Investigation of effect of antibacterial,antioxidant,and phytochemical factors of Ocimum basilicum extract on Escherichia coli and Staphylococcus aureus

Fatemeh Fazli ,Mina Owrang ,Ali Ahmadi

1Department of Biological Sciences and Technologies,Faculty of Basic Sciences,Islamic Azad University Sari Branch,Sari 4816119318,Iran.2Department of Laboratory Sciences,Faculty of Medical Sciences,Islamic Azad University Sari Branch,Sari 4816119318,Iran.3Student Research Committee,Faculty of Medical Sciences,Islamic Azad University Sari Branch,Sari 4816119318,Iran.

Abstract The use of natural antibacterial compounds in tcontroling and eliminating pathogenic microorganisms including bacteria and fungi has been extensively evaluated.Plant extracts have been studied in the past years as antibacterial agents in medicine,plant pathology,and canned food,and unlike antibiotics,they do not cause drug resistance in pathogens.BHA(Beta-hydroxy acid)is a synthetic antioxidant with good fat solubility.Most of the extracted materials have antifungal,antiviral,and antibacterial properties.Different mechanisms of these act on bacteria and prevent their growth and multiplying.Many plants’extraction has phenolic active groups in their structures.Due to the importance and antimicrobial effects of essential oil and basil extract,the present study was performed to investigate the effect of different concentrations of basil extract on the growth of two bacterial pathogens Escherichia coli and Staphylococcus aureus.Also,the chemical composition of this plant extract and its antioxidant properties have been studied.The results of this study showed that basil extract,especially in 100 and 70%concentration,had antibacterial properties against Staphylococcus aureus but it does not show sufficient antimicrobial effect against Escherichia coli in the same dose.Analysis of chemical compounds in the extraction of this plant showed a total of 15 chemical compounds,most of which are related to the compounds neofitidine with concentration (41.44%),dimethylamino pentane with concentration (14.81%) and cyclohexane with concentration (91.9%).We also compared the antioxidant properties of basil extract with BHA and showed a significant difference in comparison with the inhibition of DPPH(2,2-Diphenyl-1-Picrylhydrazyl) formation by the extract and the standard BHA,so that the inhibition of DPPH by the standard BHA sample was significantly higher than the BHA.But basil extract showed good antioxidant properties equivalent to 400 mg/mL in 800 mg/mL BHA.

Keywords: antioxidant;anti-bacterial;phytochemical; Ocimum basilicum; Escherichia coli;Staphylococcus aureus

Introduction

Today,medicinal plants used to treat various diseases.Treatment of diseases with plant extracts goes back to the past.Knowing that medicinal plants are the alternative to many diseases and that antibiotic resistance is increasing,these cases have led researchers to study the antimicrobial properties of some plants,and many articles in this field show that 250,000 to 500,000 plant species in the world.Only 1% of these plants in the world have been studied for medicinal properties [1,2].BHA (beta-hydroxy acid) is a synthetic antioxidant with good fat solubility.It has good stability in frying products.It is very effective with animal fats.But it has less effect with vegetable oils and its maximum permissible consumption is up to 200 ppm.Today,various methods used to combat the harmful effects of bacteria in the body,the most important of which is the use of antibiotics.Treatments Excessive use of these drugs has also led to drug resistance among bacteria.This factor spreads this resistant bacterium.In our country,antibiotic resistance has reached such a level that there is a serious need for alternative therapies or substances to reduce the resistance to antibiotics.Basil is one of the oldest and most popular vegetables.The scientific name of basil isOcimum basilicum,which belongs to the mint family and theOcimumcategory[3].Basil leaves and their young shoots are used for nutrition.Basil is grown by car in Iran and Afghanistan.It is grown in most parts of the world Figure 1.TheOcimumstrain belongs to the genus mint,whose ecotypes have a great morphological diversity.This genus includes at least 60 species and many of them are available.The leaves of this plant contain large amounts of phytochemical properties that prevent various diseases and improve the disease.The plant also contains large amounts of polyphenol flavonoids such as ornithine and vaccines that have tested in the laboratory to check the protective effect of their antioxidant properties against radiation-induced lipid peroxidation in rat liver.This plant has very few calories and no cholesterol[4].But it is a very rich source of many essential nutrients,minerals and vitamins.Basil contains very high amounts of beta-carotene,vitamin A,cryptoxanthin,levin and zeaxanthin.These compounds have known as protective adsorbents against oxygen-derived free radicals and reactive oxygen species,which play a very important role in aging and various diseases.This plant is available in 80% of developing countries.Since useful medicinal plants find in plenty in Iran,the study of these plant antimicrobial effects can describe in identifying the optimal use of this valuable national species.Herbs are potential sources of new antibacterial agents,even against some antibiotic-resistant strains.Plant extracts have antimicrobial activity against gram-positive and gram-negative bacteria,therapeutic agents can reduce the side effects of chemical drugs that treat some heart diseases and the large spleen.It is an anti-flatulence expectorant to relieve and stimulate stomach pain[5,6].

Figure 1 The leaves basil

Medicinal plants are a group of plants that have been used for food,medicine,treatment,clinical and pharmaceutical purposes.The presence of substances called secondary compounds in plants for their adaptation and survival in adverse environmental and biological conditions,production of herbal medicines,natural pesticides and herbicides,flavorings,aromatizers and food preservatives have a special place for a long time.Ocimum basilicumin traditional medicine as a disinfectant and used for healing since the beginning of human life [7,8].There is information about medicinal plants on papyrus from 1,500 years ago.In Europe,the Romans and Greeks conducted scientific studies on these plants.In fact,the science of recognizing the effects of herbal medicine is one of the oldest known human sciences and ancient civilizations such as Egypt,Babylon,Chaldea,Assyria,Iran,Greece,China and India after the invention of the art of writing completed it and passed it on to future generations.Recently,the use of compounds that are generally harmless has attracted a lot of attention [9,10].Biologically active natural compounds derived from plants were among the most important of these compounds because the substances obtained from essential oils and plant extracts can be used to preserve food [11].The aim of this study was to compare the antioxidant and antimicrobial effects of basil extract on the growth and survival ofStaphylococcus aureusandEscherichia coli.

Material and methods

This study is an original study with the approach of an intervention research plan that was conducted in 2020-21.In this study,basil was collected from Mazandaran province in September,and after identified and approved by the Agricultural and Natural Resources Research Center (NNRC) of Mazandaran province.

Extraction of basil extract

In this study,we cleaned and dried basil leaves a few days after washing.After drying the basil leaves and grinding them,we included 120 grams of dried and powdered basil in the study,soaked 50 grams of the sample in 5 times 70%alcohol and 250 cc of water and put it on a shaker in Erlenmeyer for 3 hours.We put it and mixed it every few minutes and then we waited for 3 to 4 days and made the solution again.We stirred the solution and put the mixture in a corner for an hour to complete the dissolution and extraction process,then smooth it.We put the solution in the oven for 30 to 34 days at a temperature of 30 degrees Celsius and we noticed that the sample did not dry.We put it back in the oven for 5 days to dry and we made sure that the drying process was complete.The solvent was then evaporated.Finally,the dry extract was obtained.The content of phenolic compounds was determined by the support method.The content of phenolic compounds was determined by the Folin Siocaltive method.

Validity and reliability of the tool

In this study,a completely randomized Duncan-based statistical method was used to evaluate the results of the data.All experiments were performed in three replications and one-way ANOVA method was used to evaluate the mean data in SPSS software version 20.

Measurement of total phenolic and flavonoid content of basil extract

We prepared a concentration of 1 mg/mL from each extract.Mix 0.5 mL of each extract with 2.5 mL of the natural culture reagent fullerene 0.2 and stir for 5 minutes,then add 2 mL of sodium carbonate solution at a concentration of 75 g/L.The rate of absorption of the samples after 2 hours at room temperature by Ultraviolet spectrophotometer was measured at 760 nm.The results were expressed as standard equivalent values of gallic acid and the result was reported as the phenolic content of the whole extract based on the equivalent value(mg gallic acid per gram of extract).We repeated the experiments for each standard extract 3 times.The flavonoid content of each extract was evaluated by colorimetric methods.We prepared a concentration of 1 mg/mL from each extract and dissolved 0.5 mL of the sample in 1.5 mL of methanol.Then we added 0.1 mL of 10% aluminum chloride,then 0.1 mL of 1 M potassium acetate solution and finally 2.8 mL of distilled water and kept it at room temperature for 30 minutes and then the resulting mixture.It was absorbed by the spectrophotometer at a wavelength of 415 nm.Visible-UV measured the number of flavonoids was reported based on the equivalent amount (mg quercetin per gram of extract).The experiments were repeated 3 times and their average was reported.

Free radical scavenging of DPPH

To measure the antimicrobial activity of basil extract by disk diffusion method,we first added a concentration of 150 μL of the desired extract to sterile disks so that the extracts were completely absorbed by the disks.From the 24-hour culture of each of the bacteria prepared in the microbial suspension,we prepared the equivalent of 0.5 McFarland and agarized it by moving it on the surface of Müller Hinton.Discs containing the extract were placed on the agar surface and the plates were incubated for 24 hours at 37°C.By measuring the diameters due to lack of growth around the discs,the sensitivity or resistance of the bacterium to the extract was determined.Stable DPPH radicals were also used to evaluate the antioxidant activity of basil extract.For this purpose,0.3 mL of basil extract with different concentrations was mixed with 2.7 mL of DPPH methanolic solution and kept in the dark place at room temperature for 60 minutes.And the adsorption was set at 517 nm.Basil-derived ascorbic acid was used as standard and the IC50 value for the extracts was determined.The concentration of each extract required radical removal was up to 50%.Finally,the percentage of DPPH radical trapping was calculated according to the following formula.

Measurement of antimicrobial activity of basil extract by disk diffusion method

In the disk diffusion method,150 microliters of the percentage of the desired extract was added to the sterile blank disks so that the extracts were completely absorbed by the disks.We prepared a uniform table culture medium on the surface of Müller Hinton agar.Discs containing the extract were placed on the agar surface and the plates were incubated for 24 hours at 37 °C.By measuring the diameters due to lack of growth around the discs,the susceptibility or resistance of the bacteria to the extract was determined.

Measurement of antimicrobial activity of basil extract by agar well diffusion method

The antibacterial properties of basil extract were determined based on bioassay test.For this purpose,bacterial suspension was prepared in sterile physiological saline solution and 0.1 mL of it was cultured on brain agar medium(nutrient agar)and Mollerington agar medium and spread completely on the medium by means of a glass rod.Wells with a diameter of 1 cm were created in which different amounts of 10,40,70 and 100 mg/mL of micro filtrated extract were poured.Finally,the plates were incubated at 37 °C for 24 hours.The difference in diameter of the auras formed by the diameter of the wells was considered as an indicator of antimicrobial activity of the extracts.

Statistical analysis

In this study,in order to evaluate the results of the data,a completely randomized statistical method based on Duncan was used.All experiments were performed in three replications and one-way ANOVA method was used in SPSS software version 20 to check the mean data.Excel 2013 software was used to draw the charts.

Results

Comparison of the mean diameter of no growth halo of two bacteria by gentamicin antibiotic

A comparison of the mean diameter of the absence halo of two bacteria with the antibiotic gentamicin is shown in Figure 2.There was a significant difference in the mean diameter of the growth inhibition zone due to the antibiotic gentamicin between the two bacteria (P=0.003).This mean diameter of growth inhibition zone forStaphylococcus aureus(24.66 ± 0.57 mm) was significantly larger thanEscherichia coli(21.66 ± 0.57 mm).

Comparison of the mean diameter of no growth halo ofStaphylococcus aureusandEscherichia coliby gentamicin in different concentrations of basil extract is shown in Figure 3.There was a significant difference in the mean diameter of the growth inhibition zone due to gentamicin antibiotic and different concentrations of the extract (P=0.00001).

Figure 2 Comparison of Mean diameter of two bacteria by gentamicin antibiotic

Figure 3 Comparison of mean diameter of lack of growth aura of Staphylococcus aureus and Escherichia coli due to antibiotics

Result of analysis of chemical compounds of basil extract

The average total phenolic and flavonoid content of basil extract is shown in Table 1.The average total phenolic content was 224.2±0.3 mg/mL,while the total flavonoid content of the plant extract was 18.8± 0.1 mg/mL.

According to of the chromatogram obtained from the analysis of basil extract,different chemical compounds have been identified in the constituents of the essential oil in Table 2.

The result of studying the antioxidant properties of basil extract

The ability to trap DPPH radicals can be seen in standard samples of BHA and basil extract in Table 3 and Table 4.

Table 1 The amount of total phenol and flavonoid content in the extract

Table 2 Chemical composition of basil extract in terms of(%)

Table 3 DPPH radical trapping rate by BHA standard

Table 4 Extent of DPPH radical trapping by the extract

Comparison of DPPH inhibition percentage with basil extract and BHA standard sample

A comparison of the percentage of DPPH inhibition by basil extract and standard BHA sample can be seen in Figure 4.There was a significant difference in the percentage of inhibition of DPPH formation by the extract and the standard sample of BHA,so that in all concentrations used the percentage of inhibition of DPPH by the standard sample of BHA was significantly higher than the extract(P<0.001).However,basil extract showed good antioxidant properties so that its concentration of 400 mg/mL was equal to the concentration of 800 mg/mL BHA(Figure 4).

Figure 4 Comparison of DPPH inhibition percentage with basil extract and BHA standard sample

Discussion

Today,the use of natural antimicrobial compounds in inhibiting pathogens,especially bacteria and fungi,is widely used.Essential oils and plant extracts have long been known as natural antimicrobial compounds in the fields of pharmacology,medical microbiology,plant pathology,food preservation,and unlike antibiotics,and do not cause drug resistance to pathogens.Most of the extracted plant essential oils have antifungal,antiviral and antibacterial properties.These compounds work on bacteria by different mechanisms and prevent them from growing and multiplying.Most essential oils and plant extracts are common in having phenolic active groups in their structure.The antibacterial activity of essential oils and plant extracts is mainly due to the presence of terpenoid and phenolic compounds such as thymol,carvacrol and eugenol.The antibacterial properties of essential oils and plant extracts and their compounds have been extensively studied against a large number of gram-negative and positive bacteria.Gram-positive bacteria are more resistant to essential oils and plant extracts due to their lipopolysaccharide membranes [9].

Basil is an herbaceous,peppermint,annual,fragrant herb that is used to treat heart disorders and spleen enlargement.Basil is an essential oil called basil essential oil or basil oil,which contains substances such as estrago.Basil essential oil has high antifungal and antibacterial properties.The main constituents of flowers and leaves are methyl cavicol and limonene.But in the stem of the plant,compounds such as apiol,methyl cavicol and cariophylene oxide are present in greater amounts.Due to the importance and significant antimicrobial effects of basil essential oil and extract,the present study was conducted to investigate the effect of different concentration of basil extract on the growth of two pathogenic bacterial species includingEscherichia coliandStaphylococcus aureus.The chemical composition of the extract of this plant and its antioxidant properties have also been studied [12].

The results of our study showed that basil extract,especially in 100% and 70% concentration,has good antibacterial properties againstStaphylococcus aureus,while it did not show a good antimicrobial effect againstEscherichia coli.Analysis of chemical compounds in the extract of this plant showed a total of 15 chemical compounds in the extract of this plant,most of which are related to the compounds of neofitadine group (41.64%),dimethyl aminopentane (14.81%) and cyclohexane (91.91%),respectively.We also found that comparing the antioxidant properties of basil extract with BHA showed a significant difference in inhibiting DPPH formation by BHA extract and standard sample,so that the percentage of DPPH inhibition by standard BHA sample was significantly higher than BHA.

The need for research is that drugs are used today in the treatment of various diseases.Treatment of diseases with plant extracts goes back to ancient times.Acceptance of medicinal plants as an alternative treatment for many diseases and the fact that antibiotic resistance is increasing,has led researchers to study the antimicrobial properties of some medicinal plants and many articles in this field.Also,due to the spread of science and technology,various methods are used to deal with the harmful effects of bacteria in the body,the most important of which is the use of antibiotic treatments.Improper use of these drugs has led to drug resistance in this bacterium,which causes the spread of this bacterium in areas with greater resistance.In our country,the resistance of these bacteria has reached such a level that there is a serious need for alternative therapies or substances that reduce the resistance to antibiotics[11,13].

However,basil extract showed good antioxidant properties.Its concentration of 400 mg/mL was equal to the concentration of 800 mg/mL BHA.In a study by Tavallai et al,Iron-urea nano-complex improves bioactive compounds in essential oils ofOcimum basilicumL.The results of this study showed that the methanolic extract of basil had a significant antibacterial effect against the three studied bacteria.The minimum inhibitory concentration forStaphylococcus aureusandBacillus subtiliswas 0.550 mg/mL and forEscherichia coliwas 1.15 mg/mL.On the other hand,the diameter of growth inhibition zone forStaphylococcus aureusandBacillus subtilisandEscherichia coliwere calculated to be 15.25,12.5 and 12 mm,respectively.Therefore,based on the results of this study,the researchers stated that basil has an effective antimicrobial activity against food-borne bacteria and can be used to protect food products.On the other hand,these researchers showed that in fact the antibacterial properties of this plant are related to flavonoids such as erintine and viscin and its essential oils include estragole,linalool,cineole,eugenol,sabinene,myrcene,limoene [13].

In another study,the antimicrobial effects of basil extract on eight gram-positive and negative bacteria and the opportunistic fungus Candida albicans were determined and the best antimicrobial effect was determined among the samples.To investigate the antimicrobial effects of basil extract,small paper discs were used on bacterial culture medium and the minimum concentration of MIC inhibitor and MBC lethality were determined and compared with the control.The results of the study of the effect of basil extract on six bacterial strains and two fungal strains indicated that despite the high resistance of gram-negative bacteria and Candida albicans,basil extract still had a significant effect on these microbes.This effect was even greater [10].

Research suggestions

(1) Comparison of antimicrobial activity of basil extract with common chemical antibiotics

(2) Investigation of effective compounds in basil extract using gas chromatography

(3) Investigating the ability to produce basil extract as a natural preservative in food

Executive suggestions

Using basil extract as an antimicrobial and natural antioxidant in the pharmaceutical industry and also in animal feed.

Conclusion

The results of this study showed that basil extract,especially in 100%and 70%,had a significant effect on inhibiting the growth ofStaphylococcus aureus,but did not show any effect on the growth ofEscherichia coli.It can be a good alternative to drugs of chemical origin to control the growth of human pathogens.This antimicrobial effect of basil extract may be associated with compounds such as neofitadine,dimethylaminopentane and cyclohexane.In addition,basil extract had good antioxidant properties,so the plant extract can be used as a natural preservative.Extensive studies can also reveal the properties of this plant extract,especially in traditional medicine,and instead of existing chemical additives and disease control,they can be used as food additives.