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Essential oils from Cannabis sativa L.

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Fitoterapia 81 (2010) 413419

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Fitoterapia
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f i t o t e

Characterization and antimicrobial activity of essential oils of industrial

hemp varieties (Cannabis sativa L.)
Lorenzo Nissen a,, Alessandro Zatta b, Ilaria Stefanini a, Silvia Grandi b, Barbara Sgorbati a,
Bruno Biavati a, Andrea Monti b
a
b

Microbiology Area, DiSTA (Department of Agroenvironmental Sciences and Technologies), Italy

GRiCI (Research Group on Industrial Crop), DiSTA, Alma Mater Studiorum, V.le Fanin 44, 40132, Bologna, Italy

a r t i c l e

i n f o

Article history:
Received 25 September 2009
Accepted in revised form 24 November 2009
Available online 4 December 2009
Keywords:
Cannabis sativa
Inorescence
GC-MS
MIC
Bacteria
Yeasts

a b s t r a c t
The present study focused on inhibitory activity of freshly extracted essential oils from three
legal (THC b 0.2% w/v) hemp varieties (Carmagnola, Fibranova and Futura) on microbial
growth. The effect of different sowing times on oil composition and biological activity was also
evaluated. Essential oils were distilled and then characterized through the gas chromatography
and gas chromatography-mass spectrometry. Thereafter, the oils were compared to standard
reagents on a broad range inhibition of microbial growth via minimum inhibitory
concentration (MIC) assay. Microbial strains were divided into three groups: i) Gram (+)
bacteria, which regard to food-borne pathogens or gastrointestinal bacteria, ii) Gram ()
bacteria and iii) yeasts, both being involved in plant interactions. The results showed that
essential oils of industrial hemp can signicantly inhibit the microbial growth, to an extent
depending on variety and sowing time. It can be concluded that essential oils of industrial
hemp, especially those of Futura, may have interesting applications to control spoilage and
food-borne pathogens and phytopathogens microorganisms.
2009 Elsevier B.V. All rights reserved.

1. Introduction
Hemp (Cannabis sativa (L.) is a C3 crop native to CentralNortheast Asia where there is evidence of its cultivation
dating back over 5000 years ago [1]. Hemp, namely industrial
hemp or bre hemp, has an incredible number of possible
applications and, especially, it has attracted the interest in
medical therapy and for textile uses [25]. It is widely know
how the worldwide commercially grown of industrial hemp
has been strongly limited because it can be easily confounded
with high-THC hemp types, namely marijuana, which are a
different breed of industrial hemp. In Europe, for example, the
cultivation of hemp is strictly ruled by the Commission
Regulation No 206/2004. Theoretically, all genotypes of
hemp, including those of industrial hemp, contain the
Corresponding author. Tel.: + 39 3289245215, + 39 0512096279;
fax: + 39 0512096274.
E-mail address: lorenzo.nissen@unibo.it (L. Nissen).
0367-326X/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.tote.2009.11.010

psychotropic agent delta9-tetrahydrocannabinol (THC),

which is a psychoactive compound. Nonetheless, unlike
marijuana, industrial hemp genotypes contains minute
amounts of THC, which does not generally exceeds 0.2%
(w/v), that is about 50 times lower than that of marijuana. To
the best of our knowledge, literature is rich of studies on
antibacterial activity of compounds extracted from high-THC
hemp types, which are known to contain powerful antibacterial agents [6]. Yet, it is somehow surprising that very few
studies focused on antimicrobial activity of essential oils
extracted from low-THC types, which, in contrast, could be
cultivated by farmers without restrictions. For example, some
authors [7,8] reported that smoking hemp may lead to a
decrease in immune functions while increasing opportunistic
infections due to excessive synthesis of Nitric Oxide from
pulmonary macrophages. A hypothesis that, however, still
remains questionable as conicting results on immunology
and cannabinoids relationships were recently released [9].
Again, hundreds of biological active compounds such as

414

L. Nissen et al. / Fitoterapia 81 (2010) 413419

cannabinoids, terpenoids, avonoids and polyunsaturated

fatty acids were found in high-THC hemp types [10,11], and
some recent ndings demonstrate that non-psychotropic
cannabinoids and their precursors are most likely antibacterial agents [6]. Basing on these results, interesting perspectives may arise from the cultivation of legal hemp varieties.
The purpose of this study was therefore to characterize and
assess the in-vitro antimicrobial activity of the essential oils
extracted from the inorescence of three legal hemp varieties
(low-THC content). In particular, we studied the antimicrobial activity against three types of microorganisms: i) Gram
(+), opportunistic and moderate pathogenic bacteria including Clostridium spp. and Enterococcus spp.; ii) Gram (),
phytopathogens bacteria including Pseudomonas spp. and
Pectobacterium spp.; and iii) yeasts, related to phytopathogens or human commensals.
2. Materials and methods
2.1. Material collection
Three hemp varieties, the Italian dioecious Carmagnola
and Fibranova [12] and the French monoecoius Futura [13],
were sown in the Po Valley at the experimental farm of the
University of Bologna (32 m a.s.l., 44 33 lat., 11 21 long.).
The eld experiment was organised according to a randomized block design with three replications. All crops were
conventionally sown in early April (04/06/2005), while
Fibranova was seeded concurrently with the other varieties
(Fibranova-I) and one month later (Fibranova-II) in order to
evaluate the effect of sowing time on oil composition and
antimicrobial activity. For all crops, the seed density was
240 seeds m 2 (13 cm row spaced), while plant densities at
the end of emergence were 112 16, 155 7, 135 6 and
140 1 for Carmagnola, Futura, Fibranova-I and -II, respectively. The harvest was carried out when occurring about 50
70% of seed maturity, corresponding to the phonological
codes 2204 and 2306 [14] for dioecious and monoecius
varieties, respectively. This stage is considered optimal to
maximize oil yield [14,15]. Phenological stages were monitored in 25 labelled plants per variety. After blooming, the
about 300 g of inorescences were collected from each plot,
stored at 20 C, distilled and nally analysed.
2.2. Determination and characterization of the essential oils
Fresh inorescences were steam distilled for 3 h in a
Clevenger-type apparatus, as given by the Italian Ofcial
Pharmacopoeia [16]. Thereafter, oils were decanted, dried
over anhydrous sodium sulfate, stored in dark glass vials and
kept at 4 C until analysis. HRGC 5160 mega gas-chromatograph (Carlo Erba, Milan, Italy) quipped with a ame
ionization detector (FID) and a Hitachi D-2000 integrator
(Hitachi, Tokyo, Japan) were used for gas-chromatography
analyses. Samples were rst diluted with ethanol absolute
(1% v/v) and then injected into a silica column (Supelco fused
SPB5, 30 0.32 mm, 0.25 m of lm thickness) operating in
a range of 70200 C through progressive increase of
5 C min 1, holding the initial temperature for 18 min. The
ow rate of the carrier gas (helium) was 1 ml min 1, while
the detector temperature was 250 C. The GC/MS analyses

were run through an Ion Trap detector (Finnigan Mat, San

Jose, Ca; model 800); set at 70 eV, equipped with specic
software and gas-chromatograph, the latter operating under
the above described conditions. The identication of the oil
components was made through the comparison of the
relative retention times of a reference, by co-elution and by
mass spectrometry. The detection of the components whose
references were not available was obtained through the
correspondence analysis of mass spectra with ITD library or as
given by Adams [17]. Quantitative data were obtained from
normalized area values.
2.3. Reagents and standards
Some major components of C. sativa oils, i.e. alpha-pinene,
beta-pinene, alpha-humulene and myrcene, were provided
by Sigma-Aldrich (St. Louis, Mo). Camphene, sabinene, alphaphellandrene, 1,8-cineole, borneol, alpha-terpineol, alloaromadendrene were purchased from Extrasynthse (Genay,
France). Limonene was provided by Carl Roth GMBH
(Karlsruhe, Deutschland), while, delta-3-carene, gammaterpinene, terpinolene, cis-beta-farnesene, trans-nerolidol
were given by Trade TCI Mark (Tokyo, Japan). Linalool,
terpinen-4-ol, beta-caryophyllene and caryophyllene-oxide
were purchased from Sigma-Aldrich. All compounds were
analytical grade standards.
2.4. Microbial strains and growth conditions
All Gram (+) lyophilised bacteria belonged to BUSCoB
(Scardovi's Collection of Bidobacteria), the Culture Collection of the University of Bologna. Streptococcus salivarius
subsp. thermophilus, Enterococcus and Clostridium spp. were
grown in De Man, Rogosa, Sharpe (MRS) (Merck, Darmstadt,
Germany) at 37 C for 48 h in aerobic and anaerobic
conditions by the use of Anaerogen (Oxoid Ltd, Hampshire,
UK). The entire Gram () bacteria were provided by the
Culture Collection of the University of Bologna (IPV-BO). They
were routinely and aerobically cultured in selective in King's
medium B (KB) at 25 C for 4 days. Finally, the yeasts were
provided by the University of Perugia (DBVPG). They were
cultured in Sabouraud broth (Merck, Germany) at 30 C for 4
6 days in aerobic conditions (Table 1). Strains were maintained at 80 C in 20% (v/v) glycerol (Merck) with the
appropriate medium. Inocula stock suspensions were prepared by diluting the cultures in phosphate-buffered saline
(PBS) to approximately 108 and 107 cfu ml 1 for bacteria and
yeasts, respectively.
2.5. Antimicrobial activity
Minimum inhibitory concentration (MIC), the lowest
essential oil concentration showing no visible microbial
growth after incubation time, was determined adopting the
broth dilution method as described by Hammer et al. [18],
and then slightly modied by Biavati et al. [19]. Briey, oils
were prepared as 10% (v/v) solutions in ethanol and stored at
4 C until analysis. Geometric solutions of the oils and the
standard components, ranging from 0.2 to 2.0% (v/v), were
diluted in an appropriate broth and distributed in a 96 well
microtiter plate with round bottom. Growth controls without

L. Nissen et al. / Fitoterapia 81 (2010) 413419

Table 1
Description of tested microorganisms.
Strain description
Gram (+) bacteria
Clostridium bifermentas
Clostridium butyricum
Clostridium sporogenes
Clostridium
tyrobutyricum
Enterococcus hirae
Enterococcus faecium
Streptococcus salivarius
subsp. thermophilus
Gram () bacteria
Pectobacterium
carotovorum subsp.
carotovorum
Pseudomonas corrugata

Cultivation method Reference/strain

type
MRS broth;
MRS broth;
MRS broth;
MRS broth;

37 C
37 C
37 C
37 C

MRS broth; 37 C
MRS broth; 37 C
MRS broth; 37 C

M151 (ATCC 8043)

M135 (DSM b 2146)
M52 (DSM 20259)

KB broth; 25 C

2731 IPV-BO
(DSM 301618)

KB broth; 25 C

2450 NCPPB
(DSM 7228)
90/1 OMP-BO
(DSM 50090)
1549 GSPB
(DSM 50282)
14B IPV-BO
(DSM 50255)
2786 IPV-BO
(DSM 11124)
2293 IPV-BO
(NCPPB d 528)

Pseudomonas uorescens

KB broth; 25 C

Pseudomonas savastonoi
pv. phaseolicola
Pseudomonas syringae
pv. atrofaciens
Pseudomonas viridiava

KB broth; 25 C

Pseudomonas campestris
pv. pruni
Yeasts
Candida sake

KB broth; 25 C
KB broth; 25 C
KB broth; 25 C

Sabouraud
broth; 30 C
Kluyveromyces marxianus Sabouraud
broth; 30 C
Pichia membranaefaciens Sabouraud
broth; 30 C
Saccharomyces cerevisiae Sabouraud
broth; 30 C
Schizosaccharomyces
Sabouraud
pombe
broth; 30 C
Schizosaccharomyces
Sabouraud
japonicus
broth; 30 C
Torulospora delbrueckii
Sabouraud
broth; 30 C
Zygosaccharomyces bailii Sabouraud
broth; 30 C

a
b
c
d

M175 (NCTC c 506)

M167 (ATCC a 14823)
M170 (NCTC 8594)
M60 (ATCC 25755)

6154 DBVPG
(DSM 70763)
6165 DBVPG
(DSM 4906)
6215 DBVPG
(DSM 70169)
6173 DBVPG
(DSM 70449)
6277 DBVPG
(DSM 2791)
6274 DBVPG
(DSM 70570)
6167 DBVPG
(DSM 70483)
6287 DBVPG
(DSM 70492)

American Type Culture Collection.

Deutsche Sammlung von Mikroorganismen.
National Collection of Type Culture.
National Collection of Plant Pathogenic Bacteria.

the ethanolic solution and with the upper limit of added

ethanol were included in each plate. Wells containing 200 l
solutions were inoculated with a microbial suspension at the
concentration of 106 cfu ml 1. A preliminary evaluation of
the microbial growth was obtained through determining the
cell mass on well bottoms and then by measuring the pH
decrease using a pH-meter. For anaerobic bacteria an
additional evaluation of pH was based on bromocresol
whose colour switches from purple to yellow parallel to the
pH decrease during fermentation. Microbial growth evaluation was then determined using a spectrophotometer plate
reader (Multiskan, Thermo Electron Oy, Vaanta, Finland) by
setting the absorbance at 600 nm. The results are the average
of three independent analyses. The minimum bactericidal
concentration (MBC), i.e. the lowest concentration able to kill

415

99.99% of microbial population, of the essential oils of Futura

versus key microbial species was also determined. The
solutions were prepared in serial dilutions ranging from the
obtained MIC values to 4.0% (v/v), as previously described by
Hammer et al. [18].
2.6. Statistical analysis
The effects of essential oils on Gram (+), yeasts and Gram
() bacteria were evaluated separately for each microbial
category through one-way analysis of variance (ANOVA). Before
ANOVA, the homogeneity of variance was tested by the
Bartlett's test. When ANOVA revealed signicant differences
among means (P 0.05), the pairwise comparison Tukey's tests
(HSD) was used to separate means into statistically different
groups (P 0.05).
3. Results
3.1. Essential oils
Among the varieties, Futura achieved the highest content
and yield of essential oils followed by Fibranova-I, while
Carmagnola and Fibranova-II resulted in a very similar amount
of essential oils (Table 2). The essential oils characterization is
reported in Table 3. Specically, 55 compounds were identied,
accounting for 95% of the whole GC prole. Most of them (28)
were sesquiterpenes in very low concentrations, with the
exception of beta-caryophyllene and alpha-humulene that
included 13% and 5% of total compounds, respectively.
Myrcene, alpha-pinene and beta-pinene were the main
compounds among the monoterpens. Generally, all varieties
revealed a consistent qualitative composition, but with some
relevant quantitative differences: Carmagnola registered the
highest myrcene content, while Futura showed the highest
amount of terpinolene; in contrast, alpha-pinene, limonene,
beta-caryophyllene and alpha-humulene were present in
similar amount in all varieties (Table 3).
3.2. Antimicrobial activity
Overall, the essential oils exhibited good antimicrobial
activities expressed as minimum inhibitory concentrations
(Tables 46). With respect to Gram (+) bacteria, which are
widely known as responsible of some pathogenesis in the
human gastrointestinal tract, essential oils resulted in an
effective control of Enterococcus hirae, Enterococcus faecium
and S. salivarius subsp. thermophilus. In contrast, only Futura
gave satisfactory results against clostridia, and moreover, it

Table 2
Production of inorescences and essential oils of industrial hemp varieties.
f.w. = fresh weight.
Variety

Carmagnola
Futura
Fibranova-I
Fibranova-II

Inorescences

Essential oils
2

% f.w.

gm

10.5
15.3
12.4
12.0

328 38
656 35
517 81
342 29

% f.w.

g m 2

0.26
0.31
0.25
0.23

86 8
201 9
132 24
82 4

416

L. Nissen et al. / Fitoterapia 81 (2010) 413419

Table 3
Characterization of the essential oils of different hemp varieties. tr = traces;
nd = not detected.
Components a

Hemp varieties
Carmagnola Fibranova-I Fibranova-II Futura

Tricyclene
Alpha-thujene
Alpha-pinene
Camphene
Sabinene
Beta-pinene
Myrcene
Alpha-phellandrene
Delta-3-carene
Alpha-terpynene
Para-cymene
Limonene
1,8-cineole
Cis-ocimene
Trans-ocimene
Gamma-terpinene
Terpinolene
Linalool
Beta-fenchol
Neo-allo-ocimene
Ipsdienol
Borneol
Terpinen-4-ol
Alpha-terpineol
Alpha-longipinene
Alfa-cubebene
Hexyl-caproate
Gamma-caryophyllene
Alpha-gurjunene
Beta-caryophyllene
Gamma-elemene
Alpha-bergamotene
trans
Alpha-guaiene
Cis-beta farnesene
Alpha-humulene
Alloaromadendrene
9-epi-caryophyllene
Drima-7,9 (11)-diene
Beta chamigrene
Gamma-muurolene
Beta-selinene
Alpha-selinene
Alpha-zingiberene
Alpha-farnesene trans
Beta-bisabolene
Gamma-cadinene
Delta-cadinene
Selina-3,7 (11)-diene
Germacrene b
Trans-nerolidol
Caryophyllene-oxide
Humulene 1,2 epoxide
Gamma-eudesmol
Alpha-bisabolol
Selin-7 (11)-en-4-ol
Total monoterpenes
Total sesquiterpenes
E.O. yield (%w/v)

0.10
0.07
15.12
0.26
tr
6.38
29.22
0.15
0.15
0.15
tr
4.87
0.28
0.20
6.91
0.14
3.42
0.34
0.16
0.17
0.12
0.06
0.08
0.09
0.07
tr
tr
0.04
tr
13.90
0.09
0.09

0.11
0.14
16.99
0.35
tr
9.33
20.33
0.24
1.79
0.20
tr
4.60
0.17
0.16
2.03
0.20
5.10
0.44
0.18
0.14
0.19
0.10
0.15
0.25
0.08
tr
tr
0.18
0.07
13.78
0.69
0.24

0.08
0.26
10.9
0.37
0.06
8.9
12.46
0.16
3.48
0.18
0.05
4.99
0.66
0.55
9.34
0.21
3.97
0.35
0.19
0.16
0.16
0.11
0.15
0.22
0.11
0.02
0.04
0.21
0.21
10.56
2.23
0.24

0.14
0.12
16.39
0.34
0.11
6.54
20.82
0.53
0.66
0.39
0.15
3.11
0.07
0.37
4.83
0.32
10.73
0.19
0.09
0.11
0.29
0.09
0.30
0.17
0.12
tr
tr
0.12
0.18
12.76
1.62
0.11

0.06
nd
5.11
0.29
tr
0.30
0.15
0.29
0.71
0.58
0.44
0.58
0.23
tr
tr
0.42
0.15
tr
0.51
0.12
0.23
0.05
0.11
68.4
26.11
0.26

tr
0.83
5.72
0.48
tr
tr
0.11
0.05
0.99
0.77
tr
0.53
0.53
tr
tr
0.52
1.13
0.06
1.05
0.25
0.36
0.07
0.17
63.24
31.51
0.25

0.38
2.67
6.71
0.57
0.05
tr
0.29
0.1
1.71
1.79
0.58
0.81
0.61
0.04
tr
1.18
0.43
0.69
1.05
0.27
0.31
tr
0.18
58.06
37.97
0.23

tr
2.21
4.84
0.68
tr
tr
0.17
tr
0.60
0.49
0.08
0.33
0.33
tr
tr
0.67
0.42
tr
1.27
0.29
0.15
tr
0.01
66.98
28.7
0.31

a
Identication of the components was obtained through the comparison
of the relative retention times of a compound and its reference substance or
using those reported by Adams [17].

registered signicantly lower MIC values than Carmagnola

and Fibranova in almost all Gram (+) bacteria (Table 4).
Among standards, alpha-pinene completely inhibited Gram

(+) bacteria growth, while alpha-humulene failed in three

cases out of seven, particularly on clostridia (Table 4). Futura
conrmed the best results even on Gram (), with MIC
values always well below the threshold limit (2.00% v/v).
As for the other varieties, it should be noted that both
Carmagnola and Fibranova-II were above the MIC limit only
in one case out of seven: Pseudomonas savastonoi and Pectobacterium carotovorum subsp. carotovorum, respectively for
the two varieties (Table 5). Importantly, sowing time showed
a signicant effect on microbial inhibitory activity. In fact, the
essential oils of Fibranova-II controlled two more bacteria
than the oils of Fibranova-I. Among standards, alpha-pinene
was again the most effective compound for contrasting Gram
() bacteria (Table 5). On the other hand, alpha-humulene
provided unsatisfactory results in six cases out of seven. With
respect to yeasts, the essential oils were unable to inhibit
Saccharomyces cerevisiae, and, with the exception of Futura,
Torulospora delbrueckii and Zygosaccharomyces bailii too
(Table 6). Carmagnola showed signicant lower MIC values
than Futura in Kluyveromyces marxianus and Pichia membranaefaciens, while the opposite was in Schizosaccharomyces
japonicus. Alpha-humulene gave the worst results among
standard compounds, while, once again, alpha-pinene was
the most effective standard compound (Table 6). Very few
compounds were capable to inhibit the entire group of Gram
(+), Gram (), or yeasts. Alpha-pinene was the only
showing MIC values below the detection limit in all the
examined cases, while the essential oils of Futura were the
unique capable to inhibit all Gram (+) and Gram () bacteria
(Fig. 1). Given that Futura provided the best essential oils
among hemp varieties, the minimum bactericidal concentration (MBC) on key Gram (+) and Gram () groups was
determined on this variety. The essential oils of Futura killed
Gram (+) and () bacteria at MBC doses about twice higher
than corresponding MIC values (Table 7).
4. Discussion
Industrial hemp is widely known to provide high quality
bres and materials for textile, paper or bio-building uses.
However, if compared to synthetic bres or other competing
crops, such as cotton, it still remains uneconomic as it
requires much labour and many passages before obtaining
marketable products [5]. Therefore, complementary products
along with bres should be taken into great account in
growing hemp as they might signicantly enhance its
competitiveness. Thanks to several sets of potentially available bio-based materials for innovative applications, hemp
has been taken as model of a multi-use crop. The oil derivates
for cosmetic industries or the essential oils and secondary
metabolites from inorescences for pharmaceutical industry
are only some examples of potential complementary products that can be derived from industrial hemp [5]. The
essential oils from inorescences, especially those of legal
hemp varieties, have received very little attention by the
researchers, in spite of the potential relevance that may have
in phyto-medical technology, the discipline studying the
plant derived pharmaceutical and nutraceutical compounds.
For example, the antimicrobial properties of hemp oils, can be
used against nosocomial, spoilage and food-borne pathogens,
such as clostridia causing gas-gangrene, food poisoning and

L. Nissen et al. / Fitoterapia 81 (2010) 413419

417

Table 4
Antimicrobial activity of the essential oils of hemp and standard compounds on Gram (+) bacteria as minimum inhibitory concentration (MIC) assay (% v/v). MIC
data above detection limit (2.00% v/v) were not included in the statistical analysis. Different letters mean signicant different means (P 0.05; Tukey's HSD test).
Three independent experiments were performed. For full names of bacteria see Table 1.
Oil a

MIC (% v/v)
C. bif.

Alpha-h
Alpha-p
Beta-p
My
Ca
F-I
F-II
Fu

C. but.

1.90
1.17
1.36
1.39
1.75
1.80
1.73
1.41

a
c
b
b
a
a
a
b

C. spo.

N 2.00
0.93
1.35
1.55
N 2.00
N 2.00
N 2.00
1.76

d
c
b

N 2.00
1.29
1.52
N 2.00
N 2.00
N 2.00
1.69
1.78

C. tyr.
b
bc

ab
a

N 2.00
1.14
N 2.00
N 2.00
N 2.00
N 2.00
N 2.00
1.66

E. hir.
b

1.69
0.80
1.16
1.56
1.80
1.81
1.69
1.40

E. fae.
a
d
c
ab
a
a
a
b

1.87
0.75
1.34
1.71
1.70
1.78
1.64
1.55

S. sal.
a
d
c
a
ab
ab
b
bc

1.53
1.26
N2.00
1.84
1.54
1.83
1.57
1.46

ab
b
a
ab
a
ab
ab

Abbreviations: alpha-h = alpha-humulene; alpha-p = alpha-pinene; beta-p = beta-pinene; My = myrcene; Ca = Carmagnola; F-I and -II = early and late
sown Fibranova, respectively; Fu = Futura.

Table 5
Antimicrobial activity of the essential oils of hemp and standard compounds on Gram () bacteria as minimum inhibitory concentration (MIC) assay (% v/v). MIC
data above detection limit (2.00% v/v) were not included in the statistical analysis. Different letters mean signicant different means (P 0.05; Tukey's HSD test).
Three independent experiments were performed. For full names of bacteria see Table 1.
Oil a

MIC (% v/v)
P. car.

Alpha-h
Alpha-p
Beta-p
My
Ca
F-I
F-II
Fu
a

Ps. u.

Ps. cor

N2.00
1.24
N2.00
N2.00
1.84
N2.00
N2.00
1.66

N2.00
1.41
1.81
1.97
1.81
1.78
1.76
1.40

N2.00
1.59
1.80
1.82
1.71
1.88
1.74
1.35

b
a
a
a
a
a
b

Ps. sav.
b
ab
ab
ab
a
ab
c

N2.00
1.05
N2.00
1.95
N2.00
N2.00
1.81
1.68

Ps. syr
c
a

ab
b

Ps. vir.

N 2.00
1.41
N 2.00
N 2.00
1.88
1.89
1.84
1.62

1.88
1.40
1.81
1.85
1.68
1.84
1.79
1.43

a
a
a
b

Ps. cam.
N 2.00
1.56
N 2.00
N 2.00
1.76
N 2.00
1.89
1.44

a
b
a
a
a
a
a
b

bc

ab
a
c

For abbreviations see Table 4.

intestinal syndromes [2022]. In this respect, the present

results show promising inhibitory activities of hemp oils against
Gram (+) opportunistic/pathogens such as Clostridium spp. and
Enterococcus spp. Moreover, other clostridia species, very
similar to C. dicile, have been also recognised as nosocomial
multi antibiotic-resistant pathogens [23,24], and some strains of
Clostridium butyricum and Clostridium sporogenes can produce
neurotoxin A and B that are associated with botulism and colitis

[24,25], whereas Clostridium bifermentas can originate bacteraemia and abscess [26,27]. In addition, there are many cases in
which clostridia are reported as food-borne pathogens and
implicated in food spoilage [28]. A number of human infections
caused by E. hirae and E. faecium are also reported in the medical
literature, while antibiotic multi resistant food-borne and
nosocomial pathogens are renowned [29,30]. Again, betacaryophyllene, which is especially concentrated in hemp

Table 6
Antimicrobial activity of the essential oils of hemp and standard compounds on yeasts as minimum inhibitory concentration (MIC) assay (% v/v). MIC data above
detection limit (2.00% v/v) were not included in the statistical analysis. Different letters mean signicant different means (P 0.05; Tukey's HSD test). Three
independent experiments were performed. For full names of yeasts see Table 1.
Oil a

Alpha-h
Alpha-p
Beta-p
My
Ca
F-I
F-II
Fu
a

MIC (% v/v)
C. sak.

K. mar.

P. mem.

N 2.00
1.17
1.26
1.45
1.87
1.81
1.70
1.63

N2.00
0.76
1.62
1.81
1.38
1.55
1.54
1.43

1.76
0.70
1.68
1.80
1.14
1.48
1.49
1.36

c
c
b
a
a
a
ab

For abbreviations see Table 4.

c
ab
ab
d
bc
bc
ab

S. cer.
a
e
ab
a
d
bc
bc
c

1.82
0.82
1.43
1.46
N2.00
N2.00
N2.00
N2.00

a
c
b
b

S. pom.

Sc. jap.

N 2.00
0.73
1.37
1.63
1.72
1.91
1.75
1.77

1.57
0.84
1.34
1.55
1.91
1.73
1.72
1.45

c
b
ab
a
a
a
a

T. del.
bc
e
d
bc
a
ab
ab
cd

N 2.00
0.91
N 2.00
N 2.00
N 2.00
N 2.00
N 2.00
1.85

Z. bai.
b

1.94
0.91
N 2.00
N 2.00
N 2.00
N 2.00
N 2.00
1.92

a
b

418

L. Nissen et al. / Fitoterapia 81 (2010) 413419

Fig. 1. Essential oils and standard compounds able to inhibit all considered
Gram (+), Gram () or yeasts (in white, grey and blank, respectively).
Essential oils and standards whose minimum inhibitory concentration (MIC)
exceeded the detection limit (2.00% v/v) for even a single bacteria or yeast
were therefore not reported. Alpha-p = alpha-pinene.

essential oil, has been shown to be involved in the mechanisms

to reduce tissue inammation in humans [31]. Among the three
hemp varieties, the qualitative composition of essential oils was
very consistent. For all varieties the main components were, in
fact, myrcene, alpha- and beta-pinene, beta-caryophyllene,
alpha-humulene, trans-ocimene, limonene and terpinolene.
Nonetheless, some important differences were found on
quantitative composition, which may somehow explain the
different behaviour of the essential oils in relation to microbial
inhibitory activity. In theory, all the varieties provided interesting oils for inhibiting specic microorganisms, but those of
Futura clearly resulted in the clearly most effective and broadest
antimicrobial activity. Moreover, though this oil only partially
controlled the yeasts, it was the only capable to inhibit all Gram
(+) and Gram () bacteria. Even including standard compounds, only alpha-pinene showed a similar broad action
spectrum (Fig. 1). The promising results of Futura oils were
corroborated by the MBC values in selected key bacterial
species. Specically, MBC was higher in Gram () than Gram
(+) bacteria that, conversely, are generally more susceptible to
the bactericidal activity of antimicrobials due to different

Table 7
Antimicrobial activity expressed as minimum bactericidal concentration
(MBC) assay (% v/v) of the essential oils of Futura and alpha-pinene on key
species of Gram (+) and Gram () bacteria. Three independent experiments
were performed.
Bacterial strains

Gram (+)
Clostridium sporogens
Enterococcus faecium
Streptococcus salivarius
subsp. thermophilus
Gram ()
Pectobacterium carotovorum
subsp. carotovorum
Pseudomonas savastanoi
pv. phaseolicola

MBC (% v/v)
Futura

Alpha-pinene

2.83 0.24
2.56 0.65
2.19 0.27

1.67 0.16
1.39 0.20
1.48 0.51

3.12 0.73

1.66 0.28

3.71 0.58

1.35 0.15

membrane structures making Gram () more resistant to

essential oils [32]. Apparently, Futura did not show signicant
differences in oil composition with respect to the other varieties,
except for the content in terpinolene, which was reported to be
more than two-fold than other varieties. Terpinolene is a
monoterpenic constituent of some essential oils of several
species which is characterized to have antifungal activity
against various pathogens [33]. Most likely, the specic
proportion among diverse compounds was the reason of the
higher performance of the essential oils of Futura. Although the
variation in the oil composition in response to sowing time was
examined in only one variety (Fibranova), it should be
emphasized that crop age determined signicant differences
on microbial inhibitory activity. Again, data were not enough
detailed to provide a mechanistic explanation of the different
behaviour of oils extracted from early and late sown plants.
Moreover, except for very few compounds such as transocimene and total sesquiterpenes, the majority of oil components, particularly alpha-pinene and myrcene, were unexpectedly higher in Fibranova-I compared to Fibranova-II as the latter
showed an overall effective inhibitory activity. The antimicrobial activity of alpha-pinene have been well documented [6,34
37], and once again they were conrmed in the present study by
the use of standards (Tables 46). It derives that the essential
oils of Fibranova-I should be expected to have a stronger
antimicrobial activity. On the other hand, as the interactions
among single components of essential oils are still an open
matter [38], a tentative hypothesis could be that the synergic
and antagonistic effects of oil compounds were the cause of
different activities of the two oils; the synergistic activity of
monoterpens, such as terpinolene and pinenes, and sesquiterpens, such as -caryophyllene, in plant essential oils has been in
fact demonstrated in previous studies [39,40]. Whatever was
the mechanistic reason behind the different behaviour of the
two oils, the fact that the microbial activity can be greatly
inuenced by sowing time and by plant age represents a new
insight in the use of essential oils of hemp, and it likely
stimulates further agronomic researches. In this view, maximizing the oil production could be not the main goal for farmers,
but priority should be given to identify the best compromise
between sowing time and oil yield for a specic application.
In conclusion, the essential oils of industrial hemp showed
interesting antimicrobial activities that strength the hypothesis
to grow hemp as a multi-use crop. Taking into account that new
forms of bacterial resistance to antimicrobials are rising
unconstrained, and that the problem of pathogenic bacteria
bearing multi-drug resistance (MRD), such as MRSA, which is
rapidly spreading worldwide accounting for more deaths per
year than AIDS [41], these ndings could be of particular
interest for human and animal health. Essential oils may
represent an economic and effective antiseptic topical treatment. They could be also successfully used against antibioticresistant strains and, thanks to the synergy with routinely used
antimicrobials, for topic applications in the treatment of
wounds and skin infections. Importantly, essential oils were
extracted from approved low-THC hemp types and not from
illegal cultivars. Although, further studies are needed, the use of
essential oils of hemp against microbial growth, especially
opportunistic and pathogenic microorganisms, seems a valuable alternative as antibiotics or antibacterial compounds,
especially in the cases of antibiotic resistance.

L. Nissen et al. / Fitoterapia 81 (2010) 413419

Acknowledgment
The research was partially funded by the European
Commission (HEMP-SYS Project QLK5-CT-2002-01363).
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