A comparative study on the anti-bacterial effect of Echinops persicus, Cardamine uliginosa and Vaccaria oxyodontha Boiss on Staphylococcus aureus and Escherichia coli bacteria in vitro

Background

Today, bacterial resistance to antibiotics has become a global problem in the treatment of diseases.Therefore,in recent years, medicinal plants have been considered due to their lesser side effects.The problem of resistance to antibiotics, occurs for various reasons,including their improper and inappropriate use.Due to the beneficial effects of medicinal plants in the treatment of diseases and their availability and greater compatibility with the human immune system, the use of plants and their active ingredients is increasing in the against of side effects caused by the use of chemical drugs [1].Among the rich flora of Iran, there are more than 7,500 species of plants, many of which are plants that are called medicinal for various reasons and are known by the local names of the places where they grow.One of the uses of medicinal plants is the use of their essential oils, which have many effects such as anti-bacterial, antiviral, antifungal, repellent of insects, and they can also be used today in aromatherapy.These properties determine chemical compounds in essential oils and extracts and their quality and effectiveness [2].Plants with a wide variety of chemicals are a possible source of anti-bacterial agents.There have been reports of anti-bacterial and antioxidant compounds in various plant components [3].The antimicrobial activity of extracts and essential oils of plants is based on applications such as food spoilage prevention,pharmacy, medicine, and natural treatment [4].Chemicals in plants with antimicrobial activity can be divided into several groups, including phenols and polyphenols, quinones, flavonoids and flavonols,tannins, terpenoids, coumarins, alkaloids, lectins and polypeptides, and compounds such as polyamines(especially spermidine),isothiocyanates, thiosulfinates,and glucosides [5].The clinical efficacy of many available antibiotics is at risk with the emergence of multidrug-resistant pathogens.Many infectious diseases have been treated with herbal medicines throughout human history, and herbs have remained the most common source of anti-bacterial agents.The use of traditional medicines is the most popular treatment in 80% of the world’s population in Asia,Latin America, and Africa and has been reported to have minimal side effects.Nearly 20% of the world’s plants have been evaluated for biological and pharmacological tests.The experiment of plant extracts reveals ongoing efforts to find new compounds that have the potential effect to act against multidrug-resistant pathogenic bacteria and fungi [6,7].

belongs to the sunflower (or chicory)family.So far,120 species have been reported worldwide, which according to the flora of Iranica, 54 species of

are distributed in different parts of Iran.Species of this genus are found in many parts of Iran,including Khuzestan,Azerbaijan,Hamedan,around Tehran,Fars, etc.[8].In Eastern and Southern Europe, Asia, and North Africa,

plants are also distributed [9].This plant is a valuable medicinal plant, some of which have long been used to treat skin diseases, abscesses, blood clotting, seizures, colds, cough relievers, and soothing esophageal irritation and eye infections[8].

(

) is a plant of the genus Caryophyllaceae.This order includes 70 genus and 2,000 species, and their highest distribution is in the Mediterranean region.Their origin is attributed to the Pliocene period.The plants of this genus are herbaceous, perennial, or in the form of woody shrubs.Some of the genus plants are grown due to their beautiful and fragrant flowers, and others are used in traditional medicine.

and

species are widely used in medicine due to their anti-cancer, analgesic, and other properties [10].From the plants of this family, various studies on the antibacterial properties of this plant family are available.Such as checking the properties of the

L.(cow cockle)plant from Falih et al.[11] or

seeds plant in the study of mao et al.[12].

belongs to the order Brassicaceae and the genus Cardamineae, which is one of the largest genera of the Cruciferae family and is one of the genera that have a global distribution.Seven species of this genus have been reported in Iran,which grows in the lowlands to the highlands of humid areas.This genus shows a great variety of karyology and morphology [13].in the study of Cilic et al.The antibacterial activity of

.essential oil was tested,

antibacterial activity againt of

and

and other bacterias the shiwed antibacterial effects[14].

This study aimed to compare the antibacterial effect of

,

, and

on

and

in vitro.

Methods

Preparation and extraction of plants

,

and

plants were prepared from the medicinal plant’s Nahavand Agriculture Jihad Education Center Section.After collecting medicinal plants, they were approved by Dr.Bostan Rudi,Professor of Botany, Damghan Azad University.

,

and

have with herbarium codes 14,605,16,862, and 23,140, respectively, transferred to the laboratory for further study.The plants were dried at room temperature and pulverized by a mill.

50 gr of plant powders were weighed and poured into sealed glass containers for extraction.Then they were mixed with 85% methanol in a ratio of 1 to 10 and placed on the heater for 24 hours.

RTCC 1885 and

PTCC 1330 were used in this study, which was lyophilized from the Faculty of Pharmacy’s Microbial Collection Tehran University of Medical Sciences.Sterile lyophilized ampoules (

and

) were transferred to nutrient broth (made by Merck, Berlin, Germany) and incubated for 24 hours at 37 °C.Broth nutrient culture medium was used to ensuring the purity of the bacteria.It was cultured linearly on differential culture medium overnight and incubated for 48 hours at 37 °C.Then a loop was taken from the bacterial colony and to prepare the new sample of culture medium, the bacteria were inoculated with broth nutrients, 24 hours before each test.Similarly, new 24-hour culture was prepared for each test to evaluate the antimicrobial effects.Finally, a microbial suspension with a concentration equal to 0.5 McFarland was prepared.

Then, to test the antibacterial properties, sterile blank paper disks were placed at a certain distance from each other and the edge of the plate on the agar.The discs were placed in concentrations made from the extract.The culture media containing the bacteria were then incubated at 37 °C for 24 hours.The diameter of the growth disk diffusion around the disks was measured with a digital caliper.The results were compared with the Clinical Laboratory Standard Institute tables [15].This experiment was performed three times for each bacterial strain in different concentrations.Standard discs of gentamicin (10 mg)and vancomycin (30 mg) antibiotics were used for positive control and dimethyl sulfoxide for negative control.

Concentrations of 1, 1/2, 1/4, 1/8, 1/16, 1/32, and 1/64 of the extract were prepared using sterile distilled water to ensure the anti-bacterial properties.Each experiment was repeated three times.

Preparation of microbial strains

The prepared extracts were then refined using Whatman filter paper (No.1).The extract was filtered using a vacuum pump in the second stage.In the next step, the extract was placed in a rotary apparatus at a temperature of 44 °C.The refined extracts were dried in sterile glass plates under absolute darkness at 37 °C for 24 hours.500 mg of dry extract of each plant was dissolved in 1 mL of sterile distilled water and considered concentration 1.Then dilution was performed from a concentration of 500 mg/mL, and concentrations of 1.2, 1.4, 1.8, 1.16, 1.32 and 1.64 were prepared with sterile distilled water.

Determination of microbial susceptibility

According to Table 1,

,

,

extracts a stronger effect than vancomycin in three concentrations 500,250,125 mg/mL.The effect of the

extract against

at a concentration of 500 mg/mL was close to gentamicin’s effect on this bacterium.

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Evaluation of the antimicrobial activity of methanolic extracts of Echinops persicus,Cardamine uliginosa and Vaccaria oxyodontha Boiss by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)method

The MIC test was performed by dilution in a liquid medium (micro broth dilution).One-tenth and one-hundredth dilutions of the 0.5-McFarland suspension prepared from the 24-hour culture of microorganisms were prepared to give an approximate number of 10

microorganisms per milliliter of suspension.To each successive dilution of 500 to 7.8 mg/mL of the prepared extracts was added 2 mL of Müller-Hinton broth medium (prepared by the company Merck, Berlin, German ).To each tube was added 20 μL of microbial suspension, and finally, all tubes were transferred to an incubator at 37 °C and evaluated for MIC after 24 hours.The test was repeated three times.To determine the extracts’MIC,a 5 mg/mL solution of triphenyl tetrazolium chloride was used.After adding tetrazolium dye, the plates were incubated for one hour.After one hour, the color of the solution in the tubes indicates the growth of bacteria.Bacteria had grown in tubes with red color,and bacteria had not grown in tubes with green and yellow color.The positive control tube contained a culture medium and bacterial suspension, and the negative control tube contained a culture medium and extract.SPSS software was used to analyze the obtained data.

For MBC testing, all turbid tubes from the previous step were cultured separately on Müller-Hinton agar medium samples were kept in a 37°C incubator for 24 hours.The lowest concentration of the extract in which the bacteria did not grow was reported as the lethal concentration.

Results

The disk diffusion method and kerby-Bauer protocol used for testing the ability of different concentrations of plant extracts in inhibitory of bacteria growth and detect the percentage of anti-bacterial properties.According to Figure 1,

extract has the strongest effect against

and

than

and

.The high inhibitory effect of

extract on

can also be expressed.

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Disk diffusion method was used to determine microbial strain’s susceptibility to methanolic extracts of

,

and

, Respectively.A concentration of 0.5 McFarland was prepared from all microbial strains in Müller-Hinton broth medium and then cultured on the surface of Müller-Hinton agar medium evenly.

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Table 2 shows the MIC and the MBC in extracts.The MICs against

strains were 125 mg/mL,31.25 mg/mL, and 15.65 mg/mL, respectively, for

,

and

.The MICs in

,

extracts and

were 31.25 mg/mL, 62.5 mg/mL and 15.62 mg/mL, respectively against

strain details MBC testing are shown in Table 2.

According to the results of Clinical Laboratory Standard Institute tables, the minimum growth inhibition zone for gentamicin antibiotic (10 μg disc)against

strain has an inhibition growth zone diameter of 19-26 mm.In the minimum growth inhibition zone for the antibiotic vancomycin (30 μg disc), the growth inhibition zone’s diameter against

strain is 14-15 mm.In Table 3, the intergroup comparison with the Kruskal-Wallis test showed that these extracts had antimicrobial properties equal to gentamicin and vancomycin antibiotics(

＜0.05).

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Discussion

Considering the widely distributed medicinal plants in Iran, studies on these plants provide a good background for medicinal and food purposes.The results of this study showed that methanolic extracts of

,

and

in the tested concentrations had anti-bacterial effects on

and

.the most effective methanolic extract was of

against of

with inhibition growth zone 23.7 ± 0.29 mm.The minimum lethal concentration was 31.25 mg/mL in the this extract.This plant also had a significant effect on

.

extract had stronger antimicrobial properties on gram-positive bacteria compared to gram-negative bacteria in the present study,which was consistent with the study by Sancholi et al.[16].In study Al-Sorchee et al.in 2010, the antimicrobial effects of ethanolic and aqueous extracts of

were investigated on four

serotypes.The results showed that the

extract had antimicrobial properties that were consistent with the results of the present study[17].In another study by Maoz et al.,the MIC of aqueous extracts of the upper parts of the

was obtained 5% (w/v) by agar dilution method for

.These findings from the above studies show that

different extracts have an effect on gram-positive and negative bacteria.And these results prove the antimicrobial effects of this plant [18].The

has a MIC of 31.25 mg/mL and a MBC (62.5 mg/mL), which showed higher anti-bacterial activity than

.Cai et al.showed effect of

inhibitory activity against oral pathogens [19].Also in the study of Hosseini et al.were investigated the anti-bacterial properties of clove essential oil against

and coliforms in raw hamburger.This study showed that with increasing the concentration of essential oil and progression of storage time, the amount of microorganisms decreased and some concentrations showed a better effect.In general, time and high concentrations of clove essential oil (at least 0.1%)were both equally effective in reducing microbial load[20].Finally, it can be noted that similar results of anti-bacterial properties were observed in the plant extract of the

as well as clove.In a report by Iranbakhsh is expressed the reason for the difference between the MIC and MBC values of different studies on different bacteria,differences in the method of extraction of medicinal plants, different methods of studying antimicrobial activity, genetic differences of plants, differences in chemical composition between species and geographical environment[21].

Another plant whose antimicrobial effects were evaluated in this study is

,a genus of perennial plants.This plant is distributed in most Iran provinces,especially Khorasan,Fars, Isfahan,Kerman,Kermanshah, Hamedan, Lorestan, Semnan,Mazandaran,and Tehran at an altitude of 1,800 meters above sea level [22].Numerous studies on the effects of

have shown that this plant has many alkaloid compounds,flavonoids,terpenoids,fats,steroids and polystylenes.Studies have shown that

has strong anti-bacterial activity,which in the present study was clearly seen on gram-positive and negative bacteria [23].The study by Hymete et al.proved the anti-bacterial effects of castor sugar in the treatment of intestinal infections caused by Enterobacteriaceae, especially

.The anti-bacterial effects of this plant can be due to the presence of flavonoid compounds [24].

had a MIC and a minimum lethal concentration of 125 mg/mL and had a lower MIC against gentamicin and vancomycin antibiotics.Plants of this family are

and

and

can be mentioned that these plants also have anti-bacterial properties.As Soltian et al.,in evaluating the antimicrobial properties of

extract observed that this plant has strong anti-bacterial properties due to the presence of phenic acid,phenol,biphenyl,thymol compounds[7,25].

In the methanolic extract of

,like

extract, the MIC was 31.25 mg/mL, and the MBC was 62.5 mg/mL.However, compared to gentamicin and vancomycin antibiotics, in the against of bacteria, it showed a stronger inhibitory effect with an inhibition growth zone of 23.8 ± 0.28 mm.A study of the antibacterial effect of different extracts of seeds

by Naderi et al.showed that aqueous, methanolic, and hydroalcoholic extracts of

seeds with different concentrations had bactericidal or at least bacteriostatic effect [26].

and

are classified in one plant family.However, according to the study,

had a higher antibacterial effect than its

seeds.

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In general, according to Table 1, methanolic extract of

, at a concentration of 500(mg/mL) showed a stronger inhibitory effect than

,

and vancomycin and gentamicin antibiotics against

and

.

extract also had a stronger inhibitory effect than

extract.

Conclusion

In general, according to the results of the antibacterial activity of different medicinal plants in this study,methanolic extract of

,

and

extract had the highest antibacterial properties, respectively.The high anti-bacterial properties of

and

require more scientific studies.

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