Syzygium aromaticum is used in combination with other plants as an al-coholic infusion by traditional practitioners to treat infections. It has been selected for evaluation for its antimicrobial properties to justify its use in traditional pharmacopoeia. The fruits were used as plant material while the microbial germs consisted of six reference strains: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 27853, Shigella flexneri ATCC 12022, Candida albicans ATCC 35659 and their clinical counterparts. The extracts were obtained by aqueous decoction, hydroethanolic and ethanolic macerations. The phytochemical screening was performed by chemical staining tests. The antibiotic susceptibility test was performed using the well diffusion m ethod and the MIC and MBC or MFC were determined using the 96-well microplate dilution method. The results showed that 66.67% of the strains tested were sensitive to the aqueous extract with inhibition diameters ranging from 15 to 21 mm and MIC and MCB or MFC between 0.0976 - 0.3906 mg/mL and 0.1953 - 07812 mg/mL respectively, thus determining bacteriostatic activity. 100% of the germs tested were sensitive to hydroethanolic and ethanolic extracts. The inhibition diameters range from 12 - 28 mm for hydroethanolic extract with MIC and MBC or MFC ranging from 0.0488 - 0.3906 mm and 0.0488 - 0.7812 mm respectively. The ethanolic extract gave inhibition diameters of 12 - 26 mm; MIC and MBC or MFC ranging from 0.0976 - 0.7812 mm. Hydroethanolic extract gave three (3) bactericidal/fungicidal activities compared to four (4) as for ethanolic extract. These results prove the use of S. aromaticum among traditional recipes for treating infections in the pharmacopoeia but further studies remain important to produce traditionally improved drugs.
The traditional use of plants for health care by the peoples of the world dates back to ancient times. Nowadays, plants continue to be used as a cure for various diseases despite advances in modern medicine; this is due to the perpetual phenomena of resistance of microbial agents to conventional antibiotics and their side effects [
The material consists of fruits (spices) of S. aromaticum, harvested in Tabligbo, Togo. The plant has been identified in the Laboratory of Botany and Plant Ecology of the Faculty of Sciences (FDS) of the University of Lomé (UL). A voucher specimen was deposited in the herbarium of this laboratory under the number TG15323. Then the fruits were washed and dried under air conditioning (20˚C ± 2˚C) in the laboratory of Physiology Pharmacology of the FDS. They were finally reduced to powder with a mortar for manual use, kept in sealed boxes protected from light for extraction and stored at room temperature.
The microorganisms consist of the reference strains and their clinical counterpart: Escherichia coli ATCC 25922, Escherichia coli, Staphylococcus aureus ATCC 29213, Staphylococcus aureus, Salmonella typhimurium ATCC 14028, Salmonella typhi, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa, Shigella flexneri ATCC 12022, Shigella flexneri, Candida albicans ATCC 35659 and Candida albicans. These germs come from the laboratory of bacteriology of the National Institute of Hygiene (INH) of Lomé except for Candida albicans ATCC 35659 which was provided by the Laboratory of Microbiology and Control of Foodstuffs (LAMICODA), ESTBA/UL. Clinical strains were isolated from urine (E. coli), pus (S. aureus, P. aeruginosa), stool (S. typhi, S. flexneri) and vaginal secretions (C. albicans) from patients (
Phytochemical compounds such as polyphenols, flavonoids, tannins, saponosides, terpenes, steroids, alkaloids, mucilages, coumarins, reducing compounds, anthocyanins, leucoanthocyanins, quinones and cyangenic derivatives, free anthracene derivatives and glycosides have been sought in plant powder using methods described by Harbon [
50 g of vegetable powder were macerated with 500 mL of ethanol 95˚ for 24 h with stirring at 1600 rpm. The residue was taken up twice in 300 mL of ethanol 95˚ before being rejected. For the hydroethanolic maceration the same technique was used with a water-ethanol mixture in equal volume (v/v). For the aqueous
extract, a decoction was obtained from 50 g of vegetable powder boiled in 500 mL of distilled water for 30 min. The decoction obtained was filtered hot on cotton and then cooled at room temperature. Decoction and macerates were then filtered through Wattman N˚ 40 filter paper. Each filtrate was then evaporated under vacuum at 40˚C using an evaporator. The three extracts were stored at −20˚C until use.
The germs collected were re-isolated on appropriate agar medium (Chapman for S. aureus, Eosin Blue Methylene (EMB) for E. coli, Hectoen for Salmonella and Shigella, Muller Hinton Agar (MHA) for Pseudomonas, Sabouraud chloramphenicol (SC) for C. albicans in order to obtain young colonies from 18 to 24 h which were used in the preparation of the inoculum. The tested extract solutions were prepared at a concentration of 200 mg/mL in distilled water and then sterilized on millipore membrane of 0.45 μm porosity and 47 mm in diameter.
1) Antibiotic susceptibility testing
The antibiotic susceptibility testing was performed by the agar well diffusion method used by Karou et al. [
The microbial suspensions used were equal to 0.5 Mac Farland (≈108 CFU/mL). They were quantified by the measurement of densitometer turbidity. The inoculum was introduced on culture medium prepared under standard conditions. These were MHA agar for bacteria and Casitone agar for Candida. The quality of these medium was evaluated by sterility and fertility tests before use. After inoculation of the medium, wells of 6 mm in diameter were made using a sterile hollow punch concentrically in the agar. Each well was flooded with 50 μL of extract at a concentration 200 mg/mL. Gentamicin solutions 10 μg (for staphylococci, Enterobacteriaceae, and Pseudomonas) and Nystatin 100 μg for C. albicans were used as reference drugs. For negative controls, sterile distilled water was used in place of the extract. After 30 minutes of pre-diffusion at laboratory temperature, the Petri dishes were incubated for 18 to 24 h at a temperature of 35˚C ± 2˚C for the bacteria and 25˚C ± 2˚C for the yeasts. The diameters of the microbial growth inhibition zones were measured using an electronic reading chart. Extracts having an inhibition diameter ≥ 12 mm (including disc) were used for the determination of MIC, MBC/MFC. The tests were repeated in triplicate.
2) Determination of Minimum Inhibitory Concentrations (MIC) and Bactericidal/Fungicidal (MBC/MFC)
The test for establishing the susceptibility curve was carried out for the extracts which gave a growth inhibition diameter of the seeds ≥ 12 mm with the previously performed antibiotic susceptibility testing (presumptive test). It was performed using the 96-well microplate dilution method [
The results were analyzed using Graph Pad Prism 6 software and then presented as the mean with standard deviation (Mean ± SD), α = 0.05.
Phytochemical screening revealed the presence of alkaloids, gallic tannins, flavonoids, anthocyanins, saponosides, terpenes, steroids, coumarins, and reducing compounds. The catechoic tannins, the leucoanthocyanins, the quinones, the cyanogenic derivatives, the mucilages, the glycosides (free, O-, C- and cardiac) being absent (
The three extracts were active on all tested germs with growth inhibition diameters ≥ 12 mm except for the aqueous extract which gave a diameter of inhibition < 12 mm for salmonella and yeasts. In general, the growth inhibition diameters of the seeds range from 0 to 21 mm for the aqueous extract, from 12 to 28 mm for the hydroethanolic extract and from 0 to 26 mm for the ethanolic extract (
Metabolites | Al | Tac | Tag | Fl | Ant | Leu | Qn | Sp | Tp | St | Cy | Mu | Cm | Cr | Hl | O-H | C-H | Hc |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
S. aromaticum | + | - | + | + | + | - | - | + | + | + | - | - | + | + | - | - | - | - |
+: Presence; -: Absence; Al: Alkaloids; Tac: Catechic Tannins; Tag: Gallic tannins; Fl: Flavonoids; Ant: Anthocyanins; Leu: Leucoanthocyanes; Qn: Quinones; Sp: Saponosides; Tp: Terpenes; St: Steroids; Cy: Cyanogenic derivatives; Mu: Mucilages; Cm: Coumarins; Cr: Reducing compounds; Hl: Free glycoside; O-H: O-Glycosides; C-H: C-Glycosides; Hc: Cardiac glycoside.
Microorganisms | S. aromaticum extracts | Positive contols | Negative control | |||
---|---|---|---|---|---|---|
Aqueous | HydroEtOH | H2O | Gentamicin | Nystatin | H2O | |
Escherichia coli ATCC 25922 | 16 ± 0 | 18 ± 0 | 12 ± 1 | 16 ± 0 | NA | 0 ± 0 |
Escherichia coli | 15 ± 1 | 17 ± 0 | 14 ± 0 | 14 ± 0 | NA | 0 ± 0 |
Staphylococcus aureus ATCC 29213 | 19 ± 0 | 22 ± 0 | 26 ± 0 | 24 ± 0 | NA | 0 ± 0 |
Staphylococcus aureus | 20 ± 0 | 21 ± 0 | 22 ± 0 | 17 ± 0 | NA | 0 ± 0 |
Salmonella typhimurium ATCC 14028 | 9 ± 0 | 12 ± 0 | 14 ± 0 | 16 ± 0 | NA | 0 ± 0 |
Salmonella typhi | 0.00 | 17 ± 1 | 16 ± 0 | 18 ± 1 | NA | 0 ± 0 |
Pseudomonas aeruginosa ATCC 27853 | 13 ± 0 | 19 ± 1 | 14 ± 0 | 15 ± 0 | NA | 0 ± 0 |
Pseudomonas aeruginosa | 15 ± 0 | 14 ± 0 | 14 ± 0 | 15 ± 0 | NA | 0 ± 0 |
Shigella flexneri ATCC 12022 | 21 ± 1 | 22 ± 0 | 14 ± 0 | 15 ± 0 | NA | 0 ± 0 |
Shigella flexneri | 21 ± 0 | 22 ± 0 | 24 ± 0 | 15 ± 0 | NA | 0 ± 0 |
Candida albicans ATCC 35659 | 0 ± 0 | 27 ± 1 | 17 ± 0 | NA | 16 ± 1 | 0 ± 0 |
Candida albicans | 0 ± 0 | 28 ± 0 | 21 ± 0 | NA | 17 ± 1 | 0 ± 0 |
HydroEtOH: Hydroethanolic; Et-OH: Ethanolic; NA: Not applicable.
MICs and MBCs/MFCs were determined for germs that were susceptible to extracts with inhibition diameters ≥ 12 mm. The bacteriostatic and bactericidal effects of the extracts on the germs were determined by the ratio of MBC/MIC or MFC/MIC ≤ 1 (Bactericidal); MBC/MIC or MFC/MIC ≥ 2 (Bacteriostatic). The results are compiled in
MICs of the aqueous extract ranged from 0.0976 to 0.3906 mg/mL for both reference and clinical microorganisms with some differences for some strains. MBC and MFC are twice as high as MICs for both germ types; which gave a ratio of MBC/MIC or MFC/MIC = 2 for the aqueous extract. For the hydroethanolic extract, the MICs and MBCs or MFCs are between 0.0488 and 0.7812 mg/mL and the MBC/MIC or MFC/MIC ratio = 2 for most germs; only three germs (E. coli ATCC 25922, S. typhimurium ATCC 14028, C. albicans ATCC 35659) had their MBC/MIC or MFC/MIC = 1. The microorganisms were also sensitive to the ethanolic extract with MIC and MBC/MFC between 0.0976 and 0.7812 mg /mL. Four germs showed a MBC/MIC or MFC/MIC = 1 ratio; this ratio is greater than or equal to 2 for the other germs and even 4 for S. flexneri. All clinical strains were inhibited with concentrations equal to or greater than those corresponding to inhibition of standard germs.
The study of the antimicrobial activity of S. aromaticum made it possible to determine the sensitivity of E. coli ATCC 25922, S. aureus ATCC 29213, S. typhimurium ATCC 14028, P. aeruginosa ATCC 27853, S. flexneri ATCC 12022, C. albicans ATCC 35659 and their clinical counterpart (E. coli, S. aureus, S. typhi, P. aeruginosa, S. flexneri, C. albicans) to aqueous, hydroethanolic and ethanolic extracts. The aqueous extract inhibited approximately 66.67% of tested germs
Reference germs | Aqueous | HydroEtOH | Et-OH | ||||||
---|---|---|---|---|---|---|---|---|---|
CMI | MBC/MFC (mg/mL) | MBC/CMI MFC/CMI | CMI | MBC/MFC (mg/mL) | MBC/CMI MFC/CMI | CMI | MBC/MFC (mg/mL) | MBC/CMI MFC/CMI | |
Escherichia coli ATCC 25922 | 0.0976 | 0.1953 | 2 | 0.0976 | 0.0976 | 1 | 0.0976 | 0.1953 | 2 |
Escherichia coli | 0.0976 | 0.1953 | 2 | 0.0976 | 0.1953 | 2 | 0.0976 | 0.1953 | 2 |
Staphylococcus aureus ATCC 29213 | 0.0976 | 0.1953 | 2 | 0.0976 | 0.1953 | 2 | 0.0976 | 0.0976 | 1 |
Staphylococcus aureus | 0.0976 | 0.1953 | 2 | 0.0976 | 0.1953 | 2 | 0.0976 | 0.1953 | 2 |
Salmonella typhimurium ATCC 14028 | - | - | - | 0.1953 | 0.1953 | 1 | 0.3906 | 0.7812 | 2 |
Salmonella typhi | - | - | - | 0.1953 | 0.7812 | 2 | 0.3906 | 0.7812 | 2 |
Pseudomonas aeruginosa ATCC 27853 | 0.3906 | 0.7812 | 2 | 0.3906 | 0.7812 | 2 | 0.3906 | 0.3906 | 1 |
Pseudomonas aeruginosa | 0.3906 | 0.7812 | 2 | 0.3906 | 0.7812 | 2 | 0.7812 | 0.7812 | 1 |
Shigella flexneri ATCC 12022 | 0.0976 | 0.1953 | 2 | 0.0976 | 0.1953 | 2 | 0.0976 | 0.3906 | 4 |
Shigella flexneri | 0.3906 | 0.7812 | 2 | 0.3906 | 0.7812 | 2 | 0.1953 | 0.3906 | 2 |
Candida albicans ATCC 35659 | - | - | - | 0.0488 | 0.0488 | 1 | 0.0976 | 0.0976 | 1 |
Candida albicans | - | - | - | 0.0488 | 0.0976 | 2 | 0.0976 | 0.1953 | 2 |
while the hydroethanolic and ethanolic extract inhibited all the germs (100%). The germs were totally susceptible to the reference drugs.
E. coli ATCC 25922 and E. coli clinical strain were susceptible to aqueous, hydroethanolic and ethanolic extracts with inhibition diameters ranging from 12 to 18 mm. The largest diameters were found with the hydroethanolic extract; diameters that are greater than those given by Gentamicin used as a reference drug. The MICs and MBCs of the aqueous and ethanolic extracts on the reference strain and the clinical strain are identical, but with MBC = 2MIC; gving a ratio MBC/MIC = 2 and determining bacteriostatic activity of these extracts on the two strains of E. coli. As for the hydroethanolic extract, the MICs and MBCs are identical to the previous ones on the clinical strain, but on the standard strain, the MIC is equal to the MBC and consequently, MBC/MIC = 1 determining its bactericidal activity. Alharbi et al. [
S. aureus ATCC 29213 and S. aureus were also sensitive to aqueous, hydroethanolic and ethanolic extracts with growth inhibition diameters ranging from 19 to 26 mm. The aqueous extract gave the best growth inhibition diameters that are even higher than those of the standard drug (Gentamicin). With the three extracts, the MBC = 2MBC on both S. aureus strains except for the aqueous extract which gave a MBC = MIC on the reference strain. The aqueous extract therefore has a bactericidal activity on S. aureus ATCC 29213 and bacteriostatic on the clinical strain. The effect of the other two extracts is bacteriostatic on both strains. Other studies carried out on S. aromaticum have shown that the pure oil of the plant has a bacteriostatic activity on strains of S. aureus Meti-R with MICs and MBCs respectively of 0.625 - 1.25 μg/mL and 1.25 - 2.50 μg/mL [
The actions of the aqueous, hydroethanolic and ethanolic extracts were irregular on S. typhimurium ATCC 14028 and S. typhi. Inhibition diameters of the aqueous extract on both strains are less than 12 mm; for this purpose it is considered inactive. The active extracts (hydroethanolic and ethanolic) have a growth inhibition diameter of between 12 and 17 mm; these diameters are smaller than those of Gentamicin used as a reference drug. The MICs and MBCs of the ethanolic extract are identical and the MBC = 2MIC; by deduction, MBC/MIC = 2, thus showing a bacteriostatic activity. The hydroethanolic extract has a bacteriostatic activity on the clinical strain and a bactericidal activity on the standard strain. The hydroethanolic extract was active against the both strains. Mohamed and Badri [
The effect of extracts on P. aeruginosa ATCC 27853 and its clinical counterpart P. aeruginosa was significant compared to that of Gentamicin with inhibition diameters between 13 and 19 mm. The ethanolic extract was more active with inhibition diameters greater than those of the reference drug. The MICs are the same except for the ethanol extract, which has a higher MIC on the clinical strain. The MBC = 2MIC except for the ethanolic extract whose MIC = MBC on the reference strain. It appears that the ethanolic extract has a bactericidal activity on P. aeruginosa ATCC 27853 and bacteriostatic on P. aeruginosa clinical strain. The aqueous and hydroethanolic extracts showed bacteriostatic activity on both clinical strains. The study of the antibacterial activity of pure oils of S. aromaticum on the clinical P. aeruginosa strains conducted by Alharbi et al. [
Shigella were also responding to the aqueous, hydroethanolic and ethanolic extracts of the plant. S. flexneri ATCC 12022 and S. flexneri were sensitive to all three extracts with growth inhibition diameters ranging from 14 to 24 mm. Both strains of Shigella were also susceptible to Gentamicin. The extracts all gave a bacteriostatic activity on the two strains with a ratio MBC/MIC = 2 and 4 exceptionally for the ethanolic extract on the standard strain. In general, the hydroethanolic extract was more active.
The yeasts showed resistance to the aqueous extract. C. albicans ATCC 35659 and C. albicans, clinical strain was sensitive to hydroethanolic and ethanolic extracts with inhibition diameters between 17 and 28 mm. Both strains were also susceptible to Nystatin as a standard. The hydroethanolic and ethanolic extracts gave a fungicidal activity on the standard and fungistatic strain on the clinical strain. The MICs of the ethanolic extract on Candida were lower than those observed with bacteria. Other studies by Mohamed and Badri [
Phytochemical compounds (alkaloids, tannins, flavonoids, anthocyanins, saponosides, terpenes, steroids, coumarins, reducing compounds) found in the fruits of S. aromaticum have been proven by some authors [
This study confirmed the antimicrobial activity of S. aromaticum fruits used in the treatment of infections by practitioners of traditional medicine. The sensitivity tests carried out on the reference strains and their clinical counterpart showed that the hydroethanolic extract was very active on all these tested germs and it would therefore be desirable to extend the tests to multidrug-resistant germs. The antimicrobial activity of aqueous extracts, hydroethanolic and ethanolic observed could be related to certain phytochemical compounds contained in the fruits of the plant. Furthermore, studies on the determination of the physicochemical constants, pharmacokinetics, pharmacodynamics and toxicity of the extracts must be pursued in order to produce traditionally improved medicinal products to replace alcoholic herbal infusions of medicinal plants since the alcohol exerts toxicity on the organism.
Our thanks to the National Institute of Hygiene (INH) of Lomé, Laboratory of Microbiology and Foodstuffs (LAMICODA) of ESTBA, UL.
No conflict of interest.
Afanyibo, Y.-G., Anani, K., Esseh, K., Sadji, Y., Idoh, K., Koudouvo, K., Agbonon, A., Améyapoh, Y., Tozo, K. and Gbeassor, M. (2018) Antimicrobial Activities of Syzygium aromaticum (L.) Merr. & L.M. Perry (Myrtaceae) Fruit Extracts on Six Standard Microorganisms and Their Clinical Counterpart. Open Access Library Journal, 5: e4951. https://doi.org/10.4236/oalib.1104951