Chemical composition of essential oil and in vitro antioxidant and antimicrobial activities of crude extracts of Commiphora myrrha resin

Industrial Crops and Products 57 (2014) 10–16

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Chemical composition of essential oil and in vitro antioxidant and antimicrobial activities of crude extracts of Commiphora myrrha resin Amal A. Mohamed a,∗ , Sami I. Ali a , Farouk K. EL-Baz a , Ahmad K. Hegazy b , Mimona A. Kord b a b

Plant Biochemistry Department, National Research Centre (NRC), Dokki, Giza, Egypt Botany Department, Faculty of Science, Cairo University, Giza, Egypt

a r t i c l e

i n f o

Article history: Received 5 November 2013 Received in revised form 15 March 2014 Accepted 18 March 2014 Keywords: Antimicrobial activity Crude extracts Commiphora myrrha DPPH Essential oil GC–MS MIC

a b s t r a c t The antioxidant and antimicrobial potential of methanol (ME-OH), ethyl acetate (ETOAC) crude extracts and essential oil (EO) of Commiphora myrrha resin were investigated. The major constituents of the essential oil identified from the resin of C. myrrha were ␣-elemene (12.86%), 7-isopropyl-1,4-dimethyl-2-azulenol (12.22%), curzerene (11.64%), and germacra-1(10)7,11-trien-15-oic acid,8,12-epoxy-6-hydroxy-c¸-lactone (6.20%). In both DPPH scavenging and Fe2+ chelating assays, the ME-OH extract exhibited the highest activity compared to ETOAC extract and EO. Concerning the reducing power ability, EO was superior to Me-OH and ETOAC extracts. The Me-OH extract manifested the highest potential of antimicrobial activity against the tested bacterial and yeast microorganisms, while ETOAC extract and EO showed moderate or no potential antibacterial activity. The Me-OH extract exhibited the highest antioxidant and antimicrobial activity as compared to ETOAC and EO. It is concluded from the present study that besides its traditional use, the C. myrrha resin could be used as a natural source for antioxidant and antimicrobial compounds for possible applications in food and nutraceutical industries. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The plant-derived medicines are based upon the premise that they contain natural substances that can promote health and alleviate illness. The demonstration of the presence of natural products such as terpenes, alkaloids, flavonoids, coumarins and other secondary metabolites in medicinal plants will provide a scientific validation for the popular use of these plants (Swayamjot et al., 2005). Many of the tropical and subtropical plants have been investigated throughout the world due to their potent antioxidant and antimicrobial activities (Mohamed et al., 2013). In the living systems, free radicals are constantly generated and when in excess, they can cause extensive damage to tissues and biomolecules leading to various pathological disorders such as aging, cancer, inflammation, Alzheimer and cardiovascular diseases (Bakkali et al., 2008). The interest in antioxidants has been increasing because of their high capacity in scavenging free radicals and protects human body from oxidative damage (Silva et al., 2007). The most commonly used synthetic antioxidants; butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) have been

∗ Corresponding author. Tel.: +20 235710098; fax: +20 235730098. E-mail address: amin [email protected] (A.A. Mohamed). http://dx.doi.org/10.1016/j.indcrop.2014.03.017 0926-6690/© 2014 Elsevier B.V. All rights reserved.

reported to cause liver damage and carcinogenesis (Politeo et al., 2007). So, there is a growing interest in naturally derived antioxidants from plants that might help attenuate oxidative damage and also overcome the deleterious effects of synthetic antioxidants (Muhammad et al., 2012). Essential oils are composed of mixtures of volatile secondary metabolites with strong odour commonly concentrated in different plant organs (Bakkali et al., 2008; Franz and Novak, 2010). Besides the antibacterial, antifungal and anti-inflammatory activities many essential oils have been confirmed to possess antioxidant activity (Prakash et al., 2012), anticancer, antinociceptive, antiphlogistic and antiviral activities (Sylvestre et al., 2006; Buchbauer, 2010). The efficiency of the essential oils depends on its chemical composition which depends on the genotypes of the plant as well as on the environmental and agronomic conditions (Mejri et al., 2010). Myrrh is an aromatic oleogum resin obtained as an exudate from the stem of Commiphora myrrha and from other plants of the family Burseraceae (Greene, 1993). It is an effective antimicrobial agent used in the treatment of mouth ulcers, gingivitis, sinusitis, glandular fever, brucellosis and as an anti-parasitic agent (Abdel-Hay et al., 2002; Abdul-Ghani et al., 2009). Moreover, myrrh volatile oils and their crude extracts exhibited diverse biological activities such as cytotoxic, anesthetic, anti-inflammatory and antimicrobial effects (Tipton et al., 2003; Massoud et al., 2004). Triterpenoids

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are major constituents isolated from Commiphora species resins while flavonoids and lignans commonly occurred in the plant stems (Shen et al., 2012). The reported chemical composition of the essential oils of several Commiphora species was characterized mainly by monoterpenes, oxygenated sesquiterpenes and sesquiterpene hydrocarbons which invariably differ from species to species (Baser et al., 2003; Marongiu et al., 2005). Hitherto, there are only limited data on the composition and antimicrobial and antioxidant activities of essential oil obtained from C. myrrha. Therefore, the present work aimed to evaluate the antioxidant and antimicrobial activities of methanol and ethyl acetate extracts as well as of essential oil of C. myrrha resin. 2. Materials and methods 2.1. Plant material The myrrha resin was purchased from Harraz Herbs Company (http://www.harrazherbs.com—Cairo, Egypt) and authenticated as resin of C. myrrha by Dr. Fathy M. Soliman by comparison with a genuine sample (based on physical appearance) kept in the Drug Museum of Pharmacognosy Dept., Faculty of Pharmacy, Cairo University, Egypt. 2.2. Isolation of the essential oil The pulverized resin of myrrha (200 g) underwent water distillation for 5 h using all-glass Clevenger apparatus (European Pharmacopoeia, 1997). The essential oil was dried over anhydrous sodium sulphate to obtain an average yield of 2.97% (v/w) on a dry weight basis and relative density of 0.98. The oil was stored at 4 ◦ C until further analysis. 2.3. Preparation of crude extracts The pulverized resin of myrrha (15 g) was macerated separately in methanol (relative polarity 0.762) and ethyl acetate (relative polarity 0.228) in glass bottles. The bottles were labeled and put in an orbital shaker (Heidolph – Unimax 2010 – Germany) for 24 h at room temperature. The extracts were filtered through Whatman No. 4 filter paper. Residues were re-extracted twice with fresh aliquots of the same solvents. Supernatants of each solvent were pooled and evaporated under vacuum (Heidolph—Germany) at 40 ◦ C to obtain methanolic extract (2.56 g, 17.1%, w/w) and ethyl acetate extract (2.58 g, 17.2%, w/w). The resulting crude extracts were re-dissolved in methanol at a concentration of 1 ␮g/␮l and used for further analysis (Sultana et al., 2009).

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time of each component (Rt ) compared with those of the Wiley9 and NIST08 mass spectra libraries (NIST, 2010).

2.5. Antioxidant activity 2.5.1. DPPH free radical scavenging assay The DPPH free radical scavenging ability of both myrrha crude extracts and essential oil dissolved in methanol was measured from the bleaching of purple-colored solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH). Quantitative measurement of radical scavenging properties of myrrha crude extracts and essential oil was carried out according to our previously published procedure (Mohamed et al., 2013). One milliliter from a 0.1 mM methanol solution of the DPPH radical was mixed to 3 ml of crude extracts at various concentrations (0.2, 0.3, 0.4 and 0.5 mg/ml) and essential oil at various concentrations (0.5, 1.0, 1.5 and 2.0 mg/ml). Discoloration was measured at 517 nm after 30 min. BHT was used as positive control. Measurements were taken in triplicate. The ability to scavenge the DPPH• radical was calculated using the following equation: DPPH• scavenging effect (%) =

A

DPPH

− AS



ADPPH

× 100

where ADPPH is the absorbance of the DPPH• solution and AS is the absorbance of the solution when the sample extract is added. The extract concentration providing 50% inhibition of radicalscavenging activity (IC50 ) was calculated and expressed as mg/ml.

2.5.2. Ferrous ions chelating assay The ferrous ion-chelating activity of both myrrha crude extracts and essential oil was determined following Zhu et al. (2011). Three milliliters of crude extracts and essential oil dissolved in methanol at different concentrations (0.5, 1.0, 1.5 and 2.0 mg/ml) were added to 60 ␮l of FeSO4 (2 mM). The reaction was started by adding 100 ␮l of ferrozine (5 mM). The mixture was shaken vigorously and kept back to stand at room temperature for 10 min. Absorbance of the solution was measured spectrophotometrically at 562 nm. EDTA was used as positive control. The inhibition percentage of ferrozineFe2+ complex formation was calculated according to the following equation: Ferrous ion-chelating activity (%) =

1 − A  1

A0

× 100

where A0 was the absorbance of the control and A1 was the absorbance in the presence of samples.

2.4. Gas chromatography/mass spectrometry (GC/MS) analysis The myrrha essential oil analysis was performed using a Thermo Scientific capillary gas chromatography (model Trace GC ULTRA) directly coupled to ISQ Single Quadruple MS and equipped with TG-5MS non polar 5% phenyl methylpolysiloxane capillary column (30 m × 0.25 mm ID × 0.25 ␮m). The operating condition of GC oven temperature was maintained as: initial temperature 40 ◦ C for 3 min, programmed rate 5 ◦ C/min up to final temperature 280 ◦ C with isotherm for 5 min. For GC–MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium was used as a carrier gas at a constant flow rate of 1.0 ml/min. Diluted sample (1:1, v/v, in diethyl ether) of 1 ␮l was injected automatically in the splitless mode. Detection was performed in the full scan mode from 40 to 500 m/z. The quantification of the components was based on the total number of fragments (total ion count) of the metabolites as detected by the mass spectrometer. The identification of the chemical components was carried out based on the retention

2.5.3. Ferric reducing power assay The reduction capacity of both myrrha crude extracts and essential oil dissolved in methanol was determined according to our previously published procedure (El-Baz et al., 2010). One milliliter of crude extracts and essential oil at different concentrations (200, 400 and 600 ␮g/ml) was mixed with 2.5 ml of sodium phosphate buffer (200 mM, pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50 ◦ C for 20 min. Aliquots (2.5 ml) of 10% trichloroacetic acid were added to the mixture. The previously mixture was then centrifuged at 10,000 rpm for 10 min. The upper layer of the solution (5.0 ml) was mixed with 5.0 ml of distilled water and 1 ml of 0.1% ferric chloride solution. The absorbance was measured at 700 nm against blank. Increased absorbance of the reaction mixture indicated increased reducing power. The BHT was used as positive control and results expressed as absorbance reading.

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2.6. Antimicrobial activity

3. Results and discussion

2.6.1. Microbial strains The microorganisms used for antimicrobial activity evaluation were obtained from the American type culture collection (ATCC; Rockville—MD—USA) as well as the culture collection of the Agricultural Microbiology Dept., National Research Centre, Egypt. The Gram-positive bacteria Streptococcus faecalis (ATCC-47077), Bacillus subtilis (ATCC-12228), Bacillus circulans (ATCC-4513), Listeria monocytogenes (ATCC-35152), Gram-negative bacteria Escherichia coli (ATCC-25922), Pseudomonas aeruginosa strain OS4 as well as two yeasts Saccharomyces cerevisiae (ATCC-9763) and Candida albicans were used in the antimicrobial assays.

3.1. Chemical composition of essential oil

2.6.2. Culture medium and inoculums The stock cultures of microorganisms used in this study were maintained on plate count agar slants at 4 ◦ C. Inoculum was prepared by suspending a loop full of bacterial cultures into 10 ml of nutrient agar broth and was incubated at 37 ◦ C for 24 h. About 60 ␮l of bacterial suspensions, adjusted to 106 –107 CFU/ml were taken and poured into Petri plates containing 6 ml sterilized nutrient agar medium. Bacterial suspensions were spread to get a uniform lawn culture.

2.6.3. Antimicrobial activity assay The agar-well diffusion method was applied to detect antimicrobial activity (Albayrak et al., 2010). Wells of 6 mm diameter were dug on the inoculated nutrient agar medium and 60 ␮l of both myrrha crude extracts and essential oil, dissolved in dimethylsulfoxide (DMSO) at concentration (400 ␮g/ml), were added in each well. The wells introduced with 60 ␮l of DMSO were used as a negative control. The plates were allowed to stand at 4 ◦ C for 2 h before incubation with the test microbial agents. The plates were incubated at 37 ◦ C overnight and examined for the zone of inhibition. The diameter of the inhibition zone was measured in mm. An extract was classified as active when the diameter of the inhibition was equal to or larger than 6 mm. All the assays were performed in triplicate and expressed as average values ± SD.

2.6.4. Minimum inhibitory concentration Based on the previous screening the minimum inhibitory concentration (MIC) of both myrrha crude extracts and essential oil were analyzed through the agar-well diffusion method. A bacterial suspension (106 –107 CFU/ml) of each tested microorganism was spread on the nutrient agar plate. The wells (6 mm diameter) were cut from agar, and 60 ␮l of both myrrha crude extracts and essential oil, dissolved in DMSO at different concentrations (20, 25, 50, 100, 150, 200, 250, 400, 600 and 1000 ␮g/ml) were delivered into them. The plates were incubated at 37 ◦ C for 24 h under aerobic conditions that followed by the measurement of the diameter of the inhibition zone expressed in millimeter. MIC was taken from the concentration of the lowest dosed well visually showing no growth after 24 h.

The GC–MS investigation led to the identification of 40 constituents representing 100% of the total oil of C. myrrha resin with average yield of 2.97% (v/w) on a dry weight basis (Table 1). Among the major constituents were ␣-elemene (12.86%), 7isopropyl-1,4-dimethyl-2-azulenol (12.22%), curzerene (11.64%), germacra-1(10)7,11-trien-15-oic acid,8,12-epoxy-6-hydroxyc¸-lactone(6.20%), ␦-elemene (5.57%), ␦-neoclovene (5.57%), germacrene B (3.97%) and eremophilene (3.35%). It is well known that the differences between the results of the present study and the chemical profile of previously investigated myrrha essential oils are in the concentrations and types of the essential components which were appeared somewhat agree with some reports in the literature of Morteza-Semnani and Saeedi (2003) in which curzerene (40.1%), furanoeudesma-1,3-diene (15.0%) and ␣-elemene (8.4%) represented the main composition of the Iranian C. myrrha essential oil. Also, Marongiu et al. (2005) confirmed that the main composition of the essential oil of Ethiopian C. myrrha were furanoeudesma-1,3-diene (38.6%), curzerene (17.5%), lindestrene (14.4%) and ␣-elemene (4.3%). On the contrary, the present results confirmed that the C. myrrha oil is devoid of furanoeudesma-1,3-diene and lindestrene components but it contain low amount of furanodiene (1.44%). These results appeared to be moderately different from those found by Baser et al. (2003) who reported that the main components of Ethiopian myrrh oil were furanoeudesma-1,3-diene (34.0%), furanodiene (19.7%) and lindestrene (12.0%). The myrrha oil of the present study was characterized by a high content of sesquiterpene hydrocarbons (45.33%), oxygenated sesquiterpenes (37.31%) and devoid of monoterpenes hydrocarbons representing the most major compounds in some reported Commiphora oils (Abegaz et al., 1989; Asres et al., 1998). Alternatively, the results of the present investigation indicated that the oxygenated monoterpenes represented only by n-octyl acetate (2.44%) and oxygenated diterpenes represented only by verticiol (1.13%). ␣-Elemene, ␦-elemene and ␦-neoclovene were the main sesquiterpenes hydrocarbons while 7-isopropyl-1,4dimethyl-2-azulenol, curzerene, germacra-1(10),7,11-trien-15-oic acid,8,12-epoxy-6-hydroxy-c¸-lactone, furanodiene, germacrone, and tau.cadinol represent the main sesquiterpenoids of myrrha resin oil (Table 1). Therefore, the present results support and extend previous reports which suggested that the structures of sesquiterpenoids from the genus Commiphora are mainly classified into germacrane, eudesmane, guaiane, cadinane, elemane, bisabolane and oplopane groups (Shen et al., 2012). The variations in the chemical composition of essential oils might be attributed to the varied environmental conditions in the region, stage of maturity and adaptive metabolism of plants (Carovic-Stanko et al., 2010). Commiphora oil was known for its medicinal properties, and exhibited interesting biological activities and this may be attributed to the presence of different groups of sesquiterpenoids in the oil. In this concern, Racine and Auffray (2005) reported that the essential oil of C. myrrha with its main constituents (curzerene, furanoeudesmadiene, and lindestrene) exhibited potent singlet oxygen quenching activity better than the control ␣-tocopherol.

2.7. Statistical analysis

3.2. Antioxidant activity

All tests were conducted in triplicate. Data are reported as means ± standard deviation (SD). Analysis of variance and significant differences among means were tested by one-way ANOVA using the COSTAT computer package (CoHort Software, 1989). The least significant difference (LSD) at P ≤ 0.05 level was calculated.

3.2.1. DPPH free radical scavenging assay The DPPH radical-scavenging activity of C. myrrha resin methanol (Me-OH) extract, ethyl acetate (ETOAC) extract and the essential oil (EO) at different concentrations (0.2–2 mg/ml) is shown in Fig. 1. A concentration dependent scavenging activity

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Table 1 Chemical constituents of the essential oil of Commiphora myrrha resin. No.

Rt a

Compounds name

1 2 3

18.54 22.14 22.41

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

23.14 23.42 23.66 24.35 24.55 24.65 24.94 25.19 25.75 25.90 26.07 26.32 26.73 26.84 26.98 27.29 27.44 27.59 27.85 28.27 29.48 30.05 30.15 30.36 30.43 30.59 30.90

31 32 33 34 35 36 37

31.13 31.54 32.65 32.86 32.92 33.55 33.73

38 39 40

33.81 34.84 37.40

n-Octyl acetate ␦-Elemene 1-H-Cyclopenta[1,3]cyclopropa-[1,2]benzene,Octahydro-7-methyl-3methylene-4-(-1-methylethyl),[3-aS(3-aà,3-bá,4-á,7-à,7-aS*)]␣-Ylangene ␣–Bourbonene ␣–Elemene Undeca4,6diyne Germacrene-d 2,10,10-Trimethyltricyclo-[7.1.1.0(2,7)]-undec-6-en-8-one Caryophyllene ␣-Caryophyllene ␥-Muurolene ␣-Cubebene Eremophilene Curzerene 6á-(-2-Methylcyclopent-1-enyl)-3,3-dimethyl-1-á-bicyclo[3.1.0]-hexan-2-one ␥-Cadinene ␦-Cadinene Guaia-3,9-diene Eudesma-4-(14),7(11)-diene Elemol Germacrene B 1-(1-Propynyl)-2-cyclohexen-1-ol 7-Isopropyl-1,4-dimethyl-2-azulenol (1RS,2RS,1 SR)-1-(1 Methoxyethyl)-2-vinylcyclobutane tau.Cadinol 4-(2 -Methoxyphenyl)]-4-methylcyclohex-3-en-1-one Furanodiene ␦-Neoclovene 6-(1,3-Dimethylbuta-1,3-dienyl)-1,5,5-trimethyl-7-oxabicyclo-[4.1.0]hept-2ene Germacrone Germacra-1(10),7,11-trien-15-oic acid,8,12-epoxy-6-hydroxy-c¸-lactone Isoledene 3-tert-Butyl-2-hydroxy-5-vinylbenzaldehyde ␥-Eudesmol 3-Ethyl-6-(methoxycarbonyl)-2-naphthol 5,8A-dimethyl-3-methylene-3A,7,8,8A,9,9A-hexahydro-3H-naphtho[2,3B]furan-2-one Methyl-7-methoxy-5-methyl-2-hydroxyl-1-naphthoate Iso-Velleral Verticiol Total identified Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Oxygenated diterpenes Other constituents

a b

Composition (%)b

Molecular formula

2.44 5.57 0.41

C10 H20 O2 C15 H24 C15 H24

0.71 2.62 12.86 2.29 0.53 3.16 0.49 1.15 1.54 1.78 3.35 11.64 0.68 0.52 1.51 1.03 1.23 0.52 3.97 2.01 12.22 2.30 1.02 1.54 1.44 5.57 1.18

C15 H24 C15 H24 C15 H24 C11 H16 C15 H24 C14 H20 O C15 H24 C15 H24 C15 H24 C15 H24 C15 H24 C15 H20 O C14 H20 O C15 H24 C15 H24 C15 H24 C15 H24 C15 H26 O C15 H24 C9 H12 O C15 H18 O C9 H16 O C15 H26 O C14 H16 O2 C15 H20 O C15 H24 C15 H22 O

1.42 6.20 0.49 0.65 0.54 0.52 0.40

C15 H22 O C15 H18 O3 C15 H24 C13 H16 O2 C15 H26 O C14 H14 O3 C15 H18 O2

0.64 0.73 1.13 100 2.44 45.33 37.31 1.13 13.79

C14 H14 O4 C15 H20 O2 C20 H34 O

Rt : retention time (min). The percentage composition was computed from the gas chromatography peak areas.

Fig. 1. DPPH scavenging activity (%) of Commiphora myrrha resin methanol (MeOH) extract, ethyl acetate (ETOAC) extract and the essential oil (EO) at different concentrations. (n = 3, value = mean ± SD).

was clearly demonstrated. At 0.5 mg/ml, the inhibition percent of Me-OH, ETOAC extracts and EO were determined as 71.1, 33.4 and 6.6%, respectively. The values of IC50 were in the ascending order BHT < Me-OH extract < ETOAC extract < EO with values of 0.07, 0.32, 0.93, and 11.33 mg/ml respectively. These results indicated that Me-OH extract exhibited the highest DPPH radical scavenging activity compared to the ETOAC extract and the EO, but it gave low DPPH radical scavenging activity compared to BHT. The highest DPPH radical scavenging activity of MeOH and ETOAC extracts compared to EO seems to be attributed to the high concentration of sesquiterpenoids, diterpenes, triterpenes and sterols in myrrha extracts which could be the electron donors and hence can react with free radicals to convert them into more stable products and terminate radical chain reactions. This is supported by previous finding of Fraternale et al. (2011) who demonstrated that the myrrha resin hexane extract exhibited the highest DPPH radical scavenging activity compared with its oils. The same authors attributed this finding to the

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Fig. 2. The Fe2+ chelating activity (%) of Commiphora myrrha resin methanol (MeOH) extract, ethyl acetate (ETOAC) extract and the essential oil (EO) at different concentrations. (n = 3, value = mean ± SD).

isolated three furano-sesquiterpenoids (myrrhone, 3-methoxyfuranogermacradien-6-one and 2-methoxy-furanogermacren-6one) from C. myrrha hexane extract that showed DPPH radical scavenging activity with IC50 values of 1.08, 4.29 and 2.56 mg/ml, respectively. Furthermore, the results of the present work are disagree with the results of Al-Harrasi et al. (2013) who reported that methanol extract of Hougari Regular (HR) grade resin of Boswellia sacra showed scavenging activity against DPPH radicals lower than the antioxidant activity of resin essential oil at the same concentration. 3.2.2. Ferrous ions chelating assay The Fe2+ chelating activity of C. myrrha resin Me-OH, ETOAC extracts and EO showed a concentration-dependent activity profile (Fig. 2). The Me-OH extract was found to be the most potent Fe2+ chelator as it caused 82.7% chelation at concentration 1 mg/ml, followed by ETOAC extract causing 79.2% chelation. At the same concentration the EO caused 28.2% as the lowest chelation activity. The Me-OH extract activity was less than that of the standard EDTA. The IC50 of the Fe2+ values for EDTA, Me-OH, ETOAC extracts and EO were 0.028, 0.238, 0.279 and 1.553 mg/ml, respectively. Similar results were also observed for the Fe2+ chelating activity of the extracts and the oil as compared to their DPPH-scavenging activities (Figs. 1 and 2). The C. myrrha resin essential oil has a lower Fe2+ chelating activity compared to its extracts and that might be due to the less iron binding capacity of essential oil components. The present results are in agreement with Roy et al. (2012) who reported that the Handia volatile components showed low metal chelating activity. The poor Fe2+ chelating activity of C. myrrha resin essential oil might be attributed to the lack of monoterpenes hydrocarbons in the oil. Previous reports confirmed that the essential oils having low content of monoterpenes hydrocarbons have poor antioxidant activity (Tepe et al., 2005; Nanyonga et al., 2013). In the present results the highest Fe2+ chelating activity of both MeOH and ETOAC extracts might be attributed to their high content of furanosesquiterpenes, triterpenes and Steroids. Similar results were found by Wang et al. (2004) isolated (Z)-guggulsterone (as a steroidal compound) from Commiphora mukul and this compound proved antioxidant activity. 3.2.3. Ferric reducing power assay The reducing power of myrrha resin extracts and its essential oil increased with the increase of concentrations (Fig. 3). The EO was superior to Me-OH and ETOAC extracts. The reducing powers of essential oil were 0.348, 0.687 and 0.864 at 200, 400 and 600 ␮g/ml, respectively. At 200 ␮g/ml concentration the essential

Fig. 3. Ferric reducing power of Commiphora myrrha resin methanol (Me-OH) extract, ethyl acetate (ETOAC) extract and the essential oil (EO) at different concentrations, BHT used as positive control. (n = 3, value = mean ± SD).

oil gave reducing power (0.348) lower than BHT (1.377) which used as positive control. The myrrha essential oil was more effective in the reducing power compared to Me-OH and ETOAC extracts. This seems to be attributed to the more hydrogen donating components in the essential oils such as different groups of monoterpenoids and sesquiterpenoids. Such hydroxyl terpene compounds can donate hydrogen atoms to transform Fe3+ to Fe2+ performing as reductones. These results are in accord with those of Racine and Auffray (2005) who reported that the essential oil of myrrha with its main sesquiterpenoids constituents including curzerene, furanoeudesmadiene and lindestrene exhibited potent singlet oxygen quenching activity better than the control ␣-tocopherol. The same authors attributed this activity to the active furan rings of the isolated furanosesquiterpenoids. The results of the present study are in agreement with Laciar et al. (2009) who reported that terpenes particularly, those with activated methylene groups in their molecules, could be the reason of the antioxidant activity shown by Artemisia echegarayi essential oils. 3.3. Antimicrobial activity The in vitro antimicrobial activity of C. myrrha resin extracts of Me-OH, ETOAC and the EO against both Gram-positive and Gram-negative bacteria as well as two yeasts were investigated. One-way ANOVA analysis showed significant differences (P ≤ 0.05) in microorganisms sensitivity among the two studied extracts and

Table 2 Antimicrobial activity of Commiphora myrrha resin methanol (Me-OH) extract, ethyl acetate (ETOAC) extract and the essential oil (EO) at 400 ␮g/ml concentration by agar well diffusion method. Microorganisms

Gram-positive S. faecalis B. subtilis B. circulans L. monocytogenes Gram-negative E. coli P. aeruginosa Yeast S. cerevisiae C. albicans LSD at P ≤ 0.5

Inhibition zone (mm) Me-OH

ETOAC

EO

12.5 ± 1.5cd 10.5 ± 1.29bc 10.3 ± 0.96bc 19 ± 1.15e

10.3 ± 0.5b 10.3 ± 0.5b 12.3 ± 0.96c NIa

10.2 ± 1.26b 10.1 ± 1.83b NIa NIa

12.3 ± 1.06cd 13.5 ± 0.5d

9.5 ± 0.58b 10.1 ± 0.1b

9.3 ± 0.5b 9.5 ± 0.5b

9.5 ± 0.58b NIa 1.73

9.3 ± 0.96b NIa 1.11

9 ± 1.83b NIa 1.96

Values are mean inhibition zone (mm) ±SD of three replicates. Data with different superscript letters in the same column were significantly different (P ≤ 0.05). The diameter of the well (6 mm) is included. NI: no inhibition zone.

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the essential oil. The results presented in Table 2 showed that the Me-OH extract at 400 ␮g/ml concentration manifested the highest in vitro potential of antibacterial activity against all the tested microorganisms except for C. albicans showed resistant to all test samples till 1000 ␮g/ml, while ETOAC extract and EO at the same concentration showed low or no antibacterial activity. In the present study the C. albicans showed more resistance to the two tested extracts and the essential oil than S. cerevisiae. In similar work, Vediyappan et al. (2010) renders the drug resistance of C. albicans to the form of biofilms which exhibit elevated intrinsic resistance to various antifungal agents. No inhibition of bacterial and yeast growth was observed with the negative control dimethylsulfoxide (DMSO). The highest potential of antibacterial activity of myrrha Me-OH extract might be attributed to the high polarity of Me-OH which is effective for more consistent extraction of different types of sesquiterpenoids particularly furanosesquiterpenoids, diterpenes, triterpenes and sterols. It is well known that the composition, structure, as well as functional groups of crude extracts and the oils play an important role in determining their antimicrobial activity. It has been demonstrated that crude extracts and the essential oils exercise their antimicrobial activity by causing structural and functional damages to the microbial cell membrane (Goni et al., 2009). In accordance with Zhu et al. (2001) who reported that Commiphora resins are rich in sesquiterpenoids and the isolated furanosesquiterpenoids or crude extracts were found to possess antibacterial and antifungal activity. Similarly, the sesquiterpenoids, epicurzerenone and (1E)-8,12-epoxygermacra-1,7,10,11tetraen-6-one isolated from Commiphora erythraea exhibited inhibitory activity against Fusarium culmorum, Phytophtora cryptogea and Alternaria solani (Fraternale et al., 2011). Mansumbinoic acid isolated from the oleo-resin of Commiphora molmol possessed potent antibacterial activity against a multidrug-resistant strain Staphylococcus aureus with a MIC value of 4 mg/ml (Rahman et al., 2008). Our results indicated that the highest activity was observed against L. monocytogenes followed by P. aeruginosa with the widest inhibition zones (19 and 13.5 mm) respectively. The findings of the present study are in line with Abdallah et al. (2009) who demonstrated that C. myrrha methanol extracts exhibited the highest antibacterial activity against S. aureus whereas the ethyl acetate extracts exhibited some degree of activity. The same authors attributed the highest antibacterial activity of methanol extracts to the presence of some active phenolic compounds, alkaloids and saponins. The test Gram-positive bacteria were found to be more susceptible to antimicrobial agents than Gram-negative bacteria (Burt, 2004; Hussain et al., 2010). The weaker antimicrobial activity against Gram-negative compared to Gram-positive bacteria is ascribed to the structure of their cellular walls mainly with regard to the presence of lipoproteins and lipopolysaccharides in Gramnegative bacteria that form a barrier to hydrophobic compounds (Inouye et al., 2001). The MIC values obtained from antimicrobial tests ranged from 25 to >1000 ␮g/ml (Table 3). The results showed that the bacterial strains S. faecalis, E. coli and B. circulans were the most sensitive to both Me-OH and ETOAC extracts with MIC value 50 ␮g/ml. Alternatively, L. monocytogenes was the least sensitive strain to both ETOAC extract and EO with MIC value >1000 ␮g/ml. The yeast S. cerevisiae was the most sensitive yeast to Me-OH, ETOAC extracts and EO with MIC values 25, 50 and 100 ␮g/ml respectively. The C. albicans was the most resistant yeast to Me-OH, ETOAC extracts and EO with MIC value >1000 ␮g/ml. According to Salvat et al. (2004), plant extracts with MIC’s less than/or around 0.5 mg/ml indicate good antibacterial activity. Accordingly, the Me-OH and ETOAC extracts, and EO of C. myrrha exhibited good antimicrobial activity against most of the tested microorganisms.

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Table 3 Minimal Inhibitory Concentration (MIC) of Commiphora myrrha resin methanol (MeOH) extract, ethyl acetate (ETOAC) extract and the essential oil (EO). Microorganisms

Gram-positive S. faecalis B. subtilis B. circulans L. monocytogenes Gram-negative E. coli P. aeruginosa Yeast S. cerevisiae C. albicans

MIC (␮g/ml) Me-OH

ETOAC

EO

50 250 100 400

50 100 50 >1000

100 200 600 >1000

50 150

100 200

100 200

25 >1000

50 >1000

100 >1000

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