1. Introduction
Aromatic and medicinal plants offer a wide range of bioactive molecules used to contrary the spread of drug resistance pathogenic bacteria and fungi that cause severe and life-threatening infections [
1,
2]. Spices and aromatic plants are commonly used in Mediterranean regions in food preparation due to their antibacterial activities against several pathogenic Gram positive and Gram negative bacteria including:
Listeria,
Vibrio,
Staphylococcus,
Pseudomonas,
Salmonella, Bacillus and
Microccocus genera with multidrug resistance profiles [
3,
4]. The increasing of multi-resistance developed in most community, and hospital acquired pathogens can be attributed to the high use of antibiotics by consumers to feat pathogens. The scientific community developed since many years an interest in aromatic and medicinal plants with antimicrobial properties [
1].
Essential oils are aromatic oily liquids with very complex nature known for their antimicrobial and medicinal properties [
1]. Nowadays, some volatile oils and their mains components are used in food industry as antimicrobials due to their ability to control natural spoilage food process and to prevent growth of foodborne pathogens acting as food preservation and food safety molecules [
3].
It is well known that both Gram positive and Gram negative pathogenic bacteria regulate their virulence traits using the quorum sensing (QS) system throughout the expression of a variety of genes as function of bacterial cell density [
5]. For example, many virulence properties are regulated by the QS system including the synthesis of violacein in
Chromobacterium violaceum ATCC 12472 [
6,
7], biofilm formation, swarming on semi-solid agar medium, production of elastase, protease and pyocyanin in the
Pseudomonas aeruginosa PAO1 [
8]. Several aromatic/medicinal essential oil and organic extract have been tested for their anti-QS activities [
9,
10]. A large variety of plant-derived molecules were described for their ability to interfere with the autoinducers molecules synthesized by Gram positive bacteria (AIP) and Gram negative bacteria (AHL) and attenuates the behavior of the QS-controlled virulence factors expression in both
C. violaceum and
P. aeruginosa PAO1 biomonitor strains [
11,
12].
The cardamom of commerce is called small green cardamom or the true cardamom (
Elettaria cardamomum). There are many other plants belonging to the
Amomum and
Aframomum genera, both belonging to the cardamom family, producing aromatic seeds. Among them, clubbed together as false cardamoms, the most important and the one that is being grown commercially is
Amomum subulatum. Most of these false cardamoms are used as flavoring plants and also as remedies for various ailments [
13].
Elettaria cardamomum (L.) Maton is a tall, perennial, reed-like herb growing wild in rainforests of South India, Sri Lanka, and other tropical countries [
9,
10]. The plant is one of the world very ancient and expensive spices and is known as “the queen of spices” [
14].
Amomum subulatum (A. Braun) P.C.M. Jensen, black cardamom or large cardamom [
15], is native to Sikkim. The Sikkim State of India alone contributes 50% of the world production of large cardamom [
16]. Korarima (
Aframomum corrorima (Braun) P.C.M. Jansen), also called Ethiopian cardamom or false cardamom, is native to Ethiopia but it is cultivated on a small scale in some West African countries. Korarima is a perennial, aromatic herb, and bearing flowers either terminally on aerial leaf shoots or from the ground level. Korarima is one of the aromatic medicinal plants used in traditional medicine by the people of southern Ethiopia [
17,
18,
19].
Specifically, Eyob and colleagues [
18] conducted an ethnobotanical survey in the southern regions of Ethiopia on the three major korarima growing wild including Gamo Gofa, Debub Omo and Kaffa. They identified 38 and 52 compounds, respectively in leaf and rhizome essential oils. The major component of the oil of the leaf was β-caryophyllene (60.7%). The rhizome oil was dominated by γ-terpinene (21.8%) and β-pinene (17.6%).
It has been reported that in the chemical composition of different species of cardamoms, monoterpenes are the main compounds [
14,
17,
20,
21,
22]. Also, antimicrobial activity of the cardamoms is reported [
23,
24]. On black cardamom, 1,8-cineole is the main component [
15,
25]. Eyob and co-workers [
17,
18] reported the essential oil composition of Ethiopian cardamom. The presence of different structures of biomolecules in the volatile oils, allows a wider spectrum of bioactivity. Recently, it has been demonstrated that organic extracts from
Amommum tsao-ko (Crevost et Lemarie) and
Elettaria cardamomum exhibited anti-quorum sensing and antibiofilm activities [
26,
27].
The aims of this paper was to compare the different essential oil composition and to clarify the potential differences in the biological activities of the three tested species (including antimicrobial, allelopathic, and anti-quorum activities) as they are considered by plant sellers as the same “cardamom” species.
2. Results
2.1. Essential Oil Composition
Components of the essential oils, obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS) technique, are divided into five class compounds based on their chemical functional groups (
Table 1).
The major class compounds were the oxygenated monoterpenes which represents 71.4%, 63.0%, and 51.0% of all compounds detected in E. cardamomum, A. corrorima and A. subulatum essential oils, respectively. This family is dominated by 1,8-cineole where its maximum percentage was observed in the green cardamom (55.4%), followed by the Ethiopian cardamom (51.8) and the black cardamom (41.7).
The monoterpene hydrocarbonss were the second dominant components in the three cardamom varieties and represent 36.9% in the green cardamom essential oil, followed respectively by the black cardamom (34.2%) and the Ethiopian cardamom (24.7%). The second class of components is dominated by α-terpinyl acetate (28.6%) in green cardamom essential oil, 4-terpineol for the Ethiopian cardamom (10.4%), and geraniol (12.5%) in the essential oil of black cardamom.
The sesquiterpenes hydrocarbons were found only in the essential oil of the A. corriroma and A. subulatum representing (0.5%) and (0.6%) of the total compounds identified, respectively. The oxygenated sesquiterpenes were also found in the black and Ethiopian cardamom essential oils representing 1.6% and 1.3% of the total compounds identified, respectively.
The principal component analysis (PCA) was carried out in order to determine the relationship between the three cardamom varieties on the basis of their essential oil composition. A better discrimination was revealed on the three dimensional visualization of the plotted scores. Results obtained from the PCA showed the existence of three well-defined groups clearly distinguished both in quality and in quantity (
Figure 1A).
These results confirm that, despite the presence of the 1,8-cineole as a main component in the three essential oils, they are both qualitatively and quantitatively chemically different. Results obtained from the cluster analysis (
Figure 1B) confirmed the existence of one well-defined group represented by the black and Ethiopian cardamom oils suggesting similar compositions. The green cardamom essential oil was clearly distinguished from the latter group both in quality and in quantity.
2.2. Antimicrobial Activities
The antibacterial activity of the three essential oils was tested using the disc diffusion assay and using the microdilution assay on twenty five Gram-positive and Gram-negative bacteria including those frequently associated with food contamination and human disorders. The results were recorded as the diameters (mm) of the growth inhibition zone and as a minimal inhibition and bactericidal concentrations (mg/mL) (
Table 2 and
Table 3).
For the antifungal activity (
Table 2), the results showed that the three essential oils were also active against the yeast strains tested with high diameters of fungal growth inhibition zone ranging from 14.33 to 21.67 mm for the green cardamom oil, from 12.67 to 34.33 mm for the Ethiopian cardamom oil, and from 6.0 to 40.33 mm for the black cardamom oil. The Ethiopian cardamom was the most active essential oil with MICs values ranging from 0.048 to 0.19 mg/mL, and MBCs values from 0.19 to 1.75 mg/mL. Using the Duncan’s test comparing the mean diameters of the microbial growth inhibition zones, independently of the microorganism tested, Corriroma essential oil was classified as the most active oil (mean diameter 17.75 mm) followed by the green cardamom oil (mean diameter 16.27 mm) and finally the Ethiopian cardamom oil (mean diameter 16.21 mm).
The essential oils showed significant antimicrobial activity against all Gram-positive and Gram-negative microorganisms tested, giving inhibition zones ranging between 6 and 41.33 mm for the green cardamom essential oil, between 11.33 and 32.00 mm for the Ethiopian cardamom essential oil, and between 6 to 43 mm for the black cardamom essential oil. The high diameters of the growth inhibition zones were obtained when Gram-positive bacteria are tested for the three volatile oils.
Indeed, the highest activity was observed on S. epidermidis ATCC 12228 (diameter 43.00 mm) for the black cardamom essential oil, on M. luteus NCIMB 8166 (diameter 41.33 mm) for the green cardamom essential oil, and on S. aureus ATCC 6816 (diameter 32.00 mm) for the Ethiopian cardamom oil.
Using the microdilution assay, the results showed that weak concentrations of the three essential oils tested were sufficient to inhibit the growth of all microorganisms tested (MICs values), and to completely stop the bacterial growth (MBCs values). In fact, the lowest MICs values were ranged from 0.048 to 0.19 mg/mL for Ethiopian cardamom oil, and from 0.048 to 0.097 mg/mL for the green and black cardamom oils. Concentrations as low as 0.39 mg/mL was sufficient to reproduce a bacteriostatic effect for the black Ethiopian cardamom oil, 0.78 mg/mL for the black cardamom oil, and 6.25 mg/mL for the green cardamom oil (
Table 3).
The results of the effects of the three essential oils and their main component 1,8-cineole on virulence factors production in
P. aeruginosa PAO1 showed an inhibition of the motility in a concentration dependent manner (
Table 4).
Under control conditions, PAO1 strain give a diameter about 54 mm and in the presence of different concentrations of the three essential oils and their main component (1,8-cineole), the bacteria were able to grow and form a colony in the center with diameter not exceeding 8.33 mm. In fact, the highest level of inhibition in the migration of PAO1 was recorded with the green and ethiopian volatile oils at 400 µg/mL. The diameter of the colony ranged from 54 ± 1 mm to 8.33 ± 0.58. 1,8-Cineole was also able to inhibit the swarming ability of the PAO1 strain with a diameter ranging from 17.33 ± 1.15 mm at 10 µg/mL to 10.33 ± 0.58 mm at 400 µg/mL.
The tested essential oils and their main component (1,8-cineole) decreased the production of the QS-controlled virulence factors (elastase and protease) in
P. aeruginosa PAO1 in a concentration dependant manner. At 10 µg/mL concentration, the three essential oils showed more than 50% of inhibition of elastolytic and proteolytic activities in
P. aeruginosa PAO1 (
Figure 2A,B).
The three tested essential oils and their main component (1,8-cineole) exhibited anti-QS activity against
C. violaceum 026 starter strain. After 24 h incubation, the weakest clear turbid halo zone of 14 mm diameter of violacein inhibition was recorded for the black cardamom and 1,8-cineole at the concentration of 2 mg/disc. The highest diameter of violacein inhibition was noted for the green cardamom about 28 mm and 16 mm with the Ethiopian cardamom essential oil (
Figure 3).
2.3. Phytotoxic Activity
In this study, the three essential oils were evaluated for their activity against germination and radicle elongation of radish (Raphanus sativus L.), garden cress (Lepidium sativum L.), and lettuce (Lactuca sativa L.).
Doses of 2.5, 1.25, and 0.625 µg/mL of
A. corrorima essential oil inhibited significantly the germination of radish, but not influenced the germination of the other species. Instead,
A. subulatum and
E. cardamomum essential oils not influenced the germination of the species considered (
Table 5). None of three oils, instead, seem to be effective against radicle elongation of all these species (data no shown).
3. Discussion
Our results showed that the tested
A. subulatum essential oil was characterized by high proportion of monoterpenes (84.6) dominated by the 1,8-cineole and geraniol. Really, in all essential oils tested, 1,8-cineole was the dominant compound found with a percentage varying from 41.7 to 55.4%. The obtained results were in agreement with those reported in the literature. In fact, in a previous work, we reported that the green cardamom was particularly rich in oxygenated monoterpenes (88.7%) with a dominance of α-terpinyl acetate (45.6%), 1,8-cineole (26%). Additionally, several reports have shown that the basic green cardamom aroma from different geographic origin is a combination of 1,8-cineole and α-terpinyl acetate with different percentages [
25]. In fact, Leela and coworkers [
28] studied the content and the chemical composition of seven cardamom genotypes from India at different maturity levels (stage of capsule development) and reported that α-terpinyl acetate and 1,8-cineole were always the main components. In 2006, Hymete and colleagues [
29] reported that the seed oil of
Aframomum corrorima from Ethiopia was particularly rich on the monoterpenes 1,8-cineole (44.3%) and sabinene (17.3%), whereas the sesquiterpenic compounds (
E)-nerolidol (17.2%), β-caryophyllene (9.7%) and caryophyllene oxide (6.9%) dominated the composition of the husk oil. Eyob and colleagues [
18] conducted an ethnobotanical survey in the southern regions of Ethiopia on the three major korarima growing wild including Gamo Gofa, Debub Omo and Kaffa. They identified 38 and 52 compounds, respectively in leaf and rhizome essential oils. The major component of the oil of the leaf was β-caryophyllene (60.7%). The rhizome oil was dominated by γ-terpinene (21.8%) and β-pinene (17.6%). Similar results have been reported by Baser and Kürkçüoglu [
30]. Kumar and co-workers [
19] reported the chemical composition of the hydrodistilled essential oil obtained from the fruits of
A. subulatum grown in northeast region of Sikkim (India) by using the capillary GC and GC-MS techniques. They reported that the hydrodistillated oil was dominated by 1,8-cineole (65.39%), α-terpineol (10.15%), β-pinene (7.23%). In 2013, Joshi and colleagues [
31] reported the GC-MS profile of the essential oils from the different locations in India. The oils collected showed qualitative and quantitative differences in composition with a dominance of oxygenated monoterpenes (65.31–75.54%), monoterpene hydrocarbons (10.53–17.12%), terpene alcohols (15.32–18.80%), and sesquiterpene hydrocarbons (5.02–9.19%). The oxygenated monoterpene 1,8-cineole (50.55–60.46%) represent the major compound in the oils collected from all regions tested.
Our results confirmed the antimicrobial activity of the three essential oils tested independently of the microorganisms used: in fact, Grădinaru and colleagues [
32] reported anti-
Staphylococcus aureus ATCC 25923 activity of the green cardamom oil at a concentration of 6.25 mg/mL. While, the methicillin-resistant clinical isolates of
S. aureus (MRSA) were most susceptible to 1,8-cineole (MIC = 1.25 mg/mL) as compared to the cardamom oil (MIC = 6.25 mg/mL). Additionally, we have previously reported that the green cardamom essential oil was active against a large panel of Gram-positive bacteria (mean diameter= 21.77 mm), cariogenic bacteria (mean diameter= 19.51 mm) and fungi (mean diameter= 39.5 mm), with MICs values ranging from 0.023 to 0.046 mg/mL for all bacterial and fungal strains tested [
25]. Recently, Teneva and coworkers [
33] reported that the green cardamom oil (chemotype α-terpinyl acetate 39.03 %, eucalyptol 31.53 %) was active against pathogenic
E. coli ATCC 25922,
E. coli ATCC 8739,
Salmonella sp.,
S. aureus ATCC 6538P, and
P. vulgaris strains with a diameters of inhibition zones between 8 and 10 mm and MICs values from 60 to >600 ppm. In 2017, Mutlu-Ingok and Karbancioglu-Guler [
34] reported that the green cardamom oil (chemotype α-terpinyl acetate 43.4%, eucalyptol 29.2%) was active against
Campylobacter jejuni and
Campylobacter coli with a diameter of growth inhibition zone ranging from 24.75 mm to 25.58, respectively. The MICs and MBCs values were low for the two
Campylobacter strains tested (0.025 µL/mL).
For the black cardamom oil, Satyal and coworkers [
35] reported an antifungal activity of the black cardamom oil against the fungus
Aspergillus niger with a MIC value about 313 μg/mL, but also a good antibacterial activity against
B. cereus,
S. aureus,
E. coli, and
P. aeruginosa strains (MICs values ranging from 313 to 625 μg/mL). A good antibacterial activity was also noticed by Naveed and coworkers [
36] on multi-drug resistant bacteria with MIC values ranging from 2.83 mg/mL against
E. coli (SS1) strain to 9.4 mg/mL against the uropathogenic
S. aureus strain. It have been also previously demonstrated that the black cardamom oil extracted from whole fruits exhibited good results against
B. pumilis,
B. subtilis,
M. luteus,
S. aureus,
S. epidermidis,
P. aeruginosa,
E. coli,
C. albicans,
A. niger and
S. cerevisiae strains [
37]. The diameter of inhibition zone ranged from 15 mm (
S. cerevisiae) to 20 mm (
B. pumilis and
S. epidermidis).
The volatile oil extracted from the fruits of Ethiopian cardamom showed promising anti-
C. albicans ATCC 90028 and anti-
S. aureus DSM 346 activity, with a diameter of inhibition zone about 35 mm and 25 mm, respectively (Bacha et al., 2016) [
9]. They also showed an antimicrobial activity against several Gram-positive and negative bacteria including
E. coli K12 DSM 498,
B. cereus ATCC 10987,
B. cereus, and
P. aeruginosa DSM 1117 ones. The MICs values ranged from 12.5 mg/mL (
E. coli K12 DSM 498) to >25 mg/mL (
P. aeruginosa DSM 1117).
The three essential oils tested exhibited pronounced antimicrobial activity against Gram-positive and Gram-negative bacteria, with the highest diameter of growth inhibition zone recorded for
M. luteus NCIMB 8166 (41.33 mm) and
S. aureus ATCC 6816 (32.67 mm) by the green cardamom oil. The Gram-positive bacterium,
B. cereus was the most sensitive bacteria to the black cardamom oil (35.67 mm). This can be explained to the difference in the structure and composition of the cell wall of bacteria belonging to two groups and the different mechanism of action of the various components in each essential oil tested [
38]. Using the two biomonitor strains
C. violaceum and
P. aeruginosa PAO1, we founded that all tested essential oils and their main component (1,8-cineole) attenuated the expression of the tested QS-controlled virulence factors (violacein pigment production, elastase and protease production, and motility) in a dose dependent manner. Previous reports have shown that the essential oil extracted by hydrodistillation from the green cardamom (chemotype α-terpinyl acetate/1,8-cineole) had anti-QS properties against the sensor plasmid pJBA132 (
E. coli strain) with a percentage of inhibition about 31% after 24 h of exposure, and a weak activity using the long-chain sensor plasmid pRK-C12 in
Pseudomonas putida strain (inhibition 21–22%). This result indicated that the chemical compounds founded in the green cardamom oil can compete with C6-HSL autoinducers molecules [
26]. Al-Haidari and colleagues [
39] reported that the green cardamom methanolic extract exhibited significant elimination of pyocyanin formation, significantly inhibited twitching and swimming motilities, and biofilm formation in
P. aeruginosa PA14 strain. It has been recently demonstrated that
Amomum tsao-ko extract exhibited a high biofilm inhibition when tested against
S. typhimurium (51.96%),
S. aureus (47.06%), and
P. aeruginosa (45.28%) at 4 mg/mL. The same authors demonstrated that this extract can inhibit the violacein production (44.59%) and anti-swarming activity by 4 mg/mL extract on
S. typhimurium and
S. aureus [
27].
For the phytotoxic activity test, different studies have reported that some essential oils and their components are potent inhibitors of seed germination and retard plant growth [
40]. Germination and seedling growth bioassays are important preliminary screening methods to determine phytotoxic potential of plant extracts and compounds [
41].
E. cardamomum,
A. corriroma and
A. subulatum essentials oils have been recognized for their wide range of physiological and pharmacological properties but no studies were carried out on their phytotoxic activities. The difference in phytotoxic activity of the oils could be attribuited to their chemical composition.
A. corrorima essential oil composition is richer in monoterpene hydrocarbons (71.4%) than other essential oils studied. Our results corroborate with De Martino and coworkers [
42] that showed monoterpenes phytotoxic activity as good inhibitory radish seed germination in a dose-dependent way.
In conclusion, this study has determined the chemical structure of Elettaria cardamomum, Aframomum corriroma and Amomum subulatum oils, and showed that these oils were mainly composed of the oxygenated monoeterpens, in particular 1,8-cineole. It is evident that the tested oils have effectively reduced/stopped the bacterial and fungal growth as well as the communication of bacterial cells. It is proposed that cardamom oils especially their extracts may have a potential use in clinical settings for microbial infections.
