Antifungal Activity of Henna Leaves (Lawsonia Inermis L.) Against Trichophyton Rubrum and Trichophyton Interdigitale
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International Journal of Research and Review Vol.7; Issue: 11; November 2020 Website: www.ijrrjournal.com Original Research Article E-ISSN: 2349-9788; P-ISSN: 2454-2237 Antifungal Activity of Henna Leaves (Lawsonia inermis L.) against Trichophyton rubrum and Trichophyton interdigitale Rabi Yakubu Bello Department of Microbiology, Faculty of Science, Yobe State University, PMB 1144, Damaturu, Yobe State, Nigeria. ABSTRACT reported to be due to dermatophytes (Tchernev et al., 2012; Kenneth et al., Onychomycosis is a term used to describe 2016). Among dermatophytes, the most fungal infection of the nails caused by some often isolated causative pathogens of dermatophytes. The present study was designed onychomycosis are Trichophyton rubrum, to assess the antifungal activity of henna leaves Trichophyton interdigitale (formerly T. (Lawsonia inermis L) extracts (n-hexane and petroleum ether) against two species of mentagrophytes), Epidermophyton dermatophytes: Trichophyton rubrum and floccosum and Trychophyton tonsurans Trichophyton interdigitale using the Agar (Tchernev et al., 2012; Piraccini and Radial Diffusion and Minimum Inhibitory Alessandrini, 2015). Concentration techniques respectively. Results The condition is worldwide in obtained revealed that n-hexane extract have no distribution and problems associated with antifungal effect against the test organisms. onychomycosis include difficulty in wearing However, crude extract of petroleum ether footwear and walking, cosmetic inhibited growth of both dermatophytes at embarrassment and lowered self-esteem concentrations of 12.5mg/ml - 200mg/ml with (Shenoy and Shenoy, 2014). Some of the the largest inhibition zone of 81±1.4mm against contributing factors causing this disease are T. rubrum. MIC values of both extracts were 50mg/ml against T. rubrum. The antifungal drug occlusive footwear, genetic predisposition Itraconazole exhibited the largest inhibition and concurrent diseases, such as diabetes zone of 71±1.8mm against T. interdigitale at and HIV infection, as well as other forms of 25mg/ml concentration. Phytochemical immunosuppression (Ameen et al., 2014). It screening of the extracts revealed the presence is a common misconception among of carbohydrates, saponins and alkaloids. This physicians that as onychomycosis is a study concludes that, henna extract possesses cosmetic problem it need not be treated antifungal activity and thus can be used to (Kenneth et al., 2016). combat the problem of drug resistance. Onychomycosis can result in disruption of integrity of the skin, providing Keywords: Lawsonia inermis, Antifungal, an entry point for bacteria leading to the Trichophyton rubrum, Trichophyton interdigitale, Itraconazole. development of foot ulcers, osteomyelitis, cellulitis and gangrene in diabetic patients INTRODUCTION (Ameen et al., 2014; Kenneth et al., 2016). Onychomycosis is a term used to Furthermore, fungal diseases are contagious describe fungal infections of the fingernails and may spread to other family members if or toenails (Ameen et al., 2014). Despite the not treated (Tchernev et al., 2012). geographical and racial variation in the However, the rate of treatment failure with aetiological agents of onychomycosis, an standard antifungal drugs is on the rise due estimated 85–90% of nail infections are to poor patient compliance, low International Journal of Research and Review (ijrrjournal.com) 314 Vol.7; Issue: 11; November 2020 Rabi Yakubu Bello. Antifungal activity of henna leaves (lawsonia inermis l.) against trichophyton rubrum and trichophyton interdigitale. bioavailability, lack of drug penetration into from ATCC 28188 catalog number SKU: the nail, drug resistance and drug 0444K and ATCC 9533 catalog number interactions (Ameen et al., 2014; Piraccini SKU: 0442K respectively were purchased and Alessandrini, 2015). from Microbiologics Inc. through 108616 According to World Health VSR INT LTD Distributor. The organisms Organisation (WHO), medicinal plants were cultured on Sabouraud Dextrose could be important resources for obtaining Emmons Agar supplemented with lactic new antimicrobial drugs (Khan et al., 2016). acid and incubated aerobically for 6 days at Today, it is estimated that about 80% of 25OC. people in developing countries rely on traditional medicines for their primary 2.3 Inoculum Preparation: a loop full of health care (Sharma and Goel, 2018). the test organisms were inoculated in Traditional medicines are becoming Sabouraud Dextrose Emmons Broth popular, due to high toxicity and adverse supplemented with lactic acid and incubated effects of orthodox medicament (Osman et for 48hours at 25OC. al., 2012; Otang and Afolayan, 2016). The name henna, refers to the dye 2.4 Extraction method (Maceration): prepared from the leaves of Lawsonia Sterile bottles were labelled with the name inermis L. (family Lythraceae) (Wagini et of each solvent to which 100grams of henna al., 2014; Rathi et al., 2017). This species powder was added. 500mls of n-hexane and has been used as medicinal plant since petroleum ether were added to respective ancient times to cure numerous diseases but bottles to make 1:5% w/v of the extract. All mainly as dye material (Rao et al., 2016). the bottles were then kept on a universal Recent studies showed that the leaves of this shaker (model HS-501 digital Werke plant have anti-inflammatory, antipyretic, Company) for 24hours. Both extracts were analgesic, antifungal and antibacterial then filtered using Whatman no. 1 (24cm) activity (Nawasrah et al., 2016). filter papers and solvents were recovered in Hence, this study involved rotary evaporator. antifungal testing of henna plant extracts against Trichophyton rubrum and 2.5 Preparation of extracts: 1g of each Trichophyton interdigitale in comparison extract was weighed, transferred into with conventional antifungal drug respective containers and dissolved in 5ml (Itraconazole). of 1% DMSO in the solvent of extraction and 1000mg/ml of itraconazole was 2. MATERIALS AND METHODS dissolved in 5ml distilled water. Thus we 2.1 Collection of plant material and have 200mg/ml of all the stock solution. 7- authentication: L. inermis Linn. plant folds serial dilutions were set up with the leaves were obtained from Adaya village least concentration of 3.125mg/ml. under Fune local Government Area of Yobe State and authenticated by a botanist at the 2.6 In vitro sensitivity tests: the antifungal department of Biological Sciences, Yobe activity of henna extracts was assessed in State University. The leaves were washed comparison with the antifungal drug thoroughly with tap water, shade dried and itraconazole. All experiments were prepared grinded using mortar and pistil. The powder in triplicates and the results shown represent was then stored in an air tight container until the mean. further use. 2.6.1 Agar Radial Diffusion: The well 2.2 Fungal strains: Trichophyton rubrum diffusion method was carried out as and Trichophyton. interdigitale (formerly described by (Kannahi and Vinotha, 2013; Trichophyton. mentagrophytes) derived International Journal of Research and Review (ijrrjournal.com) 315 Vol.7; Issue: 11; November 2020 Rabi Yakubu Bello. Antifungal activity of henna leaves (lawsonia inermis l.) against trichophyton rubrum and trichophyton interdigitale. Rao et al., 2016) with slight modification as 2.8 Statistical analysis stated below: The results are expressed as mean Nutrient agar plates were prepared standard deviation (SD) for the and allowed to solidify at room temperature. measurements of inhibition zones in A loop full of the broth culture was picked antifungal activity tests. and emulsified with distilled water. Using micropipettes, a drop of this was then 3. RESULTS aseptically transferred unto each nutrient Petroleum ether henna extract agar plates and spread using a spreader. The exhibited antifungal activity against T. plates were then allowed to sit for 30 rubrum (table 1). Zones of inhibition were minutes so as to suck up the inoculum. observed around wells containing Afterwards, 4 wells were made in the plates 12.5mg/ml that increased with increasing using a sterile cork borer of 10mm diameter concentration. and labelled. The wells were filled with respective extracts using a micropipette and Table 1: Agar radial diffusion results of henna leaves extracts 0 against Trichophyton rubrum after 48 hours of incubation at plates were then incubated at 25 C for 48 250C. Concentrations Extracts/ Zones of inhibition hours and the diameter of the zones of in mm ±SD Organism inhibition measured in millimetre. n-hexane Petroleum ether T. rubrum 3.125mg/ml N.I N.I 2.6.2 Minimum Inhibitory Concentration 6.25mg/ml N.I N.I (MIC): The MIC values of n-hexane 12.5mg/ml N.I 41±0.9 extract, petroleum ether extract and the 25mg/ml N.I 4.9±1.8 50mg/ml N.I 63±1.4 reference drug (Itraconazole) were 100mg/ml N.I 74±0.9 determined for the selected test organisms 200mg/ml N.I 81±1.4 NI: no inhibition; n=3. using tube dilution assay. This was done by adding 2ml of Both n-hexane and petroleum ether TSB into seven test-tubes. 2ml of extract extract showed little or no antifungal was added to the first test tube and mixed on activity against T. interdigitale (table 2). a whirl mixer. 2ml of the contents from tube one were removed and aseptically added to Table 2: Agar radial diffusion results of henna leaves extracts against Trichophyton interdigitale after 48 hours of incubation tube two followed by mixing. The process at 250C. was repeated up to tube six but no plant Organism Concentrations Extracts/ Zones of inhibition in mm ±SD extract was added to tube seven as it was n-hexane Petroleum kept as a positive growth control. All seven ether T. test tubes were then inoculated with a drop interdigitale 3.125mg/ml N.I N.I of overnight culture diluted in distilled 6.25mg/ml N.I N.I water. Another set of tubes were prepared 12.5mg/ml N.I N.I 25mg/ml N.I N.I in the same way but no culture added to 50mg/ml N.I N.I them as negative control. The MICs were 100mg/ml N.I 51±1.4 200mg/ml N.I 64±1.2 regarded as the lowest concentration that NI: no inhibition; n=3.