RESEARCH ARTICLE Open Access Synergistic antifungal evaluation of over- the-counter antifungal creams with turmeric essential oil or Aloe vera gel against pathogenic fungi Clement Olusola Ogidi1* , Ayokunbi Elizabeth Ojo2, Oluwatayo Benjamin Ajayi-Moses2, Oluwatoyin Modupe Aladejana3, Oluwakemi Abike Thonda3 and Bamidele Juliet Akinyele2 Abstract Background: The frequent incidence of fungal infection and widespread of antibiotic resistance are emergent concerns in public health. Hence, there is a need to harness the potential of natural bioactive compounds from plant towards treatment of fungal infection. Combination effect of antibiotic creams with natural products from plants is prospective strategy to produce new antifungal agent. This study therefore, revealed antifungal effect of combined Antifungal Creams (AFCs) with Turmeric Essential Oil (TEO) or Aloe vera Gel (AVG). Methods: Phytochemicals and bioactive compounds in TEO and AVG were revealed using GC-MS. Bioactive compounds in plant extracts were compared to known compounds in database library of National Institute of Standards and Technology (U.S.). Antifungal activity and synergistic effect of AFCs with TEO or AVG were carried out using agar well diffusion method. Results: Phenol, flavonoids, saponins, alkaloids, steroids, terpenoids and cardiac glycosides were present in TEO and AVG. GCMS revealed thirty-six (36) and eighteen (18) bioactive compounds in TEO and AVG, respectively. AFCs displayed zones of inhibition with values ranged from 5.0 to 14.3 mm, TEO was 5.0 to 11.0 mm and AVG was 8.0 to 11.7 mm against tested fungi. Minimum Inhibitory Concentration (MIC) by AFCs, TEO and AVG ranged from 1.25 to 10.0 mg/ml. Combinatory effects of AFCs with TEO or AVG revealed synergistic and indifferent properties. Conclusion: Development of novel products using bioactive ingredients from plants with commercially available AFCs will serve as potential alternative therapy to cure dermatological infections with no side effects. Keywords: Dermatophytes, -azole, Terbinafine, Curcuma longa rhizomes, Cosmeceutical, GC-MS Background Mycotic diseases are causing significant morbidity and now seen as a serious concern to public health [1, 2]. The spread of fungal diseases is increasing by overuse of broad-spectrum antibiotics, which lessening non- pathogenic bacterial population that check the growth of fungi through competition [3]. Antifungal drugs play ac- tive roles in the treatment of some fungal infections but their misuse always made the fungal infection worsen. Superficial and subcutaneous fungal infections are very dangerous if not promptly and properly treated with ap- propriate drugs. The erroneous use of antifungal drugs has contributed to frequent resistance experience over the past decades [4]. In addition, most antifungal drugs © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. * Correspondence: clementogidi@yahoo.com 1Biotechnology Unit, Department of Biological Sciences, Kings University, PMB 555, Odeomu, Nigeria Full list of author information is available at the end of the article BMC Complementary Medicine and Therapies Ogidi et al. BMC Complementary Medicine and Therapies (2021) 21:47 https://doi.org/10.1186/s12906-021-03205-5 http://crossmark.crossref.org/dialog/?doi=10.1186/s12906-021-03205-5&domain=pdf http://orcid.org/0000-0002-4154-6750 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/publicdomain/zero/1.0/ mailto:clementogidi@yahoo.com currently available to treat fungal infections have serious drawbacks, which include low concentration of active in- gredients, itching on the skin due to chemical compos- ition, development of fungi resistance and toxic side effects [5]. Conventional formulation of creams, powder, gels to treat skin or deep seated fungal infections still have various side-effects like burning, redness and swell- ing on the application site [6]. Beside different side ef- fects committed to commercially available antifungal drugs, these days, multiple drug resistance is fast rising worldwide and efficacy of single antibiotic against resist- ant microorganisms is abating. The continuous thera- peutic failure as a result of multi-drug resistance by pathogenic fungi is urgently demanding for innovative complementary approach towards production of novel, alternative and effective antimicrobial drugs. Invention of antifungal drugs with different drug delivery systems like liposomes, niosomes, ethosomes, microemulsions, microsponge, nanoparticles are now embraced as treat- ment option for fungal infection in order to achieve clin- ical efficacy [6, 7]. The increasing therapeutic potentials of drug combination has been found more efficacious than single antifungal agent aimed at a single target [8]. To overcome the drawbacks of conventional therapy and to produce antifungal agents with dose efficient, a blueprint for development of effective antifungal drugs or creams from natural herbs is now an element of con- sensus among researchers, medical practitioners and pharmaceutical companies [9]. To enhance efficacies of antibiotics (creams or drugs) and to minimize their side effects, combinatory effect of commercial antifungal drugs with natural products will demonstrate a huge success in treating fungal infections. Antifungal drugs with live lactic acid bacteria (probiotics) were designed to protect, restore the natural balance in the vagina and help to fight yeast infection without side effects [10]. Globally, combination and synergistic interaction of anti- microbial agents with multiple herbs formulation of dif- ferent natural bioactive compounds are the main therapy to cure some medical challenges [11]. Antifungal combination therapy is trending with veracity in the fields of infectious diseases and medical mycology [12]. The combination and synergistic effects of commercial antifungal drugs with natural bioactive compounds from plants will be a complementary and alternative approach to combat reoccurrence incidences of fungal infection. Medicinal plants are imperious sources of treasurable secondary metabolites and thus, contribute to availability of natural drugs in global markets [13]. Turmeric; Curcuma longa belong to ginger family; Zingiberaceae. Turmeric powder from dried ground rhizomes of C. longa possess culinary uses, act as natural product with analgesic, antibacterial, antifungal, anti-inflammatory, antioxidant, and digestive properties. Hence, biological activities of turmeric, its health promoting effects and disease prevention have attracted several applications in pharmaceutical, food and biotechnological industries [14]. Aloe vera, is another medicinal plant with immense bioactive compounds allied with some pharmacological properties like wound healing, antifungal activity, hypoglycemic or antidiabetic effects, anti-inflammatory, anticancer, immunomodulatory and gastroprotective [15]. The therapeutic potentials of A. vera in phytomedi- cine indicate its several uses in pharmaceutical and cos- metic industries [16]. Natural products in plants act as reservoirs of novel bioactive compounds and as excellent source of drug discovery with divers’ biopharmaceutical applications [17], hence, biologically active compounds in medicinal plants can be exploited and incorporated in AFCs, which will be a newsworthy option towards new prototype antifungal agents. This study therefore, re- vealed synergistic antifungal potential of over-the- counter AFCs with TEO or AVG against clinically im- portant pathogenic fungi. Methods Sample collection Turmeric was collected from a farmland in Isarun Vil- lage, Ifedore Local Government Area, Ondo State. The rhizomes were washed with distilled water before they were cut into smaller pieces. Aloe vera was obtained from a farmland in Aule, Akure South Local Govern- ment Area, Ondo state. C. longa rhizomes and Aloe vera were authenticated at the Department of Crop, Soil and Pest Management, The Federal University of Technol- ogy, Akure and was deposited in the herbarium of the same Department. Source of antifungal creams (AFCs) The commercially available AFCs namely; clotrimazole (1%), fluconazole (0.5%), ketoconazole (2%) and terbina- fine (1%) were purchased. These AFCs were certified by National Agency for Food and Drug Administration and Control (NAFDAC), a federal agency under the Federal Ministry of Health; responsible for regulation and con- trol of importation, exportation, advertisement, distribu- tion, sale and use of food, drugs, cosmetics, medical devices, chemicals and packaged water in Nigeria. Collection of tested fungi The tested fungal isolates namely: Candida tropicalis (ATCC 66029) was obtained from Nigeria Institute of Medical Research, Lagos. Candida albicans, Penicillium notatum, Aspergillus fumigatus, A. niger, A. flavus, Trichophyton rubrum, Trichophyton violceum and Trichophyton mentagrophytes were collected from Department of Medical Microbiology Laboratory, Federal Medical Centre, Ido-Ekiti, Nigeria. Ogidi et al. BMC Complementary Medicine and Therapies (2021) 21:47 Page 2 of 12 Extraction of TEO and AVG Essential oil was extracted from the turmeric rhizomes by the process of steam distillation using Clevenger ap- paratus [18]. Fresh rhizome (100 g) of turmeric was boiled with 500ml of distilled water in a Clevenger ap- paratus until oil distillation ceased after 5 h. The volume of essential oil was determined from a calibrated trap. The essential oil in the distillate were dried over anhyd- rous Na2SO4 and kept in the freezer. A. vera leaf was cleaned with ethanol, dissected and the gel; jelly-like substance found in the inner part of the A. vera leaf was aseptically collected into sterile tubes. Determination of phytochemicals and bioactive compounds in TEO and AVG Qualitative and quantitative phytochemicals in TEO and AVG were determined using the standard methods. Briefly, total phenolic and flavonoid con- tents of extracts was determined according to the method of Sofowora [19]. The methods stated by Harborne [20] and Trease and Evan [21] were used to determined alkaloids, saponins, tannins, steroid, tepernoids and cardiac glycoside. The method de- scribed by Soladoye [22] was used to determine the anthraquinone content. The bioactive compounds in the TEO and AVG were identified with the aid of gas chromatography– mass spectrometry (QP2010 plus Shimadzu, Japan), which was equipped with a split injector and an ion – trap mass spectrometer detector together with a fused – silica ca- pillary column having a thickness of 1.00 μm, dimen- sions of 20 m × 0.22 mm and temperature limits of 60 °C to 325 °C. The column temperature was programmed between 60 °C and 250 °C at a rate of 0.5 m/s with pres- sure of 100.2 Kpa. The temperature of the injector and detector were at 250 °C and 200 °C respectively. Helium gas was used as a carrier gas at flow rate of 0.46 m/s. The MS analysis was done based on comparative reten- tion times, mass and peaks of the chemical compounds using the computer-aided matching of unknown mass spectra of compounds with the known compounds stored in the software database library from the National Institute of Standards and Technology (NIST), Washing- ton, USA, having more than 62,000 patterns as the refer- ence database. The name, molecular weight and the structure of the components of the tested materials were ascertained with database library from the NIST, Wash- ington, USA. Antifungal activities of AFCs, TEO and AVG The antifungal assay was carried out using the agar well diffusion method described by CLSI [23, 24]. Suspen- sions of fungi (1.0 × 105 sfu/ml) was adjusted with the aid of spectrophotometer (UNICO S-1100 RS) to 0.5 McFarland standard. Dimethyl sulfoxide (DMSO 2% v/v) was used to reconstitute since most of AFCs and plant extracts (TEO and AVG) were not soluble in sterile dis- tilled water. The concentration of AFCs, TEO and AVG were reconstituted to 10.0 mg/ml. Plant extract was ster- ilized using a Millipore membrane filter (0.22 μm). The sterility of TEO and AVG were confirmed after Milli- pore filtration, by introducing 0.1 ml of supposed sterile extract into sterilized nutrient agar and potato dextrose agar. Each labelled plate was seeded with tested fungi by means of sterile swab stick rolled on potato dextrose agar. Sterile cork borer was used to make well (6 mm) in the Petri dishes. Aliquots of TEO, AVG and AFCs (50 μl) were dropped in each well. DMSO solution was used as the negative control. The plates were incubated at 26 °C for 48 h. The zones of inhibition around well were measured in millimeter (mm). For synergism activ- ity, concentration of each AFCs, TEO and AVG was ad- justed to 3.0 mg/ml. Determination of minimum inhibitory and fractional inhibitory concentration index (FICi) The minimum inhibitory concentration was determined by using method described by CLSI [23, 24]. The varying concentrations of 1.25, 2.5, 5.0, and 10.0 mg/ml for AFCs, TEO and AVG were prepared and in- corporated into a set of sterile tubes. Each test tube was inoculated with 0.1 ml of standardized fungal inoculum and incubated at 26 °C for 48 h. The MIC were recorded as the lowest concentration to prevent growth of macro- scopically visible colonies on plates, while there was vis- ible growth on plates without AFCs, TEO and AVG. To determine MIC of combined AFC with TEO or AVG, varying concentrations of 1.0–3.0 mg/ml was used. The synergism, indifference, and antagonism of combined AFC with TEO or AVG were screened on the studied pathogenic fungi. MICs were transformed into Fractional Inhibitory Concentration (FIC) to determine the inter- action of two samples in the following manner: FIC of AFC =MIC of AFC in presence of TEO/MIC of AFC FIC of TEO =MIC of TEO in presence of AFC /MIC of TEO or FIC of AFC =MIC of AFC in presence of AVG /MIC of AFC FIC of AVG =MIC of AVG in presence of AFC /MIC of AVG Fractional Inhibitory Concentration index (FICi) for each sample was calculated from FIC values as follows: FICi = FIC of AFC + FIC of TEO or FICi = FIC of AFC + FIC of AVG. Ogidi et al. BMC Complementary Medicine and Therapies (2021) 21:47 Page 3 of 12 The FICi was interpreted as: synergistic when FICi ≤0.5; indifferent when FICi was 0.5–4.0 and antagonistic when FICi ≥4.0 [25]. Statistical analysis Experimental studies were carried out in replicates (n = 3). Data obtained were subjected to one-way ana- lysis of variance (ANOVA) using Statistical Package for Social Sciences (SPSS) version 20, USA. Results obtained were reported as mean ± standard deviation (SD). Values were compared by Duncan’s new mul- tiple range test (MRT) and differences were consid- ered significant when P < 0.05. Results Phytochemical and bioactive compounds in TEO and AVG as revealed by GC-MS The quantity of phytochemicals of TEO and AVG was revealed in Fig. 1. Phytochemical such as phenol, flavo- noids, saponins, alkaloids, steroids, terpenoids and car- diac glycosides were present in TEO and AVG. while anthraquinones and tannins was present only in AVG. Phenol was present with values of 2.9 mg/ 100 g and 5.1 mg/ 100 g in TEO and AVG, respectively. Alkaloid in TEO and AVG were not significantly different (p = 0.05) with value of 2.3 mg/100 g and 2.1 mg/100 g. Anthraqui- nones and tannins in AVG was 3.5 mg/100 g and 2.3 mg/100 g, respectively. Cardiac glycoside has the least values of 0.30 mg/100 g in TEO and 0.48 mg/100 g in AVG. Figures 2 and 3 show chromatogram of TEO and AVG with peaks for various bioactive constituents. The peaks were shown for 36 and 18 bioactive compounds in TEO and AVG, respectively. Tables 1 and 2 show the presence of bioactive compounds in TEO and AVG, re- spectively identified with GC MS. Z-citral was the major compound in the TEO (14.02%), while Z-9-Tetradecenol (24.99%) was the most abundant compound in AVG. Bioactive compounds such as α -pinene, camphene, linalool, borneol, p-menth-1-en-8-ol, zingiberene, farne- sene, farnesol and others were found in TEO (Table 1). In AVG, cis oleic acid, dioctyl adipraate, glycerin 1,3-dis- tearate, arachidic acid methyl ester, dipentene diepox, z- 9-tetradecenol and others (Table 2). Inhibitory potentials and synergistic antifungal efficacy of AFCs with TEO or AVG against pathogenic fungi The zones of inhibition (mm) reflecting the antifungal efficacy of AFCs, TEO and AVG were reported in Table 3. AFCs namely; clotrimazole, fluconazole, ketoco- nazole and terbinafine displayed zones of inhibition against tested fungi with values ranged from 5.0 to 11.6 mm, 5.0 to 11.3 mm, 5.0 to 11.3 mm and 8.0 to 14.3 mm, respectively. TEO have inhibitory zones of 5.0 to 11.0 mm, while AVG have 8.0 to 11.7 mm against tested fungi. Varying zones of inhibition indicated antifungal activity of combined AFCs with TEO or AVG as shown Fig. 1 Quantitative constituents (mg/100 g) of phytochemicals in TEO and AVG. Error bar is SD Ogidi et al. BMC Complementary Medicine and Therapies (2021) 21:47 Page 4 of 12 in Table 4. Combinatory effect of AFCs with TEO or AVG showed better inhibitory zones against fungi. Keto- conazole + TEO, terbinafine + TEO, fluconazole + AVG and terbinafine + AVG have similar (p < 0.05) inhibitory effects against C. tropicalis (ATCC 66029). Terbinafine + AVG displayed the highest (p < 0.05) zones of inhib- ition of 12.7 mm and 13.6 mm against C. albicans and Penicillium notatum, respectively. Zones of inhibition displayed by each AFC combined with AVG against A. fumigatus were not significantly different when p < 0.05. Fig. 2 Chromatogram of TEO with peaks for various bioactive constituents Fig. 3 Chromatogram of AVG with peaks for various bioactive constituents Ogidi et al. BMC Complementary Medicine and Therapies (2021) 21:47 Page 5 of 12 Inhibitory action of each AFC combined with TEO against A. fumigatus were also similar. Ketoconazole + AVG and terbinafine + AVG respectively have similar inhibitory zones of 12.7 mm and 13.7 mm against T. mentagrophytes. Table 5 shows minimum inhibitory concentration of AFCs, TEO and AVG against fungi. AVG displayed lower range of MIC values (1.25 to 5.0 mg/ml), while other were within 1.25 to 10 mg/ml. The MIC ob- tained for combined AFCs with TEO or AVG was shown in Table 6. Combination of clotrimazole + AVG displayed lower MIC value of 1.0 to 2.0 mg/ml against tested fungi. The MICs of fluconazole + AVG, ketoconazole + AVG, and terbinafine + AVG were within 1.0 to 2.5 mg/ml against tested fungi. Table 7 shows FIC, FIC indices (FICi) as well as their Table 1 Main component and the relative contents of TEO as revealed by GCMS Peaks Retention time Area % Bioactive compounds Molecular formula Molecular weight 1 4.886 0.19 n-pentyl methyl ketone C7H14O 114 2 5.115 0.85 Amyl methyl carbinol C7H16O 116 3 5.483 0.30 Tricyclene C10H16 136 4 5.652 3.01 α -pinene C10H16 136 5 5.924 6.25 Camphene C10H16 136 6 6.251 0.25 Sabinene C10H16 136 7 6.355 1.55 6-Methyl-5-hepten-2-one C8H14O 126 8 6.452 2.01 β -Myrcene C10H16 136 9 6.625 0.55 Caprylaldehyde C8H16O 128 10 6.756 0.82 α-Phellandrene C10H16 136 11 7.222 11.41 cis-.beta.-Terpineol C10H18O 154 12 7.535 0.23 (E)-2-Octenal C8H14O 126 13 8.070 0.76 2-Carene C10H16 136 14 8.272 2.57 Linalol C10H18O 154 15 8.341 1.21 d-Verbenol C10H16O 152 16 8.871 0.41 2-Methyl- 6-methylene 2-octene C10H18 138 17 9.001 2.18 Citronellal C10H18O 154 18 9.167 1.90 Artemiseole C10H16O 152 19 9.494 6.59 Borneol C10H18O 154 20 9.584 1.09 1-Terpinen-4-ol C10H18O 154 21 9.818 3.10 p-menth-1-en-8-ol C10H18O 154 22 10.248 2.65 β -Citronellol C10H20O 156 23 10.440 11.94 cis,trans-Citral C10H16O 152 24 10.635 2.48 trans-Geraniol C10H18O 154 25 10.881 14.02 Z-Citral C10H16O 152 26 11.072 1.06 Methyl nonyl ketone C10H18 138 27 11.836 0.21 (6E)-2,6-Dimethyl-2,6-octadiene C10H18 138 28 12.247 0.57 Geraniol acetate C12H20O2 172 29 12.603 0.34 2,4-Diisopropenyl-1-methyl-1-vinylcyclohexane C15H24 204 30 13.794 1.54 α-Curcumene C15H22 202 31 13.998 7.27 Zingiberene C15H24 204 32 14.056 2.68 Farnesene C15H24 204 33 14.189 2.26 (Z)-.beta.-Farnesene C15H24 204 34 14.472 4.47 β-Sesquiphellandrene C15H24 204 35 15.064 0.92 D-nerolidol C15H26O 222 36 16.196 0.37 (2E,6E)-Farnesol C15H26O 222 Ogidi et al. BMC Complementary Medicine and Therapies (2021) 21:47 Page 6 of 12 interpretation. Clotrimazole + TEO against C. albi- cans, ketoconazole + TEO against A. niger, terbinafine + TEO against C. albicans, clotrimazole + AVG against C. albicans, fluconazole + TEO against A. fla- vus and terbinafine + AVG against C. tropicalis (ATCC 66029) displayed synergistic properties. Other combinatory effects of AFC with TEO or AVG were indifferent without antagonism. Discussion Researchers across the board have responded to con- tinuous increase of multiple antibiotic resistance by pathogenic fungal strains and thus, revealed that novel potential strategy to promote antifungal therapeutic is urgently needed to be explored [26]. In this study, the combinatory potential of AFCs with TEO or AVG was assessed. Terbinafine was efficient AFC against patho- genic fungi in vitro. Terbinafine is known as a broad spectrum antifungal agent, active against wide range of dermatophytes, moulds, yeasts and dimorphic fungi [27]. However, studies by Karri et al. [28] reported less activ- ity of terbinafine against Candida albicans. Terbinafine was considered to have potency against dermatophytes but now, there is a rise to terbinafine resistance by pathogenic fungi [29]. The efficacy of AFCs (azole creams) such as clotrima- zole, fluconazole, and ketoconazole against selected pathogenic fungi was observed, while fungi such as Table 2 Main component and the relative contents of AVG as revealed by GCMS Peaks Retention time Area % Bioactive compounds Molecular formula Molecular weight (g/mol) 1 11.049 2.18 3-Hydroxybenzhydrazide C7H8N2O2 167 2 12.905 0.88 4-Decyl methylphosphonofluoridate C11H24FO2P 182 3 14.574 2.49 Dipentene diepox C10H16O2 136 4 15.727 3.23 Arachidic acid methyl ester C21H42O2 296 5 17.08 3.05 1-Pentadecanecarboxylic acid C16H32O2 314 6 18.941 4.78 Methyl (9E,12E)-9,12-octadecadienoate C19H34O2 294 7 19.00 7.89 Methyl (10E)-10-octadecenoate C19H36O2 296 8 19.36 1.19 Methyl arachisate (Kemester 2050) C21H42O2 326 9 20.17 24.99 Z-9-Tetradecanol C14H26O 214 10 21.07 4.99 trans-13-Docosenoic acid C22H42O2 338 11 21.71 1.86 Glycerin 1,3-distearate C39H76O5 625 12 22.74 2.10 Dioctyl adipate C22H42O4 370 13 23.12 1.15 Lineoleoyl chloride C18H31ClO 300 14 23.58 9.12 cis-Oleic acid C18H34O2 282 15 23.79 1.98 Eicosanoic acid C20H40O2 312 16 24.31 18.99 n-Octyl phthalate C24H38O4 390 17 25.38 1.12 13-Tetradecenal C14H26O 210 18 25.74 8.00 Z-9-Tetradecenol C14H26O 212 Table 3 Zones of inhibition (mm) by AFCs, TEO and AVG against pathogenic fungi at 10.0 mg/ml of each tested agent Isolates Clotrimazole Fluconazole Ketoconazole Terbinafine TEO AVG Candida tropicalis (ATCC 66029) 10.6 ± 0.2 a 8.0 ± 0.0 b 11.0 ± 0.0a 8.0 ± 0.1 b 10.0 ± 0.1 a 8.3 ± 0.0 b Candida albicans 5.3 ± 0.0 c 7.0 ± 0.6 b 9.0 ± 0.0 a 9.3 ± 0.0 a 5.0 ± 0.0 c 9.7 ± 0.1 a Penicillium notatum 11.6 ± 0.1 a 11.3 ± 0.6 a 7.0 ± 0.3 b 10.7 ± 0.3 a 10.0 ± 0.3 a 10.0 ± 0.0 a Aspergillus fumigatus 8.0 ± 0.0 b 6.0 ± 0.0 c 5.0 ± 0.0 c 11.0 ± 0.0 a 9.2 ± 0.0 ab 10.0 ± 0.0 a Aspergillus niger 11.0 ± 0.2 a 8.0 ± 0.0 c 7.3 ± 0.1 c 10.0 ± 0.0 ab 8.3 ± 0.0 c 12.7 ± 0.2 a Aspergillus flavus 10.6 ± 0.2 b 9.0 ± 0.5 bc 6.0 ± 0.0 d 14.0 ± 0.0 a 11.0 ± 0.0 b 8.0 ± 0.0 c Trichophyton rubrum 5.0 ± 0.0 c 5.3 ± 0.0 c 10.3 ± 0.1b 14.3 ± 0.2 a 10.3 ± 0.2 b 11.7 ± 0.3 b Trichophyton violceum 8.0 ± 0.1 b 6.0 ± 0.0 c 11.3 ± 0.0 a 10.6 ± 0.3 a 10.6 ± 0.3 a 8.7 ± 0.1 b Trichophyton mentagrophytes 8.3 ± 0.0 b 5.0 ± 0.0 d 8.0 ± 0.0 b 12.7 ± 0.3 a 7.9 ± 0.0 bc 9.0 ± 0.5 b Value are mean ± SD of replicates (n = 3). Values with the same superscript alphabet along row are not significantly different from each other when P < 0.05 Ogidi et al. BMC Complementary Medicine and Therapies (2021) 21:47 Page 7 of 12 Candida albicans, Trichophyton rubrum, T. mentagro- phytes required higher concentration of AFCs before being inhibited. Shivamurthy et al. [30] reported that sertaconazole showed better anti-dermatophytic in clin- ical parameters than topical clotrimazole within a span of 3 weeks in the treatment of Tinea corporis. Sabatelli et al. [31] tested triazoles against wide number of clinic- ally important pathogenic fungi (19,000 yeast and mould) and found out that, species of Candida and Aspergillus exhibited resistance to fluconazole, voricona- zole, itraconazole and amphotericin B except posacona- zole that was more efficient. Azole or triazole are commonly used antifungal agents that suppress fungi growth by inhibiting a key enzyme; lanosterol 14alpha demethylase, which occurs through the binding of the free nitrogen atom of the azole ring to the iron atom of the heme-group of the enzyme [32]. In this study, in vitro antifungal effectiveness of over- the-counter AFCs and their synergism with TEO or AVG against pathogenic fungi of clinical sources was at- tributed to phytochemicals as well as bioactive ingredi- ents in TEO and AVG. Phytochemicals are biologically active, naturally occurring chemical compounds in plants that promote human health and prevent diseases [33]. The presence of these biologically active phyto- chemicals (phenol, flavonoids, saponins, alkaloids, ste- roids, terpenoids, cardiac glycosides, anthraquinones and tannins) in studied extracts make them useful for some medicinal purposes such as antimicrobial against patho- genic microorganisms. Sawant and Godghate [34] re- ported that turmeric was one of the best source to obtain a variety of drugs due to its rich phytochemical constituents. Bawankar et al. [35] reported the presence of hexadecanoic acid, 1-(phenylthioxomethyl) piperidine, 6-hydroxyhexane-3-1, octadecanoic acid, tricosane, 1- octadecanol, stigmasterol, docosane in the ethanolic extract of A. vera. Hydroxybenzhydrazide was found Table 4 Zones of inhibition (mm) displayed by combined AFCs with TEO or AVG against pathogenic fungi at 3.0 mg/ml of each tested agent Isolates Clotrimazole + TEO Fluconazole + TEO Ketoconazole + TEO Terbinafine + TEO Clotrimazole + AVG Fluconazole + AVG Ketoconazole + AVG Terbinafine + AVG C. tropicalis (ATCC 66029) 9.6 ± 0.2 bc 8.0 ± 0.0 c 10.0 ± 0.0 a 12.0 ± 0.3 a 7.7 ± 0.0 cd 10.3 ± 1.5 a 7.3 ± 0.1 cd 11.3 ± 1.2 a C. albicans 10.6 ± 0.2 b 11.0 ± 0.5 b 9.0 ± 0.0 c 8.6 ± 0.8 c 9.0 ± 1.0 c 8.0 ± 2.0 c 7.0 ± 0.0 d 12.7 ± 1.0 a Penicillium notatum 10.7 ± 0.6 b 11.3 ± 0.6 b 7.7 ± 0.3 d 9.7 ± 0.3 c 9.0 ± 1.0 c 6.0 ± 1.0 e 8.0 ± 1.0 d 13.6 ± 1.3 a A. fumigatus 9.0 ± 0.6 b 8.0 ± 0.0 b 8.0 ± 0.0 b 8.0 ± 0.0 b 11.0 ± 2.0 a 11.0 ± 1.0 a 10.6 ± 1.7 a 10.7 ± 0.5 a A. niger 10.0 ± 0.6 a 8.0 ± 0.0 b 7.0 ± 0.0 b 7.0 ± 0.0 b 7.0 ± 1.0 b 7.7 ± 0.5 b 8.6 ± 0.3 b 11.3 ± 0.3 a A. flavus 9.6 ± 0.2 b 7.0 ± 0.0 c 9.0 ± 0.5 b 11.0 ± 0.5 a 10.0 ± 1.0 a 9.0 ± 1.0 b 7.3 ± 0.3 c 10.7 ± 0.5 a T. rubrum 6.0 ± 0.5 d 10.3 ± 0.3 b 11.0 ± 0.5 b 9.6 ± 0.3 bc 8.0 ± 0.3 c 10.7 ± 1.3 b 8.0 ± 0.0 c 13.7 ± 0.3 a T. violceum 10.0 ± 0.6 a 9.3 ± 0.0 ab 9.0 ± 0.5 ab 10.3 ± 1.0 a 10.0 ± 0.0 a 8.0 ± 1.0 b 11.3 ± 0.5 a 10.0 ± 0.0 a T. mentagrophytes 8.0 ± 0.5 cd 9.0 ± 0.5 c 8.3 ± 0.3 c 7.6 ± 0.3 d 11.7 ± 1.1 b 10.0 ± 2.0 b 12.7 ± 0.0 a 13.7 ± 0.8 a Value are mean ± SD of replicates (n = 3). Values with the same superscript alphabet along row are not significantly different from each other when P < 0.05 Table 5 Minimum inhibitory concentration (mg/ml) of AFCs, TEO and AVG against pathogenic fungi Isolates Clotrimazole Fluconazole Ketoconazole Terbinafine TEO AVG C. tropicalis (ATCC 66029) 2.50 2.50 5.00 10.00 2.50 2.50 C. albicans 10.00 5.00 5.00 10.00 10.00 5.00 P. notatum 2.50 2.50 5.00 2.50 5.00 2.50 A. fumigatus 5.00 5.00 10.00 2.50 5.00 5.00 A. niger 5.00 5.00 10.00 2.50 10.00 2.50 A. flavus 2.50 5.00 10.00 1.25 5.00 5.00 T. rubrum 10.00 10.00 5.00 2.50 5.00 2.50 T. violceum 10.00 10.00 5.00 1.25 5.00 1.25 T. mentagrophytes 5.00 10.00 5.00 5.00 5.00 2.50 Ogidi et al. BMC Complementary Medicine and Therapies (2021) 21:47 Page 8 of 12 in AVG. It is a hydroxylated phenolic compound with strong and moderate antimicrobial activity [36]. Hy- droxylated phenolic compounds like pyrocatechol are known to be toxic to microorganisms [37]. The tox- icity of phenolic compounds to microorganisms are believed to be related to the number of hydroxyl groups, which inhibit microbial growth by cell membrane disruption and protein denaturation [38]. Alpha-phellandrene, α-pinene, myrcene, linalol, geraniol, ar-turmerone, turmerone, α-curcumene, zin- giberene, turmerones, curcuminiods, geraniol acetate beta-sesquiphellandrene, (2E,6E)-farnesol, camphene, tricyclen cineole were major bioactive compounds as aromatic compounds in EOs with dynamic antimicro- bial features [39, 40]. Alpha-phellandrene is a terpene-derivative metabolite, which is mostly found Table 6 Minimum inhibitory concentration (mg/ml) of combined AFCs with TEO or AVG against pathogenic fungi Isolates Clotrimazole + TEO Fluconazole + TEO Ketoconazole + TEO Terbinafine + TEO Clotrimazole + AVG Fluconazole + AVG Ketoconazole + AVG Terbinafine + AVG C. tropicalis (ATCC 66029) 1.50 1.00 2.00 3.00 1.00 1.50 1.00 1.00 C. albicans 2.50 3.00 2.00 2.00 1.00 2.50 2.00 2.00 P. notatum 2.00 2.00 1.50 1.00 1.00 1.00 1.50 1.50 A. fumigatus 2.50 2.50 3.00 1.00 1.50 1.50 2.50 1.00 A. niger 2.00 3.00 2.50 1.50 1.00 2.50 1.50 1.50 A. flavus 2.00 2.50 2.50 1.50 1.50 1.00 2.00 1.50 T. rubrum 3.00 3.00 1.50 2.50 1.50 1.50 2.50 2.50 T. violceum 2.50 2.00 2.50 1.00 1.00 2.00 1.50 2.00 T. mentagrophytes 1.50 2.50 3.00 2.00 2.00 2.00 2.00 1.50 Table 7 Fractional inhibitory concentration (FIC) and FIC indices (FICi) FIC aFICi/interpretation C. tropicalis (ATCC 66029) C. albicans P. notatum A. fumigatus A. niger A. flavus T. rubrum T. violceum T. mentagrophytes Clotrimazole 0.60 0.25 0.80 0.50 0.40 0.80 0.30 0.25 0.30 TEO 0.60 0.25 0.40 0.50 0.20 0.40 0.60 0.50 0.30 FICi 1.20/ind 0.50/syn 1.20/ind 1.00/ind 0.60/ind 1.20/ind 0.90/ind 0.75/ind 0.60/ind Fluconazole 0.40 0.60 0.80 0.50 0.60 0.50 0.30 0.20 0.25 TEO 0.40 0.30 0.40 0.50 0.30 0.50 0.60 0.40 0.50 FICi 0.80/ind 0.90/ind 1.20/ind 1.00/ind 0.90/ind 1.00/ind 0.90/ind 0.60/ind 0.75/ind Ketoconazole 0.40 0.40 0.30 0.30 0.25 0.25 0.30 0.50 0.60 TEO 0.80 0.20 0.30 0.60 0.25 0.50 0.30 0.50 0.60 FICi 1.20/ind 0.60/ind 0.60/ind 0.90/ind 0.50/syn 0.75/ind 0.60/ind 1.00/ind 1.20/ind Terbinafine 0.30 0.20 0.40 0.40 0.60 1.20 1.00 0.80 0.40 TEO 1.20 0.20 0.20 0.20 0.15 0.30 0.50 0.20 0.40 FICi 1.50/ind 0.40/syn 0.60/ind 0.60/ind 0.75/ind 1.50/ind 1.50/ind 1.00/ind 0.80/ind Clotrimazole 0.40 0.10 0.40 0.30 0.20 0.60 0.15 0.10 0.40 AVG 0.40 0.20 0.40 0.30 0.40 0.30 0.60 0.80 0.80 FICi 0.80/ind 0.30/syn 0.80/ind 0.60/ind 0.60/ind 0.90/ind 0.75/ind 0.90/ind 1.20/ind Fluconazole 0.60 0.50 0.40 0.30 0.50 0.20 0.15 0.20 0.20 AVG 0.60 0.50 0.40 0.30 1.00 0.20 0.60 1.60 0.80 FICi 1.20/ind 1.00/ind 0.80/ind 0.60/ind 1.50/ind 0.40/syn 0.75/ind 1.80/ind 1.00/ind Ketoconazole 0.20 0.40 0.30 0.25 0.15 0.20 0.50 0.30 0.40 AVG 0.40 0.40 0.60 0.50 0.60 0.40 1.00 1.20 0.80 FICi 0.60/ind 0.80/ind 0.90/ind 0.75/ind 0.75/ind 0.60/ind 1.50/ind 1.50/ind 1.20/ind Terbinafine 0.10 0.20 0.60 0.40 0.60 1.20 1.00 1.60 0.30 AVG 0.40 0.40 0.60 0.20 0.60 0.30 1.00 1.60 0.60 FICi 0.50/syn 0.60/ind 1.20/ind 0.60/ind 1.20/ind 1.50/ind 2.00/ind 3.20/ind 0.90/ind syn Synergetic, ind Indifferent aBold indicates Fractional Inhibitory Concentration index (FICi) / their interpretation Ogidi et al. BMC Complementary Medicine and Therapies (2021) 21:47 Page 9 of 12 in volatile oils and plays a role of an antimicrobial agent [41]. The presence of α-phellandrene in this study correlates with the findings of Mukesi et al. [42] who examined the bioactivity of commercial antimicrobials, EO and ethanolic extracts of Olea europaea. Another major component found in TEO is 2-carene. It is a bicyclic monoterpene that occurs in several EOs with a sweet and pungent odour. Carene and its derivatives are of modest relevance in the per- fume industry [43], hence, its presence in TEO could contributed to spicy aromatic scent. Farnesene, a volatile compound, which was identified in TEO is known to be responsible for the characteristic taste and flavour of turmeric related to peppermint [44]. TEO exhibited pronounce inhibition against Candida albicans, Penicillium notatum, species of Aspergillus and Trichophyton. Ferreira et al. [45] attributed the inhibition of A. flavus growth at 0.10%, reduction of their viable spores at 0.10% and complete inhibition at 0.50% to ar- turmerone α-turmerone and β-turmerone, being a major component of EO of C. longa. EOs components act as antifungal agents (fungistatic and fungicidal) against fungi by deactivating or disrupting the structure and function of membranes or organelles of fungal cell and/ or inhibiting the nuclear material or protein synthesis in- activation, inhibition of intracellular and extracellular enzymes [38]. The antagonistic activity of A. vera against bacteria, fungi and viruses has been expounded by some studies [46–48]. The antifungal potential of AVG against tested pathogenic fungi corresponds to the findings of Nidiry et al. [49] who reported antifungal property of bioactive constituents; aloin and aloe-emodin in A. vera against Colletotrichum gloeosporides and Cladosporium cucu- merinum. The inhibitory efficiency of AVG corroborates to the findings of Khwakhali and Shrivatava [50] who re- ported the effectiveness of A. vera against pathogenic Aspergillus spp., while Al-Snafi [51] obtained effective treatment (70% growth inhibition) of guinea pig infected with T. mentagrophytes. AVG possessed broad antifungal activities against the tested fungi. Findings of Bawankar et al. [35], Saks and Barkai-Golan [52], and Yebpella et al. [53] have reported antifungal activity of AVG against the growth of Penicillium spp., Botrytis cineria, Alternaria alternate, Aspergillus spp. and Candida albi- cans at varying concentrations. The antimicrobial poten- tial of A. vera could be attributed to anthraquinone and pyrocatechol in the gel of leaves, which are toxic to mi- croorganisms by blocking their ribosomal A site [54]. A. vera is one of the most important traditional folk and al- ternative medicine often used for the treatment of infec- tious diseases with no side effects [55]. In this study, it was observed that the combination of plant extracts; TEO or AVG with AFC was effective against all the tested fungi. Jankasem et al. [56] revealed that turmeric oil displayed better anti-dermatophytic ac- tivity with the MICs of 1.56–6.25 μg/mL when com- pared to 3.90–7.81 μg/mL of ketoconazole. Shin and Lim [57] revealed that antifungal potential of ketocona- zole was significantly improved against Trichophyton schoenleinii, T. erinacei and T. soudanense when com- bined with EO of Pelargonium graveolens. The FICi ob- tained for oils of thyme, cinnamon, clove and eucalyptus combined with amphotericin B against C. albicans and A. niger suggested that synergistic of antifungal drugs with herbs (oil or and extracts) yielded efficacious dose for the treatment of fungal infections and thus, minimiz- ing its side effects [58]. Most EO of Styrax tonkinensis, Lavandula angustifolia, Melaleuca alternifolia, Rosmari- nus officinalis, and Pelargonium graveolens and its frac- tional components; geraniol and citronellol exhibited additive effect when combined with amphotericin B and with ketoconazole against Aspergillus spp., which re- sulted to FICi ranged from 0.52 to 1.00 [53]. In the find- ings of Scalas et al. [40], EOs of Origanum vulgare (oregano), Pinus sylvestris (pine), and Thymus vulgaris (thyme red) and their components (α-pinene, carvacrol, thymol) exhibited good antifungal activity against Cryp- tococcus neoformans strains compared to fluconazole, itraconazole, and voriconazole, and thus, revealed the synergistic and additive for EO and azole (itraconazole) combination. Potential synergistic combination between two or more antimicrobial agents help in reducing re- sistant mutants, exhibit more antimicrobial action, tox- icity against pathogens and thus, serve as effective alternative traditional medicine for the treatment of vari- ous fungal infections [59–61]. The use of EOs aromatic compounds and plant extracts in formulation of topical AFCs need to be embraced to achieve optimal antifungal activity with no side effects. The availability of natural products (EOs or plant extracts) and development of combined antimicrobial agents are often an optional therapy for dermatological infections [62]. Conclusion AFCs, TEO and AVG inhibited the growth of all tested pathogenic fungi with varying degrees of zones of inhib- ition. Combinatory action of AFCs with TEO or AVG did not slow down their bioactivity against tested fungi. This indicated that bioactive compounds in plant ex- tracts can complement the activity of AFCs to improve their clinical efficacy. The antifungal properties of TEO or AVG combined with different AFCs established their importance in phytomedicine and cosmeceutical. The combination of AFCs with plant extracts will serve as al- ternative medicine in treating or combating many infec- tious fungal diseases such as dermatophytosis, which had been a widespread disease. The bioactive ingredients Ogidi et al. BMC Complementary Medicine and Therapies (2021) 21:47 Page 10 of 12 in plant extracts could argument the formulation of body and hair creams (cosmetics) to treat resistant pathogenic fungi within short time with no side effects. Acknowledgements Not applicable. Authors’ contributions OCO and BJA designed the research work. OCO, AEO, OBA, OMA and OAT carried out the experimental study. OCO and BJA supervised the work. OCO and OBA drafted the manuscript. All authors revised the manuscript. Authors read and approved the final manuscript. Funding Not applicable. Availability of data and materials The data used or analysed during the current study are available. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests Authors declared no competing interests. Author details 1Biotechnology Unit, Department of Biological Sciences, Kings University, PMB 555, Odeomu, Nigeria. 2Department of Microbiology, The Federal University of Technology, PMB 704, Akure, Nigeria. 3Microbiology Unit, Department of Biological Sciences, Kings University, PMB 555, Odeomu, Nigeria. Received: 7 September 2020 Accepted: 4 January 2021 References 1. Warnock DW. Fungal diseases: an evolving public health challenge. J Med Mycol. 2006;44(8):697–705. 2. Almeida F, Rodrigues ML, Coelho C. The still underestimated problem of fungal diseases worldwide. Front Microbiol. 2019;10:214. https://doi.org/10. 3389/fmicb.2019.00214. 3. Homei A, Worboys M. Candida: a disease of antibiotics. 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BMC Complementary Medicine and Therapies (2021) 21:47 Page 12 of 12 https://doi.org/10.1186/s12906-018-2219-4 https://doi.org/10.4103/0972-0707.9794 https://doi.org/10.5001/omj.2017.08 https://doi.org/10.5001/omj.2017.08 http://www.aaa2014.org Abstract Background Methods Results Conclusion Background Methods Sample collection Source of antifungal creams (AFCs) Collection of tested fungi Extraction of TEO and AVG Determination of phytochemicals and bioactive compounds in TEO and AVG Antifungal activities of AFCs, TEO and AVG Determination of minimum inhibitory and fractional inhibitory concentration index (FICi) Statistical analysis Results Phytochemical and bioactive compounds in TEO and AVG as revealed by GC-MS Inhibitory potentials and synergistic antifungal efficacy of AFCs with TEO or AVG against pathogenic fungi Discussion Conclusion Acknowledgements Authors’ contributions Funding Availability of data and materials Ethics approval and consent to participate Consent for publication Competing interests Author details References Publisher’s Note