nociceptive, antiprotozoal, insecticidal, wound healing, and antioxidant effects have been demonstrated previously. Research has shown the presence of phenolic, flavonoid, and terpenoid components such as thymol, menthol, carvacrol, γ-terpinene 4-ol, and p-cymene in the essential oil of Perovskia. These compounds can be responsible for the favorable antioxidant and anti-microbial activities of this plant. Hozoorbakhsh et al. reported the inhibitory effects of P. abrotanoides essential oil against Mycobacterium tuberculosis (8, 15-18). Another study also showed that the essential oils of P. abrotanoides exert antimicrobial activity against Microsporum gypseum, Candida albicans, Aspergillus fumigatus, and Salmonella typhi (30).
In a study by Mahboubi and Kazempour, essential oil from leaves of P. abrotanoides (15.2 mm on B. cereus) showed higher antimicrobial activity compared with the fixed oil from the stem (8.34 mm on S. aureus) and leaves (11.2 mm on S. aureus). The study also reported the antimicrobial activity of the essential oil against C. albicans and Gram-positive bacteria. In the mentioned study, Aspergillus niger and Gram-negative bacteria were the least susceptible tested microorganisms (17).
Phytochemical studies have indicated the presence of monoterpenes and sesquiterpenes such as α-cadinol, 1,8‐cineole (eucalyptol), myrcene, pinene, camphor, caryophyllene, humulene, camphene, and bisabolol in high concentration (31). It has been shown that n-hexane and ethyl acetate extracts of P. abrotanoides aerial parts have considerable inhibitory effects on the growth of Leishmania donovani and Trypanosoma brucei rhodesiense (8). In another study, the ethanol extract of P. abrotanoides exhibited moderate to high levels of antibacterial activity against C. albicans, S. aureus, Staphylococcus epidermidis, Bacillus cereus, and Enterococcus faecalis with MIC values of 58, 45, 53, 63, and 85 mg/ml, respectively (32).
In the present study, the highest antimicrobial activity against Y. enterocolitica was related to the ethyl acetate (MIC:200, MBC:800) and methanol (MIC:400, MBC:1600) extracts of P. abrotanoides collected from habitat 1 in 2019. It seems that these antimicrobial compounds were semi-polar and polar. Since the MBC/MIC value of these extracts was lower than the other extracts, it can be concluded that they may have bactericidal activity (33). In a similar study, MIC values below 100 μg/ml, from 100 to 500 μg/ml, from 500 to 1000 μg/ml, and over 1000 μg/ml indicated good, moderate, weak, and no antimicrobial activity, respectively (31).
The best inhibitory effects on L. monocytogenes were caused by the dichloromethane (MIC: 50, MBC:400) and methanol (MIC:100, MBC:800) extracts of P. abrotanoides collected from habitat 1 in 2019. These extract act as bacteriostatic antimicrobial compounds (MBC/MIC>4). According to the results, P. abrotanoides collected from habitat 1 in 2019 had more antibacterial potential compared with P. abrotanoides collected from habitat 2 and 3 in 2018 and 2019, or even habitat 1 in 2018. Aoyagi et al. showed that methanol extract of P. abrotanoides had various MIC values (ranging from 78-250 μg/ml) against various pathogens (31). The presence of several known abietane diterpenoids and 11‐O‐ and 12‐O‐acetylcarnosic acids in the methanol extract of P. abrotanoides has been confirmed (34).
It seems that hexane is not a suitable solvent for extracting compounds with antioxidant activity. In our study, hexane extracts (except for hexane extract of plants collected from habitat 3 in 2019) had weaker antioxidant activity. Overall, extracts of P. abrotanoides collected in 2019 had a significantly higher antioxidant potential compared with those from plants collected in 2018 (except for the hexane and dichloromethane extracts of P. abrotanoides collected from habitat 2 and 3 in 2018, respectively). It seems that both polar and non-polar compounds in P. abrotanoides extracts possess antioxidant activity. However, it is suggested that the number or concentration of polar compounds is more than non-polar compounds since the methanol and ethyl acetate extracts showed a significant DPPH scavenging activity. Similar to our findings, Ghafourian and Mazandarani study reported that P. abrotanoides extract had good antioxidant activity, especially in the DPPH method (32). In our study, the extracts of P. abrotanoides collected from habitat 3 in 2018 and 2019 showed a greater antioxidant activity than those obtained from plants collected from the other habitats.
Many studies have been carried out on the relationship of plant chemical contents with biological activity and environmental variables in natural and cultivated plant species (35-37). This valuable information has been used to determine the medicinal significance and economic importance of plant products (38). Today, it is known that exposure of plants to environmental stress such as salinity may increase the production of reactive oxygen species, which can lead to cell damage (39, 40). Salt-tolerant plants generally have a better defense mechanism against oxidative stress through antioxidant compounds, which can scavenge reactive oxygen species (41, 42). In the present study, plants from habitat 3 had the highest anti-salinity and antioxidant activity (43), indicating a possible direct relationship between antioxidant capacity and salt tolerance.
CONCLUSION
Our results confirm the antimicrobial and antioxidant activities of various extracts of P. abrotanoides grown in three different habitats. It can be concluded that both environmental and genetic factors can affect the quantity and quality of medicinal plants.
ACKNOWLEDGEMENTS
The authors would like to thank Damghan University for supporting this resarch.
DECLARATIONS
FUNDING
The authors received financial support from Damghan University, Iran.
Ethics approvals and consent to participate
Not applicable since the study did not involve human or animal samples.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest regarding the publication of this article.
Research Article: Research Article |
Subject:
Microbiology
Received: 2021/02/12 | Accepted: 2021/06/21 | Published: 2023/05/21 | ePublished: 2023/05/21
Received: 2021/02/12 | Accepted: 2021/06/21 | Published: 2023/05/21 | ePublished: 2023/05/21
References
1. Dixon RE. Control of Health-Care--Associated Infections, 1961--2011. Supplements. 2011; 60(04): 58-63. [View at Publisher] [Google Scholar]
2. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson M, Roy SL, et al. Foodborne illness acquired in the United States—major pathogens. Emerg Infect Dis. 2011;17(1):7-15. [View at Publisher] [PubMed] [Google Scholar]
3. Al-Tayyar NA, Youssef AM, Al-Hindi R. Antimicrobial food packaging based on sustainable Bio-based materials for reducing foodborne Pathogens: A review. Food Chem. 2020; 310: 125915. [View at Publisher] [DOI:10.1016/j.foodchem.2019.125915] [PubMed] [Google Scholar]
4. Jara J, Pérez-Ramos A, Del Solar G, Rodríguez JM, Fernández L, Orgaz B. Role of Lactobacillus biofilms in Listeria monocytogenes adhesion to glass surfaces. Int J Food Microbiol. 2020;334:108804. [View at Publisher] [DOI:10.1016/j.ijfoodmicro.2020.108804] [PubMed] [Google Scholar]
5. Mutz YdS, Rosario DKA, Paschoalin VMF, Conte-Junior CA. Salmonella enterica: A hidden risk for dry-cured meat consumption? Crit Rev Food Sci Nutr. 2020; 60(6): 976-90. [View at Publisher] [DOI:10.1080/10408398.2018.1555132] [PubMed] [Google Scholar]
6. Guillier L, Fravalo P, Leclercq A, Thébaut A, Kooh P, Cadavez V, et al. Risk factors for sporadic Yersinia enterocolitica infections: a systematic review and meta-analysis. Microbial Risk Analysis. 2020:100141. [View at Publisher] [DOI:10.1016/j.mran.2020.100141] [Google Scholar]
7. Ghaderi S, Ebrahimi SN, Ahadi H, Moghadam SE, Mirjalili MH. In vitro propagation and phytochemical assessment of Perovskia abrotanoides Karel.(Lamiaceae)-A medicinally important source of phenolic compounds. Biocatalysis and agricultural biotechnology. 2019;19:101113. [View at Publisher] [DOI:10.1016/j.bcab.2019.101113] [Google Scholar]
8. Tabefam M, Farimani MM, Danton O, Ramseyer J, Kaiser M, Ebrahimi SN, et al. Antiprotozoal diterpenes from Perovskia abrotanoides. Planta medica. 2018;84(12/13):913-9. [View at Publisher] [DOI:10.1055/a-0608-4946] [PubMed] [Google Scholar]
9. Jaafari MR, Hooshmand S, Samiei A, Hossainzadeh H. Evaluation of-leishmanicidal effect of Perovskia abrotanoides Karel. root extract by in vitro leishmanicidal assay using promastigotes of Leishmania major. Pharmacologyonline. 2007;1: 299-303. [View at Publisher] [Google Scholar]
10. Frankel JA. No single currency regime is right for all countries or at all times. National Bureau of Economic Research; 1999. [View at Publisher] [DOI:10.3386/w7338]
11. Prabhasankar P, Ganesan P, Bhaskar N, Hirose A, Stephen N, Gowda LR, et al. Edible Japanese seaweed, wakame (Undaria pinnatifida) as an ingredient in pasta: Chemical, functional and structural evaluation. Food Chem. 2009; 115(2): 501-8. [View at Publisher] [DOI:10.1016/j.foodchem.2008.12.047] [Google Scholar]
12. Ćujić N, Šavikin K, Janković T, Pljevljakušić D, Zdunić G, Ibrić S. Optimization of polyphenols extraction from dried chokeberry using maceration as traditional technique. Food Chem. 2016;194:135-42. [View at Publisher] [DOI:10.1016/j.foodchem.2015.08.008] [PubMed] [Google Scholar]
13. Issa AY, Volate SR, Wargovich MJ. The role of phytochemicals in inhibition of cancer and inflammation: New directions and perspectives. Journal of Food Composition and Analysis. 2006;19(5):405-19. [View at Publisher] [DOI:10.1016/j.jfca.2006.02.009] [Google Scholar]
14. Ackerknecht EH. Therapeutics from the Primitives to the 20th Century (with an Appendix: History of Dietetics): Hafner Press, A Division of MacMillan Publishing Co., New York, and Collier MacMillan Publishers, London. 1973; 194. [View at Publisher] [DOI]
15. Hozoorbakhsh F, Esfahani BN, Moghim S, Asghari G. Evaluation of the effect of Pulicaria gnaphalodes and Perovskia abrotanoides essential oil extracts against mycobacterium tuberculosis strains. Advanced Biomedical Research. 2016;5. [View at Publisher] [DOI:10.4103/2277-9175.180991] [PubMed] [Google Scholar]
16. Sadeghi Z, Alizadeh Z, Khorrami F, Norouzi S, Moridi Farimani M. Insecticidal activity of the essential oil of Perovskia artemisioides Boiss. Nat Prod Res. 2020:1-5. [View at Publisher] [DOI:10.1080/14786419.2020.1803311] [PubMed] [Google Scholar]
17. Mahboubi M, Kazempour N. The antimicrobial activity of essential oil from Perovskia abrotanoides karel and its main components. Indian journal of pharmaceutical sciences. 2009;71(3):343. [View at Publisher] [DOI:10.4103/0250-474X.56016] [PubMed] [Google Scholar]
18. Derakhshanfar A, Mehrabani D, Moayedi J, Jamhiri I. Healing Effect of Perovskia Abrotanoides Karel and Expression of VEGF and TGF-Β Genes in Burn Injury of Rats. International Journal of Nutrition Sciences. 2019; 4(4): 175-180. [View at Publisher] [Google Scholar]
19. Fereidouni A, Sabbaghian E, Ghanbari A, Khaleghian A. Effect of Hydroalcoholic Extract of Perovskia abrotanoides on Glucose, Cholesterol and HDL Levels in Diabetic Rats. Journal of Mazandaran University of Medical Sciences. 2019; 29(175): 138-44. [View at Publisher] [Google Scholar]
20. Sairafianpour M, Christensen J, Stærk D, Budnik BA, Kharazmi A, Bagherzadeh K, et al. Leishmanicidal, antiplasmodial, and cytotoxic activity of novel diterpenoid 1, 2-quinones from Perovskia abrotanoides: new source of tanshinones. J Nat Prod. 2001; 64(11): 1398-403. [View at Publisher] [DOI:10.1021/np010032f] [PubMed] [Google Scholar]
21. Jiang Z-Y, Huang C-G, Xiong H-B, Tian K, Liu W-X, Hu Q-F, et al. Perovskatone A: a novel C23 terpenoid from Perovskia atriplicifolia. Tetrahedron Lett. 2013;54(29):3886-8. [View at Publisher] [DOI:10.1016/j.tetlet.2013.05.056] [Google Scholar]
22. Iqbal J, Abbasi BA, Mahmood T, Kanwal S, Ahmad R, Ashraf M. Plant-extract mediated green approach for the synthesis of ZnONPs: Characterization and evaluation of cytotoxic, antimicrobial and antioxidant potentials. J Mol Struct. 2019;1189:315-27. [View at Publisher] [DOI:10.1016/j.molstruc.2019.04.060] [Google Scholar]
23. Jorgensen JH, Turnidge JD. Susceptibility test methods: dilution and disk diffusion methods. Manual of Clinical Microbiology, Eleventh Edition: American Society of Microbiology. 2015; 1253-73. [View at Publisher] [DOI:10.1128/9781555817381.ch71.] [Google Scholar]
24. CLSI C. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard. 2012.
25. Wikler MA. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically: approved standard. CLSI (NCCLS). 2006;26:M7-A. [View at Publisher] [DOI:10.1016/S0378-8741(01)00413-5] [PubMed] [Google Scholar]
26. Braca A, Sortino C, Politi M, Morelli I, Mendez J. Antioxidant activity of flavonoids from Licania licaniaeflora. J Ethnopharmacol. 2002;79(3):379-81. [View at Publisher] [DOI:10.1016/S0378-8741(01)00413-5] [PubMed] [Google Scholar]
27. Arumugam M, Mitra A, Jaisankar P, Dasgupta S, Sen T, Gachhui R, et al. Isolation of an unusual metabolite 2-allyloxyphenol from a marine actinobacterium, its biological activities and applications. Appl Microbiol Biotechnol. 2010; 86(1): 109-17. [View at Publisher] [DOI:10.1007/s00253-009-2311-2] [PubMed] [Google Scholar]
28. Wei X, Zhao X. Advances in typing and identification of foodborne pathogens. Current Opinion in Food Science. 2021; 37: 52-57. [View at Publisher] [DOI:10.1016/j.cofs.2020.09.002] [Google Scholar]
29. Mohammadhosseini M, Venditti A, Akbarzadeh A. The genus Perovskia Kar.: ethnobotany, chemotaxonomy and phytochemistry: a review. Toxin Rev. 2019:1-22. [View at Publisher] [DOI:10.1080/15569543.2019.1691013] [Google Scholar]
30. Safaeighomi J, Batooli H. Determination of bioac tive molecules from flowers, leaves, stems and roots of Perovskia abrotanoides Karel growing in central Iran by nano scale injection. Digest Journal of Nanomaterials and Biostructures. 2010;5:551-6. [View at Publisher] [Google Scholar]
31. Abedini A, Roumy V, Mahieux S, Gohari A, Farimani M, Rivière C, et al. Antimicrobial activity of selected Iranian medicinal plants against a broad spectrum of pathogenic and drug multiresistant micro‐organisms. Lett Appl Microbiol. 2014; 59(4): 412-21. [View at Publisher] [DOI:10.1111/lam.12294] [PubMed] [Google Scholar]
32. Ghafourian M, Mazandarani M. Ethnopharmacology, ecological requirements, antioxidant and antimicrobial activities of Perovskia abrotanoides Karel. extract for vaginal infections from semnan province. International journal of women's health and reproduction sciences. 2016;5(4):295-300 [View at Publisher] [DOI:10.15296/ijwhr.2017.50] [Google Scholar]
33. Denis F, Cattoir V, Martin C, Ploy M-C, Poyart C. Bactériologie médicale: techniques usuelles: Elsevier Masson; 2016. [View at Publisher] [Google Scholar]
34. Aoyagi Y, Takahashi Y, Satake Y, Takeya K, Aiyama R, Matsuzaki T, et al. Cytotoxicity of abietane diterpenoids from Perovskia abrotanoides and of their semisynthetic analogues. Bioorganic & medicinal chemistry. 2006;14(15):5285-91. [View at Publisher] [DOI:10.1016/j.bmc.2006.03.047] [PubMed] [Google Scholar]
35. Harper JL. Population biology of plants. Population biology of plants. 1977. [View at Publisher] [Google Scholar]
36. Pourhosseini SH, Hadian J, Sonboli A, Nejad Ebrahimi S, Mirjalili MH. Genetic and Chemical Diversity in Perovskia abrotanoides Kar.(Lamiaceae) Populations Based on ISSR s Markers and Essential Oils Profile. Chem Biodivers. 2018; 15(3): e1700508. [View at Publisher] [DOI:10.1002/cbdv.201700508] [PubMed] [Google Scholar]
37. Hayashi H, Sudo H. Economic importance of licorice. Plant Biotechnol. 2009;26(1):101-4. [View at Publisher] [DOI:10.5511/plantbiotechnology.26.101.] [Google Scholar]
38. Gende L, Maggi M, Van Baren C, Lira ADL, Bandoni A, Fritz R, et al. Antimicrobial and miticide activities of Eucalyptus globulus essential oils obtained from different Argentine regions. Spanish Journal of Agricultural Research. 2010; 3: 642-50. [View at Publisher] [DOI:10.5424/sjar/2010083-1260] [Google Scholar]
39. Ashraf M, Ali Q. Relative membrane permeability and activities of some antioxidant enzymes as the key determinants of salt tolerance in canola (Brassica napus L.). Environ Exp Bot. 2008; 63(1-3): 266-73. [View at Publisher] [DOI:10.1016/j.envexpbot.2007.11.008] [Google Scholar]
40. Karimi S, Arzani A, Saeidi G. Effect of salinity stress on antioxidant enzymes and chlorophyll content of salt-tolerant and salt-sensitive safflower (Carthamus tinctorius L.) genotypes. Journal of Plant Process and Function. 2015;4(13):25-35. [View at Publisher] [Google Scholar]
41. Koca H, Bor M, Özdemir F, Türkan İ. The effect of salt stress on lipid peroxidation, antioxidative enzymes and proline content of sesame cultivars. Environ Exp Bot. 2007;60(3):344-51. [View at Publisher] [DOI:10.1016/j.envexpbot.2006.12.005.] [Google Scholar]
42. Yildiz M, Terzi H. Effect of NaCl stress on chlorophyll biosynthesis, proline, lipid peroxidation and antioxidative enzymes in leaves of salt-tolerant and salt-sensitive barley cultivars. Journal of Agricultural Sciences. 2013; 19(2): 79-88. [View at Publisher] [DOI:10.1501/Tarimbil_0000001232] [Google Scholar]
43. Farzaneh M, Amirahmadi A, Poozesh V, Salimi F. Study on Phytochemical diversity and antioxidant properties of extracts from different populations of Perovskia abrotanoides Kar. in Eastern Alborz. Eco-phytochemical Journal of Medicinal Plants. 2021; 9(3): 16-28. [View at Publisher] [Google Scholar]
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