Screening of Chemical Analysis, Antioxidant Antimicrobial and Antitumor Activities of  Essential Oil of Oleander (Nerium oleander) Flower

Ali, H.F.M.; El-Ella, F.M.A.; Nasr, N.F.

INTRODUCTION

Nerium oleander Linn. (Syn. N. odorum Soland; N. indicum Mill), distributed in the Mediterranian region and sub-tropical Asia, is indigenous to the Indo-Pakistan subcontinent. The plant is commonly known as kaner and widely cultivated as ornamental plants, but all the parts of the plant are poisonous to man, animals and certain insects and investigations on deferent parts of the plant have revealed the presence of several glycosides, tri-terpenes and straight chain compounds (Langford and Boor, 1996; Begum et al., 1999; Soto-Blanco et al., 2006; Barbosa et al., 2007).

The chemical composition and possible biological effects of the flower essential oil of Nerium oleander has not been studied to date.

A great number of plant essential oils from different origin have been chemically analyzed and reported to possess stronger antioxidant and antimicrobial activities. The biological activity of different essential oil based on their content of phenolic compounds (Wang et al., 1998) and terpens compounds according several studies (Burt, 2004; Sacchetti et al., 2005; Wu et al., 2006; Li et al., 2009; Jaroslav et al., 2009; Oke et al., 2009; Mehmet et al., 2009).

The aim of the present study are GC/MS analysis and in vitro screening of antioxidant; antimicrobial and antitumor activities of essential oil extracted from Oleander flower.

MATERIALS AND METHODS

Plant Material
Fresh flower of Nerium oleander was collected from the different sites of Taif desert, Saudi Arabia during May to June 2009. The plant was identified and authenticated by a botanist at the Plant Department, Faculty of Science Taif university Saudi Arabia (Fig. 1).

Chemicals
Diphenylpicrylhydrazyl (DPPH), methanol, n-hexane, 2-deoxy-2-ribose, β-carotene and linoleic acid, were procured from Sigma (Sigma-Aldrich GmbH, Sternheim, Germany). Folin-Ciocalteu’s phenol reagent and tween 40 and dimethyl sulphoxide (DMSO) were from Merck (Darmstadt,Germany). All chemical reagent used were of analytical grade.

Extraction Method
Batches of 500 g of plant material were submitted to hydrodistillation for 5 h using a Clevenger-type apparatus according to the method recommended in the Council of Europe (2004), using n-hexane (10 mL) as collector solvent. After evaporation of the solvent under N₂ flow, the oil was dried over anhydrous sodium sulphate and stored in sealed vials protected from the light at -20°C before analyses by Gas Chromatography-Mass Spectroscopy (GC-MS).

GC-MS Chemical Analysis
The GC-MS analysis of the volatile oil was performed on a Varian gas chromatograph interfaced to a Finnigan SSQ 7000 Mass Selective Detector (MSD) with ICIS V2.0 data system for MS identification of the GC components.


Fig. 1:	The fresh flower of Nerium oleander

The column used was a DB-5 (J and W Scientific, Folsom, CA) cross-linked fused silica capillary column (30 m, 0.25 mm i.d.), coated with polydimethylsiloxane (0.5 mm film thickness). The oven temperature was programmed from 40°C for 3 min, isothermal, then heating by 4°C min^-1 to 250°C and isothermally for 15 min at 250°C. Injector temperature was 210°C and the volume was 0.5 μL which auto-injected. Transition-line and ion source temperatures were 250 and 150°C, respectively. The mass spectrometer had a delay of 3 min to avoid the solvent peak and then scanned from m/z 40 to m/z 550. Ionization energy was set at 70 eV. The identification of volatile components was based on computer matching with the WILEY275, NIST05 and ADAMS libraries, as well as authentic compounds for major identified compounds (Adams, 2007; NIST, 2005). The quantitative determination was carried out by peak area integrated by the analysis program.

Determination of Total Phenolic Content
Total phenolic contents of the volatile oil were determined as described by Tsai et al. (2008), with Folin-Ciocalteu reagent and gallic acid used as a standard. An aliquot (0.2 mL) of the volatile oil was added to a volumetric flask. Then, 46 mL distilled water and 1 mL Folin-Ciocalteu reagent was added and the flask was shaken thoroughly. After 3 min, a 3 mL solution of Na₂CO₃ (7.5%) was added and the mixture was allowed to stand for 2 h with intermittent shaking. Absorbance was measured at 760 nm. The results were expressed as milligrams of Gallic Acid Equivalents (GAEs) per gram of extract.

Antioxidative Assays
DPPH Assay
The antioxidant activity of oleander oil was measured in terms of hydrogen-donating or radical-scavenging ability, using the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a reagent (Sahin et al., 2004; Sharma and Bhat, 2009). Fifty microliter of oleander oil were added to 2 mL of a 60 μM buffered methanol solution of DPPH (pH = 5.5). Absorbance measurements were read at 517 nm, after 20 min of incubation time at room temperature. Absorption of a blank sample containing the same amount of buffered methanol and DPPH solution acted as the negative control, BHT and trolox (10 mg mL^-1) were used as positive control. All determinations were performed in triplicate. The percentage inhibition of the DPPH radical by the samples was calculated according to the formula:

where, A_B is the absorption of the blank sample (t = 0 min) and A_A is the absorption of the tested oil or standards substance solution (t = 20 min). The EC₅₀ value, defined as the concentration of antioxidant in the reactive system necessary to decrease the initial DPPH concentration by 50% and was calculated from the results.

β-Carotene/Linoleic Acid Bleaching Assay
In this assay, antioxidant activity was determined by measuring the inhibition of volatile organic compounds and conjugated dienehydroperoxides arising from linoleic acid oxidation. The method described by Miraliakbari and Shahidi (2008). A stock solution of β-carotene and linoleic acid was prepared with 0.5 mg of β-carotene in 1 mL chloroform, 25 μL of linoleic acid and 200 mg Tween 40. The chloroform was evaporated under vacuum and 100 mL of aerated distilled water was then added to the residue. The oleander oil was dissolved in DMSO (g L^-1) and 350 μL of solution was added to 2.5 mL of th above mixture in test tubes. The test tubes were incubated in a hot water bath at 50°C for 2 h, together with three tubes, two contained the antioxidant BHT and trolox as a positive control and the other contained the same volume of DMSO instead of the extracts. The test tubes with BHT and trolox maintained its yellow colour during the incubation period. The absorbencies were measured at 470 nm on an ultraviolet spectrometer (Cintra 6, GBC, Australia). Antioxidant Activities, (AA) (inhibition percentage, I%) of the samples. The AA of sample was evaluated in terms of bleaching of β-carotene using the following equation:

where, A_{β-carotene after 2h assay} is the absorbance of-carotene after 2 h assay remaining in the samples and A_{initial β-carotene} is the absorbance of and-carotene at the beginning of the experiments. All tests were carried out in triplicate and inhibition percentages were reported as Mean±SD of triplicates.

Ferric Reducing Antioxidant Power Assay
The reductive potential of the oils and the standards positive controls (BHT and trolox) was determined according to the method of Oyaizu (1986). The oleander oil or standards were mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide (K₃Fe(CN)₆; 2.5 mL, 1%). The mixture was then incubated at 50°C for 20 min. Afterwards, 2.5 mL of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged for 10 min at 3000 rpm. Finally, the upper layer of solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl₃ (0.5 mL, 0.1% w/v) and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. Results expressed as EC₁value, which means the concentration of antioxidant in the reactive system having ferric reducing ability equivalent to that of 1% ferric cyanide.

Antimicrobial Activity Assay
The antimicrobial activity of oleander oil was evaluated by the standard disc diffusion technique as described by Gillies and Dodds (1984) and Lngolfsdottir et al. (1997). Species; strains and cultivation conditions of used microorganism are shown in Table 1.

Table 1:	The species, strains and cultivation conditions of used microorganism

^aObtained from Department of Microbiology, Agriculture Faculty, Cairo University, ^bObtained from Plant Pathology Institute, Agricultural Research Center, Egypt, G⁺: Gram-positive bacteria, G¯: Gram-negative bacteria, TSA+YE: Trypticase Soy Agar + 0.6% Yeast Extract, PDA: Potato Dextrose Agar

Procedure
Target organisms were inoculated in melted (at 50°C) trypticase soy agar + 0.6% yeast extract medium (APHA, 1978) and mold strains were inoculated in melted (at 50°C) potato dextrose agar medium (APHA, 1978) with heavy inoculum. Then the inoculated medium were poured over a solid layer of uninoculated agar medium in sterilized Petri-dishes and left to solidify at 4°C (surface layer should be constant in volume and horizontally homogenous). Discs of Whatman No. 1 filter paper (6.0 mm in diameter) were sterilized by autoclaving at 121°C for 15 min. An accurate volume (10 μL) of undiluted essential oil was aseptically added to each disc and left to dry. Each disc was aseptically placed on the middle of agar surface (in triplicate) and left at 4°C for 1 h then plates were incubated. Gentamicin for bacterial strains and nystatin for fungal strains (10 μL disc^-1) were used as positive reference standards.

The antimicrobial activity of essential oil was evaluated by measuring the average of inhibition zone diameter against the test microorganisms and the values were expressed as Mean±SE.

Determination of the Minimum Inhibitory Concentration (MIC)
The MICs of the oleander oil against the test bacterial strains and yeast were determined by tube dilution method (Sokmen et al., 2004) using Trypticase soy broth + 0.6% yeast extract (APHA, 1978; Evans, 1996). Inocula suspensions of the microorganisms were prepared from 12 h cultures. Oleander essential oil was dissolved in 10% dimethylsulfoxide (DMSO) and serial two fold dilutions of the oil were prepared in sterilized tubes, ranging from 7.8 to 4000 μg mL^-1. The MIC was defined as the lowest concentration of the essential oil which the microorganism did not demonstrate visible growth (visible turbidity). The MIC value for each of fungus was determined by agar dilution method (Sokmen et al., 2004) using Potato Dextrose Agar (PDA) (APHA, 1978). The oil was added aseptically to sterile melted PDA medium containing 0.5% DMSO at the appropriate volume to produce concentrations ranging from 7.8 to 4000 μg mL^-1. The resulting PDA agar solutions were immediately poured into Petri dishes. The plates were spot inoculated with fungus strains. The MIC value was determined as the lowest concentration of the essential oil at which an absence of fungus growth. The MICs of the standards (Gentamicin and Nystatin) were also determined in parallel experiments as control for the microorganism's sensitivity.

Anti-Tumor Assay
Ehrlich Ascites Carcinoma(EAC) cells was used to study the anti-tumor activity of oleander oil. The tumor line is maintained in the National Cancer Institue (NCI) Cairo, Egypt in female Swiss albino mice by weekly intrapertoneal (i.p.) transplantation of 2.5x10⁶ cells. Similar line was proceeded in Biochemistry department, Cairo university. The in vitro study as described by Kandasamy et al. (2005) with slight modification. Cells were taken from tumor transplanted animals after ≈7 days of transplantation then the number of cells mL^-1 was calculated by using microscope counting technique (≈2x10⁷ cells mL^-1). The cells were centrifuged at 1000 rpm for 5 min, washed with saline then the needed numberof cells was prepared by suspending the cells in the appropriate volume of saline.

The cells culture medium was prepared using RPMI 1640, 10% fetal bovine serum, 10% L-glutamine and 0.01% dimethyl sulphoxide (DMSO). Trypan blue (0.4%) was prepared by dissolving of 0.4 g of the dye in 100 mL distilled water then kept in brown closed glass bottle.

Viability of Ehrlish Ascites Carcinoma Cells (EACC)
The viability percentages of tumor cells were measured after incubation with each oils extract as well as saline and DMSO as control. Two milliliter of media containing EACC (2x 10⁶cells) were transferred into a set of tubes each, then different concentrations (0.0, 0.2, 0.4, 0.8, 1.0, 1.4, 2,4, 8, 10 μL) oleander oil were added into the appropriate tube as well as saline. The tubes were incubated at 37°C for 2 h then centrifuged at 1000 rpm for 5 min and the separated cells were suspended in 2 mL saline.

Viable/Non-Viable Tumor Cell Percentage Count
For each examined materials (and control), a new clean, dry small test tube was used and 10 μL of cell suspension, 80 μL saline and 10 μL trypan blue were added and mixed, then the number of non-viable cells (stained) and viable cells (non-stained) counted under microscope by using a homocytometer slide in the 50 small squares.

Acute Toxicity of Oleander Oil
Toxicity of oleander essential oil was monitored in animal model system by different biochemical profiles including LD₅₀, diarrhea; GPT; LDH and createnine. Male Albino mice of 6 animals per group and weighing between 20 and 25 g were administered with graded doses of (100-400) mg kg^-1 b.wt. intra peritoneal of the oleander oil suspended in DEMSO. The toxicological effects were observed after 48 h of treatment in terms of mortality and expressed as LD₅₀. The number of animals dying during the period was noted (Ghosh, 1984). Others biochemical parameters determined after 10 days of administration according to methods of Reitman and Frankel (1957) for GPT activity; Bergmeyer (1974) for LDH activity and Husdan and Rapoport (1968) for createnine.

Statistical Analysis
Each of the measurements described was carried out in three replicate experiments and the results are recorded as mean ± standard deviation. The significantly different calculated at level of p≤0.05.

RESULTS AND DISCUSSION

Chemical Composition and Total Yield of Oleander Essential Oil
Total yield of essential oil was 0.1%. Most chemical constituents of the oleander essential oil were determined by GS/MS analysis and WILEY275, NIST05 and ADAMS libraries validation. Sixty four components, representing 94.69% of the total essential oil, were identified and listed in order of elution from capillary column in GC-MS and their retention times and area percentages (concentrations) in Table 2. Among which 34.2% were oxygenated compounds, 60.54% were terpenes compounds and alkane compounds (2.02%). The major components were, Camphore (12.76%), Eugenol (10.45%), α-, α-Campholenal (5.05%), thymol (8.43%) and which accounted for 38.12% of the essential oil.

Amount of Total Phenolics
Based on the absorbance values of the oleander essential oil solution, reacting with Folin-Ciocalteu reagent and compared with the standard solutions of gallic acid equivalents The amount of total phenolics was 136.54±3.32 mg gallic acid g^-1 essential oil.

Table 2:	Chemical composition of the essential oil of oleander

^aCompounds quantified on the DB-5 capillary column and listed in order of elution time from the same column and identified by comparison with MS libraries and cited literature except majors compounds were identified by comparison with MS libraries and with authentic compounds

Table 3:	Antioxidants activities of the essential oil of oleander

^a,b,c,dValues with different letter(s) in the same raw were significantly different (p<0.05). Each value in presented as Mean±SD (n = 3)

Antioxidant Activity
The potential antioxidant activity of the oleander essential oil was determined on the basis of three methods, the scavenging activity of the stable free radical DPPH (EC₅₀ value); inhibition of the coupled oxidation of linoleic acid and beta-carotene (AA% value) and Ferric reducing antioxidant power (EC₁ value). Since, the reaction followed a concentration-dependent pattern, only values of EC₅₀; AA% and EC₁ of oleander essential oil; BHT and Trolox are shown in Table 3. In general the lower the EC₅₀ value the higher the free radical scavenging activity of a sample.

Oleander essential oil had significantly lower EC₅₀ value (2.11±0.12) compared to trolox (6.75±0.22 μg mL^-1) and BHT (21.51±1.61 μg mL^-1). Regarding the EC₁values, the lower EC₁value the higher the ferric reducing activity of the sample. In present study, the oleander essential oil had significantly higher activity and lower EC₁(2.61±0.12 μg mL^-1) than trolox (8.35±0.22 μg mL) and BHT (4.61±0.35 μg mL^-1).

In the β-carotene linoleic acid system assay, oleander essential oil also possessed better antioxidant activity (65.73±3.11%) than trolox (54.31±2.51%) and BHT (90.20±1.81%). The efficiency of an antioxidant component to reduce DPPH essentially depends on its hydrogen donating ability, which is directly related to the number of phenolic hydroxyl moieties. In the β-carotene linoleic acid system assay, phenolics block the chain reaction of lipid peroxidation mainly by scavenging the intermediate lipid peroxyl radicals which are generated (Haslam, 1996). This also depends on the hydrogen-donating ability of antioxidants. Studies on oxidation potentials and redox reactions between polyphenols and transition metal ions have shown that the o-dihydroxyl feature is a crucial factor for the reducing efficiency (Makris and Kefalas, 2005). Regarding the three assay systems in present study, the aromatic hydroxyl groups, especially the o-dihydroxyl configuration, are important, either for hydrogen donation or interaction with Fe³⁺. The potential activity of essential oils as natural antioxidant has been studied by several authors. Wang et al. (1998) studied the antioxidant activity of phenolic compounds from sage essential oil. Ruberto and Baratta (2000) have tested about 100 pure components of essential oils from different groups of chemicals largely phenolics and terpens compounds for their antioxidant effectiveness.

Also, Ozkan et al. (2010) and Albayrak et al. (2010) recorded the same correlation in essential oil between antioxidant activities and phenolics compounds. From the major components of oleander essential oil are phenolic; Camphore (12.76%), eugenol (10.45%) and thymol (8.43%), which accounted for 31.54%. This result suggests that there may a close relationship between these three phenolics compounds and antioxidant activity specially reducing power of oleander essential oil, due to hydroxyl substitutions in aromatic ring, which possess potent hydrogen donating abilities as described by Haslam (1996).

Other compounds of oleander essential oil also seem to play an important role and have widely-varying capabilities towards all the radicals, for example α-Campholenal; Caryophyllene; Camphene and α-Humulene which represent 5.05%, 3.43, 2.75 and 2.43% of oleander essential oil.

Anti-Tumor Activity
Different concentration of oleander oil were tested for their ability to inhibit the growth of ehrlich ascites carcinoma cells line. The anti-tumor activity expressed as non-viable (dead) tumor cell percentage count is shown in Fig. 2. Anti-tumor activity gradually increase with concentration of oleander essential oil and reach to 100% with 8 μL mL^-1 of oleander essential oil. The obtained result in anti-tumor experiment in present study indicated that essential oil of oleander is very active on tested tumor cell line. This may related to some compounds present in oleander essential oil, for instance terpenic compounds and carvacrol which represent 60.54% and phenolic compounds which accounted for 31.54% from oleander essential oil. This compound recorded to has in vitro activity on tumor cell resistant to chemotherapy as well as significant antitumor effect on mice (Ulubelen et al., 2000; Mbarek et al., 2007). The effect of essential oil on tumor cell may due to toxic effect of terpenes and their ability to bonding with critical causing the death of tumor cell Omidbeygi et al. (2007).


Fig. 2:	Effect of oleander essential oil concentration (μL mL^-1) on ehrlich tumor cell death

Table 4:	The zone of inhibition (mm) and Minimum Inhibitory Concentration (MIC) values (μg mL^-1) of the essential oil of oleander; gentamicin and nystatin

Antimicrobial Activity
Antimicrobial activity of oleander oil was observed by agar disc diffusion technique against Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Salmonella typhimurium) and yeast (Saccharomyces cerevisia) and three strains of mold (Fusarium oxysporum; Rhizoctonia solani and Macrophoma mangiferae). Results from the agar disc diffusion tests for antimicrobial activity of oleander essential oil are shown in Table 4. The essential oil displayed a variable degree of antimicrobial activity against the different strains tested It was found to be active against all the microbes used for the activity compared with that of the standard antibiotics tested. According to inhibition disc zone diameter; oleander oil antimicrobial activity was in order of against B. subtilis>S. aureus>E. coli>S. cerevisia>S. typhimurium> M. mangiferae and moderately active against R. solani. Regarding the MIC values of oleander essential oil ranged ranged from 125 to 500 μg mL^-1 and 250 to 2000 μg mL^-1 for bacteria and fungi, respectively. The MIC values for standard ranged from 15.5 to 62.5 μg mL^-1 and 31.25 to 500 μg mL^-1 for Gentamicin and Nystatin, respectively. In general, the essential oil showed better antibacterial activity than antifungal activity (Table 3). The Gram-positive bacterium (Bacillus subtilis and Staphylococcus aureus) is more susceptible to the antimicrobial properties of essential oils than Gram-negative bacteria (Escherichia coli and Salmonella typhimurium) and it is considered to be due to its outer membrane which more permeable for essential oil compounds (Cox et al., 2001; Benli et al., 2007).

Several studies have been conducted to understand the mechanism of action of essential oils as antimicrobial agent. Cox et al. (2001) attributed this effect to the ability of essential oil to disrupt the permeability barrier of cell membrane structures and the accompanying loss of chemiosmotic control are the most likely reasons for its lethal action. Veldhuizen et al. (2006) attributed this function to the phenolic compounds, which has their interactions with biomembrane of microorganism and thus the antimicrobial activity. On the other hand, Cristani et al. (2007) and Omidbeygi et al. (2007) reported that the antimicrobial activity of essential oils is related to ability of terpenes to penetrating into the interior of the microorganism cells and interacting with critical interacellular sites causing the death of cells.


Fig. 3:	Effect of different doses of oleander essential oil on GOT, LDH and createnine

This studies in agreement with results of oleander oil, which has 31.54 and 60.54% of phenolic and terpenes compounds, respectively. The Gram-positive bacterium (Bacillus subtilis and Staphylococcus aureus) is more susceptible to the antimicrobial properties of essential oils than Gram-negative bacteria (Escherichia coli and Salmonella typhimurium) and it is considered to be due to its outer membrane which more permeable for essential oil compounds (Cox et al., 2001; Hanamanthagouda et al., 2010).

The variety of antimicrobial activity of the essential oil with its concentration and kind of bacteria, may due to the differences in the susceptibility of the test organisms to essential oil, this could be attributed to a variation in the rate of the essential oil constituent’s penetration through the cell wall and cell membrane structures (Cox et al., 2001).

This difference between concentrations of the essential oil and the standard antibiotic can be explained in terms of the fact that the active components in the essential oil comprise only a fraction of the oil used. Therefore, the concentration of the active components could be much lower than the standard antibiotics used Hanamanthagouda et al. (2010).

Toxicity of Oleander Essential Oil
Toxicity of oleander essential oil was monitored in animal model system by different biochemical profiles including LD₅₀, diarrhea; GPT and LDH and createnine. Obtained results are shown in (Fig. 3). Toxicity results indicated that no mortality or diarrhea observed with administration with all concentrations range of oleander oil. The GPT, an enzyme which allows determining the liver function as indicator on liver cells damage. Results indicated that no-significant effect after administration with oleander oil with different doses (in comparison with positive control). The LDH enzyme is often used as a marker of tissue breakdown as LDH is abundant in red blood cells and can function as a marker for hemolysis (Butt et al., 2002).

According to recorded results, no-significant effect after treatment with oleander oil with different doses (in comparison with positive control). Measuring serum creatinine is a simple test most commonly used indicator of renal function, increasing of createnine levels indicator of post stage of kidney failure (Delanghe et al., 1989). Results indicated that no-influence on the levels of creatinine in all treated groups with vanadyl sulphate at different doses, this indicated that the oleander oil has no side effect on kidneys tissue in animal model system under conditions of present experiments. Generally, the toxicity data indicated that the oleander essential oil has no side effect on animal model system under concentrations used in present study.

CONCLUSION

Essential oil of oleander flower had significant antioxidant activities; antimicrobial activities and antitumor activity, these biological properties without adverse effect. Therefore, it is suggested that further work be performed on the isolation and identification of the biologically active. These results indicated that the essential oil of oleander flower could be considered as a natural food preservatives and enhance the human health as natural antioxidant.

ACKNOWLEDGMENT

This study was financial supported by a grant from the Taif University, Kingdom of Saudi Arabia (grand No.1/430/458). Authors thank National Cancer Research Institute, Egypt For providing tumor cell line and experimental animals.