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Characterization and antimicrobial activity of essential oils of industrial hemp varieties (Cannabis sativa L.)
Fitoterapia 81 (2010) 413–419
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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 Inflorescence 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 significantly 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 fibre hemp, has an incredible number of possible applications and, especially, it has attracted the interest in medical therapy and for textile uses [2–5]. 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: [email protected] (L. Nissen). 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.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 conflicting results on immunology and cannabinoids relationships were recently released [9]. Again, hundreds of biological active compounds such as
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cannabinoids, terpenoids, flavonoids and polyunsaturated fatty acids were found in high-THC hemp types [10,11], and some recent findings 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 inflorescence 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 field 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 inflorescences were collected from each plot, stored at −20 °C, distilled and finally analysed. 2.2. Determination and characterization of the essential oils Fresh inflorescences were steam distilled for 3 h in a Clevenger-type apparatus, as given by the Italian Official 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 flame ionization detector (FID) and a Hitachi D-2000 integrator (Hitachi, Tokyo, Japan) were used for gas-chromatography analyses. Samples were first diluted with ethanol absolute (1% v/v) and then injected into a silica column (Supelco fused SPB5, 30 × 0.32 mm, 0.25 µm of film thickness) operating in a range of 70–200 °C through progressive increase of 5 °C min− 1, holding the initial temperature for 18 min. The flow 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 specific software and gas-chromatograph, the latter operating under the above described conditions. The identification 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 Extrasynthése (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 Bifidobacteria), 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 modified by Biavati et al. [19]. Briefly, 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) 413–419 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 fluorescens
KB broth; 25 °C
Pseudomonas savastonoi pv. phaseolicola Pseudomonas syringae pv. atrofaciens Pseudomonas viridiflava
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 µl 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
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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 significant 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. Specifically, 55 compounds were identified, accounting for 95% of the whole GC profile. 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 4–6). 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 inflorescences and essential oils of industrial hemp varieties. f.w. = fresh weight. Variety
Carmagnola Futura Fibranova-I Fibranova-II
Inflorescences
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
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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 Identification 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 significantly 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 confirmed 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 significant 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 significant 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 fibres and materials for textile, paper or bio-building uses. However, if compared to synthetic fibres 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 fibres should be taken into great account in growing hemp as they might significantly 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 inflorescences for pharmaceutical industry are only some examples of potential complementary products that can be derived from industrial hemp [5]. The essential oils from inflorescences, 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
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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 significant 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
a
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
a
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
a
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 significant 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. flu.
Ps. cor
N2.00 1.24 N2.00 N2.00 1.84 N2.00 N2.00 1.66
b
a
a
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
c
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 [20–22]. 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. dificile, 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 significant 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
a
1.94 0.91 N 2.00 N 2.00 N 2.00 N 2.00 N 2.00 1.92
a b
a
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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 inflammation 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 specific 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. Specifically, 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 significant 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 specific 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 significant 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 confirmed in the present study by the use of standards (Tables 4–6). 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 β-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 influenced 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 specific 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 findings 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.
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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 Inflorescence 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 significantly 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 fibre hemp, has an incredible number of possible applications and, especially, it has attracted the interest in medical therapy and for textile uses [2–5]. 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: [email protected] (L. Nissen). 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.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 conflicting results on immunology and cannabinoids relationships were recently released [9]. Again, hundreds of biological active compounds such as
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cannabinoids, terpenoids, flavonoids and polyunsaturated fatty acids were found in high-THC hemp types [10,11], and some recent findings 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 inflorescence 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 field 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 inflorescences were collected from each plot, stored at −20 °C, distilled and finally analysed. 2.2. Determination and characterization of the essential oils Fresh inflorescences were steam distilled for 3 h in a Clevenger-type apparatus, as given by the Italian Official 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 flame ionization detector (FID) and a Hitachi D-2000 integrator (Hitachi, Tokyo, Japan) were used for gas-chromatography analyses. Samples were first diluted with ethanol absolute (1% v/v) and then injected into a silica column (Supelco fused SPB5, 30 × 0.32 mm, 0.25 µm of film thickness) operating in a range of 70–200 °C through progressive increase of 5 °C min− 1, holding the initial temperature for 18 min. The flow 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 specific software and gas-chromatograph, the latter operating under the above described conditions. The identification 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 Extrasynthése (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 Bifidobacteria), 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 modified by Biavati et al. [19]. Briefly, 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) 413–419 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 fluorescens
KB broth; 25 °C
Pseudomonas savastonoi pv. phaseolicola Pseudomonas syringae pv. atrofaciens Pseudomonas viridiflava
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 µl 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
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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 significant 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. Specifically, 55 compounds were identified, accounting for 95% of the whole GC profile. 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 4–6). 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 inflorescences and essential oils of industrial hemp varieties. f.w. = fresh weight. Variety
Carmagnola Futura Fibranova-I Fibranova-II
Inflorescences
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
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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 Identification 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 significantly 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 confirmed 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 significant 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 significant 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 fibres and materials for textile, paper or bio-building uses. However, if compared to synthetic fibres 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 fibres should be taken into great account in growing hemp as they might significantly 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 inflorescences for pharmaceutical industry are only some examples of potential complementary products that can be derived from industrial hemp [5]. The essential oils from inflorescences, 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
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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 significant 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
a
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
a
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
a
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 significant 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. flu.
Ps. cor
N2.00 1.24 N2.00 N2.00 1.84 N2.00 N2.00 1.66
b
a
a
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
c
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 [20–22]. 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. dificile, 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 significant 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
a
1.94 0.91 N 2.00 N 2.00 N 2.00 N 2.00 N 2.00 1.92
a b
a
418
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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 inflammation 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 specific 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. Specifically, 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 significant 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 specific 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 significant 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 confirmed in the present study by the use of standards (Tables 4–6). 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 β-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 influenced 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 specific 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 findings 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.
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