Essential Oil from Origanum vulgare Completely Inhibits the Growth of Multidrug-Resistant Cystic Fibrosis Pathogens

Abstract

Essential oils (EOs) are known to inhibit the growth of a wide range of microorganisms. Particularly interesting is the possible use of EOs to treat multidrug-resistant cystic fibrosis (CF) pathogens. We tested the essential oil (EO) from Origanum vulgare for in vitro antimicrobial activity, against three of the major human opportunistic pathogens responsible for respiratory infections in CF patients; these are methicillin-resistant Staphylococcus aureus, Stenotrophomonas maltophilia and Achromobacter xylosoxidans. Antibiotic susceptibility of each strain was previously tested by the standard disk diffusion method. Most strains were resistant to multiple antibiotics and could be defined as multi-drug-resistant (MDR).The antibacterial activity of O. vulgare EO (OEO) against a panel of 59 bacterial strains was evaluated, with MIC and MBC determined at 24, 48 and 72 hours by a microdilution method. The OEO was effective against all tested strains, although to a different extent. The MBC and MIC of CEO for S. aureus strains were either lower or equal to 0.50%, v/v, for A. xylosoxidans strains were lower or equal to 1% and 0.50%, v/v, respectively; and for S. maltophilia strains were lower or equal to 0.25%, v/v. The results from this study suggest that CEO might exert a role as an antimicrobial in the treatment of CF infections.

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Essential Oil from Origanum vulgare Completely Inhibits the Growth
of Multidrug-Resistant Cystic Fibrosis Pathogens
Giovanna Pesaventoa,§, Valentina Magginib,§, Isabel Maidac, Antonella Lo Nostroa, Carmela Calonicoa,
Chiara Sassolia, Elena Perrinc, Marco Fondic , Alessio Mengonic, Carolina Chiellinid, Alfredo Vannaccib,
Eugenia Gallob, Luigi Gorib, Patrizia Boganic, Anna Rita Biliae, Silvia Campanaf, Novella Ravennif,
Daniela Dolcef, Fabio Firenzuolib and Renato Fanib,*
aDept. of Health Sciences, University of Florence, Viale G. B. Morgagni, 48, I- 50134 Florence, Italy
bCenter for Integrative Medicine, Careggi University Hospital, University of Florence, Florence, Italy
cDept. of Biology, University of Florence, Via Madonna del Piano 6, I-50019 Sesto Fiorentino (Florence), Italy
dConsiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca per l’Agrobiologia e
la Pedologia (CRA-ABP) Piazza D'Azeglio 30, I-50121 – Firenze, Italy
eDept. of Chemistry Ugo Schiff, University of Florence, Via Ugo Schiff 6, I-50019 Sesto Fiorentino (Florence),
Italy
fDept. of Paediatric Medicine Anna Meyer Children's University Hospital, Florence, Italy
renato.fani@unifi.it
§These authors contributed equally to this study
Received: November 6th, 2015; Accepted: April 11st, 2016
Essential oils (EOs) are known to inhibit the growth of a wide range of microorganisms. Particularly interesting is the possible use of EOs to treat multidrug-
resistant cystic fibrosis (CF) pathogens. We tested the essential oil (EO) from Origanum vulgare for in vitro antimicrobial activity, against three of the major
human opportunistic pathogens responsible for respiratory infections in CF patients; these are methicillin-resistant Staphylococcus aureus, Stenotrophomonas
maltophilia and Achromobacter xylosoxidans. Antibiotic susceptibility of each strain was previously tested by the standard disk diffusion method. Most strains
were resistant to multiple antibiotics and could be defined as multi-drug-resistant (MDR).The antibacterial activity of O. vulgare EO (OEO) against a panel of
59 bacterial strains was evaluated, with MIC and MBC determined at 24, 48 and 72 hours by a microdilution method. The OEO was effective against all tested
strains, although to a different extent. The MBC and MIC of OEO for S. aureus strains were either lower or equal to 0.50%, v/v, for A. xylosoxidans strains
were lower or equal to 1% and 0.50%, v/v, respectively; and for S. maltophilia strains were lower or equal to 0.25%, v/v. The results from this study suggest
that OEO might exert a role as an antimicrobial in the treatment of CF infections.
Keywords: Essential Oils, Origanum vulgare, Multidrug resistance, Cystic Fibrosis, Methicillin-resistant Staphylococcus aureus, Stenotrophomonas
maltophilia, Achromobacter xylosoxidans.
Leading causes of morbidity and mortality in cystic fibrosis (CF)
are respiratory tract infections caused by human pathogens such as
methicillin resistant Staphylococcus aureus (MRSA),
Stenotrophomonas maltophilia and Achromobacter xylosoxidans
[1]. In the United States, the prevalence of MRSA, S. maltophilia,
and A. xylosoxidans in CF patients is in the order of 22.6%, 15.8%
and 6%, respectively. Two studies in Italy have reported a
prevalence of 16% of A. xylosoxidans infection [2] and of 7% for
MRSA [3]. These bacteria often show an increasing level of
multidrug resistance (MDR) hampering the treatment of CF-
associated infections with conventional antimicrobial therapies [1].
Thus, new therapeutic strategies need to be discovered. Much
attention is paid to bioactive compounds derived by plants that are
known to possess antimicrobial properties and are used in
traditional medicine to treat several diseases. In particular, essential
oils (EOs) play an important role in the protection of plants and
have been empirically used for centuries to treat upper respiratory
tract infections such as pharyngitis, bronchitis and sinusitis [4].
Furthermore, in many cases the EO activity results from the
complex interaction between the different classes of compounds
such as phenols, aldehydes, ketones, alcohols, esters, ethers and
hydrocarbons found in EOs [5]. It is likely that it will be more
difficult for bacteria to develop resistance to the multi-component
EOs than to common antibiotics, which are often composed of only
a single molecular entity [6], since several molecular targets would
need to adapt to overcome the effects of the oil [7].
The activity of EOs against bacteria, fungi and viruses has been
tested in many studies and indeed, many bacteria, especially
pathogens, exhibit high sensitivity to EOs mainly extracted from
thyme, oregano and cloves [8-10]. We have previously performed a
preliminary analysis of EOs from six aromatic medicinal plants
(Eugenia caryophyllata, Origanum vulgare, Rosmarinus officinalis,
Lavandula hybrida, Melaleuca alternifolia and Thymus vulgaris)
revealing that, despite their different chemical composition, all of
them were able to inhibit the growth of representative strains of
members of the Burkholderia cepacia complex (Bcc) [11].
The antibacterial activity of Origanum vulgare EO (OEO) has been
attributed to the phenolic components, such as thymol and carvacrol
[5], which are able to inhibit some pathogenic bacterial strains,
including Escherichia coli, and serovars enteritidis, choleraesuis,
and typhimurium of Salmonella enterica [12]. Previous studies have
reported that OEO shows antimicrobial activity both in vitro and
in vivo against S. aureus [13-16]. On the basis of current literature
the OEO could represent a good compromise between potential
NPC Natural Product Communications 2016
Vol. 11
No. 6
861 - 864

862 Natural Product Communications Vol. 11 (6) 2016 Pesavento et al.
Table 1: Bacterial strains tested in this work.
S
p
ecies Strain Ori
g
in Reference S
p
ecies Strain Ori
g
in Reference
M
RSA* 4 CF
1
This work
A
. x
y
losoxidans* 1 CF
1
This work
5 CF
1
21 CF
2
6 CF
1
34 CF
1
9 CF
1
39 CF
2
11 CF
2
41 CF
1
12 CF
2
42 CF
3
13 CF
1
43 CF
3
15 CF
1
45 CF
2
16 CF
2
48 CF
2
17 CF
1
49 CF
2
20 CF
1
50 CF
2
23 CF
2
52 CF
1
24 CF
2
53 CF
1
25 CF
2
54 CF
1
26 CF 55 CF
1
27 CF
1
56 CF
1
28 CF
2
60 CF
1
32 CF
1
61 CF
2
33 CF
3
62 CF
1
63 CF
1
S. malto
p
hilia* 1 CF
2
This work
S. malto
p
hilia* 12 CF
1
This work
2 CF
1
13 CF
1
3 CF
1
14 CF
1
4 CF
2
15 CF
1
5 CF
1
16 CF
1
6 CF
1
17 CF
1
7 CF
2
19 CF
2
8 CF
1
20 CF
1
10 CF
1
21 CF
2
11 CF
1
22 CF
2
* Strains isolated from CF patient (CF) at the Anna Meyer Children’s Hospital
(Florence, Italy). 1 sputum; 2 throat swab; 3 bronchial
antibacterial activity [8, 9, 11, 17] and tolerability [18] if properly
formulated in specific drug delivery systems [19]. The aim of this
work was to investigate the antimicrobial potential of O. vulgare
EO, whose composition was previously determined [11], on a panel
of 59 MDR strains belonging to three of the major groups of CF
opportunistic pathogens (i.e. MRSA, Stenotrophomonas
maltophilia, and Achromobacter xylosoxidans) estimating the
bacteriostatic or bactericidal activity.
As a first step of the investigation, the molecular and/or phenotypic
characterization of all the 59 representatives of the bacterial panel
was performed revealing that the bacterial isolates of each species
corresponded to different strains (data not shown). Then, the
antibiotic resistance profile of each strain was determined using a
panel of different antibiotics (Tables S1-S3). Most of the strains
were resistant to multiple antibiotics (belonging to at least two
major classes of antibiotics) and can be therefore defined as MDR
according to the definition of the American Cystic Fibrosis
Foundation [20]. In particular:
i) A. xylosoxidans strains (Table S1) showed a resistant rate
higher than 60% for almost all categories of antibiotic tested:
between β-lactams antibiotics only the combination piperacillin/
tazobactam, imipenem and meropenem showed lower resistance
percentages. The resistant rates for colistin and minocycline were
62.5% and 50%, respectively. The percentage of antibiotics to
which each strain was resistant ranged from 50% to 100%.
ii) S. maltophilia strains (Table S2) showed a resistant rate
higher than 78% for β-lactams, aminoglycosides and
fluoroquinolones antibiotics. The resistant rates for the combination
trimethoprim/sulfamethoxazole and minocycline were 45% and
5.56%, respectively. The percentage of antibiotics to which each
strain was resistant ranged between 50% and 100%.
iii) Staphylococcus aureus strains (Table S3) were highly
resistant to β-lactams and macrolides. The resistant rate for
fluoroquinoles and lincosamides was around 74% and 83%
respectively, whilst the other rates were lower than 50% up to 0%
for linezolid, tigecyclin, trimethoprim/sulphamethoxazole and
vancomycin. One strain was resistant to teicoplanin. The percentage
of antibiotics to which each strain was resistant ranged from 31.2%
to 66.7%.
Subsequently, the antimicrobial activity of different concentrations
of OEO was tested for each of the 59 strains listed in Table 1 by
determining both MIC and MBC. Data obtained at 48 h of
incubation in the presence of OEO are shown in Tables S1-S3 and
summarized in Table 2. OEO exhibited antibacterial activity against
all the 59 bacterial strains to a different extent.
Several MRSA and A. xylosoxidans strains had MIC of 0.50% and
0.25%, v/v, EO at 48 h of incubation. Results were consistent also
at 24 h and 72 h (Table 2).
Quite interestingly, the analysis of MBC data revealed that the OEO
had a strong bactericidal activity, which in most cases was
consistent with the MIC values observed.
In general, OEO showed its inhibitory and microbicidal activity
against human pathogens, even at low concentration: after 48 h,
0.5%, v/v, OEO was able to inhibit the growth of 100% of A.
xylosoxidans, S. maltophilia, and MRSA, and to kill 100% of S.
maltophilia and of MRSA and 95% of A. xylosoxidans.
In particular, the analysis of data from Table 2 revealed that after
48h:
i) S. maltophilia strains were more sensitive than those
belonging to the other genera: no strain exhibited MIC and MBC
higher than 0.125%, v/v. The lowest OEO percentages still active
were 0.015% and 0.03, v/v, for MIC and MBC, respectively.
ii) Achromobacter strains were mostly inhibited and killed
by OEO concentrations of 0.25% and 0.50%, v/v.
iii) MRSA had MIC and MBC values ranging from 0.5% to
0.125%, v/v, of OEO.
Data obtained in this work revealed that all the 59 tested strains
were sensitive to OEO, even though to different extents. Overall the
sensitivity of S. maltophilia strains was higher than that exhibited
by MRSA and A. xylosoxidans strains. Moreover, there was no
correlation between the MDR patterns and sensitivity to the OEO of
strains belonging to the same species/group. Indeed, strains with a
(very) different MDR profile exhibited a very similar degree of
sensitivity to the OEO.
MBC values were in most cases completely consistent with the MIC
values of the same strain and indicated that the OEO had a strong
bactericidal activity on each of the 59 strains tested. Since the
strains belong to very different bacterial species/genera (both Gram
positive and Gram negative) the finding of such a broad activity of
OEO might suggest that OEO has the same cellular target(s) on
such widely different species. It is likely that the antibacterial
activity of OEO is due to the combined effect of several bioactive
molecules of the EO complex. Moreover, and quite interestingly, in
spite of the large number of experiments carried out in this work, no
mutant strain resistant to the EO tested was isolated. This finding
strongly suggests the possibility that the antimicrobial activity of
OEO is exerted toward multiple cellular targets. If this is so, the
simultaneous blocking of the activity of different molecular targets
should strongly decrease the probability of the appearance of a
mutant able to resist the essential oils, as happens for most of the
common antibiotics. For example, S. aureus is known for its
involvement in CF and nosocomial infections and is frequently
resistant to several antibiotics. OEO has been reported to reduce
lipase and coagulase activity of S. aureus [21]. The lack of
appearance of resistant strains to concentrations much lower than
2% OEO (maximum MBC value after 24 h) represents a very
interesting finding that might pave the way to the use of OEO to
fight the infections in CF patients.

Antimicrobial activity of Origanum vulgare essential oil Natural Product Communications Vol. 11 (6) 2016 863
Table 2: MIC and MBC in the twenty strains of A. xylosoxidans, S. maltophilia and MRSA.
EO concentration
(% v/v)
MIC (mg/L) (O.D.) MBC
24h 48h 72h 24h 48h 72h
Number % Number % Number %Number % Number % Number %
A. xylosoxidans
1.000 0 0 0 0 0 0 0 0 1 5 1 5
0.500 1 5 7 35 7 35 7 35 8 40 8 40
0.250 14 70 12 60 12 60 11 55 11 55 11 55
0.125 5 25 1 5 1 5 2 10 0 0 0 0
0.060 0 0 0 0 0 0 0 0 0 0 0 0
0.030 0 0 0 0 0 0 0 0 0 0 0 0
S. maltophilia
1.000 0 0 0 0 0 0 0 0 0 0 0 0
0.500 0 0 0 0 0 0 0 0 0 0 0 0
0.250 1 5 0 0 0 0 3 15 0 0 0 0
0.125 7 35 9 45 9 45 12 60 5 25 5 25
0.060 3 15 0 0 1 5 4 20 6 30 4 20
0.030 8 40 10 50 9 45 1 5 9 45 11 55
0.015 0 0 1 5 1 5 0 0 0 0 0 0
0.007 1 5 0 0 0 0 0 0 0 0 0 0
MRSA
1.000 0 0 0 0 0 0 0 0 0 0 0 0
0.500 5 26.3 5 26.3 5 26.3 7 36.8 5 26.3 5 26.3
0.250 8 42.1 8 42.1 8 42.1 7 36.8 8 42.1 8 42.1
0.125 6 31.6 6 31.6 6 31.6 5 26.4 6 31.6 6 31.6
0.060 0 0 0 0 0 0 0 0 0 0 0 0
0.030 0 0 0 0 0 0 0 0 0 0 0 0
Finally, the development of new inhaled antibiotics (or
antimicrobial-like drugs) could be of great importance to improve
the efficacy of the treatment of CF patients [22]. It is easy to
imagine that an essential oil could be developed into an inhalation
apparatus for use in patients with respiratory infections, as
previously reported for basil, rosemary and eucalyptus [23-25].
Nevertheless, toxicological screenings are mandatory before
suggesting a possible clinical use and possible additive/synergistic
effects should be investigated since EO combinations and/or EOs
plus antibiotics could boost their bactericidal effect, lowering the
concentrations needed, and minimizing in turn the risk of side
effects.
Experimental
Bacterial strains and growth conditions: The panel of 59 bacterial
strains (20 for S. maltophilia and A. xylosoxidans and 19 for
MRSA) tested in this work is reported in Table 1. The bacterial
strains were isolated from different CF patients and each strain was
maintained at -80°C under glycerol (25%, v/v) stock, and grown on
Columbia blood agar (Thermo Scientific, Oxoid SpA, Strada
Rivoltana, 20090 Rodano (MI) - Italy) at 37°C for 24 h.
Identification and typing of bacterial strains: Bacterial strains
were identified using Matrix Assisted Laser Desorption Ionization
Time-of-Flight (Maldi-Tof VITEK MS, bioMérieux Italia Spa,
Italy).
Antibiotic resistance profiling: Susceptibility was evaluated to
clinically-relevant antibiotics, specific for each pathogen [26-29]
and selected across different antimicrobial families. Minimum
Inhibitory Concentration (MIC) was evaluated for S. maltophilia by
E-Test, and for A. xylosoxidans and MRSA by an automated system
(Vitek 2, bioMérieux Italia Spa, Italy). Results were interpreted
according to the available EUCAST (b) breakpoint tables or CLSI’s
(2012).
Origanum vulgare essential oil: The O. vulgare EO, extracted by
steam distillation, was purchased from Prodotti Phitocosmetici Dott.
Vannucci di Vannucci Daniela e C. Sas, Prato, Italy. The
composition of the OEO used in this work has been already
reported [11]. MIC and MBC were determined in TSB added with
the EO in concentrations two-fold diluted from 2% to 0.007%, v/v,
and the same volume of dimethylsulphoxide (DMSO, Carlo Erba
Reagenti SpA, Milano, Italy), sterilized by filtration through filters
with a pore diameter of 0.22 µm (Sartorius Italy Srl, Monza e
Brianza, Italy).
Determination of MIC and MBC of O. vulgare EO: Determination
of MIC, in broth micro-dilutions, was performed as described in
standard protocols. Microtiter plates containing serial dilutions of
the OEO were inoculated with aliquots of 100 μL of bacterial
suspensions containing approximately 2×106 CFU/mL in a final
volume of 200 μL. The negative control contained 200 μL of TSB,
whereas the two positive controls contained TSB and DTSB (1% of
DMSO) inoculated with 100 μL of the bacterial suspension
respectively. A further negative control was set up using an
antibiotic able to inhibit the growth of the tested bacteria; different
antibiotics might be used according to the different resistance
pattern of the tested bacteria [26-29]. Microplates were incubated at
37°C aerobically. After incubation, the Infinite 200 PRO multimode
reader (Tecan), was used to detect density (using OD600).
From each tube, at time "0" a 10 μL aliquot of the suspension was
spread on TSA plates and incubated at 37°C aerobically; afterwards,
the number of CFU was determined. The effect of OEO on bacteria
was monitored at 24 h intervals up to 72 h by seeding 10 μL of the
suspensions on TSA plates. Data obtained allowed the
establishment of the bactericidal/bacteriostatic activity of OEO vs
each organism, in terms of MIC, intended as the lowest
concentration of OEO able to inhibit completely the growth of
microorganisms in tubes and on plates. The MBC was defined as
the concentration of OEO that killed at least 99.9% of the inoculum.
All assays were performed in triplicate. Colony growth was verified
even for each control. MIC and MBC were determined after
incubation for 24, 48 and 72 h.
Statistical analyses: Means, standard deviations of bacterial counts
and graphics were obtained through Microsoft Office Excel 2007
(Microsoft S.r.l., Milano, Italy).
Supplementary data: Details on the antibiotic resistance profile,
Origanum vulgare essential oil MIC and MBC of each strain are
also available.
Acknowledgments - This work was supported by Ente Cassa di
Risparmio di Firenze (project 2013.0657).

864 Natural Product Communications Vol. 11 (6) 2016 Pesavento et al.
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Natural Product Communications Vol. 11 (6) 2016
Published online (www.naturalproduct.us)
A Validated HPLC Method for Simultaneous Determination of Caffeoyl Phenylethanoid Glucosides and
Flavone 8-C-glycosides in Haberlea rhodopensis
Dimitrina Zheleva-Dimitrova, Paraskev Nedialkov and Ulrich Giresser 791
A Novel Heterodimer from Crotalaria ferruginea
Yi-Hong Zou, Xin Liu, Ye-Na Liu, Gui-Hua Tang and Sheng Yin 793
New 2-(2-Phenylethyl)chromone Derivatives and Inhibitors of Phosphodiesterase (PDE) 3A from Agarwood
Takuji Sugiyama, Yuji Narukawa, Shunsuke Shibata, Ryo Masui and Fumiyuki Kiuchi 795
Influence of Different Carbohydrates on Flavonoid Accumulation in Hairy Root Cultures of Scutellaria baicalensis
Chang Ha Park, Young Seon Kim, Xiaohua Li, Haeng Hoon Kim, Mariadhas Valan Arasu, Naif Abdullah Al-Dhabi,
Sook Young Lee and Sang Un Park 799
Pyran-2-one Derivatives from the Roots of Croton crassifolius
Weihuan Huang, Jiajian Wang, Yeyin Liang, Yaolan Li and Guocai Wang 803
New Isocoumarins and Related Metabolites from Talaromyces flavus
Jun-Wei He, Huai-Shuang Xu, Li Yang, Wei-Wei He, Chuan-Xi Wang, Feng Lin, Yang-Yun Lian, Bo-Hang Sun and Guo-Yue Zhong 805
A New Neolignan, and the Cytotoxic and Anti-HIV-1 Activities of Constituents from the Roots of Dasymaschalon sootepense
Sakchai Hongthong, Chutima Kuhakarn, Thaworn Jaipetch, Pawinee Piyachaturawat, Surawat Jariyawat, Kanoknetr Suksen,
Jitra Limthongkul, Narong Nuntasaen and Vichai Reutrakul 809
New Phenyl Propanoids from Cryptocarya bracteolata
Nurdin Saidi, Hiroshi Morita, Marc Litaudon, Mohd. Azlan Nafiah, Khalijah Awang and Mustanir 815
Anti-inflammatory Constituents from the Aerial Parts of Iris minutiaurea
Kyeong Wan Woo, Ki Ho Lee, Ji Hun Jang, Min Suk Kim, Hyun Woo Cho, Jung Hee Cho and Byeongkwan An 817
New Aminonaphthoquinone from the Sea Urchins Strongylocentrotus pallidus and Mesocentrotus nudus
Elena A. Vasileva, Natalia P. Mishchenko, Pavel A. Zadorozhny and Sergey A. Fedoreyev 821
Proteomic Analysis of the Antibacterial Mechanism of Action of Juglone against Staphylococcus aureus
Jiayi Wang, Zixue Wang, Rina Wu, Donghua Jiang, Bing Bai, Dehong Tan, Tingcai Yan, Xiyun Sun, Qi Zhang, and Zhaoxia Wu 825
A Pair of New Polyketide Enantiomers from Three Endolichenic Fungal Strains Nigrospora sphaerica, Alternaria alternata,
and Phialophora sp.
Jun-Wei He, Chuan-Xi Wang, Li Yang, Guo-Dong Chen, Dan Hu, Liang-Dong Guo, Xin-Sheng Yao and Hao Gao 829
Insight into the Presence of Stilbenes in Medicinal Plants Traditionally Used in Croatian Folk Medicine
Ivana Generalić Mekinić, Danijela Skroza, Ivica Ljubenkov and Višnja Katalinić 833
Two New Isomers of Palmityl-4-hydroxycinnamate from Flowers of Taraxacum Species
Matej Dudáš, Mária Vilková, Tibor Béres, Miroslav Repčák and Pavol Mártonfi 837
Chemical Composition, Herbicidal and Antifungal Activity of Satureja cuneifolia Essential Oils from Spain
David García-Rellán, Mercedes Verdeguer, Adele Salamone, María Amparo Blázquez and Herminio Boira 841
Composition of the Leaf Essential Oil of Phoebe formosana from Taiwan and its in vitro Cytotoxic, Antibacterial, and
Antifungal Activities
Yu-Chang Su and Chen-Lung Ho 845
Effects of Plant Density on the Number of Glandular Trichomes and on Yield and Quality of Essential Oils from Oregano
Teresa Tuttolomondo, Salvatore La Bella, Claudio Leto, Giuseppe Bonsangue, Raffaele Leone, Maria Cristina Gennaro,
Giuseppe Virga, Rosalinda Inguanta and Mario Licata 849
Antioxidant and Anti-inflammatory Activities of Essential Oils of Actinodaphne macrophylla and A. pruinosa (Lauraceae)
Wan Mohd Nuzul Hakimi Wan Salleh and Farediah Ahmad 853
Chemical Composition, Antioxidant and Cytotoxic Activities of Essential Oil of the Inflorescence of Anacamptis coriophora
subsp. fragrans (Orchidaceae) from Tunisia
Ridha El Mokni, Saoussen Hammami, Stefano Dall’Acqua, Gregorio Peron, Khaled Faidi, Jeremy Phillip Braude,
Houcine Sebei and Mohamed Hédi El Aouni 857
Essential Oil from Origanum vulgare Completely Inhibits the Growth of Multidrug-Resistant Cystic Fibrosis Pathogens
Giovanna Pesavento, Valentina Maggini, Isabel Maida, Antonella Lo Nostro, Carmela Calonico, Chiara Sassoli, Elena Perrin,
Marco Fondi, Alessio Mengoni, Carolina Chiellini, Alfredo Vannacci, Eugenia Gallo, Luigi Gori, Patrizia Bogani, Anna Rita Bilia,
Silvia Campana, Novella Ravenni, Daniela Dolce, Fabio Firenzuoli and Renato Fani 861
In Vitro Trials of Dittrichia graveolens Essential Oil Combined with Antibiotics
Dragoljub L. Miladinović, Budimir S. Ilić, Branislava D. Kocić, Marija S. Marković and Ljiljana C. Miladinović 865
Accounts/Reviews
Phytochemistry, Bioactivity and Potential Impact on Health of Juglans: the Original Plant of Walnut
Dongdong Bi, Yicheng Zhao, Rui Jiang, Yan Wang, Yuxin Tian, Xiaoyi Chen, Shaojuan Bai and Gaimei She 869

Natural Product Communications
2016
Volume 11, Number 6
Contents
Original Paper
Pubinernoid A and Apo-9'-fucoxanthinone, Secondary Metabolites from a Gorgonian Coral Pinnigorgia sp.
Hsin-Huan Chang, Yu-Chia Chang, Wu-Fu Chen, Tsong-Long Hwang, Lee-Shing Fang, Zhi-Hong Wen, Yu-Hsin Chen,
Yang-Chang Wu and Ping-Jyun Sung 707
A New Non-glucosidic Iridoid from the Roots of Strychnos nux-blanda
Jirapast Sichaem, Suttira Khumkratok, Pongpun Siripong and Santi Tip-pyang 709
New Cytotoxic Guaianolides from Centaurea aegyptiaca
Hanan G. Sary, Abdel Nasser B. Singab and Khaled Y. Orabi 711
Artemisinic Acid Serves as a Novel ORCA3 Inducer to Enhance Biosynthesis of Terpenoid Indole Alkaloids in
Catharanthus roseus Cambial Meristematic Cells
Mingxuan Wang, Jiachen Zi, Jianhua Zhu, Shan Chen, Pu Wang, Liyan Song and Rongmin Yu 715
New Antiplasmodial Diterpenes from Gutierrezia sarothrae
Qingxi Su, Seema Dalal, Michael Goetz, Maria B. Cassera and David G. I. Kingston 719
A New Cassane-type Diterpene from the Seed of Caesalpinia sappan
Hai Xuan Nguyen, Nhan Trung Nguyen, Phu Hoang Dang, Phuoc Ho Thi, Mai Thanh Thi Nguyen, Mao Van Can,
Dya Fita Dibwe, Jun-ya Ueda, Kinzo Matsumoto and Suresh Awale 723
Two New Oleanane-type Triterpenoids from Methanolyzed Saponins of Momordica cochinchinensis
Rong Fan, Rong-Rong Cheng, Hong-Tao Zhu, Dong Wang, Chong-Ren Yang, Min Xu and Ying-Jun Zhang 725
Isolation of a New Homomonoterpene from Madhuca pasquieri and Effect of Isolated Compounds on NO Production
Le Son Hoang, Manh Hung Tran, Van Thu Nguyen, Quynh Mai Thi Ngo, Joo-Sang Lee, Seong-Soo Rho, Jeong Ah Kim,
Jeong-Hyung Lee, Mi Hee Woo and Byung Sun Min 729
New Bioactive Semisynthetic Derivatives of 31-Norlanostenol and Obtusifoliol from Euphorbia officinarum
Maria Bailen, Mourad Daoubi Khamlichi, Ahmed Benharref, Rafael A. Martinez-Diaz and Azucena Gonzalez-Coloma 733
Variation of Ginsenosides in Ginseng of Different Ages
Jian-Ming He, Yi-Zhen Zhang, Jian-Ping Luo, Wen-Ju Zhang and Qing Mu 739
Colochiroside E, an Unusual Non-holostane Triterpene Sulfated Trioside from the Sea Cucumber Colochirus robustus and
Evidence of the Impossibility of a 7(8)-Double Bond Migration in Lanostane Derivatives having an 18(16)-Lactone
Alexandra S. Silchenko, Anatoly I. Kalinovsky, Sergey A. Avilov, Pelageya V. Andryjaschenko, Pavel S. Dmitrenok,
Ekaterina A. Yurchenko, Igor Yu. Dolmatov, Salim Sh. Dautov, Valentin A. Stonik and Vladimir I. Kalinin 741
Two New Steroidal Aglycones from Roots of Cynanchun paniculatum
Jia-wen Zhao, Feng-yang Chen, Li-juan Gao, Shi-fang Xu, Yi-ping Ye and Xiao-yu Li 747
Steroid Sulfates from Ophiuroids (Brittle Stars): Action on Some Factors of Innate and Adaptive Immunity
Anna K. Gazha, Lyudmila A. Ivanushko, Eleonora V. Levina, Sergey N. Fedorov, Tatyana S. Zaporozets, Valentin A. Stonik and
Nataliya N. Besednova 749
Cytotoxicity of Naturally Occurring Isoquinoline Alkaloids of Different Structural Types
Jakub Chlebek, Ivo Doskočil, Daniela Hulcová, Kateřina Breiterová, Marcela Šafratová, Radim Havelek, Klára Habartová,
Anna Hošťálková, Tereza Volštátová and Lucie Cahlíková 753
Hypoglycemic and Antioxidant Activities of the Water Extract of Aquilaria crassna Leaves in Streptozotocin-Nicotinamide-
Induced Type-2 Diabetic Mice
Suchada Manok, Bunleu Sungthong, Hitoshi Sato, Erika Sugiyama and Vilasinee Hirunpanich Sato 757
In Vitro Antifungal Activity of Kampo Medicine Water Extracts against Trichophyton rubrum
Xia Da, Hitoshi Takahashi, Kyaw Zaw Hein and Eishin Morita 763
Guarea kunthiana Bark Extract Enhances the Antimicrobial Activities of Human and Bovine Neutrophils
Natalja Jerjomiceva, Hisham Seri, Ragheda Yaseen, Nicole de Buhr, William N. Setzer, Hassan Y. Naim and
Maren von Köckritz-Blickwede 767
Red Wine Inhibits Aggregation and Increases ATP-diphosphohydrolase (CD39) Activity of Rat Platelets in Vitro
Elisabetta Caiazzo, Idolo Tedesco, Carmela Spagnuolo, Gian Luigi Russo, Armando Ialenti and Carla Cicala 771
How the Addition of Spices and Herbs to Virgin Olive Oil to Produce Flavored Oils Affects Consumer Acceptance
Manel Issaoui, Guido Flamini, Sondess Souid, Alessandra Bendini, Sara Barbieri, Ines Gharbi, Tullia Gallina Toschi,
Pier Luigi Cioni and Mohamed Hammami 775
Erythrina edulis (Pajuro) Seed Protein: A New Source of Antioxidant Peptides
Arturo Intiquilla, Karim Jiménez-Aliaga, Amparo I. Zavaleta, Inés Arnao, Carmen Peña, Elizabeth L. Chávez-Hidalgo and
Blanca Hernández-Ledesma 781
Cloning and Functional Analysis of Three Chalcone Synthases from the Flowers of Safflowers Carthamus tinctorius
Junichi Shinozaki, Hiromichi Kenmoku, Kenichi Nihei, Kazuo Masuda, Masaaki Noji, Katsuhiro Konno, Yoshinori Asakawa and
Kohei Kazuma 787
Continued inside backcover