Abstract
Sinusitis or rhinosinusitis is inflammation of the paranasal sinuses which can be due to autoimmune, allergy, and infection problems. Current study was aimed to evaluate the antibiofilm and antibacterial potential of different varieties of A sativum. Four different varieties (China white, China pink, Desi white, and Desi pink) were used and extracted with methanol and water. Results of antioxidant analysis of A sativum extracts showed that all varieties of garlic have considerable quantity of flavonoids with significant DPPH inhibition and reductive potential. Antibacterial activity of A sativum extracts was tested against different Gram negative and Gram-positive sinusitis isolates. All the sinusitis isolates were susceptible to both methanolic and aqueous extracts of different varieties of A sativum with least MIC values. Antibiofilm potential of extracts against sinusitis isolates was evaluated through crystal violet assay, and all extracts of A sativum were significantly effective against destruction of microbial biofilm. In summary, A sativum extracts possess effective antibacterial and antibiofilm activity against sinusitis isolates and can be utilized for prevention of drug resistance against sinusitis infections and further evaluation is necessary.
Introduction
Sinusitis is described as inflammation of one or more of the paranasal sinuses and the condition is considered acute if it lasts less than 4 weeks, subacute if it lasts 4–8 weeks, and chronic if it lasts more than 8 weeks. Three or more episodes of acute sinusitis per year constitute recurrent sinusitis. Viral infection is the main cause of sinusitis, but other microbes (fungi and bacteria) are also present.1,2 The bacterial invasion of the sinuses by Moraxella catarrhalis, Streptococcus pneumoniae, and Haemophilus influenzae is usually preceded by viral infections in the upper respiratory tract. These organisms, as well as Pseudomonas aeruginosa, Staphylococcus aureus, and certain anaerobes, can be identified in chronic sinusitis. 3 The normal flora of the sinusitis includes Staphylococcus epidermidis, S. aureus, Propionibacterium acnes, aerobic diphtheroid, and y-streptococci. Several sinusitis isolates have been reported from healthy nasal cavities including Streptococcus pyogenes (0–1%), H. influenzae (0–6%), M. catarrhalis (0–4%), S. pneumoniae (.5–15%) and anaerobic bacteria Prevotella spp [6–8%] and Peptostreptococcus spp. [7–16%]. 4 Fungi are becoming more widely recognized as a cause of chronic sinusitis, particularly in the southeast and southwest of the United States. 5 Sinusitis infection is increasing dramatically in both developed and developing parts of the world. More than thirty million patients have been reported from the United States alone. Sinusitis may be present in acute to chronic patients and may cause serious adverse reactions.
Medicinal plants make an outstanding contribution to modern therapeutics; around 100 plant-based new drugs were introduced in the United States drug market between 1950 and 1970, including reserpine, deserpidine, vinblastine, and vincristine. 6 A sativum (garlic) belongs to the Alliaceae family. It contains bioactive compounds that exhibit antimicrobial, anti-inflammatory, anticancer, and antioxidant activities. 7 A. sativum is considered one of the most important herbs to promote good health. Allicin (diallyl thiosulfinate) is the most important component of A. sativum, and its efficacy against a wide spectrum of Gram-negative and Gram-positive bacteria was studied. In fresh garlic cloves, allicin is not present but released after chopping and crushing of cloves due to the enzyme alliinase activity. Alliums, which are a component of garlic, are mostly cysteine sulfoxides. Cysteine sulfoxides convert alliinase to allicin, resulting in thiosulfates, which are volatile in nature. 8 Allicin, an organosulfur molecule that inhibits lipid biosynthesis, has been shown to reduce synthesis of RNA in bacteria and disrupt the cell wall of fungi. The profile of organosulfur compounds differs in different Allium species. S-amino acids including methionine and cysteine (traces), alk(en)yl cysteine sulfides (ACSOs), and g-glutamyl peptides are also present. 9 The antimicrobial activities of garlic and allicin are explored a large spectrum against Bacillus, Klebsiella, Cryptocaryon, Clostridium, Helicobacter, Escherichia, Staphylococcus, Salmonella, Proteus, Pseudomonas, Photobacterium, and Mycobacterium spp., but studies on effectiveness of A. sativum against sinusitis isolates is very scarce. Therefore, the current study was designed to evaluate the antibiofilm and antibacterial activities of different varieties of A. sativum against panel of sinusitis isolates.
Materials and Method
Collection and Maintenance of Sinusitis Isolates
Four microbial cultures of sinusitis isolates (Klebsiella pneumonia, Proteus mirabilis, S. aureus and Enterococcus faecalis) were collected from Medicinal Biochemistry Lab, Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan. For purification of bacterial culture, sterilized petri plates with nutrient agar medium were developed and plates were streaked with respective sinusitis isolate under sterilized conditions. Plates were incubated 37°C for 24 h.
Collection of Plant Material and Extract Preparation
Four different varieties of A. sativum (China white, China pink, Desi white, and Desi pink) were collected from the local market of Faisalabad, Pakistan, and identified by Department of Botany, University of Agriculture, Faisalabad, Pakistan. The plant materials were dried and blended into powder form. The dried powder of A. sativum was extracted with methanol and water as extraction solvents. For extraction, the 50 g of dried plant powder was mixed with 500 mL of solvents (methanol and water separately) and kept on orbital shaker at room temperature for 76 h. Then, extracts were filtered using Whatman filter paper 1 and solvent was reduced on rotary evaporator. 10
Total Flavonoid Contents
The total flavonoid contents of A. sativum extracts were determined using the method used by Pranuthi et al. 11 The reaction mixture was prepared by adding .15 mL of NaNO2 (5%) to 2 mL of distilled water and 1 mL of A. sativum extracts and incubated at room temperature for 6 min. After incubation, .15 mL of AlCl3 (10% solution) and 0.3 mL of NaOH (6% solution) were added to the reaction mixture. Methanol was added to make the total volume of reaction mixture up to 5 mL. Reaction mixture was incubated for 10 min, and optical density (OD) was measured at 510 nm. TFC in extracts were calculated from calibration curve of catechin.
Antioxidant Potential of Extracts
DPPH Inhibition Assay
Antioxidant activity of different varieties of A. sativum extracts was determined by measuring their ability to scavenge 1,1-diphenyl-2 picryl hydrazyl stable free radicals (DPPH). The DPPH test was performed as described by Noor et al. 12 with slight modification. A 10 μL of A. sativum extract was added to 1 mL of methanolic solution of DPPH (.0004%) (.1 mM). After 30 min of incubation in darkness at room temperature, the absorbance was recorded at 517 nm. The BHT was used as a standard. The inhibition of DPPH radical by A. sativum extracts was calculated as follows.
DPPH Inhibition (%) = 100 × (A Blank − A Sample/A Blank)
Reducing Potential of Extracts
To measure the reductive potential, reaction mixture was prepared by adding 2.5 mL of each potassium ferricyanide (1%) and phosphate buffer (pH = 6.6) and 1 mL plant extract. The blank contained 2.5 mL phosphate buffer (pH = 6.6) and 2.5 mL of 1% potassium ferricyanide solution. After this, the reaction mixture was incubated for 20 min at 50°C. Then, 2.5 mL of 1% trichloroacetic acid solution was added into reaction mixture and centrifuged for 10 min at 3000 rpm. After centrifugation, the upper layer was separated and mixed into .2 mL of .1% ferric chloride and 2.5 mL distilled water. The optical density was measured at 700 nm. 13
Antimicrobial Activity of A. sativum Against Sinusitis Isolates
Agar Well Diffusion Assay
For determination of antibacterial activity of A. sativum extracts, agar well diffusion assay was used. Nutrient agar plates were developed by pipetting fresh bacterial culture of sinusitis isolate into sterilized agar medium. After solidification, wells of 6 mm diameter were made and different extracts of A. sativum (100 μL) were added into the wells. Ampicillin was used as standard. The antimicrobial activity was calculated in terms of zone of inhibition in millimeter (mm). 14
Minimum Inhibitory Concentration
For the determination of MIC of A. sativum extracts, resazurin-based microdilution assay was performed. Briefly, 100 μL of A. sativum extracts (50 μg/mL) was added into first well of each row while nutrient broth (50 μL) was added into the 2–10 wells of each rows. Last two wells in each row contained growth control (diluted standardized inoculum) and nutrient broth only (for broth sterility control). To transfer A. sativum extracts into 2–10 wells of each row, a multichannel micropipette was used in such a way that each well has twice the concentration of final test. Bacterial suspension (50 μL) was then added into each well. Following 24 h incubation, resazurin solution (30 μL) was added into each well and followed by 2–4 h incubation. MIC was the lowest concentration before color change while column with blue color was scored as above the MIC value. Broth microdilutions were achieved precisely according to the protocol of Clinical and Laboratory Standards Institute.15,16
Microbial Biofilm Inhibition
Biofilm inhibition of A. sativum extracts was carried out using method of Nadaf et al. 17 The well of the sterile 96 well flat-bottomed plastic tissue culture plate was filled with 100 μL of sterilized nutrient broth, A. sativum extract (100 μL) and bacterial culture (20 μL). Negative control well contains only nutrient broth and bacteria. Ampicillin was used as standard. The plates were covered and incubated at 37°C for 24 h. After 24 h, content of each well was discarded, and plates were washed thrice with sterile phosphate buffer saline solution (pH, 7.4) to remove all the planktonic microbes. After washing, 200 μL of ethanol (99.9%) was added into each well and plates were incubated for 15 min. After incubation, 200 μL crystal violet stain was added to each well and plates were again incubated for 10 min. After incubation, the excess stain was discarded and plates were washed again with distilled water (200 μL) and 33% of glacial acetic acid (200 μL) was added. Absorbance of plate was measured at 600 nm. For microscopic analysis of biofilm inhibition, microscopic slides were prepared by following the same method.
Statistical Analysis
Obtained data were analyzed through statistical software SPSS, version 20. Results are expressed as Mean ± SD.
Results and Discussion
In Vitro Antioxidant Activities of Extracts
Antioxidant Potentials of Bioactive Compounds Extracted From Different Varieties of Allium sativum.
CW; China white, CP; China pink, DW; Desi white, and DP; Desi pink.
Antibacterial Potential of Extracts
Antibacterial Activities of Different Varieties of Allium sativum Against Sinusitis Isolates.
CW; China white, CP; China pink, DW; Desi white, and DP; Desi pink.
Antibacterial activity of methanolic extracts of different varieties of Allium sativum against Staphylococcus aureus and Enterococcus faecalis. Note: V1–V4 indicate varieties of garlic as China white (V1), China pink (V2), Desi white (V3), and Desi pink (V4).
Minimum Inhibitory Concentrations of Different Varieties of Allium sativum Against Sinusitis Isolates.
CW; China white, CP; China pink, DW; Desi white, and DP; Desi pink.
Anti-Biofilm Potential of Extracts
Development of microbial biofilm is a key factor involved in the virulence of microbial infections, which ensures protection and superior survival of microbial community from environmental fluctuations, antibiotics, and host defensive mechanisms.
20
It has been suggested that challenges of antibiotics treatments in eradication of pathogenic microbes are due to existence of biofilm, also known as microbial communities, adhered to host cell surfaces and covered by extra polymeric substances (EPS). Extracellular polysaccharides, DNA, and proteins make up the EPS matrix that surrounds living bacterial cells.21,22 Results of biofilm inhibition by different varieties of A. sativum extracts are presented in Table 4, and the findings of current study indicated that all the varieties of A. sativum extracts may have interfered the biofilm formation of sinusitis isolates and effective for the elimination of microbial infection by these bacteria in the sinuses. Results showed that all varieties of A. sativum have significant potential to inhibit the biofilm formation of sinusitis isolates. The maximum biofilm inhibition was shown by methanolic extract of Desi pink (DP) against P. mirabilis while the least inhibition was exhibited by water extract of China white (CW) against K. pneumonia. Results indicated that methanolic extracts of different varieties of A. sativum were more effective as compared to water extracts. Against E. faecalis, the highest inhibition (89.82%) was shown by methanolic extract of Desi pink (DP) while the least inhibition (38.67%) was observed by water extract of China white (CW). Similarly, against K. pneumonia and P. mirabilis, maximum biofilm inhibition (89.55 and 88.39%) was shown by methanolic extract of Desi pink (DP) while the least inhibition (19.08 and 37.37%) was exhibited by water extract of China white (CW). Against S. aureus, methanolic extract of Desi white (DW) and water extract of China white showed maximum and lowest inhibition of biofilm. It is also indicated that both the Desi varieties (DP, DW) of A. sativum were more effective against eradication of sinusitis isolates biofilm as compared to Chinese varieties (CP, CW). Previous studies reported that phytochemicals present in medicinal plants decrease the expression of genes involved in pathogenicity of bacteria by inhibiting the biofilm formation.
23
Observations of phase contrast and SEM results of the cell density and morphology of biofilms further supported the destructive abilities of different varieties of A. sativum extracts on biofilms of sinusitis isolates (Figure 2). Phase contrast microscopic images of biofilm inhibition of negative control (A), methanolic extracts of Desi white (B), Desi pink (C), China white (D), China pink (E), and Ampicillin (F) against Staphylococcus aureus. Antibiofilm Potential of Aqueous and Methanol Extracts of Allium Sativum Against Sinusitis Infection. CW; China white, CP; China pink, DW; Desi white, and DP; Desi pink.
Conclusion
From the current study, it was concluded that all selected varieties of A. sativum have significant antibacterial and antibiofilm activities against sinusitis isolates. Further, it can also be concluded that both Desi varieties of A. sativum are more effective against sinusitis isolates as compared to Chinese varieties. From antimicrobial and antibiofilm potential of A. sativum, we can conclude that this plant has significant potential to be utilized against sinusitis infection and this should be fully investigated prior to its clinical application. We suggest further studies directed on the bioassay-guided fractionation of methanolic extract of A. sativum to determine if different isolated compounds or a refined fraction can reach an MIC at a therapeutically appropriate concentration.
Footnotes
Acknowledgments
The authors are thankful to Higher Education Commission (HEC) of Pakistan for providing fund for this research under the project of NRPU # 4927.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
