Antibacterial , anti fungal and E . coli DNA cleavage of Euphorbia prostrata and Pelargonium graveolens extract and their combination with novel nanoparticles

As there are a lot of antibacterial and anti‐fungal resistant pathogens, researchers attempt to substitute antimicrobial drugs with various medical plants and novel nanoparticles. The present study was conducted to characterize antimicrobial activities of Euphorbia prostrata and Pelargonium graveolens extract alone and in combination with Mn‐Ni@Fe3O4‐NPs & Mn: Fe (OH)3‐NPs on the DNA cleavage of E. coli and also Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Aspergillus oryzae, and Candida albicans. The effects of antimicrobial activities on above scenarios were evaluated using disc diffusion, MIC, MBC, and E. coli DNA electrophoresis methods. The results showed that the effects of antibacterial assay values of Euphorbia prostrata & Mn: Fe(OH)3 was 21.00 mm for E. coli and while it was 19.5 mm for Euphorbia prostrata & Mn‐Ni@Fe3O4 against Pseudomonas aeruginosa at a concentration of 100mg/mL. The highest level of DNA cleavage was seen in mixed of Euphorbia prostrata & Mn: Fe(OH)3 nanoparticles. In conclusion, the combination of Euphorbia prostrata and Pelargonium graveolens extracts with nanostructures showed synergic effects on eliminating the bacteria via DNA destruction and others mechanisms. Moreover, the synergistic effect of nanoparticles with plant extracts seems to bring about new choices for the treatment of infectious diseases.


INTRODUCTION
Despite producing a number of new synthetic antibiotics by pharmacological industries, resistance to these drugs has increased.Bacteria have the genetic ability to transmit resistance to drugs.In this connection, it is necessary to develop an herbal drug to treat such bacterial diseases.Plants have an ability to produce metabolites that can be the source of antimicrobial substances.Besides, a high percentage of the peoples still use herbal drugs along with or in preference to conventional medicines (Cowan, 1999).The gramnegative cell walls, which have a complex structure, are composed of thin outer membranes that confer resistance to hydrophobic compounds detergents.Also, this layer contains lipo-polysaccharides, which are responsible for increasing the negative charge of cell membranes and their viability (Ahmad et al., 2001).Nanomaterials are among the nontraditional antibacterial treatments.Nanotechnology is a new approach to reduce the serious disease caused by bacteria and fungi and can be used as a weapon against microorganisms resistant to drugs (Ebrahiminezhad et al., 2012;Luqman et al., 2008).Iran has a large diversity of plant species containing many useful metabolites.Euphorbia prostrata and Pelargonium graveolens are two main herbs that seem to be good for microbial infections treatment.In Iran, these herbs are cultivated and collected in Fars and Chaharmahal and Bakhtiari provinces, respectively.Pelargonium graveolens has about 250 species.Essential oil of this plant contains diuretic antispasmodic properties.Medicinal values of these species show that this extract can be used for the treatment of skin diseases and as an anti-inflammatory and even an anticancer agent considering their antibacterial/antioxidant property (McGaw, Jäger, Van Staden, 2002;Lalli et al., 2008;Safaepour et al., 2009).Also, Euphorbia prostrata plant has shown anti-inflammatory and analgesic properties (Singla, Patahk, 1990).In some studies, it has been shown that combination of two antimicrobial agents (plant extract and nanomaterials) against bacterial populations, leads to enhancing their antimicrobial activity (Dorsthorst et al., 2002).Moreover, it seems that the combination of these plants and nanoparticles would have synergic effects with lower toxicity.The purpose of present study is to assess the antibacterial (Pseudomonas aeruginosa, Bacillus subtilis, E. coli and Staphylococcus aureus) as well as the anti-fungal (Candida albicans, Aspergillus oryzae) and DNA cleavage (E.coli) effects of Euphorbia prostrata and Pelargonium graveolens with/without Mn-Ni@Fe 3 O 4 -NPs nanoparticles and Mn: Fe(OH) 3 .

Extraction
The aerial parts of Euphorbia prostrata and cultivated Pelargonium graveolens were collected at the flowering stage during June 2014 respectively from Fars and Chaharmahal and Bakhtiari provinces in the southwest of Iran.The collected materials were dried in shadow and ground to coarse powder.Then, 1000 ml of ethanol (70%) was added to 50 gr of each plant and centrifuged at 10509 g for 2 h at room temperature.The supernatants were filtered through Whatman paper (NO.2) and allowed to evaporate in vacuum at 45 °C for 24 h using a rotary evaporator until 1ml.The condensed extracts were incubated at 50 °C for 12 h and dried matter's weight was measured to be 6.55 g (Hammer, Carson, Riley, 2004).

GC-MS
The Gas chromatography-Mass spectrometry (GC-MS) analyses were performed using an Agilent-7890A chromatograph interfaced to an Agilent-5975C mass spectrometer (ionization voltage 70 eV, scan time 0.5 s, scan range (40-400 Da) and equipped with a capillary column HP-5 (30 m × 0.25 mm i.d., with film thickness: 0.25 μm).The oven temperature was held at 60 °C for 5 min, then programmed from 60 to 260 °C at a rate of 5 °C/min and finally held for 3 min at 260 °C using Helium as a carrier gas (1.0 mL/min), split 1: 50.Injector and detector temperatures were 250 °C.The relative percentage of the each extract constituents was expressed as percentage with peak area normalization (Gopinath et al., 2012).

Synthesis of nanoparticles
The reaction solution for loading Mn and Ni doped Fe 3 O 4 nanoparticles (Mn-Ni@Fe 3 O 4 -NPs) on activated carbon (Ac) was prepared as follows: 5.0 g MnSO 4 , 5.0 g NiSO 4 ,6H 2 O, 5.0 g FeSO 4 ,7H 2 O and 10 g of NH 4 Fe (SO 4 ) 2 was dissolved in 20 ml deionized water using 7.0 ml conc.Then 240 ml of 2.0 M sodium hydroxide was added to the prepared mixed solution drop-by-drop along with strong stirring at room temperature in an Erlenmeyer flask.After stirring for 27 hours, washed several times and pre-dried at 70 °C for 2.5 hour.For Synthesis of Mn doped Ferric Hydroxide Fe(OH) 3 .The obtained mixed solution was stirred for 21 hours at 70 °C and finally used for antimicrobial experiments (Emamifar et al., 2010).

Apparatus
X-ray diffraction (XRD, Philips PW 1800) was preformed to characterize the phase and structure of the prepared nanoparticles using cuka radiation (40 KV and 40 mA) at angles ranging from 10 to 80 • .The morphology of the nanoparticles were observed by field emission scanning electron microscopy (FE-SEM: Hitachi S-4160) under an acceleration voltage of 15 KV.

Bacterial Strains
Various strains of the following bacteria were prepared from the Institute of Standard and Industrial Research of Iran.Overall, two gram-positive bacterium S. aureus (ATCC 2523), B. subtilis (ATCC 6633) and two Gram-negative bacteria, E. coli (ATCC 2592), P. aeruginosa (ATCC 9027) and fungi, A. oryzae (ATCC 2023), C. albicans (ATCC 1021) were studied.Bacterial strains were prepared in accordance with the manufacturer's instructions, cultured on Mueller-Hinton agar (Merck, Germany) plates, and incubated at 37 °C for 24 h before use.

Antibacterial activity Disc diffusion method
The disc diffusion method was applied to study the antibacterial activity (Carson et al., 2002) as follow: 100 µL of each bacterium including nearly 0.5×10 6 colony-forming units (CFU/mL) was speared out onto the Muller Hinton Agar medium (Merck, Germany) by a sterile swab and the stock solution was dilated to 25, 50 and 100 mg in DMSO.Then, the sterile discs (6 mm in diameter) were impregnated with trial serial solutions (including 25, 50, and 100 mg/mL of compounds per disk) and placed on the surface of the inoculated medium.At the end, plates were incubated at 37 °C for 24 h and zone of inhibitions were measured in millimeters (mm) with a caliper and compared with the control (Lalli et al., 2008).

Minimum inhibitory concentration (MIC) determination
MIC and MBC were determined using broth dilution assay method (Carson et al., 2002).For the dissection of the minimum inhibitory concentration of the extracts and nanoparticles against these bacteria, serial dilution technique was applied.The samples (0.01 mg) were dissolved in 2 ml distilled DMSO in order to obtain a stock solution (50mg/ml).Afterward, 1 ml of this solution was transferred to a test tube containing 1ml Mueller Hinton broth medium to reach a concentration of 25 mg/mL.After 24 h of incubation at 37 °C, bacterial growth was examined.The MIC of the samples was taken as the lowest concentration inhibited bacterial growth.DMSO and two antibiotics (Vancomycin, Gentamicin) were reference as negative and bactericidal drugs, respectively.

Minimum bactericidal concentration
Minimum bactericidal concentration (MBC) of samples also were investigated as same as MIC.MBC shows the lowest concentration of antibacterial agent that kills all bacterium.In this method, the bacterial growth was observed on the surface of agar medium.For this reason, a loop full of each bacterial cultured in Muller Hinton broth was cultured on MHA medium and incubated at 37 °C for 24 h (Carson, Mee, Riley, 2002).

Antifungal activity
The selected isolates were grown on Sabouraud Dextrose Agar (SDA).Next, 7 days-old culture of fungi was scraped with a sterile sculpture, macerated with sterile distilled water.Antifungal test was conducted using the disc diffusion protocol by applying 100 μL of a suspension containing (10 5 CFU/mL) of A. oryzae on the SDA medium (Oxoid, Hampshire, England).The disks (6 mm in diameter) were impregnated with samples solution (equivalent to 25, 50, and 100 mg /disc) and DMSO (as a negative control) was placed on the inoculated agar.The inoculated plates were incubated for 72 h at 25 °C and inhibition zones (antifungal activity) was measured and compared with the positive control (Clotrimazol and Amphotericin B); each assay was repeated twice (Hammer, Carson, Riley, 2004).

Preparation of culture media
DNA cleavage experiments were performed according to the literature (Lalli et al., 2008).Luria-Bertani broth (10 g/L of peptone, 5 g/L of yeast extract, and 10 g/L of NaCl) was used for the culturing of E. coli.Subsequently, 50 mL of medium was prepared and autoclaved for 20 min at 121 °C under 15 lb of pressure.The autoclaved medium was inoculated with the seed culture and incubated at 37 °C for 24 h.

Isolation of DNA
To obtain the pellet, the fresh bacterial culture of E. coli contained chromosomal DNA (1.5 mL) was centrifuged.DNA was extracted base of the Kit protocol (gram-negative extraction CINNAPURE, Iran) and chromosomal DNA was stored at -40 °C for the following test.

Agarose gel electrophoresis
Cleavage products were analyzed by the agarose gel electrophoresis method (Lalli et al., 2008).Test samples (5 mg/mL) were prepared in DMSO, added to the isolated chromosomal DNA of E. coli (5 µL/mL), and incubated for 1.5 h at 37 °C.Next, 10 μL of compound/DNA samples (mixed with bromophenol blue dye at a 5: 1 ratio) was loaded carefully into the electrophoresis chamber wells along with a standard DNA marker containing TAE buffer (4.84 g Tris base, pH 8.0; 0.5 M EDTA/1 L) and finally loaded onto the agarose gel (1% gel was stained with 10 μg/mL of ethidium bromide).A gel containing samples was connected to power supply (100 V) for 45 min, and the DNA bands under the UV transilluminator were observed to determine the extent of DNA cleavage.Also, H 2 O 2 combined/incubated with DNA were treated and used as negative control.

Identification of components
Identification of components was assigned by matching their mass spectra with Wiley Registry of Mass Spectral Data (7th edition, Agilent Technologies, Inc.) and National Institute of Standards and Technology 08 MS (NIST) library data.The identification was also confirmed by comparison of the retention indices with data in the literature (Lalli et al., 2008).The percentages of compounds were calculated by the area normalization method, without considering response factors.The GC analysis of the ethanolic extract of Euphorbia prostrata showed 14 different components.The identified components with their relative percentages and Kovats indices are given in Table (I, II).The main constituents of Euphorbia prostrata volatile oil were phytol (53.8%), stearic acid (10.1%) and palmitic acid (5.0%).Also, 24 compounds were identified in Pelargonium graveolens extract, with total abundance of 98.9% and the major constituents of the extract were β-citronellol (47.1%), trans-geraniol (6.8%) and δ-cadinene (5%).

Morphology of the nanoparticles
The morphology of the obtained Mn-Ni@Fe 3 O 4 particles investigated by FE-SEM (Figure 1).Results show that Mn-Ni@Fe 3 O 4 particles have an amorphous and porous structure.The XRD pattern of the prepared Mn-Ni@Fe 3 O 4 particles indicate very week peak at 2θ = 35.5°attributed to 311 lattice plane of magnetite (Fe 3 O 4 ) cubic structure.The XRD pattern for confirm an amorphous structure or very poor crystalline of Mn-Ni@Fe 3 O 4 particles (Figure 2).The FE-SEM images of the Mn: Fe (OH) 3 -NPs with different magnifications (Figure 1).Show that prepared Mn: Fe (OH) 3 nanoparticles are spherical with approximately size of 65 nm, It while are uniform in the shape and size.

Antibacterial activity of Plant extracts
Antibacterial activities of the nanoparticles and plant extracts were evaluated based on the disc diffusion methods (Figure 3).DMSO did not show inhibition against the tested bacteria.The most of medical extracts and nanoparticles showed moderate to high activities at 25 to 100 mg/mL concentration.Euphorbia prostrata  showed higher antibacterial activity against E. coli, P. aeruginosa and S. aureus than Pelargonium graveolens, while it was lower against B. subtilis at 25, 50 and 100 mg/ mL concentration.About nanostructures; Mn: Fe(OH) 3 showed higher antibacterial activity against E. coli and S. aureus, while it was lower for B. subtilis and P. aeruginosa at 25, 50 and 100 mg/mL concentration.Also, combination of nanoparticles with medical extract showed the highest biological activity.As seen in Table III, IV, combination of Euphorbia prostrata & Mn: Fe(OH)3 showed the highest activity with 21.00 (mm) diameter zone at 100 mg/mL concentration against E. coli.Finally, inhibitory effects of all compounds were lower than standard antibiotics.

Antifungal activity
To evaluate the antifungal activity of nanoparticles and extracts, disk diffusion method was performed.The most of the medical plants and nanoparticles showed moderate to high antibacterial activities against C. albicans.Pelargonium graveolens showed higher antifungal activity against C. albicans at 50 and 100 mg/mL concentration.Also, Mn: Fe(OH)3 showed higher antifungal activity against C. albicans at 25, 50 and    Disc diffusion method and MIC and MBC data suggest that the antimicrobial activities of the plant extract were more effective than nanoparticles and decreasing the concentration of the extract and nanoparticles had a directly proportional to reduce the zone of inhibitions.McGaw, Jäger, Van Staden, 2002).High proportions of β-citronellol and phytol in this extract make it interesting considering its high antibacterial activity.The mechanism of essential oils action may contain: 1) Damage or destruction of the cell wall; 2) Disturbances in the cytoplasmic membrane; 3) Leakage cell contents; 4) Membrane protein destruction and defeat in transports; and 5) Coagulation of cell contents (Luqman et al., 2008).The nanoparticles can either directly interact with the microbial cells (e.g.interrupting transmembrane electron transfer, disrupting/penetrating the cell envelope, and oxidizing cell components) or produce secondary products (e.g.reactive oxygen species (ROS) and dissolved heavy metal ions) that cause damage to bacteria (Emamifar et al., 2010).Anti-bacterial properties of metal oxide nanoparticles (according to the surface to volume ratio) are very different.This antibacterial activity is due to their high surface-to-volume ratio rather than to the sole effect of metal-ion release (Thukkaram et al., 2014).
Arif Khan et al. (2015a) worked on pharmacological characterization of methanol extract of Calligonum polygonoides from District Bannu Dried plant and measured growth inhibition of Aspergillus niger.They showed C. polygonoides possess significant antioxidant, antifungal and cytotoxic bioactive compounds.Also in another study conducted by Imran Khan et al. (2016), pure compound of Lonicera quinquelocularis plant were studied for antioxidant, antimicrobial and phytotoxic activities.They reported that crude extract inhibits the growth of Aspergillus fumigatus and Fusarium solani 65 and 70%, respectively.Also, antimicrobial activities of Calligonum polygonoides, Albezia lebeck and Piper nigrum were screened through the agar tube dilution method (Khan et al., 2015b).They reported that growth of the Gram-positive bacteria (S. aureus) as well as Gram-negative bacteria (E.coli) was markedly inhibited by C. polygonoides.The A. lebeck and P. nigrum extracts showed activity against Aspergillus niger followed by A. flavis; while the highest activity were shown by A. lebeck against A. niger and by P. nigrum against A. flavis.Nanoparticles can cause peroxidation of the phosphor-lipid compounds of bacterial membrane; therefore, the integrity of the cell membrane reduces, normal cellular activities in a healthy cell structure such as the respiratory activities disappear, and cell death becomes unavoidable; in agreement with the results of current research.Due to the difference between the negatively charged bacteria and the positively charged nanoparticles, the nanoparticles act as an electromagnetic absorbing the microbes causing the nanoparticles to bind cell surface.Also, the electrostatic forces or hydrophobic iron oxide nanoparticles are capable of the binding bacterial cell wall and connecting to surface bonding agents (Chifiriuc et al., 2011;Ebrahiminezhad et al., 2012).

CONCLUSION
The combination of Euphorbia prostrata and Pelargonium graveolens extracts with nanostructures showed synergic effects to eliminate the bacteria via DNA destruction and others mechanisms.Furthermore, it seems that the synergistic effect of nanoparticles with plant extracts leads to new choices for the treatment of infectious diseases.

FIGURE 3 -
FIGURE 3 -Disc diffusion methods and their hollows around discs against S. aureus.

TABLE II -
Chemical composition of the ethanolic extracts of

TABLE I -
Chemical composition of the ethanolic extracts of FIGURE 1 -FE-SEM images of the prepared Mn-Ni@Fe 3 O 4.

TABLE III -
Anti bacterial activity as diameter of zone of inhibition * (mm) around the constructed and standard discs.**All data are the mean of three measurements Combination of nanoparticles with medical extract showed the highest biological activity.As seen in TableV, combination of Pelargonium graveolens & Mn: Fe(OH)3 showed the highest antifungal activity with 17.6 (mm) diameter zone at 100 mg/mL concentration against C. albicans.Finally, inhibitory effects of all compounds were weaker than standard antibiotics.

TABLE IV -
Antibacterial effects of extract and nanoparticles performed by Broth dilution (MIC (mg/mL) and MBC (mg/mL) methods

TABLE V -
Antifungal activity as diameter of zone of inhibition * (mm) around the constructed and standard discs.* All data are the mean of three measurements