Medicinal plants are used for treatment of many diseases all around the world. Besides, some of medicines are expensive or not readily available. These situations urgently forced scientists for searching new inexpensive drugs with prolonged periods before resistance set in. Because of the side effects and the resistance that pathogenic microorganisms build against the common antibiotics, much recent attention has been m to extracts and biologically active compounds which are isolated from plants in herbal medicine. This study has carried out in order to clarify antibacterial efficacy of Euphorbia helioscopia in Sari, Iran in 2016. Gram negative (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae) and Gram positive (Staphylococcus aureus, Bacillus cereus) bacteria were used in this study. The results showed that the ethanolic extract of Euphorbia helioscopia had a promising antibacterial effect. In the present study, the best antibacterial efficacy of Euphorbia helioscopia was on Staphylococcus aureus.
Keywords: Methanol; Aqueous; Extract; Euphorbia helioscopia; Antibacterial
Infectious diseases are the major public health concern. They are responsible for considerable mortality and morbidity worldwide. It has reported that enteric diseases cause 5.8 million deaths in infants and children below 5 years all around the world . Chemotherapy is the main way for treatment of bacterial infections, but bacterial resistance to antibiotics leads to failure . Other problems relate to the antibiotics such as toxicity, low efficacy and high cost leads to research for new alternatives. These days, modern medicine appreciates traditional pharmacology and search for potential medicinal plants. This can be a new approach for treatment of many infectious diseases . There is a vast traditional usage of herbal medicine in many parts of the world. In developing countries including Iran, it is estimated that about 80% of the population using traditional medicine for their primary health care . Euphorbia (Euphorbiaceae) with about 2000 species in the world and 100 species in Iran is a cosmopolitan genus and considerable distribution in tropical regions . Species of this genus are usually herbaceous and mainly distributed in temperate areas of the Northern Hemisphere. With the exception of E. helioscopia, a widespread herb in temperate regions worldwide, the remaining species occur in the Alborz, Zagros and northwestern regions of Iran [6,7]. Traditionally, the plant is used in skin eruption, cholera, glactagogue and the leaves are useful as laxative . Antibacterial activity of E. helioscopia extract on some bacteria as Eschericha coli, Bacillus subtilis, Shigella flexenari, Staphylococcus aureu, Pseudomonas aeruginosa and Salmonella typhi in several studies has been evaluated although the results were controversial. Anti-viral cytotoxicity, anti- fungal, anthelminthic, antioxidant and vasodepressor activities, related to having terpenoids, glycosides and amino acids, make this plant interesting for studying  Many plants have been used in antimicrobial traits, because they have compounds synthesized in the secondary metabolism of plants. Therefore, more studies for using plants as therapeutic agents should design, especially those related to the control of antibiotic resistant microbes . The objective of this study was to evaluate the potential of plant extracts and phytochemicals on standard microorganism strains as well as multi-drug resistant bacteria.
Sari is a city in Mazandaran province, Iran. It is located in 36.56 latitude and in 53.06 longitude, also it is situated at elevation of 43 meters above the sea level.
Plants were gathered from Sari city in March 2016. Collected fresh plants were examined and the old, verminous and fungus-infected leaves were removed. Leaves were dried at room temperature (25°C) for one week in a purpose-built drying machine until the leaves were brittle enough to break easily. The dried plants were ground to a fine powder (diameter c. 0.1 mm) by using a laboratory grinding mill (Telemecanique/MACSALAB model 200 LAB) and stored in airtight bottles in the dark until extraction.
Separated aliquots of finely ground plant materials (50 g) were extracted with 120 ml of solvents of acetic acid and ethanol (technical grade, Merck) in conical flask for 4 h, while shaking every 3 to 5 min on a Labotec model 20.2 shaking machine. The process continued with covering the flask and incubating at 37°C for 48 h. The solution was subsequently shaken and filtered using Whatman filter paper. The filtrate was evaporated to dryness using a rotary evaporator (Model type Laborota 4010, Germany) and finally ethyl acetate and ethanolic extracts were achieved. Then the extracts stored below ambient temperature.
Preparation of plant extracts for preliminary phytochemical studies: The grinded tuber materials of 5 g were weighed separately using an electronic balance and crushed in 25 ml of sterile water, boiled at 50-600°C for 30 minutes on water bath and it was filtered through Whatman No.1 filter paper. Then filtrated fluid was centrifuged at 2500 rpm for 15 minutes and stored in sterile bottles at 50°C for further use .
Gas chromatography-mass spectrometry analysis: Gas Chromatography-Mass Spectrometry (GC-MS) was performed on Hewlett Packard 6890 series, using a DB-5 capillary column (30 m × 0.25 mm, film thickness 0.25 μm) ) which was programmed as follows: 70°C for 5min and then up to 280°C at 4°C/min. The carrier gas was helium at a flow rate of 1 mL/min; split ratio, 1: 40; ionization energy, 70 eV; scan time, 1 s; acquisition mass range, 40-400(m/z). The components of the oil were identified by their retention time, retention indices relative to C9-C28 n-alkanes, computer matching with the WILEY275.L library and as well as by comparison of their mass spectra with those of authentic samples or with data already available in the literature .
Selected test microorganisms
Pathogenic microorganisms selected for study include five bacteria, (Staphylococcus aureus) S. aureus (PTCC 1431), (Escherichia coli) E. coli (PTCC 1330), (Bacillus cereus) B. cereus (PTCC 1154), (Pseudomonas aeroginosa) P. aeroginosa (PTCC 1074) and (Klebsiella pneumoniae) K. pneumonia (PPTCC 1053). Selected microorganisms were procured from Iran Scientific and Industrial Research, Tehran, Iran. Bacterial strains were grown and maintained on Muller- Hinton Agar Medium (Oxoid, UK).
Antimicrobial screening of extracts
Disc diffusion assay (DDA) was performed for antimicrobial screening. MH (Muller-Hinton) agar base plates were seeded with the standard inoculums size of bacteria (1 × 108 CFU/ml). Sterile filter paper discs (6 mm in diameter) were impregnated with 100 μl of each concentration of both aqueous and methanol extracts (100, 75, 50, 25, 12.5, 6.25 mg/mL), left to dry to remove residual solvent, which might interfere with the determination. Extract discs were then placed on the seeded agar plates. Each concentration of extracts was tested in triplicate along with Gentamicin (1 mg/disc) as a control. The plates were kept 1h at 4°C for diffusion of extracts, there-after were incubated at 37 ± 2°C for 24 h. Finally, the zone of inhibition (IZ) or depressed growth of microorganisms in several concentrations of both ethyl acetate and ethanolic extract were measured (Figures 1-6).
The data obtained from the study were analyzed statistically using the analysis of variance (ANOVA) and GLM Univariate. Also Dancan’s multiple range test was used to separate the means .
Phytochemical screening of extract
Quantitative analysis of Euphorbia helioscopia is shown in Table 1.
|1||Methyl 5-methylfuryl sulfide||11.025||3.23||1046|
|2||Ammelide N-METHYL-PARABANIC ACID||12.551||1.24||1090|
|17||Methyl heptyl ketone||36.631||0.35||1895|
|23||2-Hexadecen-1-ol, 3,7,11,15-tetram ethyl-, [R-[R*, R*- (E)]]- (CAS) Phytol||42.687||9.7||2119|
|27||Hexadecanoic acid, 2-hydroxy-1- (hydroxymethyl)ethyl ester||48.828||0.67||2530|
|30||2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexamethyl-, (all-E)- Squalene||51.321||1.72||2698|
|35||Bicyclo[4.3.0]nonane, 3-buty l-4-hexyl-||59.191||1.57||2682|
|36||Stigmast-5-en-3-ol, (3.beta., 24S)-||59.513||8.22||2681|
Table 1: Quantitative analysis of Euphorbia helioscopia extract.
The main components including:
1. Hexadecanoic acid (28.02%).
2. 2-Hexadecen-1-ol,3,7,11,15-tetram ethyl-, [R- [R*, R*-(E)]] -(CAS) Phytol (9.7%).
3. 1,2- Benzenediol (8.35%).
4. Stigmast-5-en-3-ol, (3. beta.,24S) (8.22%).
5. Linolenic acid (7.65%).
Antibacterial activity of Euphorbia helioscopia
Disc diffusion assay: The observed antimicrobial activity of E. helioscopia at several concentrations (6.25-100 mg/mL) expressed as zone of inhibition (mm) is shown in Table 2. The results show that increase in concentration of extracts can increase the inhibition zone of growth in some of the microorganisms. Ethanolic extract of E. helioscopia (100 mg/mL) displayed good antibacterial activity against Gram negative (P. aeroginosa) bacteria (Fig 4) while ethyl acetate extract did not revealed the same efficacy. Also, ethanolic extract of E. helioscopia (100 mg/mL) showed a good inhibitory effect on S. aureus and in contrast, ethanolic extract did not show favorable efficacy on this bacteria (Fig 1). The same results by ethanolic extract for B. cereus (Figure 3), K. pneumonia and (Figure 5), E. coli (Figure 2) were achieved. Altogether, ethanolic extract seems to be more efficient in destroying both Gram positive and Gram negative bacteria while ethyl acetate extract displayed a weak inhibitory effect in all cases (Figures 1-5). The best antibacterial efficacy of Euphorbia helioscopia was on Staphylococcus aureus which revealed with the biggest zoon around the bacteria. Also our control, Gentamicin showed highest inhibitory effect on all bacteria with lowest efficacy on P. aeroginosa (Figure 6).
| Extracts and
|*=Gentamicin (Standard disc)
IZ=Inhibition Zone in mm (mean value; include 6 mm diameter of disc)
Table 2: Zone of inhibition of Euphorbia helioscopia extracts (ethil acetate and ethanolic) on test organisms.
For many years people around the world have healed the sick with herbal derived remedies and handed down through generations. Traditional medicine has an old history in health maintenance, as well as to prevent, diagnose, improve or treat physical and mental illness . Medicinal plants has a great economic value in Iran and several types of traditional medicines are using as complementary or alternative medicine . Euphorbia helioscopia (L.) (Euphorbiaceae) typically best-known as “Sun spurge ‘’ often grow in cultivated fields, pastures, rangelands and cultivated fields. It is a promising green fleshy annual herb up to 50 cm tall . Some of Euphorbia spp are invasive and noxious weeds in many regions of the world  As toxic plants, they threat grazing livestock and even humans when accidentally consume mixed with forage and harvested products. For example, Euphorbia seeds in grain crops are one of the suspected reasons for the high rate of cancers in northern Iran . In present study we evaluated antibacterial efficacy of ethyl acetate and ethanolic extracts E. helioscopia cultured in Mazandaran province, Iran on Gram negative and Gram positive pathogenic bacteria. Our results indicated that ethanolic extract has stronger antibacterial efficacy than ethyl acetate although Gentamicin as a control has better antibacterial effect than our extracts (Figures 1-6). Interestingly ethanolic extract of E. helioscopia showed good antibacterial efficacy on S. aureus, K. pneumonia, B. cereus and E. coli and the lowest efficacy on P. aeruginosa. The phytochemical constituents existing in the plant declared in a study carried out in 2013 were including: terpenoids (euphornin L,euphoscopin F, epieuphoscopin B, euphoscopin B, euphoscopin C Euphoheliosnoid D, helioscopinolide A,Euphornin N, Euphoheliosnoids A, Euphoheliosnoids B, Euphoheliosnoids C), glycosides (Quercetin-3-β-glucoside, quercetin- 3-β-galactoside,quercetin-3-β-galactoside-2”-gallate,3β,7β,15β- trihydroxy-14-oxolathyra-5E,12E-dienyl-16-O-β-d glucopyranoside) and aminoacids. Also Pharmacological studies reported anti-viral, Cytotoxic, anti-fungal, anti-bacterial, anti-tumor, wound healing affect, vassodepressor and Phytodermatitis properties of E. helioscopia . Similar to our results, researchers in 2013 indicated that methanolic extracts of E. helioscopia showed antibacterial efficacy on S. aureus, K. pneumoniae, P. multocida and E. coli . We observed that the best antibacterial efficacy of Euphorbia helioscopia was on Staphylococcus aureus. In other study carried out in 2009, Dichloromethane extract of E. helioscopia exhibited significant activity with 90% Inhibition against Fusarium solani, whereas no significant activity against Salmonella typhi and Bacillus subtilis observed. These findings were in contrast to our results that ethyl acetate and ethanolic extracts of E. helioscopia showed antibacterial efficacy on P. aeroginosa and B. cereus . Similar to our findings, in other study carried out in 2010, antimicrobial and anti fungal activities of ethanol extract of E. hirta L. leaves on S. aureus, B. ceresus, S. typhi, K. pneumoniae, P. aeuroginosa and fungal species namely Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus and Rhizopus oryzae demonstrated. It is clear that compounds in family Euphorbiaceae is responsible for antibacterial activity. Phytochemical studies on leaves of E. hirta L. revealed the presence of tannins, flavonoids alkaloids, glycosides, proteins, sterols and saponins . The antimicrobial activity of E. hirta L. may be due to one/more group of earlier mentioned phytoconstituent(s) Results from a study carried out in 2005 on Euphorbia hirta L. and Euphorbia tirucalli L. indicated that aqueous extract of Euphorbia hirta L. was inactive against both the Gram-positive bacteria, i.e. B. Subtilis and S. epidermidis, while that of Euphorbia tirucalli L. was active only against P. pseudoalcaligenes. Also results showed that (methanol) extracts revealed more consistent antimicrobial activity compared to those extracted in water . Another researchers in 2010 demonstrated that ethanol and methanol extracts of Euphorbia hirta L. were more effective in inhibition the growth of the pathogenic bacteria including B. Subtilis, S. aureus, E. coli, K. pneumoniae and P. vulgaris than aqueous and chloroform extracts . Euphorbia helioscopia L. has been employed as a conventional therapy for cancer in China. Euphornin is one of the main bioactive components of Euphorbia helioscopia L. It is believed that Euphornin is responsible for cytotoxicity and has assessed by mice lung denocarcinoma cells (LA795) .
Our study compiled all the findings about the ethanolic and ethyl acetate extracts of Euphorbia helioscopia. The aim of present study was evaluation of antibacterial efficacy of these extracts. As the plant has been in use in traditional medicine, for long time in Asian subcontinent, it has many therapeutic claims. In order to verify those claims various researchers have undertaken, various experimental research are needed. In the present study we have tried to reveal the antibacterial results, which will act as single point information source about the plant for further use by scientific community.
The authors would like to declare that there are no conflict of interest. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.