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Commiphora myrrha (Nees) Engl. extracts: Evaluation of antioxidant and antiproliferative activity and their ability to reduce microbial growth on fresh-cut salad
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Boffa, Luisa; Binello, Arianna; Boscaro, Valentina; Gallicchio, Margherita;
Amisano, Gabriella; Fornasero, Stefania; Cravotto, Giancarlo. Commiphora
myrrha (Nees) Engl. extracts: Evaluation of antioxidant and antiproliferative
activity and their ability to reduce microbial growth on fresh-cut salad.
INTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY.
51 (3) pp: 625-632.
DOI: 10.1111/ijfs.13018
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1
Commiphora myrrha (Nees) Engl. extracts: evaluation of antioxidant and antiproliferative activity and their ability to reduce microbial growth on fresh cut salad
Luisa Boffa,1 Arianna Binello,1 Valentina Boscaro,1 Margherita Gallicchio,1 Gabriella Amisano,2 Stefania Fornasero,2 Giancarlo Cravotto 1*
1 Dipartimento di Scienza e Tecnologia del Farmaco, University of Turin, Via P. Giuria, 9, 10125,
Turin, Italy
2 Dipartimento di Scienze della Sanità Pubblica e Pediatriche, University of Turin, P.za Polonia 94,
10126, Turin, Italy
*Fax: +39 11 2367684. E-mail: giancarlo.cravotto@unito.it. Summary
With the aim to develop natural preservatives displaying also chemopreventive activity, different
Commiphora myrrha (Nees) Engl. extracts were studied. Myrrh essential oils, obtained by steam
distillation and microwave-assisted hydrodistillation, and several other extracts, obtained by sequential procedures with petroleum ether (PE), ethanol, ethyl acetate and butanol have been screened for their antioxidant (DPPH• scavenging assay) and antiproliferative activity (on both non-tumor and colon cancer cell lines) without previous purification. Considering that the colon cancer cell lines were more sensitive to petroleum ether and ethanol extracts, the latter of which showed the highest antioxidant activity (EC50 = 0.160±0.008 mg/mL), both have been selected for further antibacterial/antifungal activity tests using a antimicrobial diffusion test and a growth inhibition test on salads. Results showed that the ethanol extract possessed the higher antibacterial and antifungal activity. Compared to untreated salads, fresh-cut salads treated with these two myrrh extracts displayed a significant lower bacterial growth. Although further investigation is required, these promising results offer hints as how to improve the shelf life of fresh cut salad.
2 Keywords
Antimicrobial activity, antiproliferative activity, fresh cut salad shelf life, microwave-assisted hydrodistillation, Myrrh.
Introduction
Recent years have seen great effort being invested in the search for effective natural food additives with a broad spectrum of antioxidant and antimicrobial activities that could improve the quality and shelf life of perishable foods. Food borne illnesses are currently a real problem as the consumption of packaged food has increased worldwide. The World Health Organization (WHO) has called for a reduction in the consumption of common salts as preservatives since the increasing risk of cardiovascular disorders (The World Health Report 2002). This, together with the negative consumer attitudes toward food preservatives has led to a high interest in the use of natural “green” components as alternative agents for the control of food spoilage and harmful pathogens (Calo et al., 2015). Consumption of fresh and fresh-cut leafy greens has increased over the past few decades due to the recognized health benefits of salads. Leafy green vegetables are highly susceptible to microbial contamination due to high moisture and nutrient content as well as their large surface areas; numerous food borne outbreaks (Lynch et al., 2009) have therefore shaken consumer confidence. Although washing and sanitizing can decrease microbial numbers, bacterial cells internalized in stomata or cut surfaces and those found in the hydrophobic regions, cavities, and rough surfaces of leafy greens are likely to survive sanitizer exposure (Zeng et al., 2014). Moreover chlorine, the most common decontaminant in minimally processed vegetables, is quite ineffective at the applied concentration and can lead to the formation of potentially harmful chlorinated by-products (López-Gálvez et al., 2009).
Essential oils (EOs) are among the alternatives to traditional sanitizers and their antimicrobial and antioxidant properties have led to them being tested for their ability to improve the shelf life of
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different food systems (Patrignani et al., 2015). Lamiaceae EOs, in particular oregano and thyme, are the most studied and used additives in minimally processed vegetables and can provide product shelf lives similar to chlorine (López et al., 2005; Sacchetti et al., 2005). EOs were also widely studied for their antiproliferative properties. In particular, potential for cancer treatment was found in guava leaf oil on human mouth epidermal carcinoma (KB), and sweet basil oil on murine leukemia cell lines (P388) (Manosroi et al., 2006).
Myrrh is one of the world's oldest known remedies, which has been widely used for the treatment of wounds, pain, arthritis, obesity, parasitic infections and gastrointestinal diseases in ancient Egypt (Al-Harbi et al., 1997; Abdul-Ghani et al., 2009; Langenheim, 2003). Composed of EO (myrrhol, 3-8%, soluble in ether), gum (30-60%, soluble in water) and resin (myrrhine, 25-40%, soluble in alcohol), this aromatic and resinous exudate from the bark of various Commiphora tree species (Burseraceae family) is a yellowish to reddish-brown powder with a fine bitter taste and a slightly pungent balsamic odor (Hanus et al., 2005).
Though myrrh is not listed in the generally recognized as safe list, it is freely available as a “dietary supplement” in the US under DSHEA legislation (Dietary Supplement Health and Education Act of 1994) and included in the United Farmacopeia-National Formulary (USP 26- NF 21, 2003). Thanks to its low toxicity, myrrh is used as a flavoring component for toothpaste, mouthwashes, alcoholic beverages/drinks and food (Rao, Khan, & Shah, 2001). It is noteworthy that a purified myrrh extract was formulated in soft gelatin capsules, suppositories and emulsions. This formulation, known as Mirazid®, is a new, strong schistosomicidal (Badria et al., 2001) and fasciolicidal drug (El Gohary et
al., 1999).
Several authors investigated the antiproliferative, anti-inflammatory, antimicrobial, hepatoprotective and cardiovascular properties of crude extracts and purified metabolites (Dolara et al., 2000; El Ashry
et al., 2003; Shen et al., 2012). These studies indicated that triterpenoids and diterpenoids are mainly
responsible for anti-inflammatory property, sesquiterpenoids for antimicrobial, smooth muscle-relaxing and analgesic effects, lignans for the cytotoxicity, and steroids for antiproliferative,
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inflammatory, hypolipidemic and antidiabetic activities. In 2010, Wanner et al. proved that myrrh EO furnishes good inhibitory action on Gram-positive bacteria (Bacillus cereus, Staphylococcus
aureus and S. epidermidis), while providing moderate inhibitory activity on Escherichia coli and Salmonella abony.
Myrrh oil is generally obtained by means of hydrodistillation (HD), steam distillation (SD) or solvent extraction (SE) (Baser et al., 2003; Marongiu et al., 2005). Several works have described different methods for the extraction, purification and characterization of some myrrh compounds, (Dekebo et
al., 2002; Zhu et al., 2003). An ethanol (EtOH) extract has been suspended in hot water and extracted
using petroleum ether (PE), ethyl acetate (EtOAc) and butanol (BuOH) in a sequential manner (Su et
al., 2001). All of the obtained fractions and some of the isolated compounds were tested to evaluate
cytotoxic activity on cells of human ovarian, cervical and endometrial cancers. Only the EtOH extract and PE fraction as well as abietic and dehydroabietic acids showed good antiproliferative activity. To test the possibility of creating a natural extract that could extend the shelf life and improve quality of fresh packaged food, the antioxidant, antiproliferative and antimicrobial activities of a number of
Commiphora myrrha (Nees) Engl. extracts, obtained from distillation or solvent extraction (PE,
EtOH, EtOAc, BuOH) were evaluated. The antioxidant activity of the obtained extracts has been determined using DPPH• tests, while antiproliferative activity has been screened for both non-tumor and tumor cell lines. Finally, the most promising PE and EtOH extracts have been evaluated for their antibacterial and antifungal activity using a diffusion test and a growth inhibition test on fresh salads.
Materials and methods Chemical and plant materials
All chemicals (including Trolox®) and solvents (analytical grade) were purchased from Sigma Aldrich (Milan, Italy). Myrrh was kindly supplied by MB Med. (Martin Bauer group, Rivalta, Italy).
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Distilled H2O was added to a round bottomed flask (5 L) placed on a heating mantle (Table 1, Procedure 1). Myrrh resin (250 g), grinded using a mechanical blender, was introduced on a perforated septum (flexible fine grid) in a round flask (double necked, on the top and on the bottom), which was placed on top of the round bottomed flask. Finally, a Marcusson tube and a reflux condenser were assembled to complete the distillation apparatus. EO distillation continued for 60 min starting from the moment of the boiling water. The sample was characterized by GC-MS analysis (see Table S1) and screened for antioxidant activity.
Microwave-assisted hydrodistillation (MA-HD)
Myrrh (100 g) was grinded using a mechanical blender and introduced into a round bottomed flask (250 mL) and completely immersed in distilled H2O (Table 1, Procedure 2). The flask was placed in a microwave (MW) oven (NEOS-GR-Microwave Gravity Station; Milestone Srl, Sorisole BG, Italy). A glass tube (inside the oven), a Marcusson tube and a reflux condenser were assembled on this flask to give the final distillation apparatus. MW power was gradually increased from 120 W to 150 W over 60 min to ensure constant water evaporation during the process. Distillation occurred in 2-3 min. The sample was characterized by GC-MS analysis (see Table S1) and screened for antioxidant activity.
Solvent extraction of myrrh using PE, EtOAc and BuOH Procedure 3 a.
Myrrh (200 g) was grinded using a mechanical blender and introduced into a round bottomed flask (1 L) and PE was added (500 mL) (Table 1, Procedures 3 a-c). The mixture was heated at reflux (rfx) for 2 h. After cooling, the suspension was filtered on a paper filter in a Buchner funnel and evaporated to dryness. An intense aromatic yellow oil was obtained. This sample was characterized by GC-MS analysis (see Table S1).
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The residue was placed into a round bottomed flask (1 L) and EtOAc was added (500 mL). The mixture was heated at rfx for 2 h. After cooling, the suspension was filtered on a paper filter in a Buchner funnel and evaporated to dryness, giving a dark orange powder.
Procedure 3 c.
The same procedure was repeated on the residue (after EtOAc extraction) using BuOH, affording a dark viscous oil. All the extracts were screened for antioxidant activity.
Solvent extraction of myrrh using PE and EtOH Procedure 3 d.
The residue obtained from a new extraction of myrrh using PE (see Procedure 3 a) was placed into a round bottomed flask (1 L) and EtOH was added (500 mL) (Table 1, Procedures 3 a and 3 d). The mixture was heated at rfx for 2 h. After cooling, the suspension was filtered on a paper filter in a Buchner funnel and evaporated to dryness. The dark orange powder obtained was screened for antioxidant activity.
Determination of antioxidant activity using the DPPH• radical scavenging method
The radical scavenging ability of the extracts was monitored using the stable free radical DPPH•, following the method described by Brand-Williams et al. (1995). A DPPH• solution (0.1 mM) was prepared to give absorbance at 517 nm in the 0.45-0.55 range. Preliminary tests were performed at three different concentrations for each extract (around 1, 0.1 and 0.01 mg/mL). 700 μL of the DPPH• solution were placed in cuvettes for the UV-Vis colorimetric assay. The reaction started when 700 μL of the sample diluted solutions were added to the cuvette containing the DPPH• solution. Mixtures were shaken vigorously and kept in the dark for 20 min at room temperature (time required to reach equilibrium). At this time, the UV absorbance of each sample was measured at 517 nm against a pure methanol blank, on a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA,
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USA). Results are expressed as DPPH• inhibition percentage (I %), which were calculated using the following equation:
I % = (AbsDPPH• – Abssample / AbsDPPH•) x 100.
The bleaching rate of DPPH• was monitored in the presence of different solutions of Trolox® and BuOH and EtOH myrrh extracts in order to calculate the EC50 (amount of compound/extract necessary to decrease the initial concentration of DPPH• to 50% at the equilibrium). Radical scavenging activity in the Trolox® standard solutions was measured at around 1, 2, 4, 5, 7, 10, 12, 18 µg/mL concentrations. Various concentrations of BuOH and EtOH myrrh extracts between 0.001 and 2 mg/mL were tested. Data analysis, the computation of EC50 and Probit Regressions were performed using an algorithm in Microsoft Visual Basic 6.0 (Microsoft Corporation, Redmond, WA, USA) (Locatelli et al., 2009). All samples were prepared in triplicate and DPPH• radical scavenging activity was expressed as µg or mg compound/dry extract per mL solution ± standard deviation.
Cell viability assays
HCT 116, HT-29, HUTU80, SW837 and hTERT-HME1 were purchased from American Type Culture Collection; HCA-7 cell line was purchased from Sigma-Aldrich. Immortalized human podocytes were kindly provided by Dr. G. Miglio (Dipartimento di Scienza e Tecnologia del Farmaco, University of Turin). HCT 116, HT-29, HUTU80 and podocytes were cultured in DMEM medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; PAA) and HCA-7 cells were grown in DMEM supplemented with 5% FBS. HCT-15 and SW837 cell lines were cultured in RPMI medium (Sigma-Aldrich) supplemented with 10% FBS. hTERT-HME1 cells were grown in DMEM-F12 medium (Invitrogen Corporation, Carlsbad, CA, USA), supplemented with 2% FBS, 20 ng/mL EGF, 10 µg/mL insulin, and 100 µg/mL hydrocortisone. All cell culture media were supplemented with 50 units/mL penicillin and 50 mg/mL streptomycin. All myrrh extracts were dissolved in methanol (1 mg/mL). Cells were seeded in 100 µL complete medium at an appropriate density (1500-2000 cells/well) in 96-well plastic culture plates in triplicate. The following day, 100
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µL of myrrh extracts in serum-free medium were added to cells with a multichannel pipette, after serial dilution. Vehicle and medium-only containing wells were added as controls. Plates were incubated at 37°C in 5% CO2 for 6 days, after which cell viability was assessed by ATP content using a Cell Titer-Glo Luminescent Assay (Promega Italia Srl, Milano, Italy). All luminescence measurements (indicated as relative light units) were recorded on a Victor X4 multimode plate reader (Perkin-Elmer, Waltham, MA, USA).
Antimicrobial diffusion test
The antimicrobial diffusion test was carried out to determine the antimicrobial activity of PE and EtOH extracts which had been solubilized in dimethyl sulfoxide (DMSO) (20 mg/mL). DMSO was selected because it does not show antimicrobial activity against the selected microorganisms and easily dissolves all extract components.
The selected microorganisms were Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538 P, Streptococcus pyogenes ATCC 12344, Candida albicans ATCC 10231 and Pseudomonas
aeruginosa ATCC 10145. Each of them was inoculated in Tryptic Soy Broth (TSB, Biolife S.r.l.,
Milan, Italy) and incubated at 37°C overnight to obtain the exponential growth phase (107-108 CFU/mL). About 100 µL of tested microorganism solution, obtained as described above, were spread on the entire surface of Mueller-Hinton Agar plates (Biomérieux, Craponne, France) using a spatula. Sterile paper discs (7 mm) were soaked in 20 µL of extract solutions and placed on the inoculated plates at a suitable distance. After Petri dish incubation at 37°C for 24 hours, the growth inhibition zone diameter was determined in mm via measurement with a caliber. Plate readings were repeated after 48 hours. A control test using a paper disc soaked in DMSO was also carried out.
Antimicrobial activity in salad
The antibacterial and antifungal activity of PE and EtOH extracts was tested in order to evaluate their influence on fresh cut salad microorganism growth. Twelve packages (four packs for each of three
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different production batches, same expiry date) of the same brand as the previous samples (200 g) were analyzed. After opening and sampling at T0, three packages belonging to the same production batch were sprayed, respectively, with 2 mL of EtOH extract solubilized in DMSO (20 mg/mL), PE extract solubilized in DMSO (20 mg/mL), and DMSO alone; the fourth untreated pack was used as a control test. Salads were sealed and kept refrigerated at 4°C for later analysis (T4 and Te). About 20 g of ready-to-eat lettuce were transferred to 180 mL of Trypton Soy Broth (Biolife S.r.l.), in sterile stomacher bags, and pummeled in a stomacher at 230 rpm for 2 min. Homogenized samples were serially diluted from 10-1 to 10-6 for subsequent plating on Plate Count Agar (Biolife S.r.l) (ISO 4833). Plate Count Agar was incubated for 3 days at 30ºC. Microbial counts were recorded as colony forming units per g (CFU/g) of sample. Tests were carried out in triplicate. Data are shown as mean ± standard deviation.
Results and discussion Myrrh extracts
Myrrh EOs were obtained by SD and MA-HD. This last distillation (Orio et al., 2012; Binello et al., 2014), an efficient green technique that uses volumetric dielectric heating (Chemat & Cravotto, 2013), has been carried out in a dedicated MW professional reactor where myrrh resin was immersed in H2O and irradiated with MW. With the aim to compare traditional SD and MA-HD without reaching necessarily an exhaustive extraction, a time of 1 h was set up for both techniques. MW irradiation provided a better yield than SD (1.10% instead of 0.32%, Table 1), probably due to the difficulty for the aqueous vapor to permeate a resinous matrix such as myrrh. It has to be highlighted that higher yields of conventional HD, reported in literature by Marongiu and Başer (2.8% and 2.5%, respectively), can be related to prolonged extraction times.
At the same time, myrrh resin has been subjected to solvent extraction using different solvents in sequence to obtain more active enriched extracts. A procedure which has been modified from the method reported by Su et al. (2012) was followed. The subsequent extractions were carried out under
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conventional heating at rfx with PE, EtOAc and BuOH (Table 1, Procedures 3 a-c). The PE extract was an intense aromatic yellow oil obtained in a high yield (around 5%). EtOAc extraction on residue gave a 6.83% yield calculated on starting resin, while the third step with BuOH afforded a 2.7% yield (on starting resin). In the Su et al. procedure, PE and EtOAc gave lower yields (2.3 and 4.8% on starting resin), probably due to the preliminary 85% EtOH extraction step (18% yield) which did not dissolve completely the apolar fraction.
In our case, a further extraction with EtOH was carried out on the myrrh residue after a first step with PE (Table 1, Procedures 3 a and 3 d). The so obtained extract (7.8% on starting resin) was deprived of more apolar and aromatic compounds, being different from the EtOH extract tested by Su et al.. Myrrh EOs (SD or MA-HD) and PE extracts were compared by GC-MS analyses (Table S1). It is very much worthy of note that the PE extract, obtained in a higher yield, showed almost identical composition to the EO.
Antioxidant activity
DPPH• assay was used to characterize antioxidant capacity of both EOs and extracts, as it is one of the most accurate and responsive methods for analyzing vegetal extracts. The scavenging effect of the various extracts was screened to evaluate their DPPH• inhibition percentage (I%) at different concentrations. As reported in Table 2, SD and MA-HD EOs, such as the PE extract, showed I% values lower than 20%, even at 1 mg/mL concentration. However, increasing the solvent extraction polarity, the inhibition percentage improved significantly as showed by EtOAc, BuOH and EtOH extracts, which reached respectively 48%, 71% and 88% inhibition at a concentration around 1 mg/mL.
To better analyze them, DPPH• scavenging activity was expressed as the mean of the EC50 values ± standard deviation for BuOH and EtOH extracts and compared with the Trolox® standard (Figure S1). EC50 of EtOAc extract was around 1.06 mg/mL (Table 2), while it was not possible to calculate this value for EOs and PE extract since they did not show antioxidant activity in DPPH• test. Trolox®
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showed an EC50 value of 3.94±0.50 µg/mL, while EtOH and BuOH values of 0.160±0.008 mg/mL and 0.379±0.040 mg/mL, respectively. Therefore, among all the samples screened, EtOH extract exhibited the highest antioxidant activity, even if EC50 was 40 times higher than Trolox®. BuOH and EtOAc extracts EC50 were almost 2.5 and 7 times higher than EtOH value.
Despite negative data in DPPH• scavenging activity, C. myrrha EO was found to inhibit lipid peroxidation in sunflower oil, opening the possibility to its use in pharmaceutical, cosmetic and functional foods preparations (Assimopoulou et al., 2005).
Antiproliferative activity
With the purpose of performing a prescreening on the effect of myrrh extracts on the viability of human cells, two non-tumor cell lines, hTERT-HME1 and podocytes, and six colorectal cancer cell lines, HCA-7, HCT-15, HCT-116, HT-29, HUTU-80 and SW837 have been evaluated (Figure S2). Cell viability was estimated by determining ATP content in three replicate wells. Results are normalized to the growth of the methanol treated cells and are represented as mean ± standard error of mean (SEM) over at least three independent experiments. All extracts inhibited cell viability in a dose-dependent manner in a 62.5-0.98 µg/mL concentration range. A comparison of IC50 (mean amount of compound/extract that inhibited cell viability by 50%) values obtained for each cell line seems to provide evidence for a cell specific extract response (Table 3).
In particular, hTERT-HME1 was the most sensitive to all myrrh extracts (except for the EtOH extract), irrespective of the extraction solvent used. Podocyte viability was less modified by extract treatment and podocytes were more resistant to the EtOH extract than in all other cell lines. All colon cancer cell lines were more sensitive to EtOH and PE samples, as compared to BuOH and EtOAc ones. In particular, EtOH extract showed an IC50 = 8.37±1.15 µg/mL on HCA-7 cells, which, together with HUTU-80, were also the most sensitive to the PE one (IC50 = 11.72±1.21 and 12.26±1.22 µg/mL, respectively). Moreover, HCT-15 and HT-29 were the most resistant to all myrrh extracts. A weak antiproliferative activity was evidenced by EtOH extract for which IC50 was around
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28 µg/mL for both cell lines. Our results are in agreement with data evidenced by Su et al.. As cited before, among all the samples they tested, only 85% EtOH and PE extracts significantly inhibited proliferation of various human gynecologic cancer cells with dose-dependent relation in vitro. 85% EtOH extract showed good inhibitory effects on human ovarian (A2780, SK-OV-3) and endometrial (Shikawa) cancer cells with an IC50 of 15.81, 22.08 and 20.73 µg/mL, respectively, while PE extract was less active (IC50 > 26.6 µg/mL).
Antimicrobial activity of PE and EtOH myrrh extracts
On the basis of our previous results, PE and EtOH extract were chosen to verify their antimicrobial activity. Data of antibacterial tests on E. coli, S. aureus, S. pyogenes, C. albicans and P. aeruginosa are shown in Fig. 1. Results of the diffusion test for the myrrh PE extract demonstrated antimicrobial activity against C. albicans, S. pyogenes and S. aureus and gave similar effects against all. The EtOH extract was effective against all tested strains with greater activity towards S. aureus and C. albicans (9 mm inhibition zone, 20 mg/mL conc.), confirming the potential health benefits of myrrh in the treatment of infections such as pharyngitis, sinusitis, gingivitis and phyorrhea (De Rapper et al.; 2012). It was furthermore confirmed that DMSO, used as solvent, did not show any antimicrobial activity. The good activity against C. albicans is in agreement with the anti-fungal properties demonstrated in other studies (Dolara, et al. 2000; Abd-Ulgadir, et al. 2015).
Data reported by Omer et al. (2011) are quite different although they performed the same sequential extraction on myrrh using PE and EtOH. Antimicrobial activity of their EtOH extract against E. coli,
P. aeruginosa and C. albicans resulted strongly lower. In fact, no inhibition zone was detected at 20
mg/mL (the same concentration we used), while PE extract showed a 3.7 and 5.7 mm zone for C.
albicans and for S. albus, comparable to 5 and 3 mm (S. aureus) obtained in our test. Disc diffusion
tests on pure myrrh EO, reported by Wanner et al. and discussed in the introduction, evidenced an antimicrobial activity in agreement with our data.
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The antimicrobial activity was then evaluated in ready-to-eat salad in order to explore the possibility of extending its shelf life spraying little amounts of myrrh PE or EtOH extracts. Untreated salad and salad added with only DMSO were examined as control. Figure 2 shows bacterial growth trends of the treated and non-treated samples that had been analyzed on the packaging date (T0), after 4 days (T4) and on the expiry date (Te, day 7).
At T0, all samples showed similar aerobic colony count (ACC) (mean of 5.8 log CFU/g). At expiry date, salads treated with EtOH and PE extracts had ACC of 7.7 and 7.9 log CFU/g, respectively, while the DMSO-sprayed and non-treated samples showed ACC of 9.8 and 9.9 log CFU/g, respectively. The analysis revealed 2.2 log and 2 log decreases in bacterial growth in the salad packages treated with EtOH and PE extracts, respectively, as compared to control and DMSO treated samples. Conclusions
Different myrrh extraction methods and different solvents effects have been evaluated through yields and antioxidant properties. PE extract showed an almost identical composition to EOs obtained by SD or MA-HD, but it gave a five times higher yield. EtOH extract exhibited the highest antioxidant activity among all the screened samples. Tests on cell viability of all the extracts performed on both non-tumor and tumor cell lines highlighted a cell type dependent effect, as the two non-tumor cell lines were the most sensitive, hTERT-HME1, and the most resistant, podocytes. As, among all the tested extracts, the colon cancer cell lines showed an increased sensitivity toward EtOH and PE ones, these samples were considered the most promising and chosen to test their antimicrobial activity. EtOH extract resulted to be more active than the PE one, especially against Gram-negative bacteria and C. albicans.
Fresh-cut salads treated with these two myrrh extracts revealed significant lower bacterial growth as compared to untreated salads, opening the possibility of further investigation for an extension in their shelf life. Additional studies concerning the antimicrobial action, safety and toxicity of these extracts together with sensorial analyses are of course needed to contemplate their use as an alternative natural food preservative.
14 Acknowledgments
This work has been supported by the University of Turin (fondi Ricerca locale 2014). Authors are grateful to MB Med (Rivalta, Turin) for providing myrrh resin and to Prof. S. Caramello for useful suggestions.
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18 Table 1.
Yields of the different extraction methods used on myrrh.
Procedure Technique Solvent Time (min) Yield (%)
1 SD - 60 0.32§ 2 MA-HD - 60 1.10§ 3 a Rfx (65°C) Petroleum ether 120 5.07 3 b Rfx (80°C) Ethyl acetate 120 6.83 3 c Rfx (120°C) Butanol 120 2.70 3 d Rfx (85°C) Ethanol 120 7.80 § Essential oil.
19 Table 2.
DPPH• inhibition % (I%) of the various extracts obtained from myrrh.
Sample Conc. (mg/mL) I% (±SD)
SD essential oil 0.95 12.70±0.42
0.095 11.29±0.46
0.0095 10.47±0.29
MA-HD essential oil 0.95 18.37±0.55
0.095 10.39±0.23
0.0095 8.61±0.31
Petroleum ether extract 1.020 11.15±0.35
0.1020 1.49±0.21
0.01020 1.14±0.19
Ethyl acetate extract 1.060 48.17±0.42
0.1060 9.10±0.53 0.01060 1.28±0.26 Butanol extract 1.010 71.07±0.61 0.1010 21.26±0.42 0.01010 3.59±0.16 Ethanol extract 0.910 88.19±0.37 0.0910 28.31±0.48 0.00910 3.38±0.19
20 Table 3.
IC50 values for myrrh extracts in the cell lines analyzed.
Cell lines
IC50 (µg/mL) ± SEM
Ethanol Petroelum ether Butanol Ethyl acetate
hTERT-HME1 13.64±1.58 5.34±1.47 21.81±1.61 5.83±1.41 Podocytes 48.05±1.31 33.34±1.25 > 62.5 23.24±1.25 HCA-7 8.37±1.15 11.72±1.21 34.50±1.29 25.08±1.23 HCT-15 27.74±1.34 45.12±1.38 51.32±1.29 > 62.5 HCT-116 23.20±1.32 28.62±1.25 > 62.5 42.49±1.30 HT-29 28.45±1.31 38.53±1.30 > 62.5 74.03±1.23 HUTU-80 25.89±1.29 12.26±1.22 > 62.5 41.31±1.27 SW837 20.51±1.16 19.08±1.19 38.69±1.24 25.23±1.20
21 Fig. 1. Antibacterial activity of tested extracts.
Fig. 2. Total aerobic colony count (mean) from salad samples. 0 1 2 3 4 5 6 7 8 9 10
E. coli S. aureus S. pyogenes P. aeruginosa C. albicans
In hi bi tio n di ame te r ( mm)
22 Supporting Information
Additional Supporting Information may be found in the online version of this article:
Figure S1. DPPH˙ I% curve, which depends on Trolox® concentration (EC50 is expressed in µg/mL), on myrrh EtOH and BuOH extracts concentration (EC50 is expressed in mg/mL). Figure S2. Effect of myrrh extracts on the viability of non-tumor cell lines and colorectal cancer cell lines.
Table S1. Chromatographic area percentages of the compounds identified in myrrh essential oils (SD or MA-HD) and PE extract.
Data S1. GC-MS analyses of SD, MA-HD essential oils and PE extract. Data S2. Antioxidant activity.
