|Year : 2020 | Volume
| Issue : 1 | Page : 55-60
Antidermatophyte activity of syzygium aromaticum, petroselinum crispum, and tetrapleura tetraptera
Roger Ducos Youmsi Fokouo1, Patrick Valere Tsouh Fokou2, Cedric Derick Jiatsa Mbouna3, Fabrice Fekam Boyom1
1 Department of Biochemistry, Antimicrobial and Biocontrol Agents Unit, Faculty of Science, LPMPS, University of Yaoundé; Drug Discovery and Development Unit, Laboratoire Roger Ducos, Yaounde, Cameroon
2 Department of Biochemistry, Antimicrobial and Biocontrol Agents Unit, Faculty of Science, LPMPS, University of Yaoundé, Yaoundé; Drug Discovery and Development Unit, Laboratoire Roger Ducos, Yaounde; Department of Biochemistry, Faculty of Science, University of Bamenda, Bambili, Bamenda, Cameroon
3 Department of Biochemistry, Antimicrobial and Biocontrol Agents Unit, Faculty of Science, LPMPS, University of Yaoundé , Yaoundé, Cameroon
|Date of Submission||12-Dec-2019|
|Date of Acceptance||01-Jan-2020|
|Date of Web Publication||17-Mar-2020|
Dr. Patrick Valere Tsouh Fokou
Department of Biochemistry, Faculty of Science, University of Bamenda, PO Box 39, Bambili, Bamenda
Source of Support: None, Conflict of Interest: None
Introduction: The increasing incidence of dermatophytoses in the world and the side effects of the drugs used encouraged the search of alternative drugs. Hence, the objective of this work was to determine antidermatophyte activity of Syzygium aromaticum seed, Petroselinum crispum leaves, and Tetrapleura tetraptera fruits. Methods: The extracts were prepared by maceration of plant materials into 70% ethanol and methanol. The phytochemical screening was carried out on the extracts. The antidermatophyte assay was performed by the agar dilution method. Results: The results showed that the extraction yields ranged from 9.83% to 32.96%. The phytochemical screening revealed the presence of phenol, glucosides, anthraquinones, and tannins in all extracts. The 70% ethanolic and methanolic extracts of S. aromaticum showed significant inhibition following the preliminary screening. The methanolic extract of S. aromaticum portrayed the best activity with MIC ranging from 0.3125 to 0.625 mg/ml. Conclusions: These results show that the methanolic extract of S. aromaticum seed is promising in the treatment of dermatophytosis, as an alternative in the development of a new therapy.
Keywords: Antidermatophytes activity, dermatophytes, Petroselinum crispum, Syzygium aromaticum, Tétrapleura tetraptera
|How to cite this article:|
Youmsi Fokouo RD, Tsouh Fokou PV, Jiatsa Mbouna CD, Boyom FF. Antidermatophyte activity of syzygium aromaticum, petroselinum crispum, and tetrapleura tetraptera. Biomed Biotechnol Res J 2020;4:55-60
|How to cite this URL:|
Youmsi Fokouo RD, Tsouh Fokou PV, Jiatsa Mbouna CD, Boyom FF. Antidermatophyte activity of syzygium aromaticum, petroselinum crispum, and tetrapleura tetraptera. Biomed Biotechnol Res J [serial online] 2020 [cited 2020 Jul 3];4:55-60. Available from: http://www.bmbtrj.org/text.asp?2020/4/1/55/280865
| Introduction|| |
Dermatophytes are microscopic filamentous fungi which have an affinity for keratin. They affect the skin, nails, or hair and cause superficial lesions in humans called dermatophytosis. Afflictions are among the most common forms of superficial mycosis in the world. Despite many climatic factors offering enormous potential for development, their contagiousness varies according to the species responsible. The incidence increases overnight. Dermatophytosis is the major cause of morbidity associated with superficial mycoses, with frequent relapses often refractory to therapy.
Today, many conventional medicines are used for the treatment of these lesions. However, the impact of dermatophytosis is increasing in the world., Thus, the search for new therapeutic alternatives for better management is necessary.
The World Health Organization estimates that up to 80% of the world's population in general, and in particular the African population, use traditional herbal medicine to meet their health needs due to their accessibility and reduced cost. Their use for the treatment of skin infections is a historical practice in most countries in the world. Today, more than 60% of the medicines marketed are of natural origin. Only 10%–20% of the world's flora has been studied from a therapeutic point of view, and the potential of this natural resource remains very important. Thus, with the aim of enhancing and strengthening the empirical knowledge on medicinal plants of the Cameroonian pharmacopoeia, we proposed to evaluate the antidermatophyte activity of the extracts of three plants: Petroselinum crispum, Tetrapleura tetraptera and Syzygium aromaticum.
| Methods|| |
Syzygium aromaticum 25583/SRF/cam(cloves), Petroselinum crispum 1858/SRFK (leaves), and Tetrapleura tetraptera 50616HNC (fruit) plants were used in this study. They are commonly used spices and were bought respectively from the 8th Market, Mfoundi Market and Mokolo Market provided by Dr. Youmsi (Laboratoire Roger Ducos, Yaounde, Cameroon).
The plant materials were bought from the markets of Yaounde, Cameroon, and authenticated at the National Herbarium of Cameroon where specimens are stored under reference numbers. [Table 1] summarizes information on the plant species, place and date of collection, parts used, and extraction yields.
|Table 1: Plant species, place and date of collection, parts used, and extraction yields|
Click here to view
Preparation of total extracts
Fresh plant parts were dried in the laboratory and grind using commercial millers. Hundred grams (100 g) of each plant powder were macerated either in 1L of 70% ethanol or methanol for 72 hours with mechanical stirring at room temperature. The mixture was then filtered using Whatman No. 1 paper. The process was repeated several times until the plant material was exhausted. The resulting filtrates were concentrated on a rotary evaporator (BÜCHI 011) at 80°C for ethanol extract and 60°C for methanol extract. The crude extracts obtained were weighed and their extraction yield was calculated according to the formula below.
The extracts thus obtained were stored in a refrigerator at 4°C till the experiment.
Phytochemical screening of extracts
Phytochemical screening is a qualitative analysis based on coloring and/or precipitation reactions. It was carried out according to the protocols described by Harbone, Odebiyi and Sofowora, Trease and Evans, and Sofowara.
The test for the detection of alkaloids
Fifty milligrams of extracts were diluted in 10 ml of 2% H2 SO4; the mixture was homogenized, boiled for 2 minutes, and filtered. To 1 ml of the filtrate, 5 drops of the Mayer reagent were added. The development of turbidity confirmed the presence of alkaloids.
The test for the detection of flavonoids
Five milliliters of a dilute ammonia solution were added to an aqueous solution of the extract. After the addition of the concentrated sulfuric acid to the mixture, a yellow color that disappears over time characterizes the presence of flavonoids.
The test for the detection of saponins
Twenty-five milligrams (25 mg) of the extract was mixed with 15 ml of distilled water in a test tube, and the whole was brought to the boiling water bath for 5 min. After cooling, a enough volume of solution was introduced into a test tube and was then stirred vigorously for 10 seconds by the vortex. The presence of a foam of about 1-cm thick that persists for more than a minute demonstrated the presence of saponins.
The test for the detection of tannins
To an alcoholic or aqueous solution of extract, a few drops of ferric chloride were added. The presence of tannins is manifested by a change in the color of the solution which turns to dark blue (tannins Gallic) or to the blackish-green, marking the presence of catechuic tannins.
The test for the detection of phenols
Fifty milligrams of the extracts were dissolved in 15 ml of methanol and the solution was heated in a boiling water bath for 15 minutes; to the mixture, 3 drops of a freshly prepared ferric cyanide solution were added. The formation of a green precipitate highlights the presence of phenols.
The test for the detection of steroids
Two hundred milligrams of extracts were dissolved in 10 ml of chloroform, 2 ml of acetic acid were then added to the solution, and the whole was cooled in an ice bath. Concentrated sulfuric acid was added and the formation of a gray–blue ring indicates the presence of steroids.
The test for the detection of triterpenes
To 10 ml of a 10% (w/v) extract solution, 2 ml of chloroform was added and the whole was homogenized; then, 3 ml of sulfuric acid were added to form two phases. The formation of a reddish-brown interface suggests the presence of terpenoids.
The test for the detection of glycosides
One gram of extract was dissolved in 5 ml of HCl and then neutralized with 5 ml of a 5% NaOH solution; to the mixture, a solution of Fehling (A + B) was added dropwise. The presence of glucosides is manifested by the appearance of a red brick precipitate.
The test for the detection of anthocyanins
Fifty milligrams of the extract were mixed with 15 ml of 1% HCl, and the whole was brought to the boil. The variation in the coloring of orange-red to orange-blue highlights the presence of anthocyanins.
The test for the detection of anthraquinones
Fifty milligrams of an extract were diluted in 4 ml of a mixture of chloroform and petroleum ether (v/v), homogenized, and then filtered; to 1 ml of filtrate, an equal volume of 10% NaOH was added. The development of a red color is characteristic of anthraquinones.
Evaluation of antidermatophytic activity in vitro
The antidermatophytic activity was assessed using the agar dilution method as described by Ngono in 1999.
Culture and maintenance of dermatophytes
Two strains (Trichophyton mentagrophytes ATCC 4439 and Microsporum ferruginum CBS 45780) and a fungal isolate (Trichophyton ajelloi) from the Laboratory of Biochemistry of the University of Dschang were used. They were maintained in culture by successive subculturing every 14 days in the sabouraud dextrose agar (SDA) medium supplemented with chloramphenicol and Acti-dione on slant and incubated at 25°C.
Preparation of the stock solutions
Three grams of each crude extract was diluted in 15 ml of 10% dimethylsulfoxide (DMSO) to give a stock solution of 200 mg/ml concentration. Terbinafine (Lamisil ®) was prepared at 2 mg/ml under the same conditions and used as a positive control.
Preliminary screening of antidermatophytic activities by the agar dilution method
The supercooled sterile SDA medium supplemented with chloramphenicol was mixed with each plant extract or positive control to give, respectively, a concentration of 20 mg/ml or 2 mg/ml. Each tube was homogenized and poured into an identified Petri dish More Details. The mixture was allowed to stand. The plate was seeded by depositing an explant of 7-mm diameter from a 7-day old culture. The plates were thereafter sealed and incubated for 7 days at room temperature. Each test was carried out in triplicate.
The diameter of the dermatophyte mycelial growth zone was measured in two perpendicular directions through the center of the explant to determine the percent inhibition (PI).
PI = percent inhibition expressed (%)
dt = mean colony diameter in the negative control plate (without antifungal agent)
dx = average colony diameter in the assay plate containing the extract
The ethanolic and methanolic extracts of S. aromaticum, which showed inhibition at 20 mg/ml, were selected as our interest extracts and were therefore selected for minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) determination.
Determination of minimum inhibitory concentration and minimum fungicidal concentration
The experiment was carried out as previously described. The extracts or the reference antifungal agent, terbinafine (Lamisil), two-fold serially diluted were then added to a final volume of 15 ml, with varying concentrations of 20 mg/ml to 0.03906 mg/ml and 0.07812 mg/ml to 0.00015 mg/ml, respectively. MIC was considered to be the lowest concentration of extract that showed no visible mycelia growth after 7 days of incubation. The MFC was determined by the subculture of the explants of the Petri dishes where no fungal growth was observed. The smallest concentration showing no recovery of mycelia growth was considered as MFC. The methanol extract of S. aromaticum showed promising antidermatophyte potency and was considered for the cream formulation.
The results were analyzed by ANOVA, expressed as mean ± standard error of the mean, using the Turkey test at the 5% probability threshold using SPSS 17.0 software (Armonk, NY: IBM Corp.).
| Results|| |
The results show that the extraction yields vary according to the plant used and the extraction solvent. The yield of the methanol extract of S. aromaticum (32.96%) is the highest and that of the hydroethanol extract of P. crispum (9.83%) is the lowest [Table 1]. Moreover, the yields of the different plant species extracted with methanol are higher than the yields of the hydroethanol extracts.
Phytochemical screening results
The results of the phytochemical screening of the extracts are presented in the following [Table 2].
The tests were positive with tannins, phenols, glucosides, and anthraquinones on all extracts. However, an absence of anthocyanins was observed in all extracts. Steroids are present in the methanolic extracts and absent in the hydroethanolic extracts. Methanolic extracts from three plant species have more chemical families than their hydroethanolic extracts.
Moreover, the methanolic extract of T. tetrapleura is richer in secondary metabolites. On the other hand, the hydroethanolic extract of P. crispum has fewer secondary metabolites.
Preliminary antidermatophytic activity of extracts
The mean inhibition percentages of the 20 mg/ml extracts on the experimental dermatophytes are displayed in [Table 3]. The percentage of inhibition varies according to the extracts and the microorganisms subjected to the experiment. Four extracts (SA H2O/EtOH, SA MeOH, PC H2O/EtOH, and PC MeOH) had an inhibition which varied between 73.4% and 100% on M. ferruginum, which was found to be the most susceptible species of all extracts. TT H2O/EtOH is the least active extract on M. ferruginum and T. ajelloi with 10.68% and 6.11% inhibition on its two strains, respectively. TTMeOH had no impact on the mycelial growth of T. mentagrophytes. The most active extracts are obtained from the same species: the hydroethanol and methanol extracts of S. aromaticum (SA H2O/EtOH and SA MeOH). They totally inhibit the growth of strains at 20 mg/ml.
|Table 3: Results of the anti-dermatophytic activity of the extract in percentage of inhibition|
Click here to view
These results could be derived from the quality and quantity of secondary metabolites present in the extracts, following the logic of Gottlieb, who showed that the quantity and quality of metabolites depend on the plant and the organs.
The ethanolic and methanol extracts of S. aromaticum, which showed a total inhibition at 20 mg/ml, were retained for the rest of the work.
Minimum inhibitory concentration and minimum fungicidal concentration results
The results of the MICs and MFCs of the two interest extracts are shown in [Table 4].
|Table 4: Minimum inhibitory concentrations and minimum fungicidal concentrations of Syzygium aromaticum extracts (mg/ml)|
Click here to view
The results which are displayed in [Table 4] showed that MICs ranged from 0.313 mg/ml to 1.250 mg/ml and vary according to the extract and the fungal species. The methanolic extract of S. aromaticum showed the best potency with MIC value of 0.313 mg/ml on both M. ferruginum and T. ajelloi. The smallest MICs were obtained on M. ferruginum (0.313 mg/ml for the methanol extract and 0.625 mg/ml for the hydroethanol extract) and the largest on T. mentagrophyes. Hence, M. ferruginum is the most sensitive strain and T. mentagrophytes the least susceptible.
After transplanting the explants from the Petri dishes where no fungal growth was observed, the MFCs results are shown in [Table 4]. The methanolic extract of S. aromaticum is fungicidal at 0.625 mg/ml, while its hydroethanol extract MFCs ranged from 0.625 mg/ml to 1.250 mg/ml. It is concluded that these two extracts are fungicidal with respect to these three species of dermatophytes.
| Discussion|| |
Dermatophytosis represents one of the major causes of mycoses morbidity and refractory to therapy. Medicinal plants such as S. aromaticum, P. crispum, and T. tetraptera have long history in the treatment of skin disease such as dermatophytosis. Their maceration in various solvents resulted in extraction yields varies from 9.83 to 32.96% according to the plant used and the extraction solvent. This difference in yield in relation to the solvent used is explained by the preferential solubility of the compounds as a function of the polarity of the solvent. In addition, methanol has the ability to solubilize several classes of compounds, such as ethanol, which would increase the extraction yield of methanol extracts. The preliminary antidermatophytic activity of extracts showed percentage of inhibition varying according to the extracts and the microorganisms, with M. ferruginum being the most susceptible. These results could be derived from the quality and quantity of secondary metabolites present in the extracts, following the logic of Gottlieb, who showed that the quantity and quality of metabolites depend on the plant and the organs. Both ethanol and methanol extracts of S. aromaticum with 100% inhibition at 20 mg/ml were subjected to MIC and MFC determination that showed MICs ranged from 0.313 mg/ml to 1.250 mg/ml. These results could be explained by the presence of the phenol present in the extract, thus corroborating with the work of Devi et al. and Diego et al., which demonstrated that the phenolic compounds present in the extracts of S. aromaticum are fungicidal to dermatophytes. As far as we know, no attempt has been made to test S. aromaticum organic solvent extracts. However, there are many reports on the anti-dermatophyte activity of its essential oil, and main component, eugenol against dermatophyte strains and clinical isolates. S. aromaticum essential oil study reveals the presence of eugenol, caryophyllene, eugenol acetate, and alpha-humulene, with eugenol being the main component. The essential oil showed is highly antifungal toward Microsporum gypseum and Trichophyton rubrum, with inhibition zone sizes ranging from 12–22 mm and MIC value of 9 μl/ml. Its constituents, eugenol and nerolidol, showed efficacy in a guinea pig model infected by M. gypseum suggest that could be incriminated in the observed antidermatophyte activity. Besides, Park et al. identified that eugenol is the most effective antifungal constituent against the dermatophytes T. mentagrophytes and Microsporum canis. As well, the association of S. aromaticum oleoresin with concentrated sugar demonstrated a strong fungicidal effect against T. mentagrophytes.
A study demonstrated that eugenol from S. aromaticum essential oil is inhibited T. mentagrophytes by expansion of its endoplasmic reticulum near the cell membranes of a hyphal specimen and by partially destroying the inner mitochondrial membranes, with complete destruction of the cell wall, suggesting that the antifungal activity of eugenol toward T. mentagrophytes is due to changes in fungal cell structure at the membrane level.
| Conclusion|| |
At the end of this work, which aimed to evaluate the antidermatophyte activity of P. crispum, T. tetraptera, and S. aromaticum, we found out that the methanol extract of S. aromaticum was the most active on dermatophytes and contained phenols, glucosides, anthraquinones, and tannins. However, the potent methanolic extract of S. aromaticum should be further investigated to ascertain its efficacy on T. mentagrophytes-infected animals.
We are grateful for the insightful comments offered by the anonymous peer reviewers.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Madhavi S, Rama RMV, Jyothsna K. Mycological study of dermatophytoses in rural population. Ann Biol Res 2011;2:88-93.
Havlickova B, Czaika VA, Friedrich M. Epidemiological trends in skin mycoses worldwide. Mycoses 2008;51 Suppl 4:2-15.
Gupta AK, Cooper EA. Update in antifungal therapy of dermatophytosis. Mycopathologia 2008;166:353-67.
Ostrosky-Zeichner L, Marr KA, Rex JH, Cohen SH. Amphotericin B: Time for a new “gold standard”. Clin Infect Dis 2003;37:415-25.
Babayi H, Kolo I, Okogun JI, Ijah UJJ. Antimicrobial activities of methanolic extract of Eucalytus camaldulensis
and Terminalia catappa
against some pathogenic microorganisms. Biochemistry 2004;16: -111.
World Health Organization. General Guideline for Methodologies on Research and Evaluation of Traditional Medicine. Geneva: World Health Organization; 2000.
Baby J, Sujatha S. Pharmacologically important natural products from marine sponges. J Nat Prod 2011;4:5-12.
Harborne JB. Phytochemical Methods. A Guide of Modern Techniques of Plants Analysis. London: Chapman and Hall; 1976.
Odebiyi OO, Sofowora EA. Antimicrobial alkaloids from a Nigerian chewing stick (Fagara zanthoxyloides
). Planta Med 1979;36:204-7.
Trease GE, Evans WC. Pharmacognosy. 13th
ed. London: Bailliere Tindall; 1989.
Sofowara A. Medicinal plants and traditional medicine in Africa. 2nd
ed. Ibadan, Nigeria: Spectrum Books Limited; 1993.
Ngono NR. Contribution to the study of antifungal properties and phytochemical analysis of five Cameroonian medicinal plants. Reims-Champagne: Université de Reims-Champagne-Ardenne et Université de Yaoundé; 1999. p. 103.
Reyes CR, Quiroz VRI, Jiménez EM, Navarro-Ocaňa A, Cassani HJ. Antifungal activity of selected plant secondary metabolites against Coriolus versicolor. Journal of Tropical Forest Products. 1997;3:110-3.
Kuiate JR. Biological and chemical characteristics of dermatophytes and medicinal plants with antifungal effects in two localities in the province of West Cameroon [Thèse de Doctorat d'Etat]. Yaounde: Université de Yaoundé 1; 2005. p. 163
Gottlieb OR. Phytochemicals: differentiation and function. Phytochemistry. 1990;29:1715-24.
Lekana-Douki JB, Oyegue Liabagui SL, Bongui JB, Zatra R, Lebibi J, Toure-Ndouo FS. In vitro
antiplasmodial activity of crude extracts of Tetrapleura tetraptera and Copaifera religiosa. BMC Res Notes 2011;4:506.
Ciulei I. Methodology for analysis of vegetable drugs. Pratical mannuals on the industrial utilization of medicinal and aromatic plants.. Bucharest, Romania: Arta Grafica; 1980.
Devi KP, Nisha SA, Sakthivel R, Pandian SK. Eugenol (an essential oil of clove) acts as an antibacterial agent against Salmonella typhi by disrupting the cellular membrane. J Ethnopharmacol 2010;130:107-15.
Diego F, Claudia RFS, Wanderley PO. Clove (Syzygium aromaticum): A precious spice. Asian Pac J Trop Biomed 2014;4:90-6.
Pinto E, Vale-Silva L, Cavaleiro C, Salgueiro L. Antifungal activity of the clove essential oil from Syzygium aromaticum (Eugenia caryophyllus) on Candida, Aspergillus and dermatophyte species. J Med Microbiol 2009;58:1454–62.
Ayoola GA, Lawore FM, Adelowotan T, Aibinu IE, Adenipekun E, Coker HAB, et al
. Chemical analysis and antimicrobial activity of the essential oil of Syzigium aromaticum (clove). Afr J Microbiol Res 2008;2:162–6.
Rana IS, Rana AS, Rajak RC. Evaluation of antifungal activity in essential oil of the Syzygium aromaticum (L.) by extraction, purification and analysis of its main component eugenol. Braz J Microbiol 2011;42:1269-77.
Lee SJ, Han JI, Lee GS, Park MJ, Choi IG, Na KJ, et al
. Antifungal effect of eugenol and nerolidol against Microsporum gypseum in a guinea pig model. Biol Pharm Bull 2007;30:184-8.
Park MJ, Gwak KS, Yang I, Choi WS, Jo HJ, Chang JW, et al
. Antifungal activities of the essential oils in Syzygium aromaticum (L.) Merr. Et Perry and Leptospermum petersonii Bailey and their constituents against various dermatophytes. J Microbiol 2007;45:460–5.
Nunez L, D'Aquino M, Chirife J. Antifungal properties of clove oil (Eugenia caryophylata) in sugar solution. Brazilian J Microbiol 2001;32:123–6.
[Table 1], [Table 2], [Table 3], [Table 4]