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Volume 15, Issue 4 (2023)
Iran J War Public Health 2023, 15(4): 347-352 |
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Received: 2023/08/5 | Accepted: 2023/11/21 | Published: 2023/11/28
Received: 2023/08/5 | Accepted: 2023/11/21 | Published: 2023/11/28
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Baqer G, Madhi K, Baqer F, Baqer L, Abbas B. Antibacterial Activity and Phytochemical Screening of Linum Usitatissimum L. on Bacteria Isolated from Wound Infections. Iran J War Public Health 2023; 15 (4) :347-352
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1- Department of Human Anatomy, College of Medicine, University of Basrah, Basrah, Iraq
2- Department of Microbiology, College of Medicine, University of Basrah, Basrah, Iraq
3- Department of Surgery, AL-Sader Teaching Hospital, Basrah, Iraq
4- Department of Microbiology, College of Veterinary Medicine, University of Basrah, Basrah, Iraq
2- Department of Microbiology, College of Medicine, University of Basrah, Basrah, Iraq
3- Department of Surgery, AL-Sader Teaching Hospital, Basrah, Iraq
4- Department of Microbiology, College of Veterinary Medicine, University of Basrah, Basrah, Iraq
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Introduction
Wound infection remains a difficult condition that imposes a significant healthcare burden. Early detection and successful treatments are more important for limiting its economic and health effects and antibiotic resistance [1]. Infection in a wound is a major barrier to healing and can have a negative impact on a wound's healing rate. One of the most important steps in the healing process is keeping damaged tissue free of microbial infection [2].
Continued use of antimicrobial drugs leads to the appearance of antibiotic-resistant bacteria strains and increases the costs of searching for effective antimicrobial agents [3]. Approximately 20,000 resistant genes in bacteria, which have several mechanisms for resistance to standard antimicrobial treatments, have been found [4]. The most common pathogens associated with wound infection in surgeries and burns are Staphylococcus aureus and Pseudomonas aeruginosa. Other species, like Enterococci and Enterobacteriaceae, can be found in immunocompromised patients and after abdominal surgery [5].
Plants are considered a source of natural medicinal compounds for humans that play an important role in health care since they are available and less expensive in treatment [6]. Many studies have identified antimicrobial phytochemical compounds such as phenolic acids, flavonoids, tannins, polyketides, terpenoids, glucosides, lignans, saponins, alkaloids, and steroids in plants [7, 8]. The plant seeds have promising antimicrobial activities that could be used as a natural medicinal alternative instead of chemical substances [9]. One of these important medicinal plants is the flax plant (Linum usitatissimum L.), cultivated and grown globally; the flax plant has attracted attention to its advantages as a food and medicinal benefit [10]. Linum usitatissimum is one of the 180 Linum genus species in the Lamiaceae family [11]. plant’s height is (1.2m), and leaves are 20-40mm long and 8mm wide. Have bright blue flowers with (15-22mm) diameter, and the fruit's diameter is (5-9mm) containing glossy brown seeds of (4-7mm) long [12]. Flaxseed is cultivated widely in the Australian, Asian, and Chinese regions [13]. Flaxseed is consumed as a food characterized by its high fiber content, omega-3 fatty acids, phenolic compounds, lignans, and flavonoids [14]. These seeds consist of a large amount of oil, about 35-45%, and 20-25% proteins, in addition to a small quantity of cyanogenic glycosides [15]. The major bioactive constituents in these seeds include triglycerides of α-linolenic acids (52%), oleic acids (20%), and linoleic acids (17%), in addition to the presence of many minerals such as calcium, phosphorus, and magnesium [16].
Many studies revealed important pharmacological properties of flaxseed against various diseases that have antioxidant activity [17], anticancer activity [18], anti-inflammatory [19], antibacterial [20], and antifungal activities [21]. It was revealed that flaxseed plays a role in tumor growth and decreases breast and colon cancer incidence [22]. It also treats respiratory tract infections such as cough, bronchitis, and gastrointestinal infections [12]. Flaxseed oil is an astringent in fungicidal and insecticide lotions [23]. Flaxseed oil has antimicrobial, antioxidant, and wound-healing properties. As well as Omega-3 fatty acids characterized by their anti-inflammatory benefits [24]. Certain studies suggested that the antibacterial activity that the flaxseed possesses is due to its contents of lignans and phenolic acids [25]. The lignan has antioxidant properties and efficacy for treating breast cancer [26]. The ethanol extract of flaxseed has activity against some ovary and endometrial malignant cells due to the content of phenolic compounds [27]. Also, it was confirmed that flaxseed oil has antibacterial activity due to its content of lignans, flavonoids, and phenolic acids [28].
This study aimed to determine the antibacterial activity of flaxseed aqueous, ethanol extracts, and flaxseed oil against different species of bacteria isolated from human wound infections. Furthermore, to investigate all available information about the chemical constituents of flaxseed extracts and assess the various phytochemical compositions.
Materials and Methods
Preparation of flaxseed extracts
The flaxseed and its oil were purchased from a local herbal shop in Basrah Governorate, Iraq. Seeds were cleaned and washed with distilled water and completely shade-dried for one week. Then, these seeds were ground into fine powder using a laboratory grinder. The powder was weighed and stored in a small bottle in a dry place. To make flaxseed aqueous and ethanolic extracts, 50g of seed powder was steeped separately in 500ml distilled water and ethanol. These two extracts were shaken in a rotary shaker for 24 hours and filtered through filter paper (Whatman No.1). A 0.45m micro filter was then utilized in a rotating evaporator at 50°C for additional filtration. The extracts were then kept at 4°C until they were used. These extracts were further diluted with Dimethyl Sulfoxide (DMSO) to achieve varied concentrations (200, 100, 50, and 25g/ml) [29].
Phytochemical screening
Phytochemical screening was carried out to detect the presence of active phytocomponents in flaxseed aqueous and ethanolic extracts such as alkaloids, terpenoids, tannins, flavonoids, glycosides, saponins, steroids, phenolic compounds, proteins, and carbohydrates according to different chemical tests. Flavonoids were detected using the Thamilmaraiselvi et al. method, where the appearance of a yellow tint is shown. Steroids were also tested, indicated by a color shift from violet to blue or green [30]. Mayer's reagent was used to detect alkaloids that appear as cream-colored precipitate [31]. The saponins, whose appearance was creamy and missing small bubbles, were detected according to the Edeoga et al. method. Terpenoids were detected using the Salkowski test, which is detected by a reddish-brown interface [32]. According to the method Hanaa et al. used to detect tannins and phenolic compounds [33]. As well as the method of Sood et al. was also used to detect glycosides [34]. Biuret test was used for the detection of proteins whose presence was confirmed by the formation of violet or pink color. At the same time, carbohydrates were tested using Benedict's test, which formed a reddish-brown precipitate [35].
Bacterial cultures
Flaxseed's antibacterial efficacy was tested against many species of bacteria isolated from wound infections of patients admitted to Al Sadder Teaching Hospital. Wound infections include from the head, trunk, upper and lower extremities (abdomen, leg and foot, hand, and head). Samples were collected from patients with wound infections using a sterile cotton swab and then transported using the transport medium Brain Heart Infusion (BHI); samples were then inoculated into blood agar and McConkey agar using a sterile loop (streaking method) then incubated at 37°C for 24 hours. Double wound swabs were collected from each site simultaneously to decrease the possibility of contamination. Bacterial identification was based on standard microbiological techniques such as Gram stain, colony morphology, and biochemical assays.
Antibacterial activity of flaxseed extracts
The agar well diffusion method tested the antibacterial activity of aqueous and ethanolic extracts and flaxseed oil on bacteria from wound infections. The bacterial species tested included Staphylococcus aureus, Streptococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella sp. and Proteus mirabilis. A few colonies from these bacterial species were suspended in sterile saline until the turbidity matched McFarland tube number 0.5 (1.5 108CFU/ml). Then, the bacterial inoculum was cultured on Mueller Hinton agar using the streaking method, and each strain of bacteria was inoculated on duplicate plates on agar. Then, a sterile corn borer with a diameter of 6mm was used to punch wells in the agar, where five wells were made in each plate. 50μl of each extract, including aqueous, ethyl, and oil solutions, was added to each well at different concentrations (200, 100, 50, and 25mg/ml), and the same volume of extraction solvent for control was filled in the wells. The antibacterial activities were compared with DMSO as the negative control. Then, all these plates were incubated at 37°C for 24 hours. inhibitory effect was evaluated by measuring the diameter of the bacterial growth inhibitory zone that was produced around the well, and the inhibition zone was measured in millimeters [36].
Findings
Phytochemical screening was carried out on the crude aqueous and ethanolic flaxseed extracts, revealing the presence of many active compounds. These were glycosides, flavonoids, saponins, terpenoids, alkaloids, phenols, tannins, proteins, and amino acids (Table 1). Many of these compounds were present in both extracts, except glycoside and terpenoids, which were not found in the aqueous extract, and saponins, which were not found in the ethyl extract.
Table 1. Phytochemical screening of active compounds for aqueous and ethanolic extracts of Linum usitatissimum seeds.
Varying degrees of inhibition zones for flaxseed aqueous and ethanolic crude extracts were compared with control solution DMSO, which didn’t show an inhibitory effect on all tested bacteria. Results showed that crude ethanolic extract and oil had an inhibitory effect against the growth of all gram-positive and gram-negative bacteria species. It was also revealed that aqueous extract has an inhibitory effect against some gram-positive bacteria, including Staphylococcus aureus and Streptococcus faecalis only. However, no inhibitory effect against gram-negative bacteria was shown (Table 2).
Table 2. The antibacterial activity of crude flaxseed aqueous and ethanolic extracts as well as flaxseed oil against bacteria of wound infection
Aqueous extract showed inhibition zones only for Staphylococcus aureus and Streptococcus faecalis at 50, 100, and 200mg/ml. All the concentrations of the ethanolic extract showed inhibitory effects against all tested bacteria. The maximum antibacterial effect of ethanol extract on aqueous extract was 200mg/ml. Also, 200mg/ml of flaxseed oil had the most powerful inhibitory effects on all tested bacteria (Table 3).
Table 3. The inhibition zone sizes (millimeters) of different concentrations of flaxseed aqueous extract, ethanolic extract, and oil on isolated bacteria from wound infections
Discussion
This study aimed to determine the antibacterial activity of flaxseed aqueous, ethanol extracts, and flaxseed oil against different species of bacteria isolated from human wound infections. Wound infections are still an important problem in human life. The continuing use of antibiotics resulted in the evolution of antibiotic-resistant strains and raised the problem of the side effects of using drugs for a prolonged time. The studies focused on the antibacterial effect of flaxseed against many bacterial species that cause problems in human life. In the current study, we determine the antibacterial activity of the flaxseeds by measuring the inhibitory effect of flaxseeds aqueous, ethanol extracts, and flaxseed oil against the different species of bacteria isolated from wound infections. Also investigated and assessed the various phytochemical compositions of flaxseeds, and the phytochemical analysis of crude aqueous and ethyl flaxseed extracts showed the presence of different active compounds, including glycosides, flavonoids, saponins, terpenoids, steroids, alkaloids, phenols, tannins, proteins, and carbohydrates with few differences. These findings are similar to other studies, which revealed that ethanolic extract lacks saponins, such as Hanaa [33], Amin & Thakur [37], and Alachaher et al. [38]. Similarly, other studies confirm that aqueous extract lacks glycoside and terpenoids [12, 20, 33]. The importance of phytochemical compounds in flaxseed is that they can act through various mechanisms and target areas over standard antibiotics to improve efficacy in decreasing resistance development [39]. Many studies have suggested the importance of these phytochemicals against many microorganisms by inhibiting their growth and activity through the action of phenolic acids, flavonoids, tannins, and fatty acids [40, 41]. The phenolic compounds have a role in bacterial DNA breakdown and preventing gyrase activity [42]. Terpenoids stimulate wound healing due to their astringent properties, while fatty acids improve skin moisture to aid healing [43]. Also, oleic and linoleic acids have been suggested to promote wound healing [44].
In our study, flaxseed extracts were tested on Staphylococcus aureus, Streptococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella sp., and Proteus mirabilis that were isolated from wound infections. This study included these bacteria because of their important role in causing wound infections. This agreed with other studies that recorded the most prevalent bacterial isolates in wound infections; Staphylococcus aureus and Escherichia coli, Proteus species, Klebsiella pneumoniae, and Pseudomonas aeruginosa [45, 46]. Furthermore, the most common bacterial species in wound infections were gram-positive than gram-negative bacteria [47]. Our study about the crude flaxseed aqueous, ethyl extracts, and oil revealed different inhibitory effects against tested bacteria. The aqueous extract revealed an inhibitory effect against gram-positive bacteria only. Meanwhile, ethanolic extract and the oil extract showed an inhibitory effect on all tested bacteria, with a higher inhibitory effect on gram-positive bacteria than on gram-negative bacteria. All these extracts showed different antibacterial activities regarding their various concentrations; however, their inhibitory effect was found to increase with increased extract concentration.
Several studies agree with these results as they confirmed that the flaxseed has a bactericidal effect against S. aureus, E. coli, P. aeruginosa, and K. pneumoniae [23]. Also, ethanolic extract of flaxseeds showed promising inhibitory activity against different gram-positive and gram-negative bacteria [10, 12, 37]. In addition, other studies confirmed that flaxseed oil has antibacterial activity against some bacterial species, such as Staphylococcus aureus and E. coli [46, 48]. Flaxseed oil is considered a good medication that can be used to treat wound infections, especially those caused by bacteria such as S. aureus and K. pneumoniae [36]. Another study showed that the methanol extract of flaxseed has a higher antibacterial activity than the aqueous extract. The concentration of the extracts, when increased, causes an increase in the size of the inhibition zone [20]. Several studies suggested that Linum usitatissimum L. antibacterial activity is based on the presence of natural phenolic compounds, lignans, and fatty acids [40, 49]. Flaxseed contains a high amount of secoisolariciresinoldiglucoside (SDG), a precursor of lignans that may be responsible for the antibacterial action of several flaxseed extracts. The components of lignans have antibacterial effects against gram-positive bacteria [36]. Furthermore, flavonoids play a role in inhibiting DNA synthesis in Proteus sp. and RNA production in Staphylococcus sp. [50, 51].
Conclusion
Flaxseed ethanolic extract and flaxseed oil have an inhibitory effect against different species of both gram-positive and gram-negative bacteria isolated from wound infections.
Acknowledgments: Authors acknowledge the College of Medicine and Al Sadder Teaching Hospital in Basrah for providing sample collection and laboratory facility.
Ethical Permissions: Ethical Permissions were approved by Approval Committee of College of Medicine , University of Basrah.
Conflicts of Interests: Authors declare that there is no conflict of interest.
Authors’ Contribution: Baqer GK (First Author), Introduction Writer/Assistant Researcher/Statistical Analyst (20%); Madhi KS (Second Author), Introduction Writer/Assistant Researcher (20%); Baqer FK (Third Author), Introduction Writer/Assistant Researcher (20%); Baqer LK (Fourth Author), Methodologist/Assistant Researcher/Discussion Writer (20%); Abbas BS (Fifth Author), Introduction Writer/Main Researcher/Discussion Writer (20%)
Funding/Support: Nothing has been reported.
Wound infection remains a difficult condition that imposes a significant healthcare burden. Early detection and successful treatments are more important for limiting its economic and health effects and antibiotic resistance [1]. Infection in a wound is a major barrier to healing and can have a negative impact on a wound's healing rate. One of the most important steps in the healing process is keeping damaged tissue free of microbial infection [2].
Continued use of antimicrobial drugs leads to the appearance of antibiotic-resistant bacteria strains and increases the costs of searching for effective antimicrobial agents [3]. Approximately 20,000 resistant genes in bacteria, which have several mechanisms for resistance to standard antimicrobial treatments, have been found [4]. The most common pathogens associated with wound infection in surgeries and burns are Staphylococcus aureus and Pseudomonas aeruginosa. Other species, like Enterococci and Enterobacteriaceae, can be found in immunocompromised patients and after abdominal surgery [5].
Plants are considered a source of natural medicinal compounds for humans that play an important role in health care since they are available and less expensive in treatment [6]. Many studies have identified antimicrobial phytochemical compounds such as phenolic acids, flavonoids, tannins, polyketides, terpenoids, glucosides, lignans, saponins, alkaloids, and steroids in plants [7, 8]. The plant seeds have promising antimicrobial activities that could be used as a natural medicinal alternative instead of chemical substances [9]. One of these important medicinal plants is the flax plant (Linum usitatissimum L.), cultivated and grown globally; the flax plant has attracted attention to its advantages as a food and medicinal benefit [10]. Linum usitatissimum is one of the 180 Linum genus species in the Lamiaceae family [11]. plant’s height is (1.2m), and leaves are 20-40mm long and 8mm wide. Have bright blue flowers with (15-22mm) diameter, and the fruit's diameter is (5-9mm) containing glossy brown seeds of (4-7mm) long [12]. Flaxseed is cultivated widely in the Australian, Asian, and Chinese regions [13]. Flaxseed is consumed as a food characterized by its high fiber content, omega-3 fatty acids, phenolic compounds, lignans, and flavonoids [14]. These seeds consist of a large amount of oil, about 35-45%, and 20-25% proteins, in addition to a small quantity of cyanogenic glycosides [15]. The major bioactive constituents in these seeds include triglycerides of α-linolenic acids (52%), oleic acids (20%), and linoleic acids (17%), in addition to the presence of many minerals such as calcium, phosphorus, and magnesium [16].
Many studies revealed important pharmacological properties of flaxseed against various diseases that have antioxidant activity [17], anticancer activity [18], anti-inflammatory [19], antibacterial [20], and antifungal activities [21]. It was revealed that flaxseed plays a role in tumor growth and decreases breast and colon cancer incidence [22]. It also treats respiratory tract infections such as cough, bronchitis, and gastrointestinal infections [12]. Flaxseed oil is an astringent in fungicidal and insecticide lotions [23]. Flaxseed oil has antimicrobial, antioxidant, and wound-healing properties. As well as Omega-3 fatty acids characterized by their anti-inflammatory benefits [24]. Certain studies suggested that the antibacterial activity that the flaxseed possesses is due to its contents of lignans and phenolic acids [25]. The lignan has antioxidant properties and efficacy for treating breast cancer [26]. The ethanol extract of flaxseed has activity against some ovary and endometrial malignant cells due to the content of phenolic compounds [27]. Also, it was confirmed that flaxseed oil has antibacterial activity due to its content of lignans, flavonoids, and phenolic acids [28].
This study aimed to determine the antibacterial activity of flaxseed aqueous, ethanol extracts, and flaxseed oil against different species of bacteria isolated from human wound infections. Furthermore, to investigate all available information about the chemical constituents of flaxseed extracts and assess the various phytochemical compositions.
Materials and Methods
Preparation of flaxseed extracts
The flaxseed and its oil were purchased from a local herbal shop in Basrah Governorate, Iraq. Seeds were cleaned and washed with distilled water and completely shade-dried for one week. Then, these seeds were ground into fine powder using a laboratory grinder. The powder was weighed and stored in a small bottle in a dry place. To make flaxseed aqueous and ethanolic extracts, 50g of seed powder was steeped separately in 500ml distilled water and ethanol. These two extracts were shaken in a rotary shaker for 24 hours and filtered through filter paper (Whatman No.1). A 0.45m micro filter was then utilized in a rotating evaporator at 50°C for additional filtration. The extracts were then kept at 4°C until they were used. These extracts were further diluted with Dimethyl Sulfoxide (DMSO) to achieve varied concentrations (200, 100, 50, and 25g/ml) [29].
Phytochemical screening
Phytochemical screening was carried out to detect the presence of active phytocomponents in flaxseed aqueous and ethanolic extracts such as alkaloids, terpenoids, tannins, flavonoids, glycosides, saponins, steroids, phenolic compounds, proteins, and carbohydrates according to different chemical tests. Flavonoids were detected using the Thamilmaraiselvi et al. method, where the appearance of a yellow tint is shown. Steroids were also tested, indicated by a color shift from violet to blue or green [30]. Mayer's reagent was used to detect alkaloids that appear as cream-colored precipitate [31]. The saponins, whose appearance was creamy and missing small bubbles, were detected according to the Edeoga et al. method. Terpenoids were detected using the Salkowski test, which is detected by a reddish-brown interface [32]. According to the method Hanaa et al. used to detect tannins and phenolic compounds [33]. As well as the method of Sood et al. was also used to detect glycosides [34]. Biuret test was used for the detection of proteins whose presence was confirmed by the formation of violet or pink color. At the same time, carbohydrates were tested using Benedict's test, which formed a reddish-brown precipitate [35].
Bacterial cultures
Flaxseed's antibacterial efficacy was tested against many species of bacteria isolated from wound infections of patients admitted to Al Sadder Teaching Hospital. Wound infections include from the head, trunk, upper and lower extremities (abdomen, leg and foot, hand, and head). Samples were collected from patients with wound infections using a sterile cotton swab and then transported using the transport medium Brain Heart Infusion (BHI); samples were then inoculated into blood agar and McConkey agar using a sterile loop (streaking method) then incubated at 37°C for 24 hours. Double wound swabs were collected from each site simultaneously to decrease the possibility of contamination. Bacterial identification was based on standard microbiological techniques such as Gram stain, colony morphology, and biochemical assays.
Antibacterial activity of flaxseed extracts
The agar well diffusion method tested the antibacterial activity of aqueous and ethanolic extracts and flaxseed oil on bacteria from wound infections. The bacterial species tested included Staphylococcus aureus, Streptococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella sp. and Proteus mirabilis. A few colonies from these bacterial species were suspended in sterile saline until the turbidity matched McFarland tube number 0.5 (1.5 108CFU/ml). Then, the bacterial inoculum was cultured on Mueller Hinton agar using the streaking method, and each strain of bacteria was inoculated on duplicate plates on agar. Then, a sterile corn borer with a diameter of 6mm was used to punch wells in the agar, where five wells were made in each plate. 50μl of each extract, including aqueous, ethyl, and oil solutions, was added to each well at different concentrations (200, 100, 50, and 25mg/ml), and the same volume of extraction solvent for control was filled in the wells. The antibacterial activities were compared with DMSO as the negative control. Then, all these plates were incubated at 37°C for 24 hours. inhibitory effect was evaluated by measuring the diameter of the bacterial growth inhibitory zone that was produced around the well, and the inhibition zone was measured in millimeters [36].
Findings
Phytochemical screening was carried out on the crude aqueous and ethanolic flaxseed extracts, revealing the presence of many active compounds. These were glycosides, flavonoids, saponins, terpenoids, alkaloids, phenols, tannins, proteins, and amino acids (Table 1). Many of these compounds were present in both extracts, except glycoside and terpenoids, which were not found in the aqueous extract, and saponins, which were not found in the ethyl extract.
Table 1. Phytochemical screening of active compounds for aqueous and ethanolic extracts of Linum usitatissimum seeds.
Varying degrees of inhibition zones for flaxseed aqueous and ethanolic crude extracts were compared with control solution DMSO, which didn’t show an inhibitory effect on all tested bacteria. Results showed that crude ethanolic extract and oil had an inhibitory effect against the growth of all gram-positive and gram-negative bacteria species. It was also revealed that aqueous extract has an inhibitory effect against some gram-positive bacteria, including Staphylococcus aureus and Streptococcus faecalis only. However, no inhibitory effect against gram-negative bacteria was shown (Table 2).
Table 2. The antibacterial activity of crude flaxseed aqueous and ethanolic extracts as well as flaxseed oil against bacteria of wound infection
Aqueous extract showed inhibition zones only for Staphylococcus aureus and Streptococcus faecalis at 50, 100, and 200mg/ml. All the concentrations of the ethanolic extract showed inhibitory effects against all tested bacteria. The maximum antibacterial effect of ethanol extract on aqueous extract was 200mg/ml. Also, 200mg/ml of flaxseed oil had the most powerful inhibitory effects on all tested bacteria (Table 3).
Table 3. The inhibition zone sizes (millimeters) of different concentrations of flaxseed aqueous extract, ethanolic extract, and oil on isolated bacteria from wound infections
Discussion
This study aimed to determine the antibacterial activity of flaxseed aqueous, ethanol extracts, and flaxseed oil against different species of bacteria isolated from human wound infections. Wound infections are still an important problem in human life. The continuing use of antibiotics resulted in the evolution of antibiotic-resistant strains and raised the problem of the side effects of using drugs for a prolonged time. The studies focused on the antibacterial effect of flaxseed against many bacterial species that cause problems in human life. In the current study, we determine the antibacterial activity of the flaxseeds by measuring the inhibitory effect of flaxseeds aqueous, ethanol extracts, and flaxseed oil against the different species of bacteria isolated from wound infections. Also investigated and assessed the various phytochemical compositions of flaxseeds, and the phytochemical analysis of crude aqueous and ethyl flaxseed extracts showed the presence of different active compounds, including glycosides, flavonoids, saponins, terpenoids, steroids, alkaloids, phenols, tannins, proteins, and carbohydrates with few differences. These findings are similar to other studies, which revealed that ethanolic extract lacks saponins, such as Hanaa [33], Amin & Thakur [37], and Alachaher et al. [38]. Similarly, other studies confirm that aqueous extract lacks glycoside and terpenoids [12, 20, 33]. The importance of phytochemical compounds in flaxseed is that they can act through various mechanisms and target areas over standard antibiotics to improve efficacy in decreasing resistance development [39]. Many studies have suggested the importance of these phytochemicals against many microorganisms by inhibiting their growth and activity through the action of phenolic acids, flavonoids, tannins, and fatty acids [40, 41]. The phenolic compounds have a role in bacterial DNA breakdown and preventing gyrase activity [42]. Terpenoids stimulate wound healing due to their astringent properties, while fatty acids improve skin moisture to aid healing [43]. Also, oleic and linoleic acids have been suggested to promote wound healing [44].
In our study, flaxseed extracts were tested on Staphylococcus aureus, Streptococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella sp., and Proteus mirabilis that were isolated from wound infections. This study included these bacteria because of their important role in causing wound infections. This agreed with other studies that recorded the most prevalent bacterial isolates in wound infections; Staphylococcus aureus and Escherichia coli, Proteus species, Klebsiella pneumoniae, and Pseudomonas aeruginosa [45, 46]. Furthermore, the most common bacterial species in wound infections were gram-positive than gram-negative bacteria [47]. Our study about the crude flaxseed aqueous, ethyl extracts, and oil revealed different inhibitory effects against tested bacteria. The aqueous extract revealed an inhibitory effect against gram-positive bacteria only. Meanwhile, ethanolic extract and the oil extract showed an inhibitory effect on all tested bacteria, with a higher inhibitory effect on gram-positive bacteria than on gram-negative bacteria. All these extracts showed different antibacterial activities regarding their various concentrations; however, their inhibitory effect was found to increase with increased extract concentration.
Several studies agree with these results as they confirmed that the flaxseed has a bactericidal effect against S. aureus, E. coli, P. aeruginosa, and K. pneumoniae [23]. Also, ethanolic extract of flaxseeds showed promising inhibitory activity against different gram-positive and gram-negative bacteria [10, 12, 37]. In addition, other studies confirmed that flaxseed oil has antibacterial activity against some bacterial species, such as Staphylococcus aureus and E. coli [46, 48]. Flaxseed oil is considered a good medication that can be used to treat wound infections, especially those caused by bacteria such as S. aureus and K. pneumoniae [36]. Another study showed that the methanol extract of flaxseed has a higher antibacterial activity than the aqueous extract. The concentration of the extracts, when increased, causes an increase in the size of the inhibition zone [20]. Several studies suggested that Linum usitatissimum L. antibacterial activity is based on the presence of natural phenolic compounds, lignans, and fatty acids [40, 49]. Flaxseed contains a high amount of secoisolariciresinoldiglucoside (SDG), a precursor of lignans that may be responsible for the antibacterial action of several flaxseed extracts. The components of lignans have antibacterial effects against gram-positive bacteria [36]. Furthermore, flavonoids play a role in inhibiting DNA synthesis in Proteus sp. and RNA production in Staphylococcus sp. [50, 51].
Conclusion
Flaxseed ethanolic extract and flaxseed oil have an inhibitory effect against different species of both gram-positive and gram-negative bacteria isolated from wound infections.
Acknowledgments: Authors acknowledge the College of Medicine and Al Sadder Teaching Hospital in Basrah for providing sample collection and laboratory facility.
Ethical Permissions: Ethical Permissions were approved by Approval Committee of College of Medicine , University of Basrah.
Conflicts of Interests: Authors declare that there is no conflict of interest.
Authors’ Contribution: Baqer GK (First Author), Introduction Writer/Assistant Researcher/Statistical Analyst (20%); Madhi KS (Second Author), Introduction Writer/Assistant Researcher (20%); Baqer FK (Third Author), Introduction Writer/Assistant Researcher (20%); Baqer LK (Fourth Author), Methodologist/Assistant Researcher/Discussion Writer (20%); Abbas BS (Fifth Author), Introduction Writer/Main Researcher/Discussion Writer (20%)
Funding/Support: Nothing has been reported.
Keywords:
References
1. 1- Woo K, Song J, Victoria A, Block LJ, Currie LM, Shang J, et al. Exploring prevalence of wound infections and related patient characteristics in homecare using natural language processing. Int Wound J. 2022;19(1):211-21. [Link] [DOI:10.1111/iwj.13623]
2. Al-Waili NS, Salom K, Al-Ghamdi AA. Honey for wound healing, ulcers, and burns. Sci World J. 2011;11:766-87. [Link] [DOI:10.1100/tsw.2011.78]
3. Cooper RA, Molan PC, Harding KG: The sensitivity to honey of Gram-positive cocci of clinical significance isolated from wounds. J App Microbiol. 2002;93:857-63. [Link] [DOI:10.1046/j.1365-2672.2002.01761.x]
4. Aslam B, Wang W, Arshad M I, Khurshid M, Muzammil S, Rasool MH, et al. Antibiotic resistance: a rundown of a global crisis. Infection Drug Resistance.2018;11:1645-58. [Link] [DOI:10.2147/IDR.S173867]
5. Taiwo S, Okesina A, Onile B. In vitro antimicrobial susceptibility pattern of bacterial isolates from wound infections in University of Ilorin Teaching Hospital. Afr J Clin Exp Microbiol. 2002;3(1):6-10. [Link] [DOI:10.4314/ajcem.v3i1.7342]
6. Allemailem KS. Antimicrobial potential of naturally occurring bioactive secondary metabolites. J Pharm Bioall Sci. 2021;13:155-62. [Link] [DOI:10.4103/jpbs.JPBS_753_20]
7. Álvarez-Martínez FJ, Barrajón-Catalán E, Herranz-López M, Micol V. Antibacterial plant compounds, extracts and essential oils: An updated review on their effects and putative mechanisms of action. Phytomedicine. 2021;90;15626. [Link] [DOI:10.1016/j.phymed.2021.153626]
8. Jubair N, Rajagopal M, Chinnappan S, Abdullah NB, Fatima A. Review on the antibacterial mechanism of plant-derived compounds against multidrug-resistant bacteria (MDR). Evid Based Complement Alternat Med. 2021;2021:3663315. [Link] [DOI:10.1155/2021/3663315]
9. Abu-Zaid AA, Al-Barty A, Morsy K, Hamdi H. In vitro study of antimicrobial activity of some plant seeds against bacterial strains causing food poisoning diseases. Braz J Biol. 2022;82:e256409. [Link] [DOI:10.1590/1519-6984.256409]
10. Dagnaw M. Isolation of antibacterial compounds from Linum usitatissimum and evaluation its antibacterial activity. J Med Plants Stud. 2019;7(3):94-8. [Link]
11. Ansari R, Zarshenas MM, Dadbakhsh AH. A review on pharmacological and clinical aspects of linum usitatissimum L. 2 Curr Drug Discov Technol. 2019;16(2):148-58. [Link] [DOI:10.2174/1570163815666180521101136]
12. Thamilmarai SB, Sathammaj PN, Steffi PF, Priyadarshni S. Phytochemical evaluation, GC-MS analysis of phytoactive compounds, and antibacterial activity studies from linum usitatissimum. Asian J Pharm Clin Res. 2019;12(8):141-9. [Link] [DOI:10.22159/ajpcr.2019.v12i18.34126]
13. Andronie L, Pop ID, Sobolu R, Diaconeasa Z, Truţă A, Hegeduş C, et al. Characterization of flax and hemp using spectrometric methods. Appl Sci. 2021;11(18):8341. [Link] [DOI:10.3390/app11188341]
14. Kajla P, Sharma A, Sood D. Flaxseed-a potential functional food source. J Food Sci Technol. 2015;52(4):1857-71. [Link] [DOI:10.1007/s13197-014-1293-y]
15. Coşkuner Y, Karababa E. Some physical properties of flaxseed (Linum usitatissimum L.). J Food Eng. 2007;78(3):1067-73. [Link] [DOI:10.1016/j.jfoodeng.2005.12.017]
16. Parikh M, Netticadan T, Pierce GN. Flaxseed: Its bioactive components and their cardiovascular benefits. Am J Physiol Heart Circ Physiol. 2018;314:H146-59. [Link] [DOI:10.1152/ajpheart.00400.2017]
17. Safdar B, Pang Z, Liu X, Jatoi MA, Mehmood A, Rashid MT, et al. Flaxseed gum: Extraction, bioactive composition, structural characterization, and its potential antioxidant activity. J Food Biochem. 2019;43(11):e13014. [Link] [DOI:10.1111/jfbc.13014]
18. Tannous S, Haykal T, Dhaini J, Hodroj MH, Rizk SJB, Pharmacotherapy. The anti-cancer effect of flaxseed lignan derivatives on different acute myeloid leukemia cancer cells. Biomed Pharmacother. 2020:132:110884. [Link] [DOI:10.1016/j.biopha.2020.110884]
19. Asad B, Khan T, Gul FZ, Ullah MA, Drouet S, Mikac S, et al. Scarlet flax linum grandiflorum (L.) in vitro cultures as a new source of antioxidant and anti-inflammatory lignans. Molecules. 2021;26(15):4511. [Link] [DOI:10.3390/molecules26154511]
20. Hussien ZG, Aziz RA. Chemical composition and antibacterial activity of linum usitatissimum L.(Flaxseed). Sys Rev Pharm. 2021;12(2):145-7. [Link]
21. Madheslu M, Sadasivam N, Ameerbasha SS, Ramakrishnan V. Toxicity of tungsten oxide and IAA-loaded tungsten oxide nanoparticles on linum usitatissimum germination and their antifungal activity. Cell Molec Phytotox Heavy Metal. 2020;112:403-18. [Link] [DOI:10.1007/978-3-030-45975-8_20]
22. Hosseinian SH, Muir AD, Wstcottb ND, Krol Ed S. AAPH-mediated antioxidant reactions of secoisolariciresinol and SDG. Org Biomol Chem. 2007;4:644-54. [Link] [DOI:10.1039/b617426d]
23. Borkar N, Murarkar K. Antimicrobial activity of flaxseed (L. Usitatissimum) Oil and Limestone water against pathogenic microorganisms. World J Pharmaceutical Res. 2018;7(12):841-49. [Link]
24. Mostafa M, Ayimba E. Effect of omega 3 fatty acids family in human health. Int J Adv Res. 2014;2(3):202-11 [Link]
25. Kyselka J, Rabiej D, Dragoun M, Kreps F, Burˇcová Z, Nˇemeˇcková I, et al. Antioxidant and antimicrobial activity of linseed lignans and phenolic acids. Eur Food Res Technol. 2017;243:1633-44. [Link] [DOI:10.1007/s00217-017-2871-9]
26. Basch E, Bent S, Collins J, Dacey C, Hammerness P, Harrison M, et al. Flax and flaxseed oil (Linum usitatissimum): A review by the natural standard research collaboration. J Soc Integr Oncol. 2007;5(3):92-105. [Link] [DOI:10.2310/7200.2007.005]
27. Chera EI, Pop RM, Pârvu M, Sorit,ău O, Uifălean A, Cătoi FA, et al. Flaxseed ethanol extracts' antitumor, antioxidant, and anti-inflammatory potential. Antioxidants. 2022;11(5):892. [Link] [DOI:10.3390/antiox11050892]
28. Banerjee K, Thiagarajan P. Linum usitatissimum L. (Flax) plant and its seed oil; a review. JCHPS. 2015;8(4):623-8. [Link]
29. Al-Timimi LA. Antibacterial and anticancer activities of fenugreek seed extract. Asian Pac J Cancer Prev. 2019;20(12):3771-76. [Link] [DOI:10.31557/APJCP.2019.20.12.3771]
30. Thamilmaraiselvi B, Francis PS. Investigation of phytochemical constituents in Spirulina fusiformis for antibacterial activity. Nati J Physiol Pharm Pharmacol. 2018;8:1491-5. [Link] [DOI:10.5455/njppp.2018.8.0417030072018]
31. Sathammaipriya N, Thamilmaraiselvi B, Steffi PF, Sangeetha K. Investigation of phytochemical constituents in Azolla microphylla for antibacterial activity. Nati J Physiol Pharm Pharmacol. 2018;8:1500-4. [Link] [DOI:10.5455/njppp.2018.8.0310430072018]
32. Edeoga HO, Okwu DE, Mbaebie BO. Phytochemical constituents of some Nigerian medicinal plants. Afr J Biotechnol. 2005;4(7):685-8. [Link] [DOI:10.5897/AJB2005.000-3127]
33. Hanaa MH, Ismail HA, Mahmoud ME, Ibrahim HM. Antioxidant activity and phytochemical analysis of Flaxseed (Linumusitatismum L). Minia J Agric Res Develop. 2017;37(1):129-40. [Link]
34. Sood A, Kaur P, Gupta R. Phytochemical screening and antimicrobial assay of varjous seeds extract of family. Int J Appl Biol Pharmaceutical Technol. 2012;5:401-9. [Link]
35. Ghosh S, Bhattacharyya DK, Kumar D, Ghosh M. Studies on screening of phyto chemicals of flaxseed oil and its defatted flaxseed meal and oxidative stability of the oil extracted by polar and nonpolar solvent system. Acta Sci Nutr Health. 2019;3(7):168-72. [Link]
36. Al-Mathkhury HJF, Al-Dhamin AS, Al-Taie KL. Antibacterial and antibiofilm activity of flaxseed oil. Iraq J Sci. 2016;57(2B):1086-95. [Link]
37. Amin T, Thakur M. A comparative study on proximate composition, phytochemical screening, antioxidant and antimicrobial activities of linumusitatisimum L. (Flaxseed). Int J Curr Microbiol App Sci. 2014;3(4):465-81. [Link]
38. Alachaher FZ, Dali S, Dida N, Krouf D. Comparison of phytochemical and antioxidant properties of extracts from flaxseed (Linum usitatissimum) using different solvents. Int Food Res J. 2018;25(1):75-82. [Link]
39. Abreu AC, McBain AJ, Simoes M. Plants as sources of new antimicrobials and resistance-modifying agents. Nat Prod Rep. 2012;29:1007-21. [Link] [DOI:10.1039/c2np20035j]
40. Fadzira UA, Darnisb DS, Mustafac BE, Mokhtar KI. Linum usitatissimum as an antimicrobial agent and a potential natural healer: A review. Arch Orofac Sci. 2018:13(2):55-62. [Link]
41. Górniak I, Bartoszewski R, Króliczewski J. Comprehensive review of antimicrobial activities of plant flavonoids. Phytochem Rev. 2019;18:241-72. [Link] [DOI:10.1007/s11101-018-9591-z]
42. Takó M, Kerekes EB, Zambrano C, Kotogán A, Papp T, Krisch J, et al. Plant phenolics and phenolic-enriched extracts as antimicrobial agents against food-contaminating microorganisms. Antioxidants. 2020;9(2):162. [Link] [DOI:10.3390/antiox9020165]
43. Beroual K, Agabou A, Abdeldjelil MC, Boutaghane N, Haouam S, Hamdi-Pacha Y. Evaluation of crude flaxseed (Linum usitatissimum L.) oil in burn wound healing in New Zealandrabbits. Afr J Tradit Complement Altern Med. 2017;14(3):280-86. [Link] [DOI:10.21010/ajtcam.v14i3.29]
44. Lewinska A, Zebrowski J, Duda M, Gorka A, Wnuk M. Fatty acid profile and biological activities of linseed and rapeseed oils. Molecules. 2015;20(12):2872-80. [Link] [DOI:10.3390/molecules201219887]
45. Bhatt C, Lakhey M. The distribution of pathogens causing wound infection and their antibiotic susceptibility pattern. J Nepal Health Res Council. 2006;5(1):22-6. [Link]
46. Kaithwas G, Mukerjee A, Kumar P, Majumdar DK. Linum usitatissimum (linseed/ flaxseed) fixed oil: Antimicrobial activity and efficacy in bovine mastitis. Inflammopharmacology. 2011;19(1):45-52. [Link] [DOI:10.1007/s10787-010-0047-3]
47. Haroon M, Iqbal MJ, Hassan W, Ali S, Ahmed H. Evaluation of methanolic crude extract of Linum usitatissimum for the removal of biofilm in diabetic foot isolates. Braz J Biol. 2021;6;83:e245807. [Link] [DOI:10.1590/1519-6984.245807]
48. Hady AA, Darweesh MF, Motar AA. The antibacterial of essential fatty acid semicarbazide extracted from flaxseed oil against some nosocomial infection bacteria in Iraq. Int J Curr Pharmaceutical Rev Res. 2017;8(1):31-9. [Link] [DOI:10.25258/ijcprr.v8i01.9086]
49. Narender BR, Tejaswini S, Sarika M, Karuna N, Shirisha R, Priyanka S. Antibacterial and antifungal activities of Linum usitatissimum (Flaxseeds). Int J Pharm Educ Res. 2016;3:4-8. [Link]
50. Gaafar AA, Salama ZA, Askar MS, El-Hariri DM, Bakry BA. In vitro antioxidant and antimicrobial activities of lignan flaxseed extract (Linum Usitatissimum, L.). Int J Pharm Sci Rev Res. 2013;47:291-7. [Link]
51. Tehrani MH, Batal R, Kamalinejad M, Mahbub A. Extraction and purification of flaxseed proteins and studying their antibacterial activities. J Plant Sci. 2014;2(1):70-6. [Link]