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In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae
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MYCMED-822; No. of Pages 7 Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
Available online at
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Research Paper/Document de recherche
In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae N.W. Sit a,*, Y.S. Chan a, S.C. Lai a, L.N. Lim a, G.T. Looi a, P.L. Tay a, Y.T. Tee a, Y.Y. Woon a, K.S. Khoo b, H.C. Ong c a b c
Department of Biomedical Science, Faculty of Science, Universiti Tunku Abdul Rahman, Bandar Barat, 31900 Kampar, Perak, Malaysia Department of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman, Bandar Barat, 31900 Kampar, Perak, Malaysia Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 April 2017 Received in revised form 6 July 2018 Accepted 8 July 2018 Available online xxx
Objectives. – This study was conducted to evaluate the antidermatophytic activity of 48 extracts obtained from medicinal plants (Cibotium barometz, Melastoma malabathricum, Meuhlenbeckia platyclada, Rhapis excelsa, Syzygium myrtifolium, Vernonia amygdalina) and marine algae (Caulerpa sertularioides, Kappaphycus alvarezii) against Trichophyton rubrum and Trichophyton interdigitale (ATCC reference strains), and the cytotoxicity using African monkey kidney epithelial (Vero) cells. Active plant extracts were screened for the presence of phytochemicals and tested against clinical isolates of Trichophyton tonsurans. Methods. – Six different extracts (hexane, chloroform, ethyl acetate, ethanol, methanol and water) were obtained from each plant or algae sample using sequential solvent extraction. The antidermatophytic activity for the extracts was assessed using a colourimetric broth microdilution method. The viability of Vero cells was measured by Neutral Red uptake assay. Results. – All the extracts (except the water extracts of V. amygdalina, C. sertularioides and K. alvarezii) showed antidermatophytic activity against Trichophyton spp. The minimum fungicidal concentration (MFC) ranges for the plant extracts against T. rubrum and T. interdigitale are 0.0025–2.50 and 0.005–2.50 mg/mL, respectively. The algae extracts exhibited lower potency against both species, showing MFC ranges of 0.08– 2.50 and 0.31–2.50 mg/mL, respectively. The ethanol and methanol extracts from the leaves of R. excelsa, and the methanol and water extracts from the leaves of S. myrtifolium were highly active (MFC < 0.1 mg/mL) and with high selectivity indices (SI > 2.8) against reference strains of T. rubrum and T. interdigitale, and most of the clinical isolates of T. tonsurans. Phytochemical analysis indicates the presence of alkaloids, anthraquinones, flavonoids, saponins, tannins, phenolics and triterpenoids in the extracts. Conclusions. – The medicinal plant extracts exhibited stronger antidermatophytic activity compared to the algae extracts. The leaves of R. excelsa and S. myrtifolium are potential sources of new antidermatophytic agents against Trichophyton spp. C 2018 Elsevier Masson SAS. All rights reserved.
Keywords: Minimum fungicidal concentration Minimum inhibitory concentration Trichophyton rubrum Trichophyton interdigitale Trichophyton tonsurans Vero cell
1. Abbreviations 2. Introduction ATCC CC50 CLSI MFC MIC
American Type Culture Collection 50% cytotoxic concentration Clinical and Laboratory Standards Institute Minimum fungicidal concentration Minimum inhibitory concentration
* Corresponding author. E-mail address: [email protected] (N.W. Sit).
Dermatophytosis is known as ‘‘ringworm’’ or ‘‘tinea’’ due to the classical appearance of a circular lesion with an active border that develops on the skin of the infected person [1]. The disease is clinically classified, based on the sites of infection, as tinea pedis (feet), tinea cruris (groin), tinea corporis (trunk), tinea capitis (scalp), tinea faciei (face), tinea unguium/onychomycosis (nails) or tinea manuum (hands). Among the anthropophilic dermatophytes, Trichophyton rubrum is the leading pathogen causing tinea infections on skin and nails [2] while Trichophyton interdigitale (formerly known as Trichophyton mentagrophytes) ranks second
https://doi.org/10.1016/j.mycmed.2018.07.001 C 2018 Elsevier Masson SAS. All rights reserved. 1156-5233/
Please cite this article in press as: Sit NW, et al. In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.07.001
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but is the most common infection-causing agent in some countries such as Croatia [3] and Venezuela [4]. In Malaysia, Trichophyton tonsurans is the most prevalent species among dermatophyteinduced onychomycosis in Hospital Kuala Lumpur, the largest government tertiary referral hospital in the country [5]. Dermatophytosis affects 20–25% of the world population [6], thus it poses a serious public health problem. The quality of life for the patients is sometimes impaired by the physical symptoms of dermatophytosis and associated psychological effects [7]. Several classes of antifungal drugs, such as imidazoles, triazoles and allylamines are available for the treatment of dermatophytosis. However, these drugs often have side effects, have a limited spectrum of activity and are costly. Resistance of T. rubrum to antifungal drugs terbinafine and griseofulvin has been reported [8]. These limitations justify the search for natural antifungal agents, which are safer, with higher efficacy or novel modes of action. According to the new chemical entities registered for clinical use from 1981 to 2014, more than 60% of small-molecule anti-infective drugs are natural products or derivatives or mimics of natural products [9]. Terrestrial plants and marine algae are rich sources of bioactive molecules for drug discovery and development. Biosynthetic pathways in plants and marine algae enable them to produce an array of secondary metabolites with diverse chemical structures for protection against predations and infections, and to enhance the chances of survival in the harsh environments. The secondary metabolites are extracted using solvents of different polarity. Less polar solvents such as hexane, chloroform or ethyl acetate are usually used to extract alkaloids, fatty acids, flavonoids and terpenoids while more polar solvents such as ethanol, methanol or water yield anthocyanins, polypeptides, polyphenols, quinones, saponins, tannins and terpenoids [10]. Lopez et al. [11] have compiled a list of antidermatophytic compounds isolated from non-volatile natural extracts belonging to several classes of secondary metabolites, i.e. alkaloids, coumarins, flavonoids, lignans, quinones, saponins and tannins. This highlights the potential of medicinal plants and marine algae for discovery of new antidermatophytic agents. This study was conducted in order to evaluate the in vitro antidermatophytic activity of 48 extracts obtained from six medicinal plants (Cibotium barometz, Melastoma malabathricum, Meuhlenbeckia platyclada, Rhapis excelsa, Syzygium myrtifolium, Vernonia amygdalina) and two marine algae (Caulerpa sertularioides, Kappaphycus alvarezii) from Malaysia against T. rubrum and T. interdigitale, and to test their cytotoxicity using African monkey kidney epithelial (Vero) cells. Selected active plant extracts were screened for phytochemicals and tested against clinical isolates of Trichophyton tonsurans.
3. Materials and methods 3.1. Medicinal plant and marine algae samples Six species of medicinal plants and two species of marine algae were investigated in this study. The families, common names, parts used, collection sites and specimen voucher reference numbers are listed in Table 1. The plant samples were identified by Professor Hean Chooi Ong, an ethnobotanist affiliated with Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia while the identity for the two marine algae was confirmed by Dr. Kong Soo Khoo from the Faculty of Science, Universiti Tunku Abdul Rahman, Malaysia. 3.2. Preparation of sample extracts After thorough cleaning, the fresh plant samples and the freezedried algae samples were extracted sequentially using hexane, chloroform, ethyl acetate, ethanol, methanol and distilled water. The maceration process was performed in two cycles for each solvent at room temperature, with agitation at 120 rpm. The water extract was lyophilised while other extracts were rotary-evaporated to dryness at 40 8C. All the dried extracts were kept at 20 8C prior to bioassay. 3.3. In vitro antidermatophytic assay Two species of dermatophytes, T. rubrum (ATCC128,188TM) and T. interdigitale (ATCC19533TM) were used in the study. T. rubrum and T. interdigitale were grown at 30 8C for 5 days on oatmeal agar and potato dextrose agar respectively prior to the antidermatophytic assay. The inoculum preparation and incubation conditions for both species were carried out according to the M38-A2 guidelines published by Clinical Laboratory Standards Institute [12]. A colourimetric broth microdilution method using piodonitrotetrazolium chloride as the growth indicator [13] was used to determine the minimum inhibitory concentration (MIC) of each extract towards the dermatophytes. The final concentrations tested for each extract were ranged from 0.02 to 2.50 mg/mL, obtained by two — fold serial dilution in Roswell Park Memorial Institute — 1640 medium. Dilutions beyond 0.02 mg/mL were performed whenever necessary. Positive (griseofulvin antibiotic), growth (dermatophyte only), negative (extract only) and medium controls were included in each 96-well microplate. The minimum fungicidal concentration (MFC) of active extracts was subsequently determined by spread plate method using potato dextrose agar. The assay was conducted in triplicate.
Table 1 Details of medicinal plants and marine algae investigated. Species Medicinal plants Cibotium barometz (L.) J. Sm Melastoma malabathricum L Muehlenbeckia platyclada (F.J. Mu¨ll.) Meisn Rhapis excelsa (Thunb.) Henry Syzygium myrtifolium Walp Vernonia amygdalina Delile Marine algae Caulerpa sertularioides (S. G. Gmelin) M. Howe Kappaphycus alvarezii (Doty) Doty ex P. C. Silva
Family
Common name
Part used
Collection site
Specimen voucher reference number
Cibotiaceae Melastomataceae Polygonaceae
Golden chicken fern Malabar melastome Ribbon bush, Centipede plant
Rhizome hairs Leaves Stems
Cameron Highlands, Pahang Kampar, Perak Cameron Highlands, Pahang
UTAR/FSC/12/009 UTAR/FSC/13/007 UTAR/FSC/12/011
Arecaceae Myrtaceae Compositae
Broadleaf lady palm ‘‘Kelat paya’’ Bitter leaf
Leaves Leaves Leaves
Kampar, Perak Pagoh, Johor Ipoh, Perak
UTAR/FSC/12/008 UTAR/FSC/14/001 UTAR/FSC/12/004
Caulerpaceae
Green feather alga
Whole
Port Dickson, Negeri Sembilan
UTAR/FSC/15/002
Solieriaceae
Elkhorn sea moss
Whole
Semporna, Sabah
UTAR/FSC/15/004
Please cite this article in press as: Sit NW, et al. In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.07.001
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In addition, active extracts (ethyl acetate extract of M. platyclada, ethanol and methanol extracts of R. excelsa and, methanol and water extracts of S. myrtifolium) were tested against six clinical isolates of T. tonsurans obtained from the Microbiology Unit, Pathology Department, Hospital Kuala Lumpur, Malaysia. The isolates were derived from skin scrapings and their specimen numbers were 3,106,159; 3,107,099; 3,108,460; 3,110,750; 3,111,966 and 3,11,6503. The isolates were subcultured twice on potato dextrose agar prior to the antidermatophytic assay as described above. The assay was carried out in triplicate. 3.4. Cytotoxicity assay A mammalian cell line, African monkey kidney epithelial (Vero) cell line (ATCC1CCL-81TM) was used for the cytotoxicity assay. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% foetal bovine serum, penicillinstreptomycin solution and sodium bicarbonate at 37 8C and 5% carbon dioxide [14]. Eight different extract concentrations ranging from 5 to 640 mg/mL were prepared in DMEM through a two-fold serial dilution. Vero cells (4 104 cells/well) were seeded in each well of 96-well microplates and incubated at 37 8C and 5% CO2 for 24 h. One hundrend mL (100 mL) of each of the extracts prepared was then introduced to the cells and incubated for another 72 h at 37 8C and 5% CO2. Cell and medium controls were included in each microplate. The viability of the cells after incubation was measured by Neutral Red uptake assay [15]. The assays were performed in three independent experiments in duplicates. 3.5. Phytochemical screening Active extracts (ethyl acetate extract of M. platyclada, ethanol and methanol extracts of R. excelsa and, methanol and water extracts of S. myrtifolium) were screened for alkaloids, anthraquinones, flavonoids, saponins, tannins, phenolics, steroids, triterpenoids and phytosterols using procedures described by Evans [16] and Harbone [17]. 3.6. Data analysis The MIC and MFC values are expressed as mean of three consistent replicates. For cytotoxicity assay, the percentage of cell viability was calculated based on the following formula: [(x y)/ (z y)] 100%, where x, y and z are the average absorbance of cells treated with extract, average absorbance of medium control, and average absorbance of cell control, respectively. The 50% cytotoxic concentration (CC50) of an extract was determined from the plot of percentage of cell viability against the extract concentrations. The data was analysed with one-way analysis of variance (Anova) using IBM SPSS Statistics for Windows software (Version 20). The significance level was set at P < 0.05. A post hoc test, either using the Tukey’s (equal variance assumed) or Dunnett’s (equal variance not assumed) test was conducted to determine which concentration of extract produced a significant result.
4. Results 4.1. Medicinal plants The fungistatic and fungicidal properties of an extract are indicated by their minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) respectively. These properties are dependent on the type of solvent used for extraction and the test microorganism. As shown in Table 2, all the plant
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extracts possessed inhibitory activity against T. rubrum. However, fungicidal property against T. rubrum was not observed for two extracts, which were the chloroform extract of M. malabathricum and the water extract of V. amygdalina. The MFC values for the extracts were ranged from 0.0025 mg/mL to 2.50 mg/mL. In contrast, all the plant extracts, except the water extract of V. amygdalina, exhibited both properties against T. interdigitale, and the MFC range was 0.005–2.50 mg/mL. Saraiva et al. [18] proposed a classification based on MIC values to compare the potency of antimicrobial activity in which an extract can be classified into highly active (MIC < 0.1 mg/mL), active (0.1 mg/mL MIC < 0.5 mg/mL), moderately active (0.5 mg/ mL MIC < 1 mg/mL), weakly active (1 mg/mL MIC 2 mg/ mL) and inactive (MIC > 2 mg/mL). For antidermatophytic activity, we propose that the above classification should be based on the MFC value instead of the MIC value, as fungicidal activity is more important than fungistatic activity for successful treatment of dermatophyte-related diseases. Analysis using the proposed revised classification shows that nine plant extracts derived from three medicinal plants were considered highly active against T. rubrum compared to 13 extracts from all the six plants against T. interdigitale (Table 2). There were five extracts, i.e. ethyl acetate extract of M. platyclada, ethanol and methanol extracts of R. excelsa, and methanol and water extracts of S. myrtifolium were highly active against both species of dermatophytes. The results also highlight the differences in susceptibility of the two dermatophytes towards the same plant extracts. The five active extracts were effective against six clinical isolates of T. tonsurans (Table 3). Among these, the extracts from R. excelsa and S. myrtifolium showed stronger fungicidal activity (MFC range: 0.005–0.04 and 0.0025–0.16 mg/mL, respectively) compared to the extract of M. platyclada (MFC range: 0.08– 0.63 mg/mL). In this study, griseofulvin was used as the positive control for all the antidermatophytic assay, and the MIC values for T. rubrum, T. interdigitale and T. tonsurans were 0.13–0.25, 0.5–2.0 and 0.25–0.50 mg/mL, respectively. Phytochemical analysis indicated that the strong antidermatophytic activity of R. excelsa and S. myrtifolium may be attributed to the presence of alkaloids, flavonoids, tannins, phenolics and triterpenoids in the extracts (Table 4). The cytotoxicity of all the plant extracts on Vero cells is illustrated in Fig. 1. Generally, plant extracts obtained using less polar solvents such as hexane, chloroform and ethyl acetate were more toxic to Vero cells than the extracts obtained using more polar solvents, with the exception of the water extract. None of the water extracts showed significant toxicity towards the Vero cells even at the highest concentration tested (640 mg/mL). In contrast, the chloroform and ethyl acetate extracts of M. platyclada showed high toxicity towards Vero cells in which the mean 50% cytotoxic concentration (CC50) values were 15.7 mg/mL and 38.9 mg/mL, respectively (Table 2). Taking the cytotoxicity data into consideration for the five highly active extracts against the two reference strains and the clinical isolates of dermatophytes, the selectivity index (SI) of an extract is calculated by dividing the CC50 value with the MFC value of that extract. The SI for the ethyl acetate extract of M. platyclada was 0.1–0.5, and the SI for the ethanol and methanol extracts of R. excelsa were 2.8–22.6 and 3.4–13.7, respectively. However, the SI for the methanol and water extracts of S. myrtifolium was not calculated as no significant toxicity on the Vero cells was recorded for these extracts. 4.2. Marine algae The antidermatophytic activity of the extracts obtained from C. sertularioides and K. alvarezii was weaker compared to the
Please cite this article in press as: Sit NW, et al. In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.07.001
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Table 2 Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the extracts derived from medicinal plants against dermatophytes and 50% cytotoxic concentrations (CC50) of the extracts on African monkey kidney epithelial (Vero) cells. Extract
Sample
Cibotium barometz
Trichophyton rubrum
Hexane Chloroform Ethyl acetate Ethanol Methanol Water Hexane Chloroform Ethyl acetate Ethanol Methanol Water Hexane Chloroform Ethyl acetate Ethanol Methanol Water Hexane Chloroform Ethyl acetate Ethanol Methanol Water Hexane Chloroform Ethyl acetate Ethanol Methanol Water Hexane Chloroform Ethyl acetate Ethanol Methanol Water
Melastoma malabathricum
Muehlenbeckia platyclada
Rhapis excelsa
Syzygium myrtifolium
Vernonia amygdalina
Trichophyton interdigitale
Vero cells
MICa (mg/mL)
MFCa (mg/mL)
MICa (mg/mL)
MFCa (mg/mL)
CC50b (mg/mL)
0.63 0.63 0.16 0.63 0.63 2.50 1.25 2.50 0.31 0.31 0.31 0.31 0.16 0.08 0.08 0.16 0.16 0.16 0.16 0.16 0.08 0.01 0.02 0.08 0.16 0.31 0.08 0.04 0.04 0.0025 0.04 0.63 0.31 1.25 1.25 2.50
2.50 1.25 0.16 2.50 2.50 2.50 2.50 NA 1.25 0.63 0.31 0.31 0.16 0.08 0.08 0.16 0.16 0.16 0.16 0.16 0.08 0.01 0.02 0.08 0.16 0.31 0.08 0.31 0.08 0.0025 0.16 1.25 0.63 1.25 2.50 NA
1.25 0.63 0.02 2.50 1.25 2.50 0.63 0.63 0.02 0.02 0.01 0.005 0.02 0.08 0.08 0.31 0.31 0.16 0.08 0.16 0.16 0.01 0.01 0.16 0.16 0.31 0.16 0.04 0.04 0.005 0.04 0.31 0.31 1.25 1.25 NA
1.25 0.63 0.02 2.50 2.50 2.50 1.25 0.63 0.02 0.02 0.01 0.02 0.16 0.16 0.08 0.31 0.63 0.31 0.08 0.16 0.31 0.01 0.01 0.16 0.16 0.31 0.63 0.08 0.08 0.005 0.08 0.63 0.63 2.50 2.50
NC 382.5 8.5 316.7 11.0 NC NC NC 562.6 11.0 364.7 15.1 144.7 3.9 NC NC NC 166.0 17.5 15.7 2.1 38.9 4.4 > 640 > 640 NC 615.9 24.2 161.5 19.2 132.5 5.5 113.1 6.9 68.5 4.1 NC 85.8 5.3 101.3 3.2 89.8 3.9 NC NC NC NC 131.9 8.4 167.7 18.8 272.4 6.6 485.5 2.8 NC
NA denotes no activity while NC denotes not calculated as no significant toxicity was observed. ‘’ indicates not performed due to absence of inhibitory activity. a The value is expressed as mean of three consistent replicates. b The value is expressed as mean standard deviation (n = 3).
Table 3 Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of selected active extracts from medicinal plants against clinical isolates of Trichophyton tonsurans. Specimen number
3106159
Plant
Extract
MICa
MFCa
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
Muehlenbeckia platyclada Rhapis excelsa
Ethyl acetate Ethanol Methanol Methanol Water
0.04 0.02 0.01 0.04 0.01
0.63 0.02 0.01 0.04 0.01
0.04 0.005 0.0025 0.01 0.005
0.63 0.04 0.02 0.04 0.02
0.04 0.01 0.005 0.02 0.005
0.16 0.01 0.005 0.02 0.005
0.08 0.005 0.01 0.005 0.0025
0.08 0.005 0.01 0.005 0.0025
0.31 0.01 0.02 0.02 0.01
0.31 0.01 0.02 0.08 0.02
0.16 0.01 0.01 0.08 0.02
0.31 0.02 0.02 0.16 0.02
Syzygium myrtifolium a
3107099
3108460
3110750
3111966
3116503
The value is expressed as mean of three consistent replicates in mg/mL.
medicinal plant extracts. The MFC ranges for the algae extracts against T. rubrum and T. interdigitale were 0.08–2.50 mg/mL and 0.31–2.50 mg/mL, respectively (Table 5). Neither the water extract of C. sertularioides nor the water extract of K. alvarezii showed activity against both dermatophytes. The hexane extract of C. sertularioides was the only extract that had high activity (using the proposed revised classification) against T. rubrum (MFC = 0.08 mg/mL). None of the extracts from K. alvarezii were toxic to Vero cells (Fig. 1). The cell viabilities were always more than 90%, except for the ethyl acetate extract at 640 mg/mL, which was 84.6% but not significantly different from other concentrations.
5. Discussion Malaysia is a tropical country that is rich in flora and fauna, which are valuable sources of natural products which may lead to the discovery and development of new antifungal agents. This study quantitated the antidermatophytic activity of the six medicinal plants and two marine algae against T. rubrum and T. interdigitale, and the selected extracts from medicinal plants against clinical isolates of T. tonsurans. When evaluating natural products for antidermatophytic activities, it is essential to assess the fungicidal property of an extract, as dermatophytes are true fungal pathogens. Complete
Please cite this article in press as: Sit NW, et al. In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.07.001
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Table 4 Phytochemical screening of selected active plant extracts. Plant
Muehlenbeckia platyclada
Rhapis excelsa
Syzygium myrtifolium
Extract
Ethyl acetate
Ethanol
Methanol
Methanol
Water
Alkaloids Anthraquinones Flavonoids Saponins Tannins Phenolics Steroids Triterpenoids Phytosterols
+ + + +
+
+ + + + +
+ + + + + + +
+ + + + +
‘ + ’ denotes presence while ‘’ denotes absence.
Fig. 1. Viability of African monkey kidney epithelial (Vero) cells treated with different concentrations of extracts of medicinal plants (A–F) and marine algae (G & H). The percentage is expressed as mean standard deviation of three independent experiments. The asterisk mark denotes significant difference (P < 0.05) with one-way Anova test. The x-axis is displayed as a log scale. A. Cibotium barometz. B. Melastoma malabathricum. C. Muehlenbeckia platyclada. D. Rhapis excelsa. E. Syzygium myrtifolium. F. Vernonia amygdalina. G. Caulerpa sertularioides. H. Kappaphycus alvarezii.
Please cite this article in press as: Sit NW, et al. In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.07.001
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Table 5 Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the extracts derived from marine algae against dermatophytes and 50% cytotoxic concentrations (CC50) of the extracts on African monkey kidney epithelial (Vero) cells. Sample
Caulerpa sertularioides
Kappaphycus alvarezii
Extract
Hexane Chloroform Ethyl acetate Ethanol Methanol Water Hexane Chloroform Ethyl acetate Ethanol Methanol Water
Trichophyton rubrum
Trichophyton interdigitale
Vero cells
MICa (mg/mL)
MFCa (mg/mL)
MICa (mg/mL)
MFCa (mg/mL)
CC50b (mg/mL)
0.08 0.31 0.31 0.63 1.25 NA 1.25 1.25 0.63 2.50 1.25 NA
0.08 0.31 0.31 0.63 1.25 2.50 2.50 0.63 NA 1.25
0.31 0.63 0.63 1.25 2.50 NA 0.63 0.63 0.31 2.50 0.31 NA
0.31 1.25 1.25 2.50 2.50 1.25 1.25 0.63 2.50 0.63
288.0 12.5 264.6 12.2 397.9 8.4 > 640 NC NC NC NC NC NC NC NC
NA denotes no activity while NC denotes not calculated as no significant toxicity was observed. ‘’ indicates not performed due to absence of inhibitory activity. a The value is expressed as mean of three consistent replicates. b The value is expressed as mean standard deviation (n = 3).
eradication of the dermatophyte is important for successful treatment in order to prevent relapse in patients [19]. Although the species of dermatophytes used in this study are taxonomically classified under the genus of Trichophyton, they exhibited different susceptibility towards plant or algae extracts (Table 2). Such differences have also been observed in other studies using crude extracts, fractions or isolated compounds from plants or seaweeds [20–22]. Similarly, the six clinical isolates of T. tonsurans demonstrated different susceptibility towards the fungicidal property of the selected five highly active extracts studied here (Table 3). Several classes of secondary metabolites, i.e. flavonoids, glycosides, phenols, steroids and triterpenoids have been identified in the leaves of R. excelsa [23] and S. myrtifolium [24]. Besides flavonoids, phenolics and triterpenoids, our findings indicated that alkaloids and tannins are present in the leaves of both plants (Table 4). Four flavonoids, vitexin, vicenin-2, isoorientin and orientin have been isolated from the n-butanol fraction of the leaves of R. excelsa [25]. Two flavanones ([2S]-7-hydroxy-5-methoxy-6, 8-dimethyl flavanone and [S]-5, 7-dihydroxy-6, 8-dimethyl-flavanone), two triterpenoids (betulinic and ursolic acids) and a chalcone ([E]-20 ,40 dihydroxy-60 -methoxy-30 , 50 -dimethylchalcone) have been found in the leaves of S. myrtifolium [26]. Among these compounds, only effects of orientin and betulinic acid against Trichophyton spp. have been studied. Orientin, isolated from the leaves of Piper solmsianum (Piperaceae), exhibits a MIC value of 8 mg/mL against T. rubrum and T. mentagrophytes [27]. Betulinic acid, isolated from the leaves of Hypericum lanceolatum (Guttiferae), is active against T. rubrum and T. ajelloi with MFC values of 64 and 4 mg/mL, respectively [28]. Judging from the MFC values and phytochemical analysis, it is highly likely that other compounds with antidermatophytic activity are present in these extracts. This study reported, for the first time, the phytochemicals present in the ethyl acetate extract of the stems of M. platyclada, i.e. anthraquinones, flavonoids, tannins and phenolics. Fagundes et al. [29] studied the leaves of this plant and reported that the ethanol extract contains coumarins, flavonoids, saponins, tannins, terpenoids, sterols and volatile oils. Anthraquinones, flavonoids, tannins and phenolic compounds from plants have been reported to be effective against Trichophyton spp. [30–32]. Extracts obtained using less polar solvents were found to be generally more toxic to Vero cells compared to more polar extracts. Our previous cytotoxicity study using 36 plant extracts on the same cell line recorded similar observations [14]. It is possible that less polar or hydrophobic compounds penetrate the cell membranes more readily and are therefore more toxic to cells. In a study using
the aerial parts of M. malabathricum collected from Indonesia, the CC50 value for the methanol extract on Vero (ATCC1CCL-81TM) cells was > 1000 mg/mL [33]. Our results are in agreement with their finding in which the methanol extract of the leaves of M. malabathricum was not toxic to the Vero cells (also ATCC1 CCL-81TM) even at the highest concentration (640 mg/mL) tested (Fig. 1), despite the geographical difference in the plant collection. 6. Conclusion The study evaluated 36 medicinal plant extracts and 12 marine algae extracts for antidermatophytic activity against T. rubrum and T. interdigitale, and cytotoxic activity on Vero cells. The plant extracts exhibited greater antifungal potency than the algae extracts. The ethanol and methanol extracts from the leaves of R. excelsa, and the methanol and water extracts from the leaves of S. myrtifolium were found to be highly active, with good selectivity indices against both reference strains of dermatophytes and clinical isolates of T. tonsurans. Contribution of authors The conception and design of the study: Nam Weng Sit, Kong Soo Khoo, Hean Chooi Ong. Acquisition of data, or analysis and interpretation of data: Yik Sin Chan, Shient Chen Lai, Lay Ngor Lim, Guat Teng Looi, Pooi Leng Tay, Yee Teng Tee, Yee Yan Woon. Drafting the article or revising it critically for important intellectual content: Nam Weng Sit, Kong Soo Khoo, Hean Chooi Ong. Final approval of the version to be submitted: Nam Weng Sit. Disclosure of interest The authors declare that they have no competing interest. Acknowledgments The authors would like to thank Universiti Tunku Abdul Rahman for the financial support, and Microbiology Unit, Pathology Department, Hospital Kuala Lumpur for providing the clinical isolates. The technical assistance given by Ms. Chee Kei Kong, Ms. Jun Jie Ban and Ms. Yi Hui Lim are much appreciated. The study was registered with the National Medical Research Register of Malaysia (NMRR-17-2450-38738).
Please cite this article in press as: Sit NW, et al. In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.07.001
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[20] Cavaleiro C, Salgueiro L, Gonc¸alves MJ, Hrimpeng K, Pinto J, Pinto E. Antifungal activity of the essential oil of Angelica major against Candida, Cryptococcus, Aspergillus and dermatophyte species. J Nat Med 2015;69:241–8. [21] Guedes EA, Arau´jo MA, Souza AK, de Souza LI, de Barros LD, Maranha˜o FC, et al. Antifungal activities of different extracts of marine macroalgae against dermatophytes and Candida species. Mycopathologia 2012;174:223–32. [22] Santos ML, Magalha˜es CF, da Rosa MB, de Assis Santos D, Brasileiro BG, de Carvalho LM, et al. Antifungal activity of extracts from Piper aduncum leaves prepared by different solvents and extraction techniques against dermatophytes Trichophyton rubrum and Trichophyton interdigitale. Braz J Microbiol 2013;44:1275–8. [23] Vanaja D, Kavitha S. Study on phytochemicals antioxidant activity and FT-IR analysis of Rhapis excelsa (Thumb.) A. Henry. Eur J Pharm Med Res 2016;3:390–4. [24] Memon AH, Ismail Z, Aisha AF, Al-Suede FS, Hamil MS, Hashim S, et al. Isolation, characterization, crystal structure elucidation, and anticancer study of dimethyl cardamonin, isolated from Syzygium campanulatum Korth. Evid Based Complement Alternat Med 2014;2014:470179. http://dx.doi.org/ 10.1155/2014/470179. [25] Hassanein HD, Elsayed WM, Abreu AC, Simo˜es M, Abdelmohsen MM. Polyphenolic constituents and antimicrobial activity of Rhapis excels (Arecaceae Coryphoideae). Res J Pharm Biol Chem Sci 2015;6:1714–22. [26] Memon AH, Ismail Z, Al-Suede FS, Aisha AF, Hamil MS, Saeed MA, et al. Isolation, characterization, crystal structure elucidation of two flavanones and simultaneous RP-HPLC determination of five major compounds from Syzygium campanulatum Korth. Molecules 2015;20:14212–33. [27] De Campos MP, Cechinel Filho V, Da Silva RZ, Yunes RA, Zacchino S, Juarez S, et al. Evaluation of antifungal activity of Piper solmsianum C DC. var. solmsianum (Piperaceae). Biol Pharm Bull 2005;28:1527–30. [28] Tchakam PD, Lunga PK, Kowa TK, Lonfouo AH, Wabo HK, Tapondjou LA, et al. Antimicrobial and antioxidant activities of the extracts and compounds from the leaves of Psorospermum aurantiacum Engl. and Hypericum lanceolatum Lam. BMC Complement Altern Med 2012;12:136. http://dx.doi.org/10.1186/ 1472-6882-12-136. [29] Fagundes LL, Vieira GD, de Pinho Jde J, Yamamoto CH, Alves MS, Stringheta PC, et al. Pharmacological proprieties of the ethanol extract of Muehlenbeckia platyclada (F. Muell.) Meisn. leaves. Int J Mol Sci 2010;11:3942–53. [30] Bitencourt TA, Komoto TT, Massaroto BG, Miranda CE, Beleboni RO, Marins M, et al. Trans-chalcone and quercetin down-regulate fatty acid synthase gene expression and reduce ergosterol content in the human pathogenic dermatophyte Trichophyton rubrum. BMC Complement Altern Med 2013;13:229. http://dx.doi.org/10.1186/1472-6882-13-229. [31] Chewchinda S, Wuthi-udomlert M, Gritsanapan W. HPLC quantitative analysis of rhein and antidermatophytic activity of Cassia fistula pod pulp extracts of various storage conditions. Biomed Res Int 2013;2013:821295. http:// dx.doi.org/10.1155/2013/821295. [32] Foss SR, Nakamura CV, Ueda-Nakamura T, Cortez DA, Endo EH, Dias Filho BP. Antifungal activity of pomegranate peel extract and isolated compound punicalagin against dermatophytes. Ann Clin Microbiol Antimicrob 2014;13:32. http://dx.doi.org/10.1186/s12941-014-0032-6. [33] Lohe´zic-Le De´ve´hat F, Bakhtiar A, Be´zivin C, Amoros M, Boustie J. Antiviral and cytotoxic activities of some Indonesian plants. Fitoterapia 2002;73:400–5.
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Research Paper/Document de recherche
In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae N.W. Sit a,*, Y.S. Chan a, S.C. Lai a, L.N. Lim a, G.T. Looi a, P.L. Tay a, Y.T. Tee a, Y.Y. Woon a, K.S. Khoo b, H.C. Ong c a b c
Department of Biomedical Science, Faculty of Science, Universiti Tunku Abdul Rahman, Bandar Barat, 31900 Kampar, Perak, Malaysia Department of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman, Bandar Barat, 31900 Kampar, Perak, Malaysia Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 April 2017 Received in revised form 6 July 2018 Accepted 8 July 2018 Available online xxx
Objectives. – This study was conducted to evaluate the antidermatophytic activity of 48 extracts obtained from medicinal plants (Cibotium barometz, Melastoma malabathricum, Meuhlenbeckia platyclada, Rhapis excelsa, Syzygium myrtifolium, Vernonia amygdalina) and marine algae (Caulerpa sertularioides, Kappaphycus alvarezii) against Trichophyton rubrum and Trichophyton interdigitale (ATCC reference strains), and the cytotoxicity using African monkey kidney epithelial (Vero) cells. Active plant extracts were screened for the presence of phytochemicals and tested against clinical isolates of Trichophyton tonsurans. Methods. – Six different extracts (hexane, chloroform, ethyl acetate, ethanol, methanol and water) were obtained from each plant or algae sample using sequential solvent extraction. The antidermatophytic activity for the extracts was assessed using a colourimetric broth microdilution method. The viability of Vero cells was measured by Neutral Red uptake assay. Results. – All the extracts (except the water extracts of V. amygdalina, C. sertularioides and K. alvarezii) showed antidermatophytic activity against Trichophyton spp. The minimum fungicidal concentration (MFC) ranges for the plant extracts against T. rubrum and T. interdigitale are 0.0025–2.50 and 0.005–2.50 mg/mL, respectively. The algae extracts exhibited lower potency against both species, showing MFC ranges of 0.08– 2.50 and 0.31–2.50 mg/mL, respectively. The ethanol and methanol extracts from the leaves of R. excelsa, and the methanol and water extracts from the leaves of S. myrtifolium were highly active (MFC < 0.1 mg/mL) and with high selectivity indices (SI > 2.8) against reference strains of T. rubrum and T. interdigitale, and most of the clinical isolates of T. tonsurans. Phytochemical analysis indicates the presence of alkaloids, anthraquinones, flavonoids, saponins, tannins, phenolics and triterpenoids in the extracts. Conclusions. – The medicinal plant extracts exhibited stronger antidermatophytic activity compared to the algae extracts. The leaves of R. excelsa and S. myrtifolium are potential sources of new antidermatophytic agents against Trichophyton spp. C 2018 Elsevier Masson SAS. All rights reserved.
Keywords: Minimum fungicidal concentration Minimum inhibitory concentration Trichophyton rubrum Trichophyton interdigitale Trichophyton tonsurans Vero cell
1. Abbreviations 2. Introduction ATCC CC50 CLSI MFC MIC
American Type Culture Collection 50% cytotoxic concentration Clinical and Laboratory Standards Institute Minimum fungicidal concentration Minimum inhibitory concentration
* Corresponding author. E-mail address: [email protected] (N.W. Sit).
Dermatophytosis is known as ‘‘ringworm’’ or ‘‘tinea’’ due to the classical appearance of a circular lesion with an active border that develops on the skin of the infected person [1]. The disease is clinically classified, based on the sites of infection, as tinea pedis (feet), tinea cruris (groin), tinea corporis (trunk), tinea capitis (scalp), tinea faciei (face), tinea unguium/onychomycosis (nails) or tinea manuum (hands). Among the anthropophilic dermatophytes, Trichophyton rubrum is the leading pathogen causing tinea infections on skin and nails [2] while Trichophyton interdigitale (formerly known as Trichophyton mentagrophytes) ranks second
https://doi.org/10.1016/j.mycmed.2018.07.001 C 2018 Elsevier Masson SAS. All rights reserved. 1156-5233/
Please cite this article in press as: Sit NW, et al. In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.07.001
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but is the most common infection-causing agent in some countries such as Croatia [3] and Venezuela [4]. In Malaysia, Trichophyton tonsurans is the most prevalent species among dermatophyteinduced onychomycosis in Hospital Kuala Lumpur, the largest government tertiary referral hospital in the country [5]. Dermatophytosis affects 20–25% of the world population [6], thus it poses a serious public health problem. The quality of life for the patients is sometimes impaired by the physical symptoms of dermatophytosis and associated psychological effects [7]. Several classes of antifungal drugs, such as imidazoles, triazoles and allylamines are available for the treatment of dermatophytosis. However, these drugs often have side effects, have a limited spectrum of activity and are costly. Resistance of T. rubrum to antifungal drugs terbinafine and griseofulvin has been reported [8]. These limitations justify the search for natural antifungal agents, which are safer, with higher efficacy or novel modes of action. According to the new chemical entities registered for clinical use from 1981 to 2014, more than 60% of small-molecule anti-infective drugs are natural products or derivatives or mimics of natural products [9]. Terrestrial plants and marine algae are rich sources of bioactive molecules for drug discovery and development. Biosynthetic pathways in plants and marine algae enable them to produce an array of secondary metabolites with diverse chemical structures for protection against predations and infections, and to enhance the chances of survival in the harsh environments. The secondary metabolites are extracted using solvents of different polarity. Less polar solvents such as hexane, chloroform or ethyl acetate are usually used to extract alkaloids, fatty acids, flavonoids and terpenoids while more polar solvents such as ethanol, methanol or water yield anthocyanins, polypeptides, polyphenols, quinones, saponins, tannins and terpenoids [10]. Lopez et al. [11] have compiled a list of antidermatophytic compounds isolated from non-volatile natural extracts belonging to several classes of secondary metabolites, i.e. alkaloids, coumarins, flavonoids, lignans, quinones, saponins and tannins. This highlights the potential of medicinal plants and marine algae for discovery of new antidermatophytic agents. This study was conducted in order to evaluate the in vitro antidermatophytic activity of 48 extracts obtained from six medicinal plants (Cibotium barometz, Melastoma malabathricum, Meuhlenbeckia platyclada, Rhapis excelsa, Syzygium myrtifolium, Vernonia amygdalina) and two marine algae (Caulerpa sertularioides, Kappaphycus alvarezii) from Malaysia against T. rubrum and T. interdigitale, and to test their cytotoxicity using African monkey kidney epithelial (Vero) cells. Selected active plant extracts were screened for phytochemicals and tested against clinical isolates of Trichophyton tonsurans.
3. Materials and methods 3.1. Medicinal plant and marine algae samples Six species of medicinal plants and two species of marine algae were investigated in this study. The families, common names, parts used, collection sites and specimen voucher reference numbers are listed in Table 1. The plant samples were identified by Professor Hean Chooi Ong, an ethnobotanist affiliated with Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia while the identity for the two marine algae was confirmed by Dr. Kong Soo Khoo from the Faculty of Science, Universiti Tunku Abdul Rahman, Malaysia. 3.2. Preparation of sample extracts After thorough cleaning, the fresh plant samples and the freezedried algae samples were extracted sequentially using hexane, chloroform, ethyl acetate, ethanol, methanol and distilled water. The maceration process was performed in two cycles for each solvent at room temperature, with agitation at 120 rpm. The water extract was lyophilised while other extracts were rotary-evaporated to dryness at 40 8C. All the dried extracts were kept at 20 8C prior to bioassay. 3.3. In vitro antidermatophytic assay Two species of dermatophytes, T. rubrum (ATCC128,188TM) and T. interdigitale (ATCC19533TM) were used in the study. T. rubrum and T. interdigitale were grown at 30 8C for 5 days on oatmeal agar and potato dextrose agar respectively prior to the antidermatophytic assay. The inoculum preparation and incubation conditions for both species were carried out according to the M38-A2 guidelines published by Clinical Laboratory Standards Institute [12]. A colourimetric broth microdilution method using piodonitrotetrazolium chloride as the growth indicator [13] was used to determine the minimum inhibitory concentration (MIC) of each extract towards the dermatophytes. The final concentrations tested for each extract were ranged from 0.02 to 2.50 mg/mL, obtained by two — fold serial dilution in Roswell Park Memorial Institute — 1640 medium. Dilutions beyond 0.02 mg/mL were performed whenever necessary. Positive (griseofulvin antibiotic), growth (dermatophyte only), negative (extract only) and medium controls were included in each 96-well microplate. The minimum fungicidal concentration (MFC) of active extracts was subsequently determined by spread plate method using potato dextrose agar. The assay was conducted in triplicate.
Table 1 Details of medicinal plants and marine algae investigated. Species Medicinal plants Cibotium barometz (L.) J. Sm Melastoma malabathricum L Muehlenbeckia platyclada (F.J. Mu¨ll.) Meisn Rhapis excelsa (Thunb.) Henry Syzygium myrtifolium Walp Vernonia amygdalina Delile Marine algae Caulerpa sertularioides (S. G. Gmelin) M. Howe Kappaphycus alvarezii (Doty) Doty ex P. C. Silva
Family
Common name
Part used
Collection site
Specimen voucher reference number
Cibotiaceae Melastomataceae Polygonaceae
Golden chicken fern Malabar melastome Ribbon bush, Centipede plant
Rhizome hairs Leaves Stems
Cameron Highlands, Pahang Kampar, Perak Cameron Highlands, Pahang
UTAR/FSC/12/009 UTAR/FSC/13/007 UTAR/FSC/12/011
Arecaceae Myrtaceae Compositae
Broadleaf lady palm ‘‘Kelat paya’’ Bitter leaf
Leaves Leaves Leaves
Kampar, Perak Pagoh, Johor Ipoh, Perak
UTAR/FSC/12/008 UTAR/FSC/14/001 UTAR/FSC/12/004
Caulerpaceae
Green feather alga
Whole
Port Dickson, Negeri Sembilan
UTAR/FSC/15/002
Solieriaceae
Elkhorn sea moss
Whole
Semporna, Sabah
UTAR/FSC/15/004
Please cite this article in press as: Sit NW, et al. In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.07.001
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In addition, active extracts (ethyl acetate extract of M. platyclada, ethanol and methanol extracts of R. excelsa and, methanol and water extracts of S. myrtifolium) were tested against six clinical isolates of T. tonsurans obtained from the Microbiology Unit, Pathology Department, Hospital Kuala Lumpur, Malaysia. The isolates were derived from skin scrapings and their specimen numbers were 3,106,159; 3,107,099; 3,108,460; 3,110,750; 3,111,966 and 3,11,6503. The isolates were subcultured twice on potato dextrose agar prior to the antidermatophytic assay as described above. The assay was carried out in triplicate. 3.4. Cytotoxicity assay A mammalian cell line, African monkey kidney epithelial (Vero) cell line (ATCC1CCL-81TM) was used for the cytotoxicity assay. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% foetal bovine serum, penicillinstreptomycin solution and sodium bicarbonate at 37 8C and 5% carbon dioxide [14]. Eight different extract concentrations ranging from 5 to 640 mg/mL were prepared in DMEM through a two-fold serial dilution. Vero cells (4 104 cells/well) were seeded in each well of 96-well microplates and incubated at 37 8C and 5% CO2 for 24 h. One hundrend mL (100 mL) of each of the extracts prepared was then introduced to the cells and incubated for another 72 h at 37 8C and 5% CO2. Cell and medium controls were included in each microplate. The viability of the cells after incubation was measured by Neutral Red uptake assay [15]. The assays were performed in three independent experiments in duplicates. 3.5. Phytochemical screening Active extracts (ethyl acetate extract of M. platyclada, ethanol and methanol extracts of R. excelsa and, methanol and water extracts of S. myrtifolium) were screened for alkaloids, anthraquinones, flavonoids, saponins, tannins, phenolics, steroids, triterpenoids and phytosterols using procedures described by Evans [16] and Harbone [17]. 3.6. Data analysis The MIC and MFC values are expressed as mean of three consistent replicates. For cytotoxicity assay, the percentage of cell viability was calculated based on the following formula: [(x y)/ (z y)] 100%, where x, y and z are the average absorbance of cells treated with extract, average absorbance of medium control, and average absorbance of cell control, respectively. The 50% cytotoxic concentration (CC50) of an extract was determined from the plot of percentage of cell viability against the extract concentrations. The data was analysed with one-way analysis of variance (Anova) using IBM SPSS Statistics for Windows software (Version 20). The significance level was set at P < 0.05. A post hoc test, either using the Tukey’s (equal variance assumed) or Dunnett’s (equal variance not assumed) test was conducted to determine which concentration of extract produced a significant result.
4. Results 4.1. Medicinal plants The fungistatic and fungicidal properties of an extract are indicated by their minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) respectively. These properties are dependent on the type of solvent used for extraction and the test microorganism. As shown in Table 2, all the plant
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extracts possessed inhibitory activity against T. rubrum. However, fungicidal property against T. rubrum was not observed for two extracts, which were the chloroform extract of M. malabathricum and the water extract of V. amygdalina. The MFC values for the extracts were ranged from 0.0025 mg/mL to 2.50 mg/mL. In contrast, all the plant extracts, except the water extract of V. amygdalina, exhibited both properties against T. interdigitale, and the MFC range was 0.005–2.50 mg/mL. Saraiva et al. [18] proposed a classification based on MIC values to compare the potency of antimicrobial activity in which an extract can be classified into highly active (MIC < 0.1 mg/mL), active (0.1 mg/mL MIC < 0.5 mg/mL), moderately active (0.5 mg/ mL MIC < 1 mg/mL), weakly active (1 mg/mL MIC 2 mg/ mL) and inactive (MIC > 2 mg/mL). For antidermatophytic activity, we propose that the above classification should be based on the MFC value instead of the MIC value, as fungicidal activity is more important than fungistatic activity for successful treatment of dermatophyte-related diseases. Analysis using the proposed revised classification shows that nine plant extracts derived from three medicinal plants were considered highly active against T. rubrum compared to 13 extracts from all the six plants against T. interdigitale (Table 2). There were five extracts, i.e. ethyl acetate extract of M. platyclada, ethanol and methanol extracts of R. excelsa, and methanol and water extracts of S. myrtifolium were highly active against both species of dermatophytes. The results also highlight the differences in susceptibility of the two dermatophytes towards the same plant extracts. The five active extracts were effective against six clinical isolates of T. tonsurans (Table 3). Among these, the extracts from R. excelsa and S. myrtifolium showed stronger fungicidal activity (MFC range: 0.005–0.04 and 0.0025–0.16 mg/mL, respectively) compared to the extract of M. platyclada (MFC range: 0.08– 0.63 mg/mL). In this study, griseofulvin was used as the positive control for all the antidermatophytic assay, and the MIC values for T. rubrum, T. interdigitale and T. tonsurans were 0.13–0.25, 0.5–2.0 and 0.25–0.50 mg/mL, respectively. Phytochemical analysis indicated that the strong antidermatophytic activity of R. excelsa and S. myrtifolium may be attributed to the presence of alkaloids, flavonoids, tannins, phenolics and triterpenoids in the extracts (Table 4). The cytotoxicity of all the plant extracts on Vero cells is illustrated in Fig. 1. Generally, plant extracts obtained using less polar solvents such as hexane, chloroform and ethyl acetate were more toxic to Vero cells than the extracts obtained using more polar solvents, with the exception of the water extract. None of the water extracts showed significant toxicity towards the Vero cells even at the highest concentration tested (640 mg/mL). In contrast, the chloroform and ethyl acetate extracts of M. platyclada showed high toxicity towards Vero cells in which the mean 50% cytotoxic concentration (CC50) values were 15.7 mg/mL and 38.9 mg/mL, respectively (Table 2). Taking the cytotoxicity data into consideration for the five highly active extracts against the two reference strains and the clinical isolates of dermatophytes, the selectivity index (SI) of an extract is calculated by dividing the CC50 value with the MFC value of that extract. The SI for the ethyl acetate extract of M. platyclada was 0.1–0.5, and the SI for the ethanol and methanol extracts of R. excelsa were 2.8–22.6 and 3.4–13.7, respectively. However, the SI for the methanol and water extracts of S. myrtifolium was not calculated as no significant toxicity on the Vero cells was recorded for these extracts. 4.2. Marine algae The antidermatophytic activity of the extracts obtained from C. sertularioides and K. alvarezii was weaker compared to the
Please cite this article in press as: Sit NW, et al. In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.07.001
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Table 2 Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the extracts derived from medicinal plants against dermatophytes and 50% cytotoxic concentrations (CC50) of the extracts on African monkey kidney epithelial (Vero) cells. Extract
Sample
Cibotium barometz
Trichophyton rubrum
Hexane Chloroform Ethyl acetate Ethanol Methanol Water Hexane Chloroform Ethyl acetate Ethanol Methanol Water Hexane Chloroform Ethyl acetate Ethanol Methanol Water Hexane Chloroform Ethyl acetate Ethanol Methanol Water Hexane Chloroform Ethyl acetate Ethanol Methanol Water Hexane Chloroform Ethyl acetate Ethanol Methanol Water
Melastoma malabathricum
Muehlenbeckia platyclada
Rhapis excelsa
Syzygium myrtifolium
Vernonia amygdalina
Trichophyton interdigitale
Vero cells
MICa (mg/mL)
MFCa (mg/mL)
MICa (mg/mL)
MFCa (mg/mL)
CC50b (mg/mL)
0.63 0.63 0.16 0.63 0.63 2.50 1.25 2.50 0.31 0.31 0.31 0.31 0.16 0.08 0.08 0.16 0.16 0.16 0.16 0.16 0.08 0.01 0.02 0.08 0.16 0.31 0.08 0.04 0.04 0.0025 0.04 0.63 0.31 1.25 1.25 2.50
2.50 1.25 0.16 2.50 2.50 2.50 2.50 NA 1.25 0.63 0.31 0.31 0.16 0.08 0.08 0.16 0.16 0.16 0.16 0.16 0.08 0.01 0.02 0.08 0.16 0.31 0.08 0.31 0.08 0.0025 0.16 1.25 0.63 1.25 2.50 NA
1.25 0.63 0.02 2.50 1.25 2.50 0.63 0.63 0.02 0.02 0.01 0.005 0.02 0.08 0.08 0.31 0.31 0.16 0.08 0.16 0.16 0.01 0.01 0.16 0.16 0.31 0.16 0.04 0.04 0.005 0.04 0.31 0.31 1.25 1.25 NA
1.25 0.63 0.02 2.50 2.50 2.50 1.25 0.63 0.02 0.02 0.01 0.02 0.16 0.16 0.08 0.31 0.63 0.31 0.08 0.16 0.31 0.01 0.01 0.16 0.16 0.31 0.63 0.08 0.08 0.005 0.08 0.63 0.63 2.50 2.50
NC 382.5 8.5 316.7 11.0 NC NC NC 562.6 11.0 364.7 15.1 144.7 3.9 NC NC NC 166.0 17.5 15.7 2.1 38.9 4.4 > 640 > 640 NC 615.9 24.2 161.5 19.2 132.5 5.5 113.1 6.9 68.5 4.1 NC 85.8 5.3 101.3 3.2 89.8 3.9 NC NC NC NC 131.9 8.4 167.7 18.8 272.4 6.6 485.5 2.8 NC
NA denotes no activity while NC denotes not calculated as no significant toxicity was observed. ‘’ indicates not performed due to absence of inhibitory activity. a The value is expressed as mean of three consistent replicates. b The value is expressed as mean standard deviation (n = 3).
Table 3 Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of selected active extracts from medicinal plants against clinical isolates of Trichophyton tonsurans. Specimen number
3106159
Plant
Extract
MICa
MFCa
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
Muehlenbeckia platyclada Rhapis excelsa
Ethyl acetate Ethanol Methanol Methanol Water
0.04 0.02 0.01 0.04 0.01
0.63 0.02 0.01 0.04 0.01
0.04 0.005 0.0025 0.01 0.005
0.63 0.04 0.02 0.04 0.02
0.04 0.01 0.005 0.02 0.005
0.16 0.01 0.005 0.02 0.005
0.08 0.005 0.01 0.005 0.0025
0.08 0.005 0.01 0.005 0.0025
0.31 0.01 0.02 0.02 0.01
0.31 0.01 0.02 0.08 0.02
0.16 0.01 0.01 0.08 0.02
0.31 0.02 0.02 0.16 0.02
Syzygium myrtifolium a
3107099
3108460
3110750
3111966
3116503
The value is expressed as mean of three consistent replicates in mg/mL.
medicinal plant extracts. The MFC ranges for the algae extracts against T. rubrum and T. interdigitale were 0.08–2.50 mg/mL and 0.31–2.50 mg/mL, respectively (Table 5). Neither the water extract of C. sertularioides nor the water extract of K. alvarezii showed activity against both dermatophytes. The hexane extract of C. sertularioides was the only extract that had high activity (using the proposed revised classification) against T. rubrum (MFC = 0.08 mg/mL). None of the extracts from K. alvarezii were toxic to Vero cells (Fig. 1). The cell viabilities were always more than 90%, except for the ethyl acetate extract at 640 mg/mL, which was 84.6% but not significantly different from other concentrations.
5. Discussion Malaysia is a tropical country that is rich in flora and fauna, which are valuable sources of natural products which may lead to the discovery and development of new antifungal agents. This study quantitated the antidermatophytic activity of the six medicinal plants and two marine algae against T. rubrum and T. interdigitale, and the selected extracts from medicinal plants against clinical isolates of T. tonsurans. When evaluating natural products for antidermatophytic activities, it is essential to assess the fungicidal property of an extract, as dermatophytes are true fungal pathogens. Complete
Please cite this article in press as: Sit NW, et al. In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.07.001
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Table 4 Phytochemical screening of selected active plant extracts. Plant
Muehlenbeckia platyclada
Rhapis excelsa
Syzygium myrtifolium
Extract
Ethyl acetate
Ethanol
Methanol
Methanol
Water
Alkaloids Anthraquinones Flavonoids Saponins Tannins Phenolics Steroids Triterpenoids Phytosterols
+ + + +
+
+ + + + +
+ + + + + + +
+ + + + +
‘ + ’ denotes presence while ‘’ denotes absence.
Fig. 1. Viability of African monkey kidney epithelial (Vero) cells treated with different concentrations of extracts of medicinal plants (A–F) and marine algae (G & H). The percentage is expressed as mean standard deviation of three independent experiments. The asterisk mark denotes significant difference (P < 0.05) with one-way Anova test. The x-axis is displayed as a log scale. A. Cibotium barometz. B. Melastoma malabathricum. C. Muehlenbeckia platyclada. D. Rhapis excelsa. E. Syzygium myrtifolium. F. Vernonia amygdalina. G. Caulerpa sertularioides. H. Kappaphycus alvarezii.
Please cite this article in press as: Sit NW, et al. In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.07.001
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Table 5 Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the extracts derived from marine algae against dermatophytes and 50% cytotoxic concentrations (CC50) of the extracts on African monkey kidney epithelial (Vero) cells. Sample
Caulerpa sertularioides
Kappaphycus alvarezii
Extract
Hexane Chloroform Ethyl acetate Ethanol Methanol Water Hexane Chloroform Ethyl acetate Ethanol Methanol Water
Trichophyton rubrum
Trichophyton interdigitale
Vero cells
MICa (mg/mL)
MFCa (mg/mL)
MICa (mg/mL)
MFCa (mg/mL)
CC50b (mg/mL)
0.08 0.31 0.31 0.63 1.25 NA 1.25 1.25 0.63 2.50 1.25 NA
0.08 0.31 0.31 0.63 1.25 2.50 2.50 0.63 NA 1.25
0.31 0.63 0.63 1.25 2.50 NA 0.63 0.63 0.31 2.50 0.31 NA
0.31 1.25 1.25 2.50 2.50 1.25 1.25 0.63 2.50 0.63
288.0 12.5 264.6 12.2 397.9 8.4 > 640 NC NC NC NC NC NC NC NC
NA denotes no activity while NC denotes not calculated as no significant toxicity was observed. ‘’ indicates not performed due to absence of inhibitory activity. a The value is expressed as mean of three consistent replicates. b The value is expressed as mean standard deviation (n = 3).
eradication of the dermatophyte is important for successful treatment in order to prevent relapse in patients [19]. Although the species of dermatophytes used in this study are taxonomically classified under the genus of Trichophyton, they exhibited different susceptibility towards plant or algae extracts (Table 2). Such differences have also been observed in other studies using crude extracts, fractions or isolated compounds from plants or seaweeds [20–22]. Similarly, the six clinical isolates of T. tonsurans demonstrated different susceptibility towards the fungicidal property of the selected five highly active extracts studied here (Table 3). Several classes of secondary metabolites, i.e. flavonoids, glycosides, phenols, steroids and triterpenoids have been identified in the leaves of R. excelsa [23] and S. myrtifolium [24]. Besides flavonoids, phenolics and triterpenoids, our findings indicated that alkaloids and tannins are present in the leaves of both plants (Table 4). Four flavonoids, vitexin, vicenin-2, isoorientin and orientin have been isolated from the n-butanol fraction of the leaves of R. excelsa [25]. Two flavanones ([2S]-7-hydroxy-5-methoxy-6, 8-dimethyl flavanone and [S]-5, 7-dihydroxy-6, 8-dimethyl-flavanone), two triterpenoids (betulinic and ursolic acids) and a chalcone ([E]-20 ,40 dihydroxy-60 -methoxy-30 , 50 -dimethylchalcone) have been found in the leaves of S. myrtifolium [26]. Among these compounds, only effects of orientin and betulinic acid against Trichophyton spp. have been studied. Orientin, isolated from the leaves of Piper solmsianum (Piperaceae), exhibits a MIC value of 8 mg/mL against T. rubrum and T. mentagrophytes [27]. Betulinic acid, isolated from the leaves of Hypericum lanceolatum (Guttiferae), is active against T. rubrum and T. ajelloi with MFC values of 64 and 4 mg/mL, respectively [28]. Judging from the MFC values and phytochemical analysis, it is highly likely that other compounds with antidermatophytic activity are present in these extracts. This study reported, for the first time, the phytochemicals present in the ethyl acetate extract of the stems of M. platyclada, i.e. anthraquinones, flavonoids, tannins and phenolics. Fagundes et al. [29] studied the leaves of this plant and reported that the ethanol extract contains coumarins, flavonoids, saponins, tannins, terpenoids, sterols and volatile oils. Anthraquinones, flavonoids, tannins and phenolic compounds from plants have been reported to be effective against Trichophyton spp. [30–32]. Extracts obtained using less polar solvents were found to be generally more toxic to Vero cells compared to more polar extracts. Our previous cytotoxicity study using 36 plant extracts on the same cell line recorded similar observations [14]. It is possible that less polar or hydrophobic compounds penetrate the cell membranes more readily and are therefore more toxic to cells. In a study using
the aerial parts of M. malabathricum collected from Indonesia, the CC50 value for the methanol extract on Vero (ATCC1CCL-81TM) cells was > 1000 mg/mL [33]. Our results are in agreement with their finding in which the methanol extract of the leaves of M. malabathricum was not toxic to the Vero cells (also ATCC1 CCL-81TM) even at the highest concentration (640 mg/mL) tested (Fig. 1), despite the geographical difference in the plant collection. 6. Conclusion The study evaluated 36 medicinal plant extracts and 12 marine algae extracts for antidermatophytic activity against T. rubrum and T. interdigitale, and cytotoxic activity on Vero cells. The plant extracts exhibited greater antifungal potency than the algae extracts. The ethanol and methanol extracts from the leaves of R. excelsa, and the methanol and water extracts from the leaves of S. myrtifolium were found to be highly active, with good selectivity indices against both reference strains of dermatophytes and clinical isolates of T. tonsurans. Contribution of authors The conception and design of the study: Nam Weng Sit, Kong Soo Khoo, Hean Chooi Ong. Acquisition of data, or analysis and interpretation of data: Yik Sin Chan, Shient Chen Lai, Lay Ngor Lim, Guat Teng Looi, Pooi Leng Tay, Yee Teng Tee, Yee Yan Woon. Drafting the article or revising it critically for important intellectual content: Nam Weng Sit, Kong Soo Khoo, Hean Chooi Ong. Final approval of the version to be submitted: Nam Weng Sit. Disclosure of interest The authors declare that they have no competing interest. Acknowledgments The authors would like to thank Universiti Tunku Abdul Rahman for the financial support, and Microbiology Unit, Pathology Department, Hospital Kuala Lumpur for providing the clinical isolates. The technical assistance given by Ms. Chee Kei Kong, Ms. Jun Jie Ban and Ms. Yi Hui Lim are much appreciated. The study was registered with the National Medical Research Register of Malaysia (NMRR-17-2450-38738).
Please cite this article in press as: Sit NW, et al. In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.07.001
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Please cite this article in press as: Sit NW, et al. In vitro antidermatophytic activity and cytotoxicity of extracts derived from medicinal plants and marine algae. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.07.001