Antifungal Activity of Allium Sativum L. And Mentha Piperita L. against C.
Albicans from Clinical Samples
Hayfaa Almabrook1, *Harison Masih1
1Department of Industrial Microbiology, JJBB, SHUATS, Prayagraj (India) Abstract
Candida species are one of the most fatal fungal species which has detrimental effects on humans.
Antifungal agents against these fungal species are slowly restricting their effect due to development of resistant species. Nowadays, antifungal agents of plant or microbial origin are gaining pace due to their easy development methods, less side effects and less complex metabolites produced y them. This study also focuses on two plant sources whose antifungal potency has been reported earlier against some fungal species. Allium sativum L bulbs and Mentha piperita L. leaves were extracted with ethanol and their active constituents were studied using GC-MS analysis. Antifungal activity of constituents and their time kill kinetic study have been done to evaluate their efficacy as antigungal agents. The results showed that Mentha piperita L. extract activity was strongest against C.albicanswith ZOI of 25.9±1.31mm quite comparable ZOI (34.9±0.36mm) of the positive control. This was followed by activity against C.glabrata(23.97±0.06). Allium sativum L. extract showed higher activity against C.albicans (23.8 ±1.31mm) and lesser effective against C.glabrata (20.57±0.67mm).MICs for Allium sativum L. and Mentha piperita L. extracts were determined to be 6.25 mg/ml and MFC 12.5 mg/ml in all cases.
Keywords: GC-MS profiling, CLSI, MFC, Time-kill curve, colony forming unit, bioactive, retention time
1. Introduction
Candida species are one of the major group of fungus which affects human extensively, the category of infections it causes are collectively named as Candidiasis. Immunocompromised individuals are majorly affected in these cases (Ferreira et al., 2009). In recent times, it is seen that fungal infections are majorly caused due to candida and non-albicans Candida (NAC) besides other yeast of this category. The issue also grabs attention due to gradually developing resistant species among this category (Li et al., 2015). Researchers are high to develop products of plant origin that could be used to inhibit their growth and thus slow down their speedy spread.Candida albicans are the prevailing species in causing candiasis among other Non-albicans Candida (NAC) species which hold 25%, 8%, 7%, and 4% by C. tropicalis, C. glabrata, C. parapsilosis and C. krusei respectively (Mukherjee and Chandra, 2015). There are several factors in these species that determine the intensity of the effect on individuals which includes the formation of the germ tube, adherence capacity of the pathogen, variation in phenotypic properties, formation of biofilm, and secretion of lytic enzymes (Deorukhkar et al., 2014).Thus these factors ultimately define their capacity of pathogenesis.
Five well-known categories of compounds which are known to possess antifungal properties are polyenes, allylamines, azoles, fluoropyrimidines, and echinocandins (Rabes et al., 2015). The increasing drug resistances against common antifungal agents like fluconazole and voriconazole due to excessive usage have raised concerns among researchers to find better antifungal agents.
Plant sources are seen as better alternatives, owing to their cheap source and fewer side effects.
Therefore, they have replaced the chemically synthesized compounds almost completely which is evident from the fact that among the marketed antibiotics that are used for clinical purposes, about 80% of them are derived from natural sources.
In this study, Allium sativum L. and Mentha piperita L. have been focused to identify their antifungal activity on Candida species, majorly emphasizing on Candida albicans. Both the plants are of utmost importance from a medicinal and economical point of view. Therefore, they are thoroughly studied in literature and it is reported to possess many properties like antiviral, antibacterial, and antifungal (Mimica-Dukić et al., 2003; Iscan et al., 2002; Singh et al., 2015). The present scenario presents a minacious picture where Candidiasis numbers are increasing at an alarming rate, the reason being the incidence of immunosuppressive diseases among people. As the usage of existing drugs increased heavily, they are soon acquiring a resistant label. As plants are the most reliable sources to obtain bioactive components, they play a central role in most studies in recent times. We in this study, aim to determine more such active components from these well-known plants that could possess better potential to control Candida albicans and Non-Albicans Candida (NAC) species.
2. Material and Methodology 2.1 Strains and media
The strains used in the study were Candida albicans and Candida glabrata. The isolates were obtained from clinical samples, identified and characterized using molecular method of Amplification and sequencing ITS region of the fungus in our earlier work. The obtained sequence were submitted to NCBI/DDBJ and can be accessed from official website (www.ncbi.nlm.nih.gov) for Candida albicans and Candida glabrata. All the media and chemicals used in the study were obtained from HiMedia Laboratories Pvt. Ltd.
2.2Extraction of Allium sativum L. and Mentha piperita L.
Extraction of the active constituents frombulbs of Allium sativum Land leaves of Mentha piperita L. were done using ethanol, cold water and hot water. For this leaves of Mentha piperitaL.were collected from local area, both parts were rinsed with distilled water thoroughly to remove dust paticles and other contaminants. Leaves were then dried in shade, ground to powder using electric blender white in case of garlic, bulbs were freshly peeled and then powdered. These powders were then separately extracted with ethanol, hot water and cold water using conventional method. Each powdered sample was weighed 10gm and mixed with the solvents in 3 separate flasks. These flasks were then placed on shaker for 72hours. After completion of incubation period, the extracts were
filtered using whatman filter paper no. 1 and filtrate was collected in separate tubes. The extracts were then dried using rotary evaporator (ZEXTER Pvt. Ltd., India) and dried powder obtained were stored at 4˚C for further processing.
2.3 Thin-Layer Chromatography (TLC)
The dried extracts were tested for their phytochemical components using Thin Layer Chromatography following method given by Marica et al. (2010). Samples of Allium sativum L and Mentha piperita L extracts were prepared in separate solvents (described in section 3.1). The mixture was placed on the silica gel coated plates (pre-activated at 105˚C) leaving 1cm distance from base. The sample was allowed to absorb on the surface. The plates were then placed in chamber saturated with the solvent system (described in section 3.1). After completion of the process, plates were developed and visualized under UV light. Rf were calculated for the observed spots. These extracts were then further analyzed using GC-MS profiling.
2.4 GC-MS Profiling of the extract
The extracts were analyzed for their using GC-MS Analysis equipment (Thermo Scientific Co.) Thermo GC-TRACE ultra ver.: 5.0, Thermo MS DSQ II.
Experimental set up conditions for GC-MS system
TR 5-MS capillary standard polar column, dimension: 30Mts, ID: 0.25 mm
Film thickness: 0.25μm.
Flow rate of mobile phase (carrier gas: He): 5.0 ml/min.
Temperature programme (oven temperature): 40°C- 350°C at 5°C/min
Injection volume: 1 μl.
Solvent used for sample preparation:Methanol
Range: 50-650 m/z
Results obtained were compared from Wiley Spectral Library Search Programme available in the system.
2.5 Antifungal Activity against characterized candida isolates
Extract were assessed for their antifungal activity against C.albican and C.glabrata using Agar well diffusion method standardized by the Clinical and Laboratory Standards Institute (NCCLS/ CLSI, 1997; Fothergill, 2011).
2.6 Minimum inhibitory and fungicidal concentrations of the extracts
MIC for the extracts was determined using standard broth micro-dilution method recommended by the Clinical and Laboratory Standards Institute/The National Committee for Clinical Laboratory Standards (CLSI/ NCCLS, 2002). MFC was evaluated from MIC concentrations which
showed no visible growth onto plates. Results for MFC were mentioned as no growth concentration on plate.
2.7 Time-Kill Activity
Time Kill kinetics assay was performed by first preparing concentration of MIC, twice MIC and four times MIC for the extracts (ethanolic extracts of Allium sativum L. bulbs and Mentha piperita ). Innoculum size of 1.0x106 was added to the extracts and incubated at 37°C. 1 ml of media was collected from each tube at regular intervals of 0, 5, 10, 15, 20, 25, 30, 35, 40 hours.
These were then spread on Nutrient Agar media and plates were incubated at 25-27°C. CFU was recorded for each case and graph was plotted between Log10CFU/ml vs Time.
2.8 Statistical Analysis
All the experiments were performed in triplicates, and the results’ statistical significance was expressed with the help of IBM SPSS Statistics 20.
3. Result & Discussion
3.1 Chemical constituents in the Extracts
3.1.1 Screening of components using TLC:Identification of chemical constituents inethanolic and aqueous extracts of Allium sativum L. bulbs and Mentha piperita L. leaves were primarily done based on the thin layer chromatography summarized in the table (table 1) and illustrated in the figure (figure 1).
Allium sativum L.extracts were run on silica gel with solvent system of toluene and ethyl acetate in a proportion of 76:24, 50:50 and 24:76. The plates were developed in 1% vanillin in glacial acetic acid and images of the plates were visualized in presence of white light and UV light. The spots which appeared more prominent in this case were yellow, green and brown in colour. Distances covered by the spots were measured and Rf values were calculated. The Rf values corresponding to the spots are summarized in the table (table 1). The table shows presence of various compounds in the extract, majorly sulphur compounds were extracted in ethanolic extracts, defined by presence of spot with Rf of 0.45, referring to allicin and also compounds with very low Rf of 0.062 referring to cepaenes and azoens. Less number of spots were visible in case of hot and cold water extracts.
The result were found similar to Libbieet al. (2008)work where identification of peppermint leaves extracts constituents were based on TLC and spots’ appearance showed similar pattern as in observed in this study.Lilianaet al. (2004) work revealed similar pattern for garlic extracts in their work for TLC.
In case of Mentha piperita L. extract, constituents were separated on silica gel using developing solvent system of toluene and ethyl acetate in proportion of 76:24, 50:50 and 24:76. Samples were prepared for TLC by extracting powdered leaves with toluene (at 10%). The slurry was then
centrifuged and liquid portion were spotted onto silica plates. The reagent used for development of the plate was 1% vanillin in mixture of H2SO4 methanol (40:10). The spots which prominently appeared in this case were of menthol (Rf= 0.4), 1,8 Cineole (Rf= 0.54) and Piperitenone oxide (Rf= 0.6) and also common pigment of chlorophyll (Rf= 0.4) were observed in its extract. In this case also, ethanolic extract proved better solvent as compared to water;more number of compounds were identified using ethanolic extract. Therefore ethanolic extracts of both plants were used in GC-MS analysis for further identification of compounds.
3.1.2 GC-MS Analysis of Extracts
A profile of bioactive constituents in Allium sativum L. and Mentha piperita L. ethanolic extractswere assessed based on GC-MS results. The chromatograms of GC-MS for Allium sativum L. (fig 1) and Mentha piperita L. (fig 2) are reveals the set of compounds extracted using ethanol (75%). Both aqueous (cold and hot water)extracts were screened for active constituents using TLC, as observed in the results they showed feeble appearance of spots and less effective identification, therefore not analyzed using GC-MS. Ethanolic extract showed better identification and more number phytoactive components, therefore its further analysis was done using GC-MS profiling.
Some of the major identified components in Mentha piperita L. ethanolic extracts were á- Terpinene (RT: 3.666 min), L-Menthone (RT: 11.156 min), Menthol (RT: 15.780 min), Piperitone oxide (RT: 24.389 min) and Thymol (RT: 30.288 min). Many studied have been conducted earlier and reported the antimicrobial activity of active constituents of Mentha piperita L. extracts viz., menthol, menthone and limonene, etc. Deans and Baratta (1998) and Flemming (1998) in their work have reported such activity.
In case ofAllium sativum L. ethanolic extracts, identified compounds were 1-propenyl methyl disulphide (RT: 7.442 min), Trisulfide, allyl trisulfide (RT:24.58 min). Propanone (RT: 12.08 min) while other general acidic components which provide it acidic nature were Acetaldehyde (RT:12.54 min), Acetic acid (RT: 16.51 min), Propanoic acid (RT:16.76 min). These are compounds which are obtained due to degradation of alline (Mochizuki et al., 1995; Block, 1985;
Stoll and Seebeck, 2006). They impart medically important properties like antioxidant and anticancer to the plant (Park et al., 2015; Lawson and Wang, 2003; Sparnins et al., 1986; Fukao et al., 2004; Rattanachaikunsopon and Phumkhachorn, 2008; Enders and Balensiefer, 2004; Son and Lee, 2010; Wu et al., 2001)
Fig 1: A typical GC-MS Chromatogram of Allium sativum L. bulbs ethanolic extracts
Fig 2: A typical GC-MS Chromatogram of Mentha piperita L. leaves ethanolic extracts
Table 3: GC-MS profile representing chemical composition of the Allium sativum L. bulbs ethanolic extracts
S.No. Peak No. Compound name Retention time (RT)
Mentha piperita L. leaves ethanolic extract
1 Peak 2 á-Terpinene 3.666
2 Peak 12 L-Menthone 11.156
3 Peak 14 neo-Menthol acetate 13.351
4 Peak 15 neo-Menthol 15.338
5 Peak 16 5-Methyl-2-(1-methylethylidene)-
cyclohexanone 15.727
6 Peak 16 Menthol 15.780
7 peak 24 Piperitone oxide 24.389
8 peak 30 Thymol 30.288
9 Peak 40 Methyl linolenate 40.624
10 Peak 41 3,3-Isopropyledenebis(1,5,8,11-
tetraoxacyclotridecane) 41.808
11 Peak 43 Octadecanoic acid 43.507
Allium sativum L. bulbs ethanolic extracts
1 Peak 4 1-propenyl methyl disulphide 7.442
2 Peak 7 Propanone 12.08 2
3 Peak 8 Acetaldehyde 12.54
4 Peak 11 Acetic acid 16.51
5 Peak 12 Propanoic acid 16.76
6 Peak 25 Trisulfide, allyl trisulfide 24.58
7 Peak 35 1,3-Dihydroxyacetone dimer 30.46
8 Peak 36 2-Hydroxy-gamma-butyrolactone 32.12
9 Peak 39 4H-Pyran-4-one 33.86
10 Peak 47 Hexadecanoic acid 44.2
11 Peak 51 Heptadecane 47.54
3.2Antifungal activity and MIC/MFC of the extracts
Three extracts of Allium sativum L. bulbs and Mentha piperita L. leaves prepared in ethanol (EAE), hot water (HWE) and cold water (CWE) were assessed for their antifungal activity against C.albicans and C.glabrata characterized earlier in this work. Susceptibility of these microorganisms is tabulated in the table given below (table 4).
Table 4: Antifungal activity of Allium sativum L. and Mentha piperita L. extracts against C.albicans and C.glabrata isolates
Extracts
Zone of Inhibition (Mean ± S.D.)
Allium sativum L. Mentha piperita L.
C.albicans C.glabrata C.albicans C.glabrata
EAE 23.8 ±1.31 20.57±0.67 25.9±1.31 23.97±0.06
CWE 9.1 ± 0.22 9.98±0.57 10.07±0.31 7.43±1.11
HWE 12.63 ± 0.60 14.2±0.60 19.83±0.29 11.6±0.78
PC 37.53 ± 0.99 32.33±0.58 34.9±0.36 30.03±0.35
NC 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
*EAE: Ethanolic extract; CWE: Cold water extract; HWE: Hot water extract; PC: Positive control (Clotrimazole :10mg/ml); NC: Negative control (DMSO); zone of inhibition are recorded excluding well diameter -6mm; Data are expressed as means±standard deviation independent experiments in
triplicates.
Fig 3: Antibacterial activity of Extracts against Candida species; ASCA: A.sativum against C.albicans; ASCG:
A.sativum against C.glabrata; MPCA:Mentha.piperita against C.albicans; MPCG: Mentha.piperita against C.glabrata; EAE: Ethanolic extract; CWE: Cold water extract; HWE: Hot water extract; PC: Positive control
(Clotrimazole :10mg/ml)
The results revealed that both extracts(Allium sativum L. and Mentha piperita L.) had no significant antifungal activity in aqueous extracts. Whereas ethanolic extracts of both plants had considerably higher inhibitory action against test fungal strains.
Cold water extract showed lowestantifungal activity of 9.1±0.22 and 9.98±0.57mm (C.albicans and C.glabrata)by Allium sativum L. extract while 10.07±0.31mm and 7.43±1.11mm (C.albicans and C.glabrata) by Mentha piperita L. extract. Hot water extracts had intermediate potency with 12.63
± 0.60 mm and 14.2±0.60 mm(C.albicans and C.glabrata) by Allium sativum L. extract while 19.83±0.29 mm and 11.6±0.78 mm(C.albicans and C.glabrata) by Mentha piperita L. extract.
Susceptibility to ethanolic extracts was found comparable to the positive control. Mentha piperita L. extract activity was strongest against C.albicanswith ZOI of 25.9±1.31mm quite comparable ZOI (34.9±0.36mm) of the positive control. This was followed by activity against C.glabrata(23.97±0.06). Allium sativum L. extract showed higher activity against C.albicans (23.8
±1.31mm) and lesser effective against C.glabrata (20.57±0.67mm). Our study was found in accordance to the earlier reported antifungal potency of Allium sativum L. and Mentha piperita L.
extracts. Abdallah (2017) reported significant activity of garlic bbulb extract against C.albicans.
Inhibitory zone observed against this fungus was 28.0±1.0 mm.
MIC and MFC were performed for only ethanolic extracts as aqueous extracts did not show very significant results for both Allium sativum L. and Mentha piperita L. extracts. It was done to evaluate the minimum concentration at which this extract could inhibit/kill the fungal strains.In case of MIC, each extracts were prepared in concentration ranging from 3.125 mg/ml - 100 mg/ml while positive control (Clotrimazole) concentrations were in range of 0.25 to 128 μg/mL. MICs for
23.8 20.57
25.9 23.97
9.1 9.98 10.07
7.43
12.63 14.2
19.83
11.6 37.53
32.33 34.9
30.03
ASCA ASCG MPCA MPCG
Antibacterial activity of Extracts against Candida species
EAE CWE HWE PC
Allium sativum L. and Mentha piperita L. extracts were determined to be 6.25 mg/ml and MFC 12.5 mg/ml in all cases. The number of viable cells of C.albicans and C. glabrata ar different point of time were assessed using Time-Kill activity plot between Log10CFU of Cells/ml and time intervals (in hours). The plot (shown in fig 4) shows that nubber of viable cells reduce in case of antifungal agents (PC, Allium sativum L. and Mentha piperita L. extracts) as compared to untreated cells (UC).
Fig 4: Time Kill plot of Allium sativum L. and Mentha piperita L. ethanolic extracts at different time intervals; PC: Positive control; ASCA: A.sativum against C.albicans; ASCG: A.sativum against C.glabrata;
MPCA:Mentha.piperita against C.albicans; MPCG: Mentha.piperita against C.glabrata;UC: untreated control
Antifungal activity of methanolic and choloroform extracts from Mentha piperita L. leaves has been reported by Wenji et al. (2019) in his work. His results showed that both methanolic and chloroform extracts possess antifungal activity against C. albicans.Diba and Alizadeh (2018) in their study revealed antifungal activity of Allium sativum L. against C.tropicalis.These two plants have been widely studied for their antifungal potential, against various Albicans and Non-Albbican species. Our study aimed to explore their potency against C.albicans and C.glabrata species.
0 2 4 6 8 10 12 14 16
0 5 10 15 20 25 30 35 40 45
Log 10CFU of Cells/ml
Time (Hrs)
PC ASCA ASCG MPCA MPCG UC
Conclusion
This study dealt with extraction and identification of active constituents fromAllium sativum L. and Mentha piperita L. extracts. The results in the study suggested that ethanol turns out to be a better solvent for extraction of active constituents rather than water. Also, in aqueous extract hot water extracts had significant activity than cold water extract. The preliminary identification based on TLC results showed presence of many constituents and their confirmation was done using GC-MS analysis. Antifungal activities of these two extracts on C.alicans and C.glabrata showed Mentha piperita L. extracts more potent than garlic extract. Many studies have been reported earlier for C.albicans inhibition but C.glabrata is less studied and not many antifungal agents are yet well known. Since natural products are in high demand owing to their low complexity, less side effects and less harmful metabolites. Therefore, this study provides an insight into beneficial roles of Allium sativum L. and Mentha piperita as potential natural active agents for future use in field of therapeutics and drugs.
Acknowledgment
We are sincerely thankful to the Department of Clinical Laboratory Sciences, Faculty of Health Sciences, SHUATS, Allahabad (U.P.) to provide facilities to carry out this work. I am also thankful to my advisor for his valuable suggestions to improve this manuscript.
Financial support and sponsorship Nil
Conflicts of interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.
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