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. hours and the diameter of the zones of Concentrations Extracts/ Zones of inhibition 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. inhibited visible growth of the test 0 organisms after incubation at 25 C for 48 The reference drug itraconazole hours in comparison with the controls. exhibited antifungal activity against both organisms at all concentrations (table 3). 2.7 Phytochemical screening: In order to Interestingly, the largest zone of inhibition determine the bioactive compounds of of 71±1.8mm was observed against T. henna extracts, phytochemical screening interdigitale at the concentration of was carried out as described by (Tiwari et 25mg/ml. al., 2011).
International Journal of Research and Review (ijrrjournal.com) 316 Vol.7; Issue: 11; November 2020 Rabi Yakubu Bello. Antifungal activity of henna leaves (lawsonia inermis l.) against trichophyton rubrum and trichophyton interdigitale.
Table 3: Agar radial diffusion results of itraconazole against reactions mostly in children (Namiranian et Trichophyton rubrum and Trichophyton interdigitale after 48 hours of incubation at 250C. al., 2019). Ointment and oil from henna flowers has been prepared by ancient Concentrations Zones of inhibition in mm ±Standard Deviation Egyptians for making the limbs supple (Dinesh Babu and Subhasree 2009). The Trichophyton. Rubrum Trichophyton. interdigitale main chemical components of this plant are 3.125mg/ml 32±0.9 38±0.9 Lawsone (2-hydroxy-1,4-naphthoquinone) 6.25mg/ml 37±0.5 42±0.8 12.5mg/ml 44±0.9 47±0.9 which is responsible for the reddish brown 25mg/ml 50±0.5 71±1.8 coloration, tannic acid, gallic acid, mannite 50mg/ml 58±1.2 54±0.8 100mg/ml 62±0.9 57±0.8 and mucillage (Al-Rubiay et al., 2008; Gull 200mg/ml 67±0.5 67±2.1 et al., 2013; Al-Snafi et al., 2019). N=3. The n-hexane henna extract used in this study exhibited no antifungal activity For petroleum ether extract, T. against both dermatophytes. This may be interdigitale seems to be more sensitive with due to the polarity of this solvent resulting MIC value of 12.5mg/ml. Petroleum ether in the precipitation of the active components and n-hexane have the same MIC values of of the plant during the extraction process. 50mg/ml against T. rubrum. However, According to (Arun, 2010), hexane henna Itraconazole showed the lowest MIC value extract did not possess antibacterial effect of less than 3.125mg/ml for both test against Staphylococcus aureus, Escherichia organisms. coli, Klebsiella pneumonia, Proteus and Pseudomonas aeruginosa even at Table 4: Minimum Inhibitory Concentration (MIC) results of henna extracts and itraconazole against Trichophyton rubrum concentration of 1000 μg/ml. This is and Trichophyton interdigitale after 48 hours of incubation at contrary to the findings of (Padron-Marquez 250C. Organisms Extracts/ MIC (mg/ml) et al., 2012) who observed the best n- Petroleum Itraconazole antifungal activity with n-hexane which hexane ether Trichophyton 50 50 < 3.125 inhibited the growth of all the tested rubrum dermatophytes. Trichophyton 100 12.5 < 3.125 interdigitale Furthermore, petroleum ether henna extract inhibited the growth of both T. The results of phytochemical rubrum and T. interdigitale at a varying screening carried out on both n- hexane and degree. At the highest concentration of petroleum ether henna extracts is shown in 200mg/ml, Petroleum ether showed table 5. inhibition zone of 81±1.4mm against T. rubrum and 64±1.2mm against T. Table 5: Phytochemical screening of n-hexane and petroleum interdigitale. According to the study carried ether henna extracts Phytochemicals n-hexane Petroleum ether out by (Kavitha and Vidyasagar, 2013), Alkaloids + + petroleum ether extract inhibited 100% Carbohydrates + + growth of T. rubrum at 40000 μg/ml. Glycosides - - Saponins + + Furthermore, the antifungal drug Phytosterols - - itraconazole exhibited inhibition zone of Phenols - - Tannins - - 67±0.5 and 67±2.1 against T. rubrum and T. Flavonoids - - interdigitale respectively. Surprisingly, at Present (+); Absent (-). 25mg/ml concentration, itraconazole
possess the highest zone of inhibition of 4. DISCUSSION 71±1.8 against T. interdigitale. This could Henna, has been used anciently as a be due to the structure of the fungus and medicinal plant (Rao et al., 2016; Rathi et also the mode of action of the drug. al., 2017). It is safe to use with no health In another study, the antifungal effect (Nawasrah et al.,2016) except for drugs fluconazole, ketoconazole and some few reported cases of allergic
International Journal of Research and Review (ijrrjournal.com) 317 Vol.7; Issue: 11; November 2020 Rabi Yakubu Bello. Antifungal activity of henna leaves (lawsonia inermis l.) against trichophyton rubrum and trichophyton interdigitale. nystatin showed little or no antifungal effect certain phytochemicals exhibit their against Candida albicans and Microsporum antimicrobial action only with other spp. at all concentrations. However, mixing phytoconstituents in a synergistic way. antifungal drug and henna plant extracts Furthermore, the susceptibility of T. showed increased antifungal activity interdigitale at 25mg/ml concentration of (Elmanama et al., 2011). itraconazole instead of higher concentration Both petroleum ether and n-hexane needs to be investigated. henna extracts have the same MIC values of 50mg/ml against T. rubrum. Petroleum ether ACKNOWLEDGMENT have MIC value of 12.5mg/ml against T. This study was sponsored by interdigitale while that of n-hexane henna Tertiary Education Trust Fund (TET fund) extract is 100mg/ml. The reference drug through the Institutional Based Research itraconazole has the lowest MIC value of < Grant of Yobe State University. 3.125 against both dermatophytes. Phytochemical screening technique REFERENCES was carried out to test for the presence of 1. Abdelraouf A. Elmanama, Amany A. eight (8) bioactive components. In both Alyazji and Nedaa A. Abu Gheneima extracts only alkaloids, carbohydrates and (2011). Antibacterial, Antifungal and saponins were present. Among the three (3) Synergistic Effect of Lawsonia inermis, Punica granatum and Hibiscus sabdariffa. components, only alkaloid (Tiwari et al., Annals of Alquds Medicine Volume/Issue 2011) have been reported to have 7:33-41 (1432, 2011). antimicrobial activity hence the little or no 2. Ali Esmail Al-Snafi (2019) A review on antifungal activity observed from the lawsonia inermis: a potential medicinal extracts. A similar study carried out by (El plant International Journal of Current Massoudi et al., 2019) showed the presence Pharmaceutical Research issn- 0975-7066 of phenols, proteins, lipids, flavonoids and vol 11, issue 5, 2019 tannins. Variation in bioactive compounds 3. Amal Nawasrah, Amani Al-Nimr and in henna leaves can be related to factors like Aiman A. Ali (2016). Antifungal Effect of leaf maturity, climatic conditions, extraction Henna against Candida albicans Adhered to method, solvents used in extraction as well Acrylic Resin as a Possible Method for Prevention of Denture Stomatitis. Int. J. as season of harvest and storage conditions Environ. Res. Public Health 2016, 13, 520; (Tiwari et al., 2011; El Massoudi et al., doi:10.3390/ijerph13050520, 2019; Neeraj, et al., 2019). www.mdpi.com/journal/ijerph 4. Arun P. (2010) In vitro antibacterial activity 5. CONCLUSION of Lawsonia inermis (henna) leaves. PhD This study showed that extracts of thesis, Educational and Research Institute Lawsonia inermis L. leaves exhibit University, Chennai; 2010. promising antifungal activity against 5. Bianca Maria Piraccini and Aurora Trichophyton rubrum and Trichophyton Alessandrini (2015): Onychomycosis: A interdigitale. The response of the test Review, J. Fungi 2015, 1, 30-43; organisms against the extracts in this study doi:10.3390/jof1010030 Journal of Fungi ISSN 2309-608X seems to be dose dependent as larger zones www.mdpi.com/journal/jof of inhibition were observed with increasing 6. Georgi Tchernev, Plamen Kolev Penev, concentration. This suggests that extracts of Pietro Nenoff, Liliya Georgieva Zisova, Lawsonia inermis can be used to combat the José Carlos Cardoso, Teodora Taneva, problem of drug resistance. However, there Gabriele Ginter-Hanselmayer, Julian is the need for toxicity tests and also Ananiev, Maya Gulubova, Reni Hristova, synergistic activities of henna should be Desislava Nocheva, Claudio Guarneri, tested in combination with other plant Nobuo Kanazawa, Wien Med Wochenschr extracts or antifungal drugs. This is because (2012): Onychomycosis: modern diagnostic
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