Antifungal activity of Gracilaria corticata methanol extract against Trichophyton mentagrophytes, Microsporum canis, and Microsporum gypseum on rat dermatophytosis models

Introduction

Dermatophytoses are superficial fungal infections caused by dermatophytes that invade and feed on keratinized tissues, like the epidermis, hair, and nails, causing an infection. Incidence rate of these pathogens is higher in hot and humid climates areas with poor hygienic conditions and over-populated regions [ 1 , 2 ] .

Dermatophytosis is caused by a group of fungi known as dermatophytes belonging to the Trichophyton, Microsporum, or Epidermophyton genera [ 3 - 6 ]. Microsporum canis is a zoophilic dermatophyte and is the most common dermatophyte pathogen in cats and dogs [ 7 , 8 ]. It is a geophilic dermatophyte that typically accompanies inflammatory reactions [ 9 ].

Most members of the Trichophyton genus are anthropomorphic. In addition, dermatophytosis caused by Trichophyton mentagrophytes, which is a zoophilic fungus, mainly causes disease in rodents and rabbits, compared to cats, dogs, and other animals [ 9 ]. Usage of topical and systemic drugs to treat dermatophytosis is often associated with fungal resistance, high cost, and side effects. Therefore, it is necessary to conduct studies on effective and safe alternative medicine based on plant compounds [ 10 , 11 ].

Seaweeds belonging to a group of plants known as algae have attracted a lot of attention in recent years. These plants are used for many applications, such as pharmaceuticals, cosmetics, functional foods, and food packaging [ 12 ]. Marine algae are classified as red algae (Rhodophyta), brown algae (Phaeophyta), and green algae (Chlorophyta) [ 13 ]. Among red algae, the genus Gracilaria is a source of important bioactive metabolites and exhibits many biological activities, including antioxidant, anti-inflammatory, antimicrobial, anti-ulcer, anti-cancer, anti-lipid, and anti-diabetic activities [ 14 ]. Wide distribution of G. corticata in the coastal areas of Iran requires more research to be conducted about it from different perspectives [ 15 ].

Due to the higher prevalence rate of common types of dermatophytosis and the growing popularity of keeping pets, this study, aimed to investigate the in vitro antifungal effects of G. corticata methanol extract against T. mentagrophytes, M. canis, and M. gypseum. Since in vivo studies using animal models are pivotal for testing the efficacy of antifungal compounds, their efficacy was also evaluated in rat dermatophytosis models [ 7 , 16 ].

Materials and Methods

Dermatophytes

Trichophyton mentagrophytes (PTCC5054), M. canis (PTCC5069), and M. gypseum (PTCC5057) that were used in this study were purchased from the Persian Type Culture Collection (PTCC, Tehran, Iran).

Antifungal agent

In this study, G. corticata was collected from the Chabahar coast in Baluchistan province, Iran. The algae were washed; afterward, the cleaned algae were air-dried and powdered. The powdered sample was extracted by soaking it in methanol according to the method proposed by Saideni et al. [ 17 ]. Briefly, the extract was filtered and centrifuged, then the supernatant was concentrated and dissolved in methanol. Afterward, the cconcentrated extract was filtered and stored at 4 °C for further use and analysis.

Animal

In total, 40 adult male Wistar rats (200-250 g) were obtained from the laboratory animal house of the Faculty of Veterinary Medicine of the Science and Research Branch, Islamic Azad University, Tehran, Iran. The rats were housed in a room under controlled temperature (22±2 °C) and humidity (50 °C±10%) with a 12/12 h light/dark cycle and free access to food and water.

All methods were carried out following relevant guidelines and regulations. Rats were humanely treated following the Animal Research: Reporting of in Vivo experiments guidelines for animal care [ 18 ]. Moreover, all in vivo experimental procedures were approved by the Research Ethics Committee of the Science and Research Branch, Islamic Azad University (IR.IAU.SRB.REC.1399.196).

Preparation of fungal suspension

Trichophyton mentagrophytes, M. canis, and M. gypseum were used in this study. Each sample was cultured on Sabouraud Dextrose Agar (Ibresco, Iran) and the plates were incubated at 30 °C for up to two weeks. Afterward, a suspension was prepared from the colony of dermatophytes using normal saline solution and Tween 80 with a final concentration of 1×10⁶ spores mL^-1.

In vitro assays

Antimicrobial susceptibility testing

The minimum inhibitory concentration (MIC) of G. corticata and terbinafine against the tested dermatophytes was determined using the broth microdilution method recommended by the Clinical and Laboratory Standards Institute M38-A2 method [ 19 ]. Serial dilutions of antifungal agents starting at 70 µg ml^-1 of G. corticata were prepared and compared with the reference antifungal drug, which was terbinafine. Concentrations of G. corticata and terbinafine were within the ranges of 78-0.125 and 4-0.008 µg ml^-1, respectively [ 20 ].

Well diffusion method

The agar well diffusion method was used to assess the inhibitory activity of G. corticata methanol extract. Effectiveness of the active solution was calculated by measuring the inhibition zone around three wells. Moreover, the percentage of dermatophyte growth inhibition was calculated as follows [ 21 ]:

$\mathrm{FI\; (\%)}=\mathrm{(IR/GR)}\times 100$

FI: fungal inhibition, IR: inhibition radius, GR: growth radius

In vivo assays

Dermatophytosis model

The rats were anesthetized by intraperitoneal injection of ketamine/xylazine. Backs of the animals were shaved and their skins were gently rubbed with sterile fine sandpaper to make them susceptible to infection.

Afterward, their skins were inoculated with fungal suspension [ 16 ]. After 4 days of inoculation, the samples were collected and cultured. Moreover, to confirm the infection, direct microscopic examination was performed in 8-10 days.

Experimental group

On day 10 after the infection, the rats were randomly divided into four groups, and the wound areas were covered with each of the assigned treatments once a day. It should be noted that this procedure continued until complete recovery was achieved. The study groups included 1) negative control: inoculated rats that were treated with Vaseline ointment; 2) positive control: inoculated rats that were treated with terbinafine ointment; 3) inoculated rats that were treated with 5% G. corticata ointment; and 4) inoculated rats that were treated with 10% G. corticata ointment. Gracilaria corticata ointment was prepared by mixing 5 and 10 ml of G. corticata methanol extract (MIC value) with 100 g of Vaseline ointment.

Wound surface area measurement

To evaluate the therapeutic efficacy of G. corticata ointment, the wound area was assessed daily and measured manually using a transparent grid throughout the treatment period to determine the recovery rate of the infected site. The percentage of wound closure was calculated based on the following equation: wound closure (%)=(A₀–A_n/A₀)×100

A₀ represents the wound area at day 0 and A_n refers to the wound surface area at a different time point [ 22 ].

Statistical analysis

The results were expressed as mean values of three independent replicates. The results were analyzed using analysis of variance followed by Tukey's honestly significant difference. P values of less than 0.05 were considered statistically significant.

Results

In vitro antidermatophytic effect of Gracilaria corticata

The in vitro antifungal effects of G. corticata methanol extract was evaluated against T. mentagrophytes, M. canis, and M. gypseum, and their MIC, Minimum fungicidal concentration (MFC), and percentage of growth inhibition were determined. It was found that all tested dermatophytes were highly sensitive to G. corticata methanol extract (Table 1).

In the microdilution assay, the MIC and MFC values were within the range of 4-78 µg mL^-1. Moreover, it was revealed that G. corticata methanol extract showed the highest inhibitory activity against T. mentagrophytes with a MIC value of 4 µg mL^-1.

Table 1 also represents the antidermatophytic potential of G. corticata methanol extract, compared to synthetic anti-dermatophyte drugs. The MIC and MFC values of terbinafine ranged from 0.1 to 9, which are much less than the MIC of G. corticata methanol extract. The fungus T. mentagrophytes was found to be more sensitive than M. canis and M. gypseum. The well diffusion method showed that the greatest growth inhibition rate of the extract was observed against T. mentagrophytes (52±0.056%), followed by M. canis (34±0.028%) and M. gypseum (28±0.028) at their MIC concentrations.

In vivo antidermatophytic activity of Gracilaria corticata

To evaluate the efficacy of G. corticata ointment, different degrees of skin lesions among various treatment groups were assessed and recorded throughout the study period. All rats infected with dermatophyte showed signs of inflammation on the third day. Severity of lesions reached the highest level almost on day 10, which indicated the growth of dermatophyte fungus in the infected areas.

Visual examination of skin lesions after treatment showed improvement in the clinical signs of infection, while patches of hair loss and scaly skin were observed in the negative control group. The first signs of recovery were observed in animals infected with M. gypseum followed by animals infected with T. mentagrophytes and M. canis (Figure 1). Both concentrations of G. corticata led to a significant decrease in lesion size and hair growth in the infected sites, compared to the negative control. However, 10% G. corticata ointment significantly accelerated skin wound healing.

Figure 1. Time course of recovery of dermatophytes infection in animal groups treated with terbinafine as the positive control, tested ointments (5 and 10 % Gracilaria corticata methanolic extract), and Vaseline as the negative control.

In the M. gypseum-infected rats (Figure 1A), 100% wound healing was achieved on days 19 and 25 in 10% and 5% G. corticata groups, respectively, compared to terbinafine as a positive control on day 11. It was revealed that the wound closure rate on day 11 in the 10% G. corticata group was significantly lower, compared to the terbinafine group (50% vs. 100%, P<0.0001), while it was significantly higher than 5% G. corticata (50% vs. 25 %, P<0.01). It was also indicated that 5% G. corticata ointment showed higher therapeutic effects on the dermatophyte infection, compared to Vaseline (P<0.0001).

As can be seen (Figure 1B), in T. mentagrophytes-infected rats, wound healing reached 100% on day 14 in rats treated with terbinafine. It was also indicated that complete recovery from the infection was observed 25 days after the treatment in rats treated with 10% G. corticata topical ointment, whereas those treated with 5% extract ointment were completely cured on the 30th day of treatment.

In the case of M. canis-infected rats (Figure 1C), no sign of lesion was observed in the terbinafine treatment group on day 16, while 100% recovery was observed on the 38^th day of treatment in the 10% G. corticata topical ointment group and 5% G. corticata topical ointment group showed clear signs of recovery on 42^nd day. Antidermatophytic activities of different treatments had highly significant differences in terms of time and dose-dependence in the following order: terbinafine > 10% G. corticata > 5% G. corticata > Vaseline.

Discussion

There have been several reports of antibiotic resistance in dermatophyte pathogens which has made the commercialization of alternative antidermatophytic agents a necessity. The herbal-based medicines have attracted great attention as favorable candidates for antidermatophytic therapy [ 23 ]. The present study investigated the in vitro and in vivo antidermatophytic activities of G. corticata methanol extract. In this study, it was found that G. corticata exhibited strong in vitro antidermatophytic activities against T. mentagrophytes, M. canis, and M. gypseum.

It was also demonstrated that the therapeutic effects of G. corticata ointments in the rat model are dose- and time-dependent. To our knowledge, this is the first report of the antifungal activity of G. corticata methanol extract against dermatophyte fungi. There have been some studies on the antifungal effects of seaweed extracts [ 24 - 26 ]; however, there are not many published papers regarding their antidermatophytic effects.

It has been reported that the MIC values of the Bifurcaria bifurcata and brown algae against T. mentagrophytes, M. canis, and M. gypseum were 100, 400, and 800 µg mL^-1, respectively [ 27 ]. But these seaweed extracts were less effective in comparison to G. corticata methanol extract against T. mentagrophytes, M. canis, and M. gypseum in the present study with MIC values of 4, 19, and 78 µg mL^-1, respectively.

However, it should be noted that comparison was difficult as the used seaweeds were not the same. Nevertheless, T. mentagrophytes showed the highest sensitivity to both seaweeds followed by M. canis and M. gypseum. The same results have been reported for the antifungal activity of Lobophora variegata and brown alga against T. mentagrophytes. However, the methanol extract of L. variegata exhibited higher antifungal activity with an inhibition zone diameter (IZD) of 11.42±0.002 mm, 62.64% at 100 μg mL^-1, compared to other solvent extracts [ 28 ].

Another previous study has investigated the antifungal efficacy of methanolic extract from marine brown seaweed, Colopomenia peregrine, at 30 µg ml^-1 concentration against M. gypseum and reported an IZD value of 12 mm [ 29 ]. In another study performed on the antifungal efficiency of various solvent extracts of red algae, Acanthaphora spicifera, it was revealed that the methanol extracts showed maximum antifungal activity and the zone of inhibition was 12 mm against M. gypseum at a concentration of 50 mg/ml^-1 [ 30 ].

In contrast to the results of the antidermatophytic activity of selected algae against the tested dermatophytes, the green, brown, and red algae obtained from Brazil showed strong activity with low MIC values ranging from 0.03 to 16.00 µg ml^-1. Microsporum canis showed a MIC value of 0.03 µg ml^-1 with an IZD value of 10-25 mm. The ethanol extract was found to be the most effective growth inhibitor of the fungi [ 31 ].

The terpenoids, tannins, and phenolic compounds were present in the different extracts of G. corticata isolated from the Persian Gulf [ 32 ]. It has been reported that the possible antimicrobial mechanism of the seaweeds is related to their phlorotannins extracts. This mechanism acts by affecting the fungal cell wall and membrane composition as well as the mitochondrial function [ 33 ]. A series of bromophenols isolated from red alga showed MIC values of 1.56, 12.5, 25, 50, and >100 µg ml^-1 against T. mentagrophytes [ 34 ].

Effects of anti-fungal activity greatly depend upon the factors influencing the geographical region, sampling period, species variation, environmental variations and climatic conditions, extraction method, and chemical composition of the seaweed [ 35 ]. Considering the potential in vitro activity of algae, the efficacy of its in vivo application against induced dermatophytosis in the rat model was also investigated. There are several clinical reports on in vitro anti-dermatophytic efficacy of natural sources [ 35 - 40 ], while just some limited investigations have been performed on the in vivo effects of algae.

In contrast to in vitro results, in vivo results showed that M. gypseum was the most sensitive species to the methanol extracts of G. corticata, while M. canis was less effective. This finding might be attributed to the immune response of animals to dermatophytes which leads to self-healing. In this way, the average area of skin lesions of rats infected by M. gypseum, T. mentagrophytes, and M. canis in negative control was slightly decreased in a time-dependent manner.

Results of the present study are in agreement with those obtained by other investigators which have shown the in vivo anti-dermatophytic activity of algae against dermatophytes. The ethanol extract of the algae showed promising antifungal activity against T. mentagrophytes with a MIC value of 62.5 μg mL^-1. In vivo evaluation of the ethyl acetate soluble fraction of the ethanol extract (MIC: 15.8 μg mL^-1) in a multi-infection fungal model in Swiss albino mice provided 60% protection at 50 mg/kg p.o. with a reduced CFU [ 41 ].

Yu-Xia reported in vivo antifungal activity of different concentrations (0.1%, 1%, and 5%) of chitooligosaccharides (COS) derived from chitosan, against T. rubrum using a guinea pig model. In the aforementioned study, the 5% COS group showed a significant reduction in skin lesions, compared to the positive control group that received 1% fluconazole. Similar to the results of the present study, skin lesion reduction revealed the dose-dependent therapeutic effect of COS [ 42 ]. Several reports have shown that chitosan, which is derived from chitin and is present in algae, can induce morphological change in the fungal hyphae and acts as a chelating agent to limit nutrients for fungal growth [ 43 , 44 ].

It is well known that the presence of functional bioactive compounds in seaweed extract is responsible for its high anti-dermatophytic activity. Results of a study conducted by Radhika et al. [ 45 ] have reported the effectiveness of green algae, Chlorella vulgaris, and ointment on M. canis-infected rats after the production of a thermal lesion on its back. They showed that C. vulgaris, which is rich in phenolic compounds, was effective and also revealed the normal histological situation of the skin tissues of rats after the treatment [ 46 ]. In the present study, it seemed that the high amount of total phenolics in G. corticata might be the reason for its high anti-dermatophytic activity [ 45 , 47 ].

It has been reported that lectins isolated from some red algae exhibited analgesic, anti-microbial, anti-ulcerogenic, and anti-inflammatory effects [ 48 ]. Topical treatment of lectin isolated from Bryothamnion seaforthii on induced skin wounds in mice showed a significant healing potential [ 49 ]. Although the used wound-induced model organism was not the same as the one in the present study, the results of both studies showed that the topical application of the investigated algae may increase their penetration into the skin and lead to a faster wound-healing process.

Conclusion

The results obtained in the present study provide scientific validation for the anti-dermatophytic activity of G. corticata methanol extract at low concentrations. Moreover, the findings also indicated its in vivo efficacy in rat models infected with T. mentagrophytes, M. canis, and M. gypseum.

The collected data confirmed the potential applications of algae extracts against tested dermatophytes which can motivate future studies on its active ingredients, mechanisms of action, and randomized clinical trials. As various algal species widely grow in different tropical regions of the world, they may be recommended as natural resources in the treatment of dermatophyte infections as well as an alternative to a synthetic drug for topical applications in a dose-dependent manner.

Acknowledgments

Not declared.

Authors’ contribution

The authors declare that all listed authors have made equal contributions to the conceptualization, formal analysis, methodology, review preparation, and edition of the present research. All authors have read and approved the submitted final manuscript.

Conflicts of interest

The author states that there is no conflict of interest.

Financial disclosure

No financial interests related to the material of this manuscript have been declared.

References

1. AL-Khikani FH. Dermatophytosis a worldwide contiguous fungal infection: Growing challenge and few solutions. Biomed Biotechnol Res J. 2020; 4:117.
2. AL-Khikani FH, Ayit AS. Major challenges in dermatophytosis treatment: current options and future visions. Egypt J Dermatol Venerol. 2021; 41(1):1-9.
3. Paryuni AD, Indarjulianto S, Widyarini S. Dermatophytosis in companion animals: A review. Vet World. 2020; 13(6):1174-81.
4. Gautam SS, Babu N, Kumar S. Current perspective of dermatophytosis in animals. Springer: Fungal Diseases in Animals; 2021.
5. Moriello KA. Dermatophytosis. Springer: Feline Dermatology; 2020.
6. Gräser Y, Monod M, Bouchara JP, Dukik K, Nenoff P, Kargl A, et al. New insights in dermatophyte research. Med Mycol. 2018; 56(1):2-9.
7. Mao L, Zhang L, Li H, Chen W, Wang H, Wu S, et al. Pathogenic fungus Microsporum canis activates the NLRP3 inflammasome. Infect Immun. 2014; 82(2):882-92.
8. Aneke CI, Otranto D, Cafarchia C. Therapy and antifungal susceptibility profile of Microsporum canis. J Fungi. 2018; 4(3):107.
9. Dolenc-Voljc M, Gasparic J. Human infections with Microsporum gypseum complex (Nannizzia gypsea) in Slovenia. Mycopathologia. 2017; 182(11):1069-75.
10. Lopes G, Pinto E, Salgueiro L. Natural products: an alternative to conventional therapy for dermatophytosis?. Mycopathologia. 2017; 182:143-67.
11. Soares LA, Sardi JD, Gullo FP, Pitangui ND, Scorzoni L, Leite FS, et al. Anti dermatophytic therapy: prospects for the discovery of new drugs from natural products. Braz J Microbiol. 2013; 44:1035-41.
12. Polat S, Trif M, Rusu A, Simat V, Cagalj M, Alak G, et al. Recent advances in industrial applications of seaweeds. Crit Rev Food Sci Nutr. 2021;1-30.
13. Silva A, Silva SA, Carpena M, Garcia-Oliveira P, Gullón P, Barroso MF, et al. Macroalgae as a source of valuable antimicrobial compounds: Extraction and applications. Antibiotics. 2020; 9(10):642.
14. Aziz E, Batool R, Khan MU, Rauf A, Akhtar W, Heydari M, et al. An overview on red algae bioactive compounds and their pharmaceutical applications. J Complement Integr Med. 2020; 17
15. Rosemary T, Arulkumar A, Paramasivam S, Mondragon-Portocarrero A, Miranda JM. Biochemical, micronutrient and physicochemical properties of the dried red seaweeds Gracilaria edulis and Gracilaria corticata. Molecules. 2019; 24(12):2225.
16. Sharma B, Kumar P, Joshi SC. Topical treatment of dermatophytic lesion on mice (Mus musculus) model. Indian J Microbiol. 2011; 51:217-22.
17. Saidani K, Bedjou F, Benabdesselam F, Touati N. Antifungal activity of methanolic extracts of four Algerian marine algae species. Afr J Biotechnol. 2012; 11:9496-500.
18. Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol. 2020; 18(7):e3000410.
19. CLSI. Reference method for broth dilution antifungal susceptibility testing of filamentous Fungi. Approved Standard: CLSI document M38–A2, 2nd. Clinical and Laboratory Standards Institute: Wayne, PA; 2008.
20. Falahati M, Fateh R, Nasiri A, Zaini F, Fattahi A, Farahyar S. Specific identification and antifungal susceptibility pattern of clinically important dermatophyte species isolated from patients with dermatophytosis in Tehran, Iran. Arch Clin Infect Dis. 2018; 13(3):e63104.
21. Mohammadi R, Abbaszadeh S, Sharifzadeh A, Sepandi M, Taghdir M, Youseftabar Miri N, et al. In vitro activity of encapsulated lactic acid bacteria on aflatoxin production and growth of Aspergillus Spp. Food Sci Nutr. 2021; 9:1282-8.
22. Kordjazi M, Shabanpour B, Zabihi E, Faramarzi MA, Gavlighi HA, Feghhi SMA, et al. Investigation of effects of fucoidan polysaccharides extracted from two species of Padina on the wound-healing process in the rat. Turk J Vet Anim Sci. 2017; 41:106-117.
23. Savarirajan D, Ramesh VM, Muthaiyan A. In vitro antidermatophytic activity of bioactive compounds from selected medicinal plants. J Anal Sci Technol. 2021; 12(1):1-13.
24. De Almeida CL, Falcão DS, Lima DM, Gedson R, Montenegro DA, Lira NS, et al. Bioactivities from marine algae of the genus Gracilaria. Int J Mol Sci. 2011; 12(7):4550-73.
25. Kumar P, Selvi SS, Govindaraju M. Seaweed-mediated biosynthesis of silver nanoparticles using Gracilaria corticata for its antifungal activity against Candida spp. Appl Nanosci. 2013; 3:495-500.
26. Perez MJ, Falque E, Dominguez H. Antimicrobial action of compounds from marine seaweed. Mar Drugs. 2016; 14(3):52.
27. Carvalho GLc, Silva R, Goncalves JM, Batista TM, Pereira L. Extracts of the seaweed Bifurcaria bifurcata display antifungal activity against human dermatophyte fungi. J Oceanol Limnol. 2019; 37:848-54.
28. Subbiah M, Thennarasan S, Vajiravelu S. Effect of marine brown alga lobophora variegata (j.v.lamouroux) womersley exe.c.oliveir various solvents extracts on dermatophytes. Int J Pharm Sci Res. 2019; 10:672-7.
29. Manam D, Kiran V. Anti-Fungal efficacy and secondary metabolite analysis of the methanolic extract from Colpomenia peregrina towards opportunistic fungal pathogens. Int J Innov Eng Manag Res. 2021; 10:89-100.
30. Pandian P, Selvamuthukumar S, Manavalan R, Parthasarathy V. Screening of antibacterial and antifungal activities of red marine algae Acanthaphora spicifera (Rhodophyceae). J Biomed Sci Res. 2011; 3(3):444-8.
31. Guedes EA, dos Santos Araújo MA, Souza AK, de Souza LI, de Barros LD, et al. Antifungal activities of different extracts of marine macroalgae against dermatophytes and Candida Species. Mycopathologia. 2012; 174:223-32.
32. Ghannadi A, Shabani L, Yegdaneh A. Cytotoxic, antioxidant and phytochemical analysis of Gracilaria species from Persian Gulf. Adv Biomed Res. 2016; 5:139.
33. Lopes G, Pinto E, Andrade PB, Valentao P. Antifungal activity of phlorotannins against dermatophytes and yeasts: approaches to the mechanism of action and influence on Candida albicans virulence factor. PLoS One. 2013; 8(8):e72203.
34. Oh KB, Lee JH, Chung SC, Shin J, Shin HJ, Kim HK, et al. Antimicrobial activities of the bromophenols from the red alga Odonthalia corymbifera and some synthetic derivatives. Bioorg Med Chem Lett. 2008; 18(1):104-8.
35. Toreyhi H, Lotfali E, Fattahi A, Rezaee Y, Ghasemi R, Sabour ES. A Review on anti dermatophytosis potential of medicinal plants: in-vitro, in-vivo and important components. NBM. 2021; 9(2):71-100.
36. Prasad CS, Shukla R, Kumar A, Dubey NK. In vitro and in vivo antifungal activity of essential oils of Cymbopogon martini and Chenopodium ambrosioides and their synergism against dermatophytes. Mycoses. 2010; 53(2):123-9.
37. Njateng GSS, Gatsing D, Mouokeu RS, Lunga PK, Kuiate JR. In vitro and in vivo antidermatophytic activity of the dichloromethane-methanol (1: 1 v/v) extract from the stem bark of Polyscias fulva Hiern (Araliaceae). BMC Complement Altern Med. 2013; 13:1-10.
38. Ayatollahi Mousavi SA, Kazemi A. In vitro and in vivo antidermatophytic activities of some Iranian medicinal plants. Med Mycol. 2015; 53:852-9.
39. Wagini N, Abbas MS, Soliman AS, Hanafy YA, Badawy El-Saady M. In vitro and in vivo anti dermatophytes activity of Lawsonia inermis L.(henna) leaves against ringworm and its etiological agents. Am J Clin Exp Med. 2014; 2(3):51-8.
40. Sun K, Song X, Jia R, Yin Z, Zou Y, Li L, et al. In vivo evaluation of Galla Chinensis solution in the topical treatment of dermatophytosis. Evid Based Complement Alternat Med. 2017; 2017:3843595.
41. Lakshmi V, Srivastava S, Mishra SK, Khan ZK. Antifungal activity of marine algae of the Indian coast. Indian Drugs. 2010; 47:54-6.
42. Mei YX, Dai XY, Yang W, Xu XW, Liang YX. Antifungal activity of chitooligosaccharides against the dermatophyte Trichophyton rubrum. Int J Biol Macromol. 2015; 77:330-5.
43. Lopez-Moya F, Suarez-Fernandez M, Lopez-Llorca LV. Molecular mechanisms of chitosan interactions with fungi and plants. Int J Mol Sci. 2016; 20(2):332.
44. Egusa M, Iwamoto R, Izawa H, Morimoto M, Saimoto H, Kaminaka H, et al. Characterization of chitosan nanofiber sheets for antifungal application. Int J Mol Sci. 2015; 16(11):26202-10.
45. Radhika D, Mohaideen A. Fourier transform infrared analysis of Ulva lactuca and Gracilaria corticata and their effect on antibacterial activity. Asian J Pharm Clin Res. 2015; 8:209-12.
46. El-Sheekh MM, El-Shafay SM, El-Ballat EM. In vivo evaluation of antimicrobial effect of methanolic extract of Chlorella vulgaris on impetigo and some dermatophytes. EJBO. 2016; 56(2):423-37.
47. Narasimhan MK, Pavithra SK, Krishnan V, Chandrasekaran M. In vitro analysis of antioxidant, antimicrobial and antiproliferative activity of Enteromorpha antenna, Enteromorpha linza and Gracilaria corticata extracts. Jundishapur J Nat Pharm Prod. 2013; 8(4):151-9.
48. Singh RS, Walia AK. Lectins from red algae and their biomedical potential. J Appl Phycol. 2018; 30(3):1833-58.
49. do Nascimento-Neto LG, Carneiro RF, Da Silva SR, Da Silva BR, Vassiliepe Sousa Arruda F, Carneiro VA, et al. Characterization of isoforms of the lectin isolated from the red algae Bryothamnion seaforthii and its pro-healing effect. Mar Drugs. 2012; 10(9):1936-54.