Chemical Composition and Antimicrobial Properties of Elettaria cardamomum Extract

Currently, the phenomenon of bacterial resistance is known in all antibiotic families and affects almost all bacterial species. The resistance extends both quantitatively and qualitatively. For more than 20 years, many determinants of resistance have been described with the emergence of increasingly resistant bacteria.1 This has generated considerable interest in researching new drugs or preparations from natural sources, including plants.2,3


INTRODUCTION
Currently, the phenomenon of bacterial resistance is known in all antibiotic families and affects almost all bacterial species. The resistance extends both quantitatively and qualitatively. For more than 20 years, many determinants of resistance have been described with the emergence of increasingly resistant bacteria. 1 This has generated considerable interest in researching new drugs or preparations from natural sources, including plants. 2,3 The use of plant extracts and compounds of plant origin are valuable sources for traditional medicine in the treatment and prevention of infectious diseases. 4 Thus, they can become the basis of drug development, a natural model for the development of new drugs. 5 Similarly, according to the WHO, 6 more than 80% of the world's population use medicinal plants to treat several diseases. 7 Indeed, natural substances of plant origin are endowed with several biological activities such as antioxidant, anti-inflammatory, anticancer, antimicrobial activity.
In addition, many herbs used by Ayurvedic practitioners have promising results and may be appropriate for larger randomized trials. It is assumed that the broad-spectrum efficacy of these spices can provide an appropriate basis for new antimicrobial therapies. 8 The main groups of antimicrobial phytocompounds include alkaloids, anthraquinones, cardiac glycosides, saponins, tannins and polyphenols. 9 Some of these herbs are those of the genus:

Elettaria;
herbaceous perennial, Elettaria cardamomum (EC), revered as the "queen of spices", belongs to the ginger family, Zingiberaceae. 10,11 The seed contains phytochemical compounds such as phenols, starch, tannins, terpinoids, flavonoids, proteins and sterols. 12 It has been used to treat infections of the gums, teeth and throat and to treat pulmonary congestion, tuberculosis, high blood pressure, heart disease and digestive disorders. 13 Anti-inflammatory, anti-proliferative, pro-apoptotic 14 and antioxidant activities have been recognized as mechanisms underlying the anticancer properties of cardamom. 13 Recently, it has been found that cardamom extract acts as a powerful modulator of macrophages 15 and as a protective factor against uranium risks. 16 Elgayyar and Sekine [17][18] reported that cardamom extract has well-recognized antimicrobial and antifungal properties. Analgesic, antidepressant, anticonvulsive and antispasmodic activities have been attributed to this plant. 19,20 This study aims to identify the chemical composition of the extract of Elettaria cardamomum, to characterize the mode of action of extract with antimicrobial activity and to determine the minimum inhibitory concentration of this extract towards pathogenic microorganisms.

Drugs and reagents
The TFA, EtOH, acetonitrile, formic acid, were obtained from Sigma-Aldrich (St Louis, MO, USA). The Mueller Hinton agar was also purchased from Merck.

Extraction of Elettaria cardamomum
The fruits of E.cardamomum, locally called "Hab el Hal" were bought dry from an herbalist in the city of Mostaganem in northwest Algeria. They were, identified and authenticated by the botanist Pr Larid Mohamed in Laboratory of Biodiversity and Water and Soil Conservation from the Mostaganem University. They were crushed with a mortar until a fine powder was obtained, then placed in hermetically sealed jars and stored dry (room temperature) and protected from humidity.
Extraction of the plant material was carried out according to the protocol of Masoumi-Ardakani 21 , which consists of placing 200 g of powdered plant material in contact with 1000 mL of absolute ethanol. The preparation was left to macerate at room temperature for 72 hours in a dark place. After filtration with a Whatman No. 1 paper, the recovered ethanolic extract was concentrated using rotavapor to evaporate the ethanol. The ethanolic extract of E. cardamomum (EEC) was recovered in a dark glass vial and stored at 4 ° C.
The sample was prepared by diluting EEC with methanol at 1:100 (v/v). Cardamom components were identified by comparing their retention times and UV spectra with different phenolic standards (transcinnamic acid, gallic acid, benzoic acid, ferulic acid, m-coumaric acid, caffeic acid, rosmarinic acid and ellagic acid), flavonoids (catechin, hesperidin, thymol, galangin, tectochrysin, pinocembrin, acetine, rutin, chrysin, apigenin, kaempferol and quercetin) and other compounds (ascorbic acid, menthol). The standards were dissolved in methanol to give stock solutions at 1 mg.mL -1 . The quantification of the constituents was determined using standard curves expressed in mg per 1 g of crude cardamom.

Microbial strains and culture preparations
Ten microbial strains were selected and used in this study given their degree of pathogenicity and virulence. Four gram-positive bacterial strains (Bacillus cereus ATCC 10876, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 33862 and Carnobacterium maltoaromaticum 20722) and four gram-negative bacterial strains (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Enterobacter sp. and Schigella ssp.), plus two fungal strains (Candida albicans ATCC 10231 and Aspergillus niger ATCC 106404).
The strains were maintained on Mueller Hinton agar tilts. Antimicrobial activity was evaluated in vitro by two methods; the disc diffusion method for determination of inhibition zone diameters and the dilution series method for determination of minimum inhibitory concentrations (MIC).

Disc diffusion methods
Petri dishes containing Mueller Hinton agar were aseptically inoculated with 100 µL of microbial suspension adjusted to 10 8 CFU.mL -1 turbidity. After a 15 min rest, filter discs (CT0998B, Oxoid, France) containing different concentrations of E. cardamomum extract (12.5, 25, 50 and 100 µg.mL -1 ) were placed on the agar of each Petri plate. The plates were incubated at 37°C for 24 hours in aerobic conditions. After 24 h incubation, the diameters of the inhibition zones were measured using a ruler (Andrews, 2001).

Determination of Minimum Inhibitory Concentration (MIC)
The Minimum Inhibitory Concentration (MIC) of cardamom extract (EEC) was determined by the liquid-media dilution technique coupled with solid-media spreading as described by Boufadi. 22 In a test tube, variable EEC concentrates were added, so that the concentration gradient extracts follow a progression geometry of 2 and 0 to 30 mg/ mL.
Each tube in one of the series is then inoculated with 1 ml of an inoculum of 10 8 CFU/mL. Then, all the tubes were incubated in an oven at 37°C for 24 h. One millilitre of the contents of each tube was then inoculated in a solid medium. The lowest concentration that completely inhibits the test germ is considered the MIC.

Statistical analysis
SigmaStat software (SPSS, 3.0, SPSS, Inc., Chicago, IL) was used for the statistical analysis. The data were presented as mean ± standard deviation and were evaluated by a one-way ANOVA with Dunnett's post-hoc test. The ranked-based ANOVA analysis with Dunn's posthoc test was used where appropriate.

Chemical composition of EEC extract
Chromatographic analysis at 270 and 320 nm of the ethanolic extract of Elettaria cardamomum (EEC) ( Figure 1A and B) allowed to identify and quantify the phenolic compounds ( Table 1). Comparison of EEC extract retention times with those of different standards revealed the presence of 23 phenolic compounds.
Catechin, luteolin and myricetin were detected as the major flavonoids of EEC extract, with a content of 1.78, 1.41 and 1.25 mg.g -1 EEC, respectively. In addition, the highest phenolic acid content was that of gallic acid (2.83 mg.g -1 EEC). Ellagic acid and syringic acid were found at low concentrations. Table 2 shows the results of the antimicrobial activity of EEC extract. The extract reacted positively to virtually all microbial strains tested. Wide variations in the diameters of the inhibition zones obtained ranging from 0.33 to 33.67 mm were observed.

Antimicrobial activity
It can be noted that the diameters of EEC extract inhibition zones corresponding to concentrations 100 and 50 μg.mL -1 are higher than those observed with lower concentrations; 25 and 12.5 μg.mL -1 except for diameter of Aspergillus niger. Therefore, the EEC extract inhibitory activity seems to be dose-dependent.    It can also be noted that the EEC extract showed varying degrees of antimicrobial activity against all microbial strains tested. EEC extract showed significant activity (P<0.05) against Pseudomonas aeruginosa with an inhibition zone diameter of (31 mm) at a concentration of 50 μg.mL -1 .

Minimum inhibitory concentration (MIC)
Microbial growth is indicated by the presence of a white pellet forming at the bottom of the well. Table 3 shows the MIC of the pathogenic strains. Although antimicrobial susceptibility of various strains varied, cardamom extract was highly effective in inhibiting the growth of all tested strains of most pathogens, with MIC ranging from 6.25 to 12.50 mg.mL -1 .

DISCUSSION
Some authors, Elguindy 23 reported the presence of polyphenolic compounds in the cardamom extract analyzed by HPLC/UV, such as gallic acid, tannic acid, caffeic acid and 4.5-dicaffeoyl quinic acid. In addition, Rahman 24 noted that the ethanolic extract of cardamom consists of: epicatechin, vanillin, p-coumaric acid, trans-ferulic acid and ellagic acid.
Previous work by Hong and Beltran-Ramirez [25][26] showed the presence of several phenolic components in cardamom seed such as gallic acid, caffeic acid and 4,5-dicaffeoyl quinic acid.
In addition, Kikuzaki and Jessie [27][28] reported that 1,8-cein, alphaterpineol, Protocatechualdehyde and protocatechic acid present in cardamom seeds show antioxidant activity and have one of the potential health benefits by inhibiting lipid peroxidation. Phytochemical analysis of the aqueous extract of E. cardamomum fruit shows the presence of alkaloids, flavonoids, tannins, terpenoids, reducing sugars, steroids and phenols. 29 The results of this study are in agreement with the work of Masoumi-Ardakania 21 , who showed that the methanolic extract of cardamum seeds contains high concentrations of kaempferol, rutin and quercetin.
The same results were confirmed by El Malti and Rajan. [30][31] The authors reported that the differences observed in the inhibition zone may be due to the different sensitivity of different bacteria to gold nanoparticles (colloids) synthesized by the aqueous extract of Elettaria cardamomum seeds. In addition, Mahady 32 showed that E. cardamomum seeds have antibacterial activity against Gram-negative bacteria.
In another study, the antibacterial effect of various Elettaria cardamomum fruit extracts was studied on oral bacteria. It was shown that the most significant effect was on S. aureus and that its ethanol and acetone extracts were a potential antibacterial source. 33 The results of the present study are in agreement with those of Arora and Kaur 34 , who reported that the aqueous extract of E. cardamomum was effective against several pathogenic bacteria in inhibition zones ranging from 15 to 28 mm. Whereas, Nanasombat and Lohasuthawee 35 found that the ethanolic extract of cardamom seed showed an inhibition zone between 7 to 12 mm on all strains tested. However, their results were in contrast to the study by Ahmad 36 who found no antibacterial activity using the aqueous extract; this was due to either the extraction method or strain differences.  Furthermore, Goze 37 supported this hypothesis by demonstrating that antibacterial extracts could be due to the presence of different compounds in the extracts, which are also influenced by factors such as geographical location, temperature, plant growth phase, harvest period, plant, soil factor, and plant-related genetic and environmental factors.
The results obtained in this study are in agreement with those of El Malti 30 , who showed that the MIC of the cardamom extract ranged from 9.4 to 18.75 mg.mL -1 for all strains tested, with the exception of E. coli, Bacillus cereus and Enterobacter cloacae which were highly sensitive to the extract (MIC <2.34 mg.mL -1 ).
The antibacterial effect of the EEC extract sample can be attributed to their phenolic compounds. The presence of these compounds can increase permeability and eliminate cytoplasmic content by attacking cell membrane phospholipids. In addition, these compounds can affect the enzymes of the bacterial cell walls. 38,39 CONCLUSION This study was taken to identity the chemical composition and antimicrobial potential of Elettaria cardamomum ethanolic extract (EEC). The antimicrobial effect of the EEC extract was studied on ten pathogenic reference strains.HPLC / UV analysis identified and quantified nine phenolic acids and fourteen flavonoids from cardamom extract (EEC). The EEC extract showed strong antimicrobial activity against all of the microbial strains tested. Its inhibitory effect is spectacular, and is due to its richness in phenolic compounds.
All of these results constitute a scientific justification for the use of this spice in traditional pharmacopoeia in the treatment of infectious diseases and confirms once again the relevance of traditional remedies.

ACKNOWLEDGMENTS
We want to thank Abdelhamid Ibn Badis (Algeria) University and University ULB, CPO Laboratory (Belgium).

FINANCIAL SUPPORT
There has been no significant financial support for this work that could have influenced its outcome.

CONFLICTS OF INTEREST
We wish to confirm that there are no known conflicts of interest associated with this publication

DECLARATION OF COMPETING INTERESTS
None.