Yasser Shahbazi

Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Razi University, Kermanshah, Iran.

Abstract

Scrophulariastriatabelongs to theScrophulariaceaefamily andwidely grows in the several regions throughout the world especially Iran, Turkey and Azerbaijan. The aims of the present study were to evaluate antibacterial activity of theS.striatamethanolic extract collected from west part of Iran by micro-broth dilution and agar disk diffusion assays, and also determine its antioxidant properties using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and thiobarbituric acid (TBA) methods. The most antibacterial activity was observed against Bacillus cereus, followed byB. subtilis, S. aureus. Listeria monocytogenes, Salmonellatyphimurium and Escherichia coli O157:H7, respectively. Moreover, the scavenging properties on DPPH radical scavenging and TBA of S. striatamethanolic extract were found to be 0.92 ± 0.21and 7.98 ± 0.23,respectively. The strong in vitroantibacterial and antioxidant activities of S. striatamethanolic extract supports its traditional application in the treatments and/or prevention of different diseases.

Keywords: Scrophulariastriata;Antibacterial; Antioxidant; Iran.

INTRODUCTION

In the third world and developing countries, consumptionof foods contaminated with some microorganismssuch as Listeria monocytogenes, Salmonella spp., Escherichia coliO157:H7, Bacillus spp. and Staphylococcus aureusrepresents serious health risks to humans1. In addition, the subsistence andgrowth of microorganisms in foods usuallyresult in spoilage, toxin production and quality deterioration of vulnerable food products such as raw meat, fish, shrimp and salad vegetables 2,3.  Since ancient times, medicinal plants and spices have been incorporated todifferent types of food not only to improve the organoleptic properties (aroma, flavor and taste), but alsoas food preservatives 4. In general term, essential oils and extracts are active compounds,which areoften concentratedin a particularorgan of plants including bud, seed, root, leave, stem, wood, bark and flower5. An estimated more than 3000 essential oils and extracts are recognized, which approximately 300 are commercially important for the flavor and fragrances markets as well as medicinal properties 6. In the last decades, the essential oils and the herbalextracts from various species of edible and medicinal plants haveattracted a great deal of scientific interest due to their potentialas a source of natural agents to increase the safety and shelf lifeof foods and of natural biologically active compounds7. Especially, the antimicrobialactivity of plant extracts have formed the basis of many applications,including fresh and processed food preservation,pharmaceuticals, alternative medicine and natural therapies8,9.

ScrophulariastriataBoiss.belongs to thefamily of Scrophulariaceaeandwidely growsas wild in the several regions all over the world especially Iran, Turkey and Azerbaijan10.It has square stems, opposite leaves and open two-lippedflowers forming clusters at the end of their stems11. In Iran, the species commonly known as “Tashnehdari” is abundant in the Zagros Mountainous Range12. Different parts of this plant has been used as Iranian folk remedies because of its medicinal properties for treatment of various diseases likescrophulas, scabies, tumors, eczema, psoriasis, rheumatics andchronic inflammatory diseases11,13,14.Earlier studies are focused on its application as a natural anticancer agent and indicated that methanolic extract of S.striatamightcontain various polar compounds that inhibittumor invasion, metastasis and angiogenesis11,14. In addition, it has been reported that the oil extracted by steam distillation of leavescould inhibit numerous inflammatory factors such as PGE-2, leukotriene B4, NO,IL-1β, IL-4, INF-γ, but did not have any effect on the productionof IL-1015.

The biological effects of the plant extracts and essential oils can vary greatly depending upon its chemical compositions which depends on the geographical and climate conditions, variety of species and genotypes of the plant1,16,17. Therefore, studying the biological properties of S.striatacollected from each endemic area may have an important role in identification andintroduction of new germplasm in order to be used in food and pharmaceutical industries18. Based on our findings, there is no comprehensive study on the antioxidant and antibacterial properties of S. striatacollected from Kermanshah, west part of Iran.  Therefore, the aims of the present study were to evaluate antibacterial activity of theS. striatamethanolic extract by broth-micro dilution and agar disk diffusion assays, and also determine its antioxidant property using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and thiobarbituric acid (TBA) methods.

MATERIALS AND METHODS

Collection of plant material
The fresh leaves of S. striataplant was collected from Kermanshah, west part of Iran during full flowering stage in April 2016. The plant was identified as S. striataBoiss.by a botanical taxonomist (Dr. Seyed Mohammad Masoumi, Faculty of Agriculture,Razi University, Kermanshah, Iran). Then, the fresh leaves of collected plant was washed with distilled water and air-dried indoor in a shady place at room temperature for twelve days.

Preparation of plant extract
To prepare S. striatamethanolic extract, 5 g of fine-powdered plant was dissolved in 20 ml methanol and extracted with a shaker at room temperature for 24 h. The extract was filtered through Whatman filter paper no. 3, concentrated in a rotary evaporator at 40 ºC and stored at refrigerated temperature till further use 19,20.

Bacterial strains
The antibacterial activity of S. striatamethanolic extract was studied against sixpathogenic bacteria including S. aureus(ATCC 6538), B. subtilis(ATCC 6633), B. cereus (ATCC 11774), L. monocytogenes (ATCC 19118), S. typhimurium (ATCC 14028) and E. coli O157:H7 (ATCC 10536).All aforementioned bacterial strains were purchased from the Iranian Research Organization for Science and Technology (IROST), Tehran, Iran.The strains werecultured overnight at 37°C in Brain Heart Infusion broth (BHI), adjusted to a final density (5 log CFU/ml) using a spectrophotometer at 600nm and used as an inoculum dose.

Determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC)
The MIC of the methanolic extract of S. striata was determined using micro-broth dilution assay descried by Shahbazi et al. (2015)21 with some minor modifications. Different concentrations of dried plant extract ranging from 0.05 to 10 mg/ml were set up in Brain Heart Infusion (BHI; Merck, Darmstadt, Germany) broth containing dimethyl sulfoxide(DMSO; 0.5% v/v) and filtered by 0.45 µm filters for sterilization. The 96-well sterile micro-titer plate was prepared by pouring 180µl BHI broth medium containing specified concentrations of the plant extract and 20µl of fresh overnight bacterial cultures (5 log CFU/ml) into each well. Parallel positive (BHI broth containing inoculum without the tested materials) and negative controls (BHI broth containing the tested materials) were maintained in the last well of each strip. The microplates were shaked at 300 rpm for 20s and incubated at 37 ºC for 24h. The MIC was defined as the lowest concentration of the plant extract that completely inhibited the growth of microorganisms. Referring to results of the MIC, 20µl of each well without any invisible growth was sub-cultured on BHI (Merck, Darmstadt, Germany)agar plates, incubated at 37°C for 24h. MBC was determined as the highest dilution atwhich no growth occurred on the plates1.

Agar disk diffusion assay
For agar disk diffusion assay, 100 µl of each bacterial suspension (8 log CFU/ml) was uniformly spread on BHI agar medium using a sterile cotton swab. Then, the sterile paper discs with 6 mm in diameter wereimpregnated with 10 µl of each designated concentration of plant extractand placed on the surface of the inoculated BHI agar plates. These plates were incubated for 24 h under appropriate cultivation temperature (37°C). The area of the inhibition zone (millimeter) was calculated as πr2 20.All tests were performed in triplicate.

1,1-Diphenyl-2-picrylhydrazyl (DPPH) assay
The antioxidant activity of the S. striata extract was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay through a UV-vis spectrophotometer 22,23. An aliquot of 50 µl of various concentrations of the S. striata extract was added to 5 ml of methanolic DPPH solution and the absorbance was measured at 517 nm. The percentage of DPPH radical scavenging activity of S. striata extract was calculated as follows 20,23:

I (%) = ([Ab-AS])/Ab × 100

Where I% is the capability to scavenge the DPPH radical or to inhibit free radicals, Ab is the absorbance of the control reaction (containing all reagents except the S. striata extract), and As is the absorbance of the S. striata extract sample.

Thiobarbituric acid reactive substances (TBA) assay
The thiobarbituric acid reactive substances (TBA) value, a secondary product of lipid peroxidation, of S. striata extract was evaluated according to the method of Singh et al., (2010). The TBA value (Meq of malondialdehyde/g) of S. striata extractwas calculated as following formula 4,24:

TBA value = ([50 ×(A-B)])/M

Where A is the absorbance of test sample, B is the absorbance of reagent blank and M is the mass of the sample (mg).

Table 1. Antibacterial activity of S.striataextract indicated as Minimum Inhibitory/ Bactericidal Concentrations-MIC/MBC (mg/ml)

    Extract Tetracycline
Bacteria MIC MBC MIC MBC
S. aureus 0.8 0.8 2 2.5
B. subtilis 0.4 0.4 2.5 2.5
B. cereus 0.1 0.1 2 2
L. monocytogenes 2 2 2.5 2.5
S. typhimurium 4 8 2.5 2.5
E. coli O157:H7 8 10 2.5 2.5

 

RESULTS AND DISCUSSION

Bacteria are the most common cause of foodborne diseases and exist in a variety of shapes, types and properties. Some pathogenic bacteria are capable of spore formation and thus, highly heat-resistant (e.g. Bacillus subtilus, Bacillus cereus). Some are capable of producing heat-resistant toxins (e.g.Staphylococcus aureus, Clostridium botulinum). Overall, these outbreaks caused 45,874 cases of illness (209 more than 2014), 3,892 hospitalisations (2,546 less than 2014) and 17 deaths (10 less than 2014). The overall reporting rate of food-borne outbreaks in the EU was 0.95 per 100,000 population, which represents a slight decrease compared with data provided for 201425. Epidemiological studies have consistently shown that there is aclear significant positive association between the intake of medicinal plants and a reduced rate of food borne diseases 19. In the present study, antibacterial and antioxidant activities of theS. striatamethanolic extract was examined using broth-microdilution assay, agar disk diffusion method, DPPH and TBA.

Table 2. Antibacterial effect of S.striataextract by agar disk diffusion assay

Inhibition zone (mm)  
S. aureus B. subtilis B. cereus L. monocytogenes S. typhimurium E. coli O157:H7
Extract 8.4 ± 0.1 10.1  ± 0.1 10.9  ± 0.2 7.8 ± 0.5 3.1 ± 0.0 3.1 ± 0.0
Tetracycline 10.2 ± 0.1 11.5  ± 0.7 14 ± 0.0 13 ± 0.1 12 ± 0.0 10 ± 0.2

The antibacterial effects of S. striata extractagainst common food-borne pathogenic bacteria including S. aureus, B. subtilis, B. cereus, L. monocytogenes, S. typhimurium and E. coli O157:H7 are exhibited in Table 1and 2.Based on our findings, themost antibacterialactivity was observed against B. cereus, followed byB. subtilis, S. aureus, L. monocytogenes,S. typhimurium and E. coli O157:H7, respectively. Indeed, Gram-negative bacteria (S. typhimurium and E. coli O157:H7) were more resistant to the presence of methanolicS. striata extract than Gram-positive bacteria (S. aureus, B. subtilis, B. cereus and L. monocytogenes). It can beattributed to the hydrophobic outer membrane surrounding thecell wall of Gram-negative bacteria, which restricts diffusion of lipophilic compounds such asessential oils and extracts 26,27. In agreement with our findings, Mahboubi and Haghi (2008) examined antibacterial effects of the essential oil and extract of Menthaspicata,and reported Gram-positive bacteria including S. aureus, L. monocytogenes, B.cereuswere more sensitive than Gram-negative including E. coli O157:H7 and Yersinia enterocolitica28. In another study, Mahboubi et al., (2013) evaluated antibacterial activity of S. striataBoissextract against some pathogenic and spoilage microorganisms and reported that Staphylococcus epidermidis, Streptococcus sobrinus, Klebsiellapneumoniae, B. subtilisand B.cereus had more sensitivity to aqueous extract of S. striataBoiss11, which is good in agreement with our findings.Monsef-Esfahani et al., (2010)demonstratedthat cinnamic acid, quercetine, isorhamnetin-3-O-rutinoside, nepitrin, phenyl propanoid glycoside (acteoside 1) are the most abundant compounds isolated from S. striataextract 10. These data also are in agreement with Abbasiet al. (2007), using the same antimicrobial method, they haveshown thatS. striataextract was effective againstS. aureus and Pesudomonasaeroginosa29. In agreement with our results are those ofSharafati-Chaleshtori and Rafieian-kopaei, (2014) who reported thatS. striataethanolicextract had inhibitory effect against the E. coli O157:H7 in two methods of sink diffusion and macrodilution30. The action mode the phenolic compounds is related to their hydroxyl group ofthe phenolic ring which plays a significant role in the formation of hydrogenbonds and also in the presence of delocalized electronsand subsequently dissipate the pH gradient overthe bacterial cytoplasmic membrane31. It disturbs the proton motiveforce (PMF), depletes the amount of intracellular ATP (ATPin) pooland leads to impairment of essential process in the bacterial cell3,32.

 Table 3. Antioxidant activity of S.striataextract(mg/ml; mean ± SD)

  Extract BHT
DPPH radical-scavenging activity (IC50a) 0.92 ±0.21 0.19 ± 0.12
TBA (EC50b) 7.98 ± 0.23 0.001 ± 0.000

a IC50, concentration (g/l) for a 50% inhibition.
b EC50, concentration (g/l) for a 50% inhibition.

The scavenging properties on DPPH radical and TBA of S. striatamethanolic extract were found to be 0.92 ± 0.21and 7.98 ± 0.23, respectively(Table 3).DPPH scavengingability of the S. striatamethanolic extract was significantly higher than that of synthetic antioxidant BHT, indicating the presenceof specific bioactive components in this plant that can be responsible for its antioxidant activity. Similar results were also found in a previous study where reported that all parts of S. striatahad high antioxidant activities33. According to these results, the IC50 of S. striata extracts were ranged 0.98 and 0.99 mg/ml, which is good in agreement with our findings. The anti-inflammatory, antioxidant and immunomodulatoryactivities of some species of Scrophularia have also been reported by other researchers12,34. Differences in the results of antioxidant activities of Scrophulariaspp. extracts among these studies could be mostly explained by variations in the phenolic concentrations of plant and used antioxidant method 34. In accordance to the antibacterial property, the antioxidant effect of S. striataextractisdue to phytochemical contents especially compounds, including flavonoids, cinamic acid, phenylpropanoid,nepitrin, flavonoid glycoside, acteoside1 and phenylpropanoid glycoside10. In confirmation with our findings, Mahboubi et al., (2013) 11 and Monsef-Esfahaniet al., (2010)10 reported that free radical-scavenging activity is greatly influencedby the phenolic composition of the sample and remarkable positive correlation between antioxidant property of S. striata extracts and its phenolic compounds. The antioxidant activity of the phenolic compoundswere attributed to its redox properties, which allowthem to act as reducing agents, hydrogen donators, singlet oxygenquenchers and have also metal chelating properties31,35. However, further investigations about the total phenols (TP), total flavonoids (TFO) and total flavan-3-ols (TFL) contents of S. striataextract is required.

CONCLUSION

The present study indicated that S. striatamethanolicextract showed remarkable antibacterial activity against common food-borne bacteria associated with outbreaks (S. aureus, B. subtilis, B. cereus, L. monocytogenes, S. typhimurium, and E. coli O157:H7) and also antioxidant property. Further research is required to evaluate the combination of S. striata extract with other antibacterial constituents such as nisin, lysozyme, monolaurin and other essential oils. The strong in vitroantibacterial and antioxidant activities of S. striatamethanolic extract supports its traditional application in the treatments and/or prevention of different diseases.

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