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Industrial Crops and Products 57 (2014) 10–16

Contents lists available at ScienceDirect

Industrial Crops and Products

jo u r n al homep age: www.elsev ier .com/ locate / indcrop

hemical composition of essential oil and in vitro antioxidant andntimicrobial activities of crude extracts of Commiphora myrrha resin

mal A. Mohameda,∗, Sami I. Alia, Farouk K. EL-Baza,hmad K. Hegazyb, Mimona A. Kordb

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

r t i c l e i n f o

rticle history:eceived 5 November 2013eceived in revised form 15 March 2014ccepted 18 March 2014vailable online 5 April 2014

eywords:ntimicrobial activity

a b s t r a c t

The antioxidant and antimicrobial potential of methanol (ME-OH), ethyl acetate (ETOAC) crudeextracts and essential oil (EO) of Commiphora myrrha resin were investigated. The major con-stituents 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-oicacid,8,12-epoxy-6-hydroxy-c -lactone (6.20%). In both DPPH scavenging and Fe2+ chelating assays, theME-OH extract exhibited the highest activity compared to ETOAC extract and EO. Concerning the reduc-ing power ability, EO was superior to Me-OH and ETOAC extracts. The Me-OH extract manifested the

rude extractsommiphora myrrhaPPHssential oilC–MSIC

highest potential of antimicrobial activity against the tested bacterial and yeast microorganisms, whileETOAC extract and EO showed moderate or no potential antibacterial activity. The Me-OH extract exhib-ited the highest antioxidant and antimicrobial activity as compared to ETOAC and EO. It is concluded fromthe present study that besides its traditional use, the C. myrrha resin could be used as a natural source forantioxidant and antimicrobial compounds for possible applications in food and nutraceutical industries.

. Introduction

The plant-derived medicines are based upon the premise thathey contain natural substances that can promote health and allevi-te illness. The demonstration of the presence of natural productsuch as terpenes, alkaloids, flavonoids, coumarins and other sec-ndary metabolites in medicinal plants will provide a scientificalidation for the popular use of these plants (Swayamjot et al.,005). Many of the tropical and subtropical plants have been inves-igated throughout the world due to their potent antioxidant andntimicrobial activities (Mohamed et al., 2013). In the living sys-ems, free radicals are constantly generated and when in excess,hey can cause extensive damage to tissues and biomoleculeseading to various pathological disorders such as aging, can-er, inflammation, Alzheimer and cardiovascular diseases (Bakkalit al., 2008). The interest in antioxidants has been increasingecause of their high capacity in scavenging free radicals and pro-

ects human body from oxidative damage (Silva et al., 2007). The

ost commonly used synthetic antioxidants; butylated hydrox-toluene (BHT) and butylated hydroxyanisole (BHA) have been

∗ Corresponding author. Tel.: +20 235710098; fax: +20 235730098.E-mail address: amin amal@yahoo.com (A.A. Mohamed).

ttp://dx.doi.org/10.1016/j.indcrop.2014.03.017926-6690/© 2014 Elsevier B.V. All rights reserved.

© 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 antiox-idants from plants that might help attenuate oxidative damageand also overcome the deleterious effects of synthetic antioxidants(Muhammad et al., 2012).

Essential oils are composed of mixtures of volatile secondarymetabolites with strong odour commonly concentrated in differentplant organs (Bakkali et al., 2008; Franz and Novak, 2010). Besidesthe antibacterial, antifungal and anti-inflammatory activities manyessential oils have been confirmed to possess antioxidant activity(Prakash et al., 2012), anticancer, antinociceptive, antiphlogisticand antiviral activities (Sylvestre et al., 2006; Buchbauer, 2010).The efficiency of the essential oils depends on its chemical compo-sition which depends on the genotypes of the plant as well as onthe environmental and agronomic conditions (Mejri et al., 2010).

Myrrh is an aromatic oleogum resin obtained as an exudate fromthe stem of Commiphora myrrha and from other plants of the familyBurseraceae (Greene, 1993). It is an effective antimicrobial agentused in the treatment of mouth ulcers, gingivitis, sinusitis, glan-dular fever, brucellosis and as an anti-parasitic agent (Abdel-Hay

et al., 2002; Abdul-Ghani et al., 2009). Moreover, myrrh volatileoils and their crude extracts exhibited diverse biological activitiessuch as cytotoxic, anesthetic, anti-inflammatory and antimicrobialeffects (Tipton et al., 2003; Massoud et al., 2004). Triterpenoids

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re major constituents isolated from Commiphora species resinshile flavonoids and lignans commonly occurred in the plant stems

Shen et al., 2012). The reported chemical composition of the essen-ial oils of several Commiphora species was characterized mainlyy monoterpenes, oxygenated sesquiterpenes and sesquiterpeneydrocarbons which invariably differ from species to species (Basert al., 2003; Marongiu et al., 2005).

Hitherto, there are only limited data on the composition andntimicrobial and antioxidant activities of essential oil obtainedrom C. myrrha. Therefore, the present work aimed to evaluatehe antioxidant and antimicrobial activities of methanol and ethylcetate extracts as well as of essential oil of C. myrrha resin.

. Materials and methods

.1. Plant material

The myrrha resin was purchased from Harraz Herbs Companyhttp://www.harrazherbs.com—Cairo, Egypt) and authenticated asesin of C. myrrha by Dr. Fathy M. Soliman by comparison with aenuine sample (based on physical appearance) kept in the Druguseum of Pharmacognosy Dept., Faculty of Pharmacy, Cairo Uni-

ersity, Egypt.

.2. Isolation of the essential oil

The pulverized resin of myrrha (200 g) underwent water dis-illation for 5 h using all-glass Clevenger apparatus (Europeanharmacopoeia, 1997). The essential oil was dried over anhydrousodium sulphate to obtain an average yield of 2.97% (v/w) on a dryeight basis and relative density of 0.98. The oil was stored at 4 ◦Cntil further analysis.

.3. Preparation of crude extracts

The pulverized resin of myrrha (15 g) was macerated separatelyn methanol (relative polarity 0.762) and ethyl acetate (relativeolarity 0.228) in glass bottles. The bottles were labeled and put

n an orbital shaker (Heidolph – Unimax 2010 – Germany) for 24 ht room temperature. The extracts were filtered through What-an No. 4 filter paper. Residues were re-extracted twice with

resh aliquots of the same solvents. Supernatants of each solventere pooled and evaporated under vacuum (Heidolph—Germany)

t 40 ◦C to obtain methanolic extract (2.56 g, 17.1%, w/w) and ethylcetate extract (2.58 g, 17.2%, w/w). The resulting crude extractsere re-dissolved in methanol at a concentration of 1 �g/�l andsed for further analysis (Sultana et al., 2009).

.4. Gas chromatography/mass spectrometry (GC/MS) analysis

The myrrha essential oil analysis was performed using a Thermocientific capillary gas chromatography (model Trace GC ULTRA)irectly coupled to ISQ Single Quadruple MS and equipped withG-5MS non polar 5% phenyl methylpolysiloxane capillary column30 m × 0.25 mm ID × 0.25 �m). The operating condition of GC ovenemperature was maintained as: initial temperature 40 ◦C for 3 min,rogrammed rate 5 ◦C/min up to final temperature 280 ◦C with

sotherm for 5 min. For GC–MS detection, an electron ionizationystem with ionization energy of 70 eV was used. Helium was useds a carrier gas at a constant flow rate of 1.0 ml/min. Diluted sam-le (1:1, v/v, in diethyl ether) of 1 �l was injected automatically inhe splitless mode. Detection was performed in the full scan mode

rom 40 to 500 m/z. The quantification of the components was basedn the total number of fragments (total ion count) of the metabo-ites as detected by the mass spectrometer. The identification ofhe chemical components was carried out based on the retention

and Products 57 (2014) 10–16 11

time of each component (Rt) compared with those of the Wiley9and NIST08 mass spectra libraries (NIST, 2010).

2.5. Antioxidant activity

2.5.1. DPPH free radical scavenging assayThe DPPH free radical scavenging ability of both myrrha crude

extracts and essential oil dissolved in methanol was measured fromthe bleaching of purple-colored solution of 2,2-diphenyl-1-picryl-hydrazyl (DPPH). Quantitative measurement of radical scavengingproperties of myrrha crude extracts and essential oil was carriedout according to our previously published procedure (Mohamedet al., 2013). One milliliter from a 0.1 mM methanol solution ofthe DPPH radical was mixed to 3 ml of crude extracts at variousconcentrations (0.2, 0.3, 0.4 and 0.5 mg/ml) and essential oil at var-ious concentrations (0.5, 1.0, 1.5 and 2.0 mg/ml). Discoloration wasmeasured at 517 nm after 30 min. BHT was used as positive control.Measurements were taken in triplicate. The ability to scavenge theDPPH• radical was calculated using the following equation:

DPPH• scavenging effect (%) =[

ADPPH − AS

ADPPH

]× 100

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

2.5.2. Ferrous ions chelating assayThe ferrous ion-chelating activity of both myrrha crude extracts

and essential oil was determined following Zhu et al. (2011). Threemilliliters of crude extracts and essential oil dissolved in methanolat different concentrations (0.5, 1.0, 1.5 and 2.0 mg/ml) were addedto 60 �l of FeSO4 (2 mM). The reaction was started by adding 100 �lof ferrozine (5 mM). The mixture was shaken vigorously and keptback to stand at room temperature for 10 min. Absorbance of thesolution was measured spectrophotometrically at 562 nm. EDTAwas used as positive control. The inhibition percentage of ferrozine-Fe2+ complex formation was calculated according to the followingequation:

Ferrous ion-chelating activity (%) =(

1 − A1

A0

)× 100

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

2.5.3. Ferric reducing power assayThe reduction capacity of both myrrha crude extracts and essen-

tial oil dissolved in methanol was determined according to ourpreviously published procedure (El-Baz et al., 2010). One milliliterof crude extracts and essential oil at different concentrations (200,400 and 600 �g/ml) was mixed with 2.5 ml of sodium phosphatebuffer (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 previ-ously mixture was then centrifuged at 10,000 rpm for 10 min. Theupper layer of the solution (5.0 ml) was mixed with 5.0 ml of dis-tilled water and 1 ml of 0.1% ferric chloride solution. The absorbance

was measured at 700 nm against blank. Increased absorbance ofthe reaction mixture indicated increased reducing power. The BHTwas used as positive control and results expressed as absorbancereading.

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.6. Antimicrobial activity

.6.1. Microbial strainsThe microorganisms used for antimicrobial activity evaluation

ere obtained from the American type culture collection (ATCC;ockville—MD—USA) as well as the culture collection of the Agri-ultural Microbiology Dept., National Research Centre, Egypt. Theram-positive bacteria Streptococcus faecalis (ATCC-47077), Bacil-

us subtilis (ATCC-12228), Bacillus circulans (ATCC-4513), Listeriaonocytogenes (ATCC-35152), Gram-negative bacteria Escherichia

oli (ATCC-25922), Pseudomonas aeruginosa strain OS4 as wells two yeasts Saccharomyces cerevisiae (ATCC-9763) and Candidalbicans were used in the antimicrobial assays.

.6.2. Culture medium and inoculumsThe stock cultures of microorganisms used in this study were

aintained on plate count agar slants at 4 ◦C. Inoculum was pre-ared by suspending a loop full of bacterial cultures into 10 ml ofutrient agar broth and was incubated at 37 ◦C for 24 h. About 60 �lf bacterial suspensions, adjusted to 106–107 CFU/ml were takennd poured into Petri plates containing 6 ml sterilized nutrient agaredium. Bacterial suspensions were spread to get a uniform lawn

ulture.

.6.3. Antimicrobial activity assayThe agar-well diffusion method was applied to detect antimi-

robial activity (Albayrak et al., 2010). Wells of 6 mm diameterere dug on the inoculated nutrient agar medium and 60 �l of

oth myrrha crude extracts and essential oil, dissolved in dime-hylsulfoxide (DMSO) at concentration (400 �g/ml), were added inach well. The wells introduced with 60 �l of DMSO were used as

negative control. The plates were allowed to stand at 4 ◦C for 2 hefore incubation with the test microbial agents. The plates were

ncubated at 37 ◦C overnight and examined for the zone of inhibi-ion. The diameter of the inhibition zone was measured in mm. Anxtract was classified as active when the diameter of the inhibitionas equal to or larger than 6 mm. All the assays were performed in

riplicate and expressed as average values ± SD.

.6.4. Minimum inhibitory concentrationBased on the previous screening the minimum inhibitory con-

entration (MIC) of both myrrha crude extracts and essential oilere analyzed through the agar-well diffusion method. A bacterial

uspension (106–107 CFU/ml) of each tested microorganism waspread on the nutrient agar plate. The wells (6 mm diameter) wereut from agar, and 60 �l of both myrrha crude extracts and essen-ial oil, dissolved in DMSO at different concentrations (20, 25, 50,00, 150, 200, 250, 400, 600 and 1000 �g/ml) were delivered intohem. The plates were incubated at 37 ◦C for 24 h under aerobiconditions that followed by the measurement of the diameter ofhe inhibition zone expressed in millimeter. MIC was taken fromhe concentration of the lowest dosed well visually showing norowth after 24 h.

.7. Statistical analysis

All tests were conducted in triplicate. Data are reported as

eans ± standard deviation (SD). Analysis of variance and signif-

cant differences among means were tested by one-way ANOVAsing the COSTAT computer package (CoHort Software, 1989). The

east significant difference (LSD) at P ≤ 0.05 level was calculated.

and Products 57 (2014) 10–16

3. Results and discussion

3.1. Chemical composition of essential oil

The GC–MS investigation led to the identification of 40 con-stituents representing 100% of the total oil of C. myrrha resinwith average yield of 2.97% (v/w) on a dry weight basis (Table 1).Among the major constituents were �-elemene (12.86%), 7-isopropyl-1,4-dimethyl-2-azulenol (12.22%), curzerene (11.64%),germacra-1(10)7,11-trien-15-oic acid,8,12-epoxy-6-hydroxy-c -lactone(6.20%), �-elemene (5.57%), �-neoclovene (5.57%),germacrene B (3.97%) and eremophilene (3.35%). It is well knownthat the differences between the results of the present study andthe chemical profile of previously investigated myrrha essen-tial oils are in the concentrations and types of the essentialcomponents which were appeared somewhat agree with somereports in the literature of Morteza-Semnani and Saeedi (2003) inwhich curzerene (40.1%), furanoeudesma-1,3-diene (15.0%) and�-elemene (8.4%) represented the main composition of the IranianC. myrrha essential oil. Also, Marongiu et al. (2005) confirmedthat 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, thepresent results confirmed that the C. myrrha oil is devoid offuranoeudesma-1,3-diene and lindestrene components but it con-tain low amount of furanodiene (1.44%). These results appearedto be moderately different from those found by Baser et al. (2003)who reported that the main components of Ethiopian myrrh oilwere furanoeudesma-1,3-diene (34.0%), furanodiene (19.7%) andlindestrene (12.0%).

The myrrha oil of the present study was characterized bya high content of sesquiterpene hydrocarbons (45.33%), oxy-genated sesquiterpenes (37.31%) and devoid of monoterpeneshydrocarbons representing the most major compounds in somereported Commiphora oils (Abegaz et al., 1989; Asres et al., 1998).Alternatively, the results of the present investigation indicatedthat the oxygenated monoterpenes represented only by n-octylacetate (2.44%) and oxygenated diterpenes represented only byverticiol (1.13%). �-Elemene, �-elemene and �-neoclovene werethe main sesquiterpenes hydrocarbons while 7-isopropyl-1,4-dimethyl-2-azulenol, curzerene, germacra-1(10),7,11-trien-15-oicacid,8,12-epoxy-6-hydroxy-c -lactone, furanodiene, germacrone,and tau.cadinol represent the main sesquiterpenoids of myrrharesin oil (Table 1). Therefore, the present results support and extendprevious reports which suggested that the structures of sesquiter-penoids from the genus Commiphora are mainly classified intogermacrane, eudesmane, guaiane, cadinane, elemane, bisabolaneand oplopane groups (Shen et al., 2012). The variations in the chem-ical composition of essential oils might be attributed to the variedenvironmental conditions in the region, stage of maturity and adap-tive metabolism of plants (Carovic-Stanko et al., 2010).

Commiphora oil was known for its medicinal properties, andexhibited interesting biological activities and this may be attributedto the presence of different groups of sesquiterpenoids in theoil. In this concern, Racine and Auffray (2005) reported that theessential oil of C. myrrha with its main constituents (curzerene,furanoeudesmadiene, and lindestrene) exhibited potent singletoxygen quenching activity better than the control �-tocopherol.

3.2. Antioxidant activity

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 andthe essential oil (EO) at different concentrations (0.2–2 mg/ml) isshown in Fig. 1. A concentration dependent scavenging activity

A.A. Mohamed et al. / Industrial Crops and Products 57 (2014) 10–16 13

Table 1Chemical constituents of the essential oil of Commiphora myrrha resin.

No. Rta Compounds name Composition (%)b Molecular

formula

1 18.54 n-Octyl acetate 2.44 C10H20O2

2 22.14 �-Elemene 5.57 C15H24

3 22.41 1-H-Cyclopenta[1,3]cyclopropa-[1,2]benzene,Octahydro-7-methyl-3-methylene-4-(-1-methylethyl),[3-aS(3-aà,3-bá,4-á,7-à,7-aS*)]-

0.41 C15H24

4 23.14 �-Ylangene 0.71 C15H24

5 23.42 �–Bourbonene 2.62 C15H24

6 23.66 �–Elemene 12.86 C15H24

7 24.35 Undeca4,6diyne 2.29 C11H16

8 24.55 Germacrene-d 0.53 C15H24

9 24.65 2,10,10-Trimethyltricyclo-[7.1.1.0(2,7)]-undec-6-en-8-one 3.16 C14H20O10 24.94 Caryophyllene 0.49 C15H24

11 25.19 �-Caryophyllene 1.15 C15H24

12 25.75 �-Muurolene 1.54 C15H24

13 25.90 �-Cubebene 1.78 C15H24

14 26.07 Eremophilene 3.35 C15H24

15 26.32 Curzerene 11.64 C15H20O16 26.73 6á-(-2-Methylcyclopent-1-enyl)-3,3-dimethyl-1-á-bicyclo[3.1.0]-hexan-2-one 0.68 C14H20O17 26.84 �-Cadinene 0.52 C15H24

18 26.98 �-Cadinene 1.51 C15H24

19 27.29 Guaia-3,9-diene 1.03 C15H24

20 27.44 Eudesma-4-(14),7(11)-diene 1.23 C15H24

21 27.59 Elemol 0.52 C15H26O22 27.85 Germacrene B 3.97 C15H24

23 28.27 1-(1-Propynyl)-2-cyclohexen-1-ol 2.01 C9H12O24 29.48 7-Isopropyl-1,4-dimethyl-2-azulenol 12.22 C15H18O25 30.05 (1RS,2RS,1′SR)-1-(1 Methoxyethyl)-2-vinylcyclobutane 2.30 C9H16O26 30.15 tau.Cadinol 1.02 C15H26O27 30.36 4-(2′-Methoxyphenyl)]-4-methylcyclohex-3-en-1-one 1.54 C14H16O2

28 30.43 Furanodiene 1.44 C15H20O29 30.59 �-Neoclovene 5.57 C15H24

30 30.90 6-(1,3-Dimethylbuta-1,3-dienyl)-1,5,5-trimethyl-7-oxabicyclo-[4.1.0]hept-2-ene

1.18 C15H22O

31 31.13 Germacrone 1.42 C15H22O32 31.54 Germacra-1(10),7,11-trien-15-oic acid,8,12-epoxy-6-hydroxy-c -lactone 6.20 C15H18O3

33 32.65 Isoledene 0.49 C15H24

34 32.86 3-tert-Butyl-2-hydroxy-5-vinylbenzaldehyde 0.65 C13H16O2

35 32.92 �-Eudesmol 0.54 C15H26O36 33.55 3-Ethyl-6-(methoxycarbonyl)-2-naphthol 0.52 C14H14O3

37 33.73 5,8A-dimethyl-3-methylene-3A,7,8,8A,9,9A-hexahydro-3H-naphtho[2,3-B]furan-2-one

0.40 C15H18O2

38 33.81 Methyl-7-methoxy-5-methyl-2-hydroxyl-1-naphthoate 0.64 C14H14O4

39 34.84 Iso-Velleral 0.73 C15H20O2

40 37.40 Verticiol 1.13 C20H34OTotal identified 100Oxygenated monoterpenes 2.44Sesquiterpene hydrocarbons 45.33Oxygenated sesquiterpenes 37.31Oxygenated diterpenes 1.13Other constituents 13.79

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

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

.

was clearly demonstrated. At 0.5 mg/ml, the inhibition percent ofMe-OH, ETOAC extracts and EO were determined as 71.1, 33.4and 6.6%, respectively. The values of IC50 were in the ascend-ing order BHT < Me-OH extract < ETOAC extract < EO with valuesof 0.07, 0.32, 0.93, and 11.33 mg/ml respectively. These resultsindicated that Me-OH extract exhibited the highest DPPH radi-cal scavenging activity compared to the ETOAC extract and theEO, but it gave low DPPH radical scavenging activity comparedto BHT. The highest DPPH radical scavenging activity of Me-OH and ETOAC extracts compared to EO seems to be attributedto the high concentration of sesquiterpenoids, diterpenes, triter-penes and sterols in myrrha extracts which could be the electrondonors and hence can react with free radicals to convert theminto more stable products and terminate radical chain reac-

tions. This is supported by previous finding of Fraternale et al.(2011) who demonstrated that the myrrha resin hexane extractexhibited the highest DPPH radical scavenging activity comparedwith its oils. The same authors attributed this finding to the

14 A.A. Mohamed et al. / Industrial Crops and Products 57 (2014) 10–16

F 2+

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Fig. 3. Ferric reducing power of Commiphora myrrha resin methanol (Me-OH)

of Me-OH, ETOAC and the EO against both Gram-positive andGram-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 2Antimicrobial activity of Commiphora myrrha resin methanol (Me-OH) extract, ethylacetate (ETOAC) extract and the essential oil (EO) at 400 �g/ml concentration by agarwell diffusion method.

Microorganisms Inhibition zone (mm)

Me-OH ETOAC EO

Gram-positiveS. faecalis 12.5 ± 1.5cd 10.3 ± 0.5b 10.2 ± 1.26b

B. subtilis 10.5 ± 1.29bc 10.3 ± 0.5b 10.1 ± 1.83b

B. circulans 10.3 ± 0.96bc 12.3 ± 0.96c NIa

L. monocytogenes 19 ± 1.15e NIa NIa

Gram-negativeE. coli 12.3 ± 1.06cd 9.5 ± 0.58b 9.3 ± 0.5b

P. aeruginosa 13.5 ± 0.5d 10.1 ± 0.1b 9.5 ± 0.5b

YeastS. cerevisiae 9.5 ± 0.58b 9.3 ± 0.96b 9 ± 1.83b

C. albicans NIa NIa NIa

ig. 2. The Fe chelating activity (%) of Commiphora myrrha resin methanol (Me-H) extract, ethyl acetate (ETOAC) extract and the essential oil (EO) at differentoncentrations. (n = 3, value = mean ± SD).

solated three furano-sesquiterpenoids (myrrhone, 3-methoxy-uranogermacradien-6-one and 2-methoxy-furanogermacren-6-ne) from C. myrrha hexane extract that showed DPPH radicalcavenging activity with IC50 values of 1.08, 4.29 and 2.56 mg/ml,espectively. Furthermore, the results of the present work are dis-gree with the results of Al-Harrasi et al. (2013) who reported thatethanol extract of Hougari Regular (HR) grade resin of Boswellia

acra showed scavenging activity against DPPH radicals lower thanhe antioxidant activity of resin essential oil at the same concen-ration.

.2.2. Ferrous ions chelating assayThe Fe2+ chelating activity of C. myrrha resin Me-OH, ETOAC

xtracts and EO showed a concentration-dependent activity pro-le (Fig. 2). The Me-OH extract was found to be the most potente2+ chelator as it caused 82.7% chelation at concentration 1 mg/ml,ollowed by ETOAC extract causing 79.2% chelation. At the sameoncentration the EO caused 28.2% as the lowest chelation activ-ty. The Me-OH extract activity was less than that of the standardDTA. The IC50 of the Fe2+ values for EDTA, Me-OH, ETOACxtracts and EO were 0.028, 0.238, 0.279 and 1.553 mg/ml, respec-ively.

Similar results were also observed for the Fe2+ chelating activityf the extracts and the oil as compared to their DPPH-scavengingctivities (Figs. 1 and 2). The C. myrrha resin essential oil has a lowere2+ chelating activity compared to its extracts and that might beue to the less iron binding capacity of essential oil components.he present results are in agreement with Roy et al. (2012) whoeported that the Handia volatile components showed low metalhelating activity. The poor Fe2+ chelating activity of C. myrrha resinssential oil might be attributed to the lack of monoterpenes hydro-arbons in the oil. Previous reports confirmed that the essentialils having low content of monoterpenes hydrocarbons have poorntioxidant activity (Tepe et al., 2005; Nanyonga et al., 2013). Inhe present results the highest Fe2+ chelating activity of both Me-H and ETOAC extracts might be attributed to their high contentf furanosesquiterpenes, triterpenes and Steroids. Similar resultsere found by Wang et al. (2004) isolated (Z)-guggulsterone (as a

teroidal compound) from Commiphora mukul and this compoundroved antioxidant activity.

.2.3. Ferric reducing power assayThe reducing power of myrrha resin extracts and its essential

il increased with the increase of concentrations (Fig. 3). The EOas superior to Me-OH and ETOAC extracts. The reducing pow-

rs of essential oil were 0.348, 0.687 and 0.864 at 200, 400 and00 �g/ml, respectively. At 200 �g/ml concentration the essential

extract, ethyl acetate (ETOAC) extract and the essential oil (EO) at different con-centrations, BHT used as positive control. (n = 3, value = mean ± SD).

oil gave reducing power (0.348) lower than BHT (1.377) which usedas positive control. The myrrha essential oil was more effective inthe reducing power compared to Me-OH and ETOAC extracts. Thisseems to be attributed to the more hydrogen donating componentsin the essential oils such as different groups of monoterpenoidsand sesquiterpenoids. Such hydroxyl terpene compounds candonate hydrogen atoms to transform Fe3+ to Fe2+ performing asreductones. These results are in accord with those of Racine andAuffray (2005) who reported that the essential oil of myrrha withits main sesquiterpenoids constituents including curzerene, fura-noeudesmadiene and lindestrene exhibited potent singlet oxygenquenching activity better than the control �-tocopherol. The sameauthors attributed this activity to the active furan rings of the iso-lated furanosesquiterpenoids. The results of the present study are inagreement with Laciar et al. (2009) who reported that terpenes par-ticularly, those with activated methylene groups in their molecules,could be the reason of the antioxidant activity shown by Artemisiaechegarayi essential oils.

3.3. Antimicrobial activity

The in vitro antimicrobial activity of C. myrrha resin extracts

LSD at P ≤ 0.5 1.73 1.11 1.96

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

Crops and Products 57 (2014) 10–16 15

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

Microorganisms MIC (�g/ml)

Me-OH ETOAC EO

Gram-positiveS. faecalis 50 50 100B. subtilis 250 100 200B. circulans 100 50 600L. monocytogenes 400 >1000 >1000

Gram-negativeE. coli 50 100 100P. aeruginosa 150 200 200Yeast

A.A. Mohamed et al. / Industrial

he essential oil. The results presented in Table 2 showed thathe Me-OH extract at 400 �g/ml concentration manifested theighest in vitro potential of antibacterial activity against all theested microorganisms except for C. albicans showed resistant toll test samples till 1000 �g/ml, while ETOAC extract and EO athe same concentration showed low or no antibacterial activity.n the present study the C. albicans showed more resistance to thewo tested extracts and the essential oil than S. cerevisiae. In sim-lar work, Vediyappan et al. (2010) renders the drug resistance of. albicans to the form of biofilms which exhibit elevated intrinsicesistance to various antifungal agents. No inhibition of bacterialnd yeast growth was observed with the negative control dimethyl-ulfoxide (DMSO). The highest potential of antibacterial activity ofyrrha Me-OH extract might be attributed to the high polarity ofe-OH which is effective for more consistent extraction of differ-

nt types of sesquiterpenoids particularly furanosesquiterpenoids,iterpenes, triterpenes and sterols. It is well known that the com-osition, structure, as well as functional groups of crude extractsnd the oils play an important role in determining their antimicro-ial activity. It has been demonstrated that crude extracts and thessential oils exercise their antimicrobial activity by causing struc-ural and functional damages to the microbial cell membrane (Gonit al., 2009).

In accordance with Zhu et al. (2001) who reported that Com-iphora resins are rich in sesquiterpenoids and the isolated

uranosesquiterpenoids or crude extracts were found to possessntibacterial and antifungal activity. Similarly, the sesquiter-enoids, epicurzerenone and (1E)-8,12-epoxygermacra-1,7,10,11-etraen-6-one isolated from Commiphora erythraea exhibitednhibitory activity against Fusarium culmorum, Phytophtora cryp-ogea and Alternaria solani (Fraternale et al., 2011). Mansumbinoiccid isolated from the oleo-resin of Commiphora molmol possessedotent antibacterial activity against a multidrug-resistant straintaphylococcus aureus with a MIC value of 4 mg/ml (Rahman et al.,008).

Our results indicated that the highest activity was observedgainst L. monocytogenes followed by P. aeruginosa with the widestnhibition zones (19 and 13.5 mm) respectively. The findings of theresent study are in line with Abdallah et al. (2009) who demon-trated that C. myrrha methanol extracts exhibited the highestntibacterial activity against S. aureus whereas the ethyl acetatextracts exhibited some degree of activity. The same authorsttributed the highest antibacterial activity of methanol extractso the presence of some active phenolic compounds, alkaloids andaponins. The test Gram-positive bacteria were found to be moreusceptible to antimicrobial agents than Gram-negative bacteriaBurt, 2004; Hussain et al., 2010). The weaker antimicrobial activ-ty against Gram-negative compared to Gram-positive bacteria isscribed to the structure of their cellular walls mainly with regardo the presence of lipoproteins and lipopolysaccharides in Gram-egative bacteria that form a barrier to hydrophobic compoundsInouye et al., 2001).

The MIC values obtained from antimicrobial tests ranged from5 to >1000 �g/ml (Table 3). The results showed that the bacterialtrains S. faecalis, E. coli and B. circulans were the most sensitive tooth Me-OH and ETOAC extracts with MIC value 50 �g/ml. Alterna-ively, L. monocytogenes was the least sensitive strain to both ETOACxtract and EO with MIC value >1000 �g/ml. The yeast S. cerevisiaeas the most sensitive yeast to Me-OH, ETOAC extracts and EO withIC values 25, 50 and 100 �g/ml respectively. The C. albicans was

he most resistant yeast to Me-OH, ETOAC extracts and EO with MICalue >1000 �g/ml. According to Salvat et al. (2004), plant extracts

ith MIC’s less than/or around 0.5 mg/ml indicate good antibacte-

ial activity. Accordingly, the Me-OH and ETOAC extracts, and EOf C. myrrha exhibited good antimicrobial activity against most ofhe tested microorganisms.

S. cerevisiae 25 50 100C. albicans >1000 >1000 >1000

4. Conclusions

The various solvent extracts and essential oil of C. myrrha resinshowed the potential for use as antioxidant and antimicrobial activ-ities. The methanol extract exhibited the highest antioxidant andantimicrobial activity when compared to ethyl acetate extract andessential oil. All the tested microorganisms except C. albicans weresensitive to the Me-OH extract. Further investigations are rec-ommended including extra isolation and structure elucidation ofbioactive pure compounds from two extracts and essential oil aswell as in vitro and in vivo studies to establish which components ofthe essential oil or extracts offers the best antioxidant and antimi-crobial activity. In addition, C. myrrha resin needs to be explored forfunctional food as capsules or pills and nutraceutical applicationsbesides its traditional uses.

Conflict of interest

The authors declare that there are no conflicts of interest.

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