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An ancient plant Lawsonia inermis (henna): Determination of in vitro antifungal activity against dermatophytes species
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MYCMED-490; No. of Pages 6 Journal de Mycologie Médicale (2014) xxx, xxx—xxx
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
An ancient plant Lawsonia inermis (henna): Determination of in vitro antifungal activity against dermatophytes species ´ ) : de ´ termination Une ancienne plante Lawsonia inermis (henne ´ antifongique in vitro contre les espe ` ces de de l’activite dermatophytes G.S. Gozubuyuk a, E. Aktas a, N. Yigit b,* a
Microbiology and Clinical Microbiology Department, Ataturk University Medical Faculties, Erzurum, Turkey Medical Laboratory Department, Ataturk University Health Services Vocational Training School, 25070 Erzurum, Turkey
b
Received 7 April 2014; received in revised form 29 May 2014; accepted 3 July 2014
KEYWORDS Henna paste; Dermatophytes; Antifungal activity
Summary World is endowed with a rich wealth of medicinal plants. There is a widespread belief that green medicines are healthier and more harmless or safer than synthetic ones. Medicinal plants have been used to cure a number of diseases. The ancient plant Lawsonia inermis or henna is used as medicinal plant because of its attributed strong fungicidal, antiinflammatory, analgesic, antibacterial, virucidal, antiparasitic, antiamoebiasis, astringent, antihemorrhagic, hypotensive, sedative, anticancer effect and possible anti-sweating properties. In this study, we investigated antifungal activity of L. inermis against clinical dermatophytes species. This study was carried out using 70 clinical isolates of dermatophytes representing six different species; 44 Trichophyton rubrum, 8 Trichophyton mentagrophytes, 6 Microsporum canis, 6 Trichophyton tonsurans, 4 Epidermophyton floccosum, and 2 Trichophyton violaceum. The antifungal activity of L. inermis (henna) was determined by agar diffusion method and henna was used as paste form. Henna paste showed the high antifungal activity against all dermatophytes species (20 to 50 mm inhibition zone). # 2014 Elsevier Masson SAS. All rights reserved.
* Corresponding author. Medical Laboratory Department, Ataturk Universtiy Health Services Vocational Training School, 25070 Erzurum, Turkey. E-mail address: [email protected] (N. Yigit). http://dx.doi.org/10.1016/j.mycmed.2014.07.002 1156-5233/# 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Gozubuyuk GS, et al. An ancient plant Lawsonia inermis (henna): Determination of in vitro antifungal activity against dermatophytes species. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.07.002
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MOTS CLÉS Pâte de henné ; Dermatophyte ; L’activité antifongique
Re ´sume ´ Le monde dispose d’un riche patrimoine de plantes médicinales. Une majorité des gens croient bien que la plante médicinale est plus saine, plus inoffensive et plus sûre que les médicaments de synthèse. Cette dernière est utilisée pour le traitement d’un certain nombre de maladies. L’ancienne plante Lawsonia inermis ou henné est utilisé comme une plante médicinale parce qu’elle est créditée d’un fort pouvoir fongicide, anti-inflammatoire, antalgique, antibiotique bactéricide, parasiticide, antiamibien, astringent antihémorragique et des effets de sédation, d’hypotension et d’anticancer ainsi que de propriétés possibles anti-transpiration. Dans cette étude, nous avons étudié l’activité antifongique de L. inermis contre des espèces de dermatophytes cliniques. Cette étude a été réalisée en utilisant 70 dermatophytes cliniques représentant six espèces différentes à savoir 44 Trichophyton rubrum, 8 Trichophyton mentagrophytes, 6 Microsporum canis, 6 Trichophyton tonsurans, 4 Epidermophyton floccosum, et 2 Trichophyton violaceum. L’activité antifongique de L. inermis (henné) a été déterminée par la méthode de diffusion en gélose et le henné a été utilisé sous forme de pâte. Celle-ci a montré l’activité antifongique la plus élevée contre toutes les espèces de dermatophytes (zone d’inhibition de 20 et 50 mm). # 2014 Elsevier Masson SAS. Tous droits réservés.
Introduction For thousands of years, medicine and natural products have been closely linked through the use of traditional medicines and natural drugs. Plants have played a significant role in maintaining human health and improving the quality of human life and have served humans well as valuable components of medicines, seasonings, beverages, cosmetics and dyes. Herbal medicine is based on the premise that plants contain natural substances that can promote health and alleviate illness [18,27]. Plants are used as medicines since time immemorial. According to World Health Organization (WHO), medicinal plants would be the greatest source to obtain a variety of drugs. About 80% of the populations of developing countries being unable to afford pharmaceutical drugs rely on traditional medicines, mainly plant-based, to sustain their primary health care needs. Herbal medicines are in great demand in the developed as well as developing countries for primary healthcare because of their wide biological and medicinal activities, higher safety margins and lesser cost [2,15]. Therefore, such plants should be investigated to better understand their properties, safety and efficiency [30]. Lawsonia inermis, belonging to the family Lythraceae, is grown in dry tropical and subtropical zones, including North Africa, India, Sri Lanka and the Middle East. L. inermis is commercially cultivated in Western India, Pakistan, Morocco, Yemen, Iran, Sudan and Libya [2,11,12]. This plant is commonly called as henna. It is known by various names in different languages; in English (henna, samphire), in Sanskrit (mendhi, mendika), in Arabic (alhenna, hinna), in French (henné or alcana d’Orient), in Hindi (Hena, mhindi), in Greek (kypros) [2,10,37] and in Turkish (k½na). Henna is a tall shrub or small tree, 2—6 m high, it is glabrous, multi-branched with spine tipped branchlets. Leaves are opposite, entire, glabrous, sub-sessile, elliptical, and broadly lanceolate, having depressed veins on dorsal surface. Henna flowers have four sepals. Petals are obviate, white or red stamens inserted in pairs on the rim of the calyx tube. Fruits are small, brownish capsules, 4—8 mm in diameter, and open irregularly into four splits [1,2,6,10,34,37].
Phytochemical investigations have shown the presence of the following constituents: b-sitosterolglucosides, flavonoids, quinoids, naphthalene derivatives, luteolin, betulin, lupeol gaic acid, coumarins, xanthones, and phenolic glycosides. The plant contains 25—33% water-soluble matter; aqueous solutions are orange in color and show a green fluorescence. The principal coloring matter is lawsone, 2hydroxy-1,4-naphtoquinone, which is present in dried leaves in a concentration of 0.4—1.5% [2,11,26,28,29,33,34]. The two pentacyclic triterpenes, namely, 3b, 30-dihydroxylup20(29)-ene (hennadiol) and (20S) 3b, 30-dihydroxylupane have been isolated from the bark [28]. Henna leaf has an orange-red dye and leaf paste or powder is widely used for decorating hands, nails and feet with patterns. The plant is traditionally used to develop a red or black coloring to hands, feet and hair in some occasions, such as weddings and religious festivals [1]. Henna leaves are used as a remedy in skin diseases in the form of paste or decoction against burns, bruises and skin inflammation. Leaves in the form of paste have been used as external application in headache and rubbed over the soles of the feet in burning feet. Decoction of the leaves is used as gargle in sore throat [9]. The essential oil obtained from the flowers finds use in perfumery due to its b-ionone content [34]. The bark is applied in the form of decoction to burns and scalds. It is given internally in a variety of affections, such as jaundice, enlargement of the spleen, calculus, as an alternative in leprosy and obstinate skin affections. Root is considered as a potent medicine for gonorrhoea and herpes infection. Root is astringent and may be pulped and used for sore eyes. Pulped root may also be applied to the heads of children for boils. The root is supposed to be useful in the treatment of hysteria and nervous disorders [2,17]. Henna is believed to have a cooling effect on the body and the paste is used to bring down fever. As a medicinal plant, because of its attributed antibacterial effects specially for Gram positive bacteria, antifungal activity against dermatophytes and wound healing, antitumoral effects in rat, antiamoebiasis, astringent, antihemmoragic, hypotensive, and sedative effects [6,8,14,19,30]. Dermatophytes are a group of fungi adapted to keratin of human and animal skins; they are very contagious and
Please cite this article in press as: Gozubuyuk GS, et al. An ancient plant Lawsonia inermis (henna): Determination of in vitro antifungal activity against dermatophytes species. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.07.002
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In vitro antifungal activity of henna against dermatophytes species responsible for numerous outbreaks. In the past decade, the incidence of dermatophytosis has risen dramatically. The humid weather, over population and poor hygenic conditions, and the increase in number of immunocompromised hosts are ideally suited for the growth of dermatophytes [20,32,36]. Although several synthetic antimycotic drugs are available in the market, at present, the use of these drugs has been minimized because of a number of factors which includes low potency, poor solubility, development of resistant strains, drug toxicity and side effects, like gastrointestinal disturbances, cutaneous reaction, hepatotoxicity, leukopenia. The poor availability of efficient non-toxic antifungals and increasing number of treatment failures have motivated current searches for therapeutic alternatives to include the testing of medicinal plant and essential oils as potential antimicrobial agents [32]. Research on the antimicrobial activity of L. inermis has been focused on this plant extraction, such as methanol, ethanol, water, and chloroform [1,3,6,12,14,19— 21,30,32,35]. The study presented here is, to our knowledge, the f½rst to analyse the antifungal activity of powdered leaves of this plant, in the form of a paste against clinical dermatophytes species.
Materials and methods Sample collection Seventy patients diagnosed with dermatomycosis at Dermatology Department of University Hospital in Erzurum between 2012 to 2013 were included in this study. The age and gender of the patients, duration of the disease, the region where the patient lives (urban/rural), contact in with animal and the clinical type of the disease were recorded. The clinical specimens, including cutaneous skin scales, hair and nail scrapings or eclipses were collected from all patients for mycological examination by conventional technique. Both hairs and scales were placed in clean, labelled, sterile petri dishes and sent to Mycology Laboratory, Microbiology Department of Medical Faculty for investigation.
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Plant material Henna was purchased in powdered leaves form from local herbalist. The dry powder henna was mixed with sterile saline solution to make a preparation with toothpaste-like consistency (Henna paste; traditional usage among people in our countries for hands, feet and hair) and was sterilized at 121 8C for 15 min in autoclave.
Antifungal activity The antifungal activity of the henna was determined by employing agar diffusion technique [32]. For each isolate, a suspension of mycelia from a 7-day culture was prepared in sterile saline to a concentration of 106 cells/mL. The suspensions were swabbed evenly on the surface of the petri plates containing solidified Sabouraud Dextrose agar. Well of 4 mm depth and 6 mm diameter were made in the centre of the agar plate with a sterile cork borer. The well was then filled with the henna paste and allowed to diffuse at room temperature for an hour. Agar plates were incubated at 28 8C for 96 h and the diameter of the zones of inhibition around the well that was filled with henna paste were measured and recorded. Commercially available disks miconazole (10 mg/disk) (rosco NeoSensitabs; Key scien, TX) were used as control.
Results This study was conducted on 70 patients with dermatomycosis of skin, hair and nail. They were 20 (28.6%) female and 50 (71.4) male. Their age ranged from 18 to 76 years. The majority 63 (90.0%) of patients went from a rural area. Nine (12.9%) patients were contact with animal Table 1. The patients were clinically diagnosed and mycologically confirmed as having tinea pedis (27 cases), tinea unguium (23 cases), tinea corporis (10 cases), tinea capitis (5 cases) and tinea cruris (5 cases) Table 2. In this study, a total of 70 dermatophytes strains were isolated from all patients. The most frequently isolated species was Trichophyton rubrum, comprising 44 (63.0%) isolates, followed by Trichophyton mentagrophytes (8 isolated, 11.4%),
Laboratory procedures and identification Media used for primary isolation of dermatophytes, maintenance, and identification were according to conventional reported procedures. The plucked hairs and scraped scales were evaluated by direct microscopic examination for fungal elements in 30% potassium hydroxide solution. For culture examination, each specimen was cultured on Sabouraud glucose agar (Difco, Detroit, MI, USA) with chloramphenicol and Mycobiotic agar (Difco, Detroit, MI, USA). All cultures were incubated at 25 8C for 30 days and checked twice weekly. Species were identified using conventional method, which emphasized colony morphology, microscopy, and other miscellaneous tests. Cultures were examined macroscopically for morphology, texture, and color from the top and reverse sides of the plate. Then, using a sterile straight loop, the colony was examined by placing a sample on drop of lactophenol solution on a glass slide. The preparation was observed under the microscope for the presence of microconidia, macroconidia and other structures.
Table 1 Demographic and exposure characteristic of cases (total number of patients: 70). ´ mographiques et cliniques des cas (nom´ ristiques de Caracte bre des patients : 70). Character Urban Rural Male Female Patients in contact with animals Patients no contact with animals Ages in years 18—35 35—50 50
Cases (%) (n) 10.0% 90.0% 71.4% 28.6% 12.9% 87.1%
(7) (63) (50) (20) (9) (61)
44.3% (31) 28.6% (20) 27.1% (19)
Please cite this article in press as: Gozubuyuk GS, et al. An ancient plant Lawsonia inermis (henna): Determination of in vitro antifungal activity against dermatophytes species. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.07.002
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G.S. Gozubuyuk et al. Table 2 The number and percentages of dermatophytes species according to the type of tinea. ` ces de dermatophytes selon les types de teignes. Nombre et pourcentage des espe Types of tinea (n)
T. pedis (27) T. unguium (23) T. corporis (10) T. capitis (5) T. cruris (5) Total (70)
Dermatophytes species n (%) T. rubrum
T. mentagrophytes
M. canis
T. tonsurans
E. floccosum
T. violaceum
21 (77.8) 18 (78.3) 3 (30.0) — 2 (40.0) 44 (63.0)
1 (3.7) 1 (4.3) 3 (30.0) 3 (60.0) — 8 (11.4)
— 2 (8.7) 2 (20.0) 2 (40.0) — 6 (8.6)
3 (11.1) — — — 3 (60.0) 6 (8.5)
2 (7.4) 2 (8.7) — — — 4 (5.7)
— — 2 (20.0) — — 2 (2.8)
Microsporum canis (6 isolated, 8.6%), Trichophyton tonsurans (6 isolated, 8.5%), Epidermophyton floccosum (4 isolated, 5.7%), and Trichophyton violaceum (2 isolated, 2.8%) as presented in Table 2. In present study, were investigated antifungal activity of henna paste against clinical dermatophytes species by using agar diffusion methods and the zones of inhibition were calculated. The zone of inhibition exhibited by the effect of henna paste on dermatophytes species is shown in Table 3, and illustrated in Fig. 1. Henna paste showed the high antifungal activity against 15 T. rubrum strains (20 to 30 mm inhibition zone), 29 T. rubrum strains (40 to 50 mm inhibition zone), 4 T. mentagrophytes strains (20 to 30 mm inhibition zone), 4 T. mentagrophytes strains (40 to 50 mm inhibition zone), 5 M. canis strains (25 mm inhibition zone), 1 M. canis strain (30 mm inhibition zone), 1 T. tonsurans strain (30 mm inhibition zone), 5 T. tonsurans strains (40 mm inhibition zone), 4 E. floccosum strains (40 mm inhibition zone), and 2 T. violaceum strains (20 mm inhibition zone) Table 3. The inhibition zone diameter range (IZD range) and mean inhibition zone diameter standard error (Mean IZD SE) of miconazole (as control antifungal) against dermatophytes species were as follows respectively; T. rubrum (n: 44) 15 to 30 mm, 24.54 4.67, T. mentagrophytes (n: 8) 20 to 40 mm, 22.50 5.97, M. canis (n: 6) 15 to 40 mm, 32.50 9.35, T. tonsurans (n: 6) 20 to 40 mm, 29.16 8.01, E. floccosum (n: 4) 33 to 60 mm, 47.00 11.22, T. violaceum (n: 2) 15 and 30 mm Table 4.
Discussion Dermatophytes are specialized group of fungi, which effect keratinous tissue of humans and other vertebrates, causing superficial infections. The organisms belong to three genera, Trichophyton, Epidermophyton, and Microsporum. Infections caused by these fungi are among the most prevalent cutaneous infections globally and the recent increase in the number of patients with immunocompromised states, such as AIDS, diabetes mellitus, cancer and organ transplantation have given these infections more prominence [5,13,23]. The treatment of dermatophytosis is based on the use of topical and systemic antifungal agents. In recent years, a number of safe and highly effective antifungal agents have been introduced into clinical practice [5,13,23]. The increasing number of problems, such as antibiotic resistance and toxicity due to long-term treatment pose a real challenge to their clinical treatment. These challenges prompt scientists to find out new broad-spectrum antifungals for the effective management of dermatomycosis. Despite great advances in modern medicine, plants play an important role and make a major contribution to health care. Since early times, a majority of the medicinal preparations were of plant origin, either in the simple form of raw plant material or in the more refined forms of the crude extract, mixtures. This resulted from the recognition of the value of traditional medicinal practices; these traditional medical practices have since been adopted in many countries as an integral part of their culture [25].
Table 3 In vitro antifungal activity of henna paste against dermatophytes species. ´ antifongique in vitro de la pa ˆ te de henne ´ contre les espe ` ces de dermatophytes. Activite Species (n)
Inhibitory diameter zone (mm) 20 mm
T. rubrum (44) T. mentagrophytes (8) M. canis (6) T. tonsurans (6) E. floccosum (4) T. violaceum (2) Total (70)
25 mm
30 mm
40 mm
45 mm
50 mm
n
%
n
%
n
%
n
%
n
%
n
%
3 1 — — — 2 6
6.8 12.5 — — — 100 8.6
4 1 5 — — — 10
9.1 12.5 83.3 — — — 14.3
8 2 1 1 — — 12
18.2 25.0 16.7 16.7 — — 17.2
23 1 — 5 4 — 33
52.3 12.5 — 83.3 100 — 47.1
3 1 — — — — 4
6.8 12.5 — — — — 5.7
3 2 — — — — 5
6.8 25.0 — — — — 7.1
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In vitro antifungal activity of henna against dermatophytes species
Figure 1 Inhibition zone of henna paste against T. rubrum. ˆ te de henne ´ contre T. rubrum. Zone d’inhibition de la pa
L. inermis is a tropical and subtropical shrub, cultivated in the Middle East, along the African coast of the Mediterranean Sea. The powder made of dried crushed leaves is called henna. When applies in a form of paste onto hair or skin, it imparts a reddish brown coloration lasting for up to twelve weeks. It was used as a hair dye as early as ancient times; for instance, the hair and nails of Egyptian mummies was dyed with henna and is one of the oldest cosmetics still in use [11,29]. Besides its use in cosmetics, henna was also used in Medieval Persian, Arab, Turkish and Jewish medicine to treat headaches, skin and teeth diseases, as well as animal bites. In many countries, it is still used in folk medicine to treat different skin disorders [29]. Use of henna as a natural dye is related with cultural practices, rituals, arts and crafts, fabrics and for personal embodiment. It has been in use as hair colour and for the decoration of soles of the feet and palm of hands. Henna is widely used in cosmetology for its dyeing properties, due to thr strong binding of lawsone to hair and skin, which probably can be attributed to the reaction of thiol group with keratin. It is also used in the form shampoo and hair lotion. L. inermis is a natural colorant with antimicrobial effect. It is environmental friendly as it can exhibit better biodegradability and have a higher compatibility with the environment [24]. Quinones, and particularly naphthoquinones are widespread among the secondary metabolites of henna plant. They
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can also be prepared synthetically and are widely produced by the chemical industry as organic dyes. The interest of naphthoquinones is not restricted to the chemistry of dyes; a wide spectrum of biological activities is described for them, including antitumor, antibacterial, anti-inflammatory, antiparasitic, cytotoxic activities and antifungal activity [27]. In Turkey, L. inermis are not cultivated. Henna is imported from other countries as henna powder, which is made by drying the henna leaves and milling them to powder, and then the powder is sifted. This powder is purchased from the local herbalist and is mixed with usually water, in less lemon juice or tea. This is mixed to a toothpaste consistency (henna paste) and applied with traditional tools. Henna is used in the treatment of various skin diseases, which is made by applying the henna paste on the affected skin. In present study, are investigated antifungal activity of henna paste against clinical dermatophytes species by using agar diffusion methods and the inhibition zones were calculated. Henna was used as a paste form according to traditional use in our study. Henna paste showed the high antifungal activity against all dermatophytes species in this study. Henna paste exhibited widely inhibition zone (40 to 50 mm inhibition zone) against 29 (65.9%) T. rubrum strains, 4 (50.0%) T. mentagrophytes strains, 5 (83.3%) T. tonsurans strains, 4 (100%) E. floccosum strains and showed inhibition zone against; 15 (34.1%) T. rubrum strains (20 to 30 mm), 4 (50.0%) T. mentagrophytes strains (20 to 30 mm), 5 (83.3%) M. canis strains (25 mm), 1 (16.7%) M. canis strain (30 mm), 1 (16.7%) T. tonsurans strain (30 mm) and 2 (100%) T. violaceum strains (20 mm). Henna has been used to treat skin infections, such as tinea and it is known to have antimicrobial properties which have been attributed to naphthoquinones, including lawson [6]. During antifungal screening of higher plants, the leaves of L. inermis were found to exhibit strong fungitoxicity where naphthoquinones were found to be the active factor. L. inermis exhibited absolute toxicity against ringworm causing fungal species, such as M. gypseum and T. mentagrophytes. The ethanol extract of the whole plant of L. inermis showed antifungal activity against T. mantagrophytes, Candida albicans, C. neoformans, Aspergillus niger and M. canis [4,7,16,20,25,31,32,36]. Bark extract of L. inermis showed fungitoxic effect against ringworm fungi [35]. In a study realized against the strains T. mentagrophytes, T. rubrum, T. tonsurans, T. violaceum, Trichophyton verrucosum, Trichophyton schoenleinii, E. floccosum, Microsporum ferrugineum, M. canis, Sporothrix schenckii, sensitivity toward henna was strong [22]. The present study identifies L. inermis (henna) as a potential source of biological antimicrobial, since it showed
Table 4 In vitro antifungal activity of miconazole against dermatophytes species. ´ ces de dermatophytes. ´ antifongique in vitro de miconazole contre les espe Activite Species (n)
Antifungal agent
IZD range (mm)
Mean IZD SE (mm)
T. rubrum (44) T. mentagrophytes (8) M. canis (6) T. tonsurans (6) E. floccosum (4) T. violaceum (2)
Miconazole Miconazole Miconazole Miconazole Miconazole Miconazole
15—30 20—40 15—40 20—40 33—60 15 and 20
24.54 4.67 22.50 5.97 32.50 9.35 29.16 8.01 47.00 11.22 —
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high activity against wide spectrum of dermatophytes species. These findings have also been mentioned in literature [7,16,20,22,25,31,32,35,36]. It is important later to reinforce further our investigation in more than one model of fungal species in order to establish a basis for taking an extract or preparation into in vivo or clinical tests.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
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[16] Gupta S, K½ran K, Data A. Bio-control of clinical dungal isolates associated with fungal keratitis using medicinal plant extract. Int J Pharm Pharm Sci 2012;4:544—7. [17] Jeyaseelan EC, Jenothiny S, Pathmanthan MK, Jeyadevan JP. Antibacterial activity of sequentially extracted organic solvent extracts of fruits, flowers and leaves of Lawsonia inermis L. from Jaffna. Asian Pacific J Tropic Biomed 2012;798—802. [18] Kamal M, Jawaid T. Pharmacological activities of Lawsonia inermis Linn: a review. Int J Biomed Res 2010;2:62—8. [19] Karpe ST, Kulkarni SR, Shaikh S, Manikrao AM. Bactericidal and bacteriostatic activity of isolated naphthoquinone fraction of Lawsonia inermis and synthetic lawsone on Staphylococcus epidermidis. Pharmacologyonline 2011;2:156—63. [20] Mansour-Djaalab H, Kahlouche-Riachi F, Djerrou Z, SeraktaDelmi M, et al. In vitro evaluation of antifungal effects of Lawsonia inermis, Pistacia lentiscus, and Juglans regia. Int J Med Arom Plants 2012;2:263—8. [21] Muhammad HS, Muhammad S. The use of Lawsonia inermis Linn (henna) in the management of burn wound infections. Afr J Biotech 2005;4:934—7. [22] Natarajan MR, Lalitha DK. Leaf extracts of Lawsonia inermis as antifungal agent. Curr Sci 1987;56:1021—2. [23] Pakshir K, Bahaedinie L, Rezaei Z, et al. In vitro activity of six antifungal against clinically important dermatophytes. Jundishapur J Microb 2009;2:158—63. [24] PhirkeSS, Saha M. An overview of Lawsonia inermis L: a natural dye plant. Bionano Frontier 2013;6:181—4. [25] Ponnusamy K, Petchiammal C, Mohankumar R, Hopper W. In vitro antifungal activity of indirubin isolated from a South Indian ethnomedicinal plant Wrightia tinctoria R. Br J Etnopharm 2010;132:349—54. [26] Rahiman FA, Taha RM. Plant regeneration and induction of coloured callus from henna (Lawsonia inermis syn Lawsonia alba). J Food Agric Environ 2011;9:397—9. [27] Rahmoun NM, Boucherit-Otmani Z, Benabdallah M, Boucherit K, et al. Antimicrobial activities of henna extract and some synthetic naphthoquinones derivatives. Am J Med Bio Res 2013;1: 16—22. [28] Rahmoun N, Boucherit-Otmani Z, Boucherit K, Benabdallah M, Choukchou-Braham N. Antifungal activity of the Algerian Lawsonia inermis (henna). Pharmaceutical Biol 2013;51:131—5. [29] Raja W, Ovais M, Dubey A. Phytochemical screening and antibacterial activity of Lawsonia inermis leaf extract. Int J Microb Res 2013;4:33—6. [30] Rayavarapu KA, Kaladhar DSVGK, Santhos Kumar S. Evaluation of antimicrobial activity of Lawsonia inermis (henna) on aquapathogens. J Pharm B½omed Sci 2011;7:1—3. [31] Sagar K, Vidyasagar GM. Anti-dermatophytic activity of some traditionally used medicinal plants of North Karnataka region. J Applied Pharm Sci 2013;3:77—8. [32] Sharma KK, Saikia R, Kotoky J, Kalita JC, Devi R. Antifungal activity of Solanum melongena L., Lawsonia inermis L., and Justicia gendarussa B. against dermatophytes. Int J PharmTech Res 2011;3:1635—40. [33] Siddiqui BS, Kardar MN, Khan S. Two new triterpenoids from Lawsonia inermis. Z Naturforsch 2005;60b:37—40. [34] Siddiqui BS, Kardar MN, Tarig Ali S, Khan S. Two new and a known compound from Lawsonia inermis. Helvetica Chemica Acta 2003;86:2164—9. [35] Sowjanya NC, Chary CM. Effect of plant extracts on the growth of Microsporum gypseum. J Phytology 2012;4:41—4. [36] Vaijayanthimala J, Rajendra Prasad N, Anandi C, Pagalendi KV. Anti-dermatophytic activity of some Indian medicinal plants. J Nat Remedies 2004;4:26—31. [37] Yadav S, Kumar A, Dora J, Kumar A. Essential perspectives of Lawsonia inermis. Int J Pharm Chemical Sci 2013;2:888—96.
Please cite this article in press as: Gozubuyuk GS, et al. An ancient plant Lawsonia inermis (henna): Determination of in vitro antifungal activity against dermatophytes species. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.07.002
MYCMED-490; No. of Pages 6 Journal de Mycologie Médicale (2014) xxx, xxx—xxx
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
An ancient plant Lawsonia inermis (henna): Determination of in vitro antifungal activity against dermatophytes species ´ ) : de ´ termination Une ancienne plante Lawsonia inermis (henne ´ antifongique in vitro contre les espe ` ces de de l’activite dermatophytes G.S. Gozubuyuk a, E. Aktas a, N. Yigit b,* a
Microbiology and Clinical Microbiology Department, Ataturk University Medical Faculties, Erzurum, Turkey Medical Laboratory Department, Ataturk University Health Services Vocational Training School, 25070 Erzurum, Turkey
b
Received 7 April 2014; received in revised form 29 May 2014; accepted 3 July 2014
KEYWORDS Henna paste; Dermatophytes; Antifungal activity
Summary World is endowed with a rich wealth of medicinal plants. There is a widespread belief that green medicines are healthier and more harmless or safer than synthetic ones. Medicinal plants have been used to cure a number of diseases. The ancient plant Lawsonia inermis or henna is used as medicinal plant because of its attributed strong fungicidal, antiinflammatory, analgesic, antibacterial, virucidal, antiparasitic, antiamoebiasis, astringent, antihemorrhagic, hypotensive, sedative, anticancer effect and possible anti-sweating properties. In this study, we investigated antifungal activity of L. inermis against clinical dermatophytes species. This study was carried out using 70 clinical isolates of dermatophytes representing six different species; 44 Trichophyton rubrum, 8 Trichophyton mentagrophytes, 6 Microsporum canis, 6 Trichophyton tonsurans, 4 Epidermophyton floccosum, and 2 Trichophyton violaceum. The antifungal activity of L. inermis (henna) was determined by agar diffusion method and henna was used as paste form. Henna paste showed the high antifungal activity against all dermatophytes species (20 to 50 mm inhibition zone). # 2014 Elsevier Masson SAS. All rights reserved.
* Corresponding author. Medical Laboratory Department, Ataturk Universtiy Health Services Vocational Training School, 25070 Erzurum, Turkey. E-mail address: [email protected] (N. Yigit). http://dx.doi.org/10.1016/j.mycmed.2014.07.002 1156-5233/# 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Gozubuyuk GS, et al. An ancient plant Lawsonia inermis (henna): Determination of in vitro antifungal activity against dermatophytes species. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.07.002
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MOTS CLÉS Pâte de henné ; Dermatophyte ; L’activité antifongique
Re ´sume ´ Le monde dispose d’un riche patrimoine de plantes médicinales. Une majorité des gens croient bien que la plante médicinale est plus saine, plus inoffensive et plus sûre que les médicaments de synthèse. Cette dernière est utilisée pour le traitement d’un certain nombre de maladies. L’ancienne plante Lawsonia inermis ou henné est utilisé comme une plante médicinale parce qu’elle est créditée d’un fort pouvoir fongicide, anti-inflammatoire, antalgique, antibiotique bactéricide, parasiticide, antiamibien, astringent antihémorragique et des effets de sédation, d’hypotension et d’anticancer ainsi que de propriétés possibles anti-transpiration. Dans cette étude, nous avons étudié l’activité antifongique de L. inermis contre des espèces de dermatophytes cliniques. Cette étude a été réalisée en utilisant 70 dermatophytes cliniques représentant six espèces différentes à savoir 44 Trichophyton rubrum, 8 Trichophyton mentagrophytes, 6 Microsporum canis, 6 Trichophyton tonsurans, 4 Epidermophyton floccosum, et 2 Trichophyton violaceum. L’activité antifongique de L. inermis (henné) a été déterminée par la méthode de diffusion en gélose et le henné a été utilisé sous forme de pâte. Celle-ci a montré l’activité antifongique la plus élevée contre toutes les espèces de dermatophytes (zone d’inhibition de 20 et 50 mm). # 2014 Elsevier Masson SAS. Tous droits réservés.
Introduction For thousands of years, medicine and natural products have been closely linked through the use of traditional medicines and natural drugs. Plants have played a significant role in maintaining human health and improving the quality of human life and have served humans well as valuable components of medicines, seasonings, beverages, cosmetics and dyes. Herbal medicine is based on the premise that plants contain natural substances that can promote health and alleviate illness [18,27]. Plants are used as medicines since time immemorial. According to World Health Organization (WHO), medicinal plants would be the greatest source to obtain a variety of drugs. About 80% of the populations of developing countries being unable to afford pharmaceutical drugs rely on traditional medicines, mainly plant-based, to sustain their primary health care needs. Herbal medicines are in great demand in the developed as well as developing countries for primary healthcare because of their wide biological and medicinal activities, higher safety margins and lesser cost [2,15]. Therefore, such plants should be investigated to better understand their properties, safety and efficiency [30]. Lawsonia inermis, belonging to the family Lythraceae, is grown in dry tropical and subtropical zones, including North Africa, India, Sri Lanka and the Middle East. L. inermis is commercially cultivated in Western India, Pakistan, Morocco, Yemen, Iran, Sudan and Libya [2,11,12]. This plant is commonly called as henna. It is known by various names in different languages; in English (henna, samphire), in Sanskrit (mendhi, mendika), in Arabic (alhenna, hinna), in French (henné or alcana d’Orient), in Hindi (Hena, mhindi), in Greek (kypros) [2,10,37] and in Turkish (k½na). Henna is a tall shrub or small tree, 2—6 m high, it is glabrous, multi-branched with spine tipped branchlets. Leaves are opposite, entire, glabrous, sub-sessile, elliptical, and broadly lanceolate, having depressed veins on dorsal surface. Henna flowers have four sepals. Petals are obviate, white or red stamens inserted in pairs on the rim of the calyx tube. Fruits are small, brownish capsules, 4—8 mm in diameter, and open irregularly into four splits [1,2,6,10,34,37].
Phytochemical investigations have shown the presence of the following constituents: b-sitosterolglucosides, flavonoids, quinoids, naphthalene derivatives, luteolin, betulin, lupeol gaic acid, coumarins, xanthones, and phenolic glycosides. The plant contains 25—33% water-soluble matter; aqueous solutions are orange in color and show a green fluorescence. The principal coloring matter is lawsone, 2hydroxy-1,4-naphtoquinone, which is present in dried leaves in a concentration of 0.4—1.5% [2,11,26,28,29,33,34]. The two pentacyclic triterpenes, namely, 3b, 30-dihydroxylup20(29)-ene (hennadiol) and (20S) 3b, 30-dihydroxylupane have been isolated from the bark [28]. Henna leaf has an orange-red dye and leaf paste or powder is widely used for decorating hands, nails and feet with patterns. The plant is traditionally used to develop a red or black coloring to hands, feet and hair in some occasions, such as weddings and religious festivals [1]. Henna leaves are used as a remedy in skin diseases in the form of paste or decoction against burns, bruises and skin inflammation. Leaves in the form of paste have been used as external application in headache and rubbed over the soles of the feet in burning feet. Decoction of the leaves is used as gargle in sore throat [9]. The essential oil obtained from the flowers finds use in perfumery due to its b-ionone content [34]. The bark is applied in the form of decoction to burns and scalds. It is given internally in a variety of affections, such as jaundice, enlargement of the spleen, calculus, as an alternative in leprosy and obstinate skin affections. Root is considered as a potent medicine for gonorrhoea and herpes infection. Root is astringent and may be pulped and used for sore eyes. Pulped root may also be applied to the heads of children for boils. The root is supposed to be useful in the treatment of hysteria and nervous disorders [2,17]. Henna is believed to have a cooling effect on the body and the paste is used to bring down fever. As a medicinal plant, because of its attributed antibacterial effects specially for Gram positive bacteria, antifungal activity against dermatophytes and wound healing, antitumoral effects in rat, antiamoebiasis, astringent, antihemmoragic, hypotensive, and sedative effects [6,8,14,19,30]. Dermatophytes are a group of fungi adapted to keratin of human and animal skins; they are very contagious and
Please cite this article in press as: Gozubuyuk GS, et al. An ancient plant Lawsonia inermis (henna): Determination of in vitro antifungal activity against dermatophytes species. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.07.002
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In vitro antifungal activity of henna against dermatophytes species responsible for numerous outbreaks. In the past decade, the incidence of dermatophytosis has risen dramatically. The humid weather, over population and poor hygenic conditions, and the increase in number of immunocompromised hosts are ideally suited for the growth of dermatophytes [20,32,36]. Although several synthetic antimycotic drugs are available in the market, at present, the use of these drugs has been minimized because of a number of factors which includes low potency, poor solubility, development of resistant strains, drug toxicity and side effects, like gastrointestinal disturbances, cutaneous reaction, hepatotoxicity, leukopenia. The poor availability of efficient non-toxic antifungals and increasing number of treatment failures have motivated current searches for therapeutic alternatives to include the testing of medicinal plant and essential oils as potential antimicrobial agents [32]. Research on the antimicrobial activity of L. inermis has been focused on this plant extraction, such as methanol, ethanol, water, and chloroform [1,3,6,12,14,19— 21,30,32,35]. The study presented here is, to our knowledge, the f½rst to analyse the antifungal activity of powdered leaves of this plant, in the form of a paste against clinical dermatophytes species.
Materials and methods Sample collection Seventy patients diagnosed with dermatomycosis at Dermatology Department of University Hospital in Erzurum between 2012 to 2013 were included in this study. The age and gender of the patients, duration of the disease, the region where the patient lives (urban/rural), contact in with animal and the clinical type of the disease were recorded. The clinical specimens, including cutaneous skin scales, hair and nail scrapings or eclipses were collected from all patients for mycological examination by conventional technique. Both hairs and scales were placed in clean, labelled, sterile petri dishes and sent to Mycology Laboratory, Microbiology Department of Medical Faculty for investigation.
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Plant material Henna was purchased in powdered leaves form from local herbalist. The dry powder henna was mixed with sterile saline solution to make a preparation with toothpaste-like consistency (Henna paste; traditional usage among people in our countries for hands, feet and hair) and was sterilized at 121 8C for 15 min in autoclave.
Antifungal activity The antifungal activity of the henna was determined by employing agar diffusion technique [32]. For each isolate, a suspension of mycelia from a 7-day culture was prepared in sterile saline to a concentration of 106 cells/mL. The suspensions were swabbed evenly on the surface of the petri plates containing solidified Sabouraud Dextrose agar. Well of 4 mm depth and 6 mm diameter were made in the centre of the agar plate with a sterile cork borer. The well was then filled with the henna paste and allowed to diffuse at room temperature for an hour. Agar plates were incubated at 28 8C for 96 h and the diameter of the zones of inhibition around the well that was filled with henna paste were measured and recorded. Commercially available disks miconazole (10 mg/disk) (rosco NeoSensitabs; Key scien, TX) were used as control.
Results This study was conducted on 70 patients with dermatomycosis of skin, hair and nail. They were 20 (28.6%) female and 50 (71.4) male. Their age ranged from 18 to 76 years. The majority 63 (90.0%) of patients went from a rural area. Nine (12.9%) patients were contact with animal Table 1. The patients were clinically diagnosed and mycologically confirmed as having tinea pedis (27 cases), tinea unguium (23 cases), tinea corporis (10 cases), tinea capitis (5 cases) and tinea cruris (5 cases) Table 2. In this study, a total of 70 dermatophytes strains were isolated from all patients. The most frequently isolated species was Trichophyton rubrum, comprising 44 (63.0%) isolates, followed by Trichophyton mentagrophytes (8 isolated, 11.4%),
Laboratory procedures and identification Media used for primary isolation of dermatophytes, maintenance, and identification were according to conventional reported procedures. The plucked hairs and scraped scales were evaluated by direct microscopic examination for fungal elements in 30% potassium hydroxide solution. For culture examination, each specimen was cultured on Sabouraud glucose agar (Difco, Detroit, MI, USA) with chloramphenicol and Mycobiotic agar (Difco, Detroit, MI, USA). All cultures were incubated at 25 8C for 30 days and checked twice weekly. Species were identified using conventional method, which emphasized colony morphology, microscopy, and other miscellaneous tests. Cultures were examined macroscopically for morphology, texture, and color from the top and reverse sides of the plate. Then, using a sterile straight loop, the colony was examined by placing a sample on drop of lactophenol solution on a glass slide. The preparation was observed under the microscope for the presence of microconidia, macroconidia and other structures.
Table 1 Demographic and exposure characteristic of cases (total number of patients: 70). ´ mographiques et cliniques des cas (nom´ ristiques de Caracte bre des patients : 70). Character Urban Rural Male Female Patients in contact with animals Patients no contact with animals Ages in years 18—35 35—50 50
Cases (%) (n) 10.0% 90.0% 71.4% 28.6% 12.9% 87.1%
(7) (63) (50) (20) (9) (61)
44.3% (31) 28.6% (20) 27.1% (19)
Please cite this article in press as: Gozubuyuk GS, et al. An ancient plant Lawsonia inermis (henna): Determination of in vitro antifungal activity against dermatophytes species. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.07.002
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G.S. Gozubuyuk et al. Table 2 The number and percentages of dermatophytes species according to the type of tinea. ` ces de dermatophytes selon les types de teignes. Nombre et pourcentage des espe Types of tinea (n)
T. pedis (27) T. unguium (23) T. corporis (10) T. capitis (5) T. cruris (5) Total (70)
Dermatophytes species n (%) T. rubrum
T. mentagrophytes
M. canis
T. tonsurans
E. floccosum
T. violaceum
21 (77.8) 18 (78.3) 3 (30.0) — 2 (40.0) 44 (63.0)
1 (3.7) 1 (4.3) 3 (30.0) 3 (60.0) — 8 (11.4)
— 2 (8.7) 2 (20.0) 2 (40.0) — 6 (8.6)
3 (11.1) — — — 3 (60.0) 6 (8.5)
2 (7.4) 2 (8.7) — — — 4 (5.7)
— — 2 (20.0) — — 2 (2.8)
Microsporum canis (6 isolated, 8.6%), Trichophyton tonsurans (6 isolated, 8.5%), Epidermophyton floccosum (4 isolated, 5.7%), and Trichophyton violaceum (2 isolated, 2.8%) as presented in Table 2. In present study, were investigated antifungal activity of henna paste against clinical dermatophytes species by using agar diffusion methods and the zones of inhibition were calculated. The zone of inhibition exhibited by the effect of henna paste on dermatophytes species is shown in Table 3, and illustrated in Fig. 1. Henna paste showed the high antifungal activity against 15 T. rubrum strains (20 to 30 mm inhibition zone), 29 T. rubrum strains (40 to 50 mm inhibition zone), 4 T. mentagrophytes strains (20 to 30 mm inhibition zone), 4 T. mentagrophytes strains (40 to 50 mm inhibition zone), 5 M. canis strains (25 mm inhibition zone), 1 M. canis strain (30 mm inhibition zone), 1 T. tonsurans strain (30 mm inhibition zone), 5 T. tonsurans strains (40 mm inhibition zone), 4 E. floccosum strains (40 mm inhibition zone), and 2 T. violaceum strains (20 mm inhibition zone) Table 3. The inhibition zone diameter range (IZD range) and mean inhibition zone diameter standard error (Mean IZD SE) of miconazole (as control antifungal) against dermatophytes species were as follows respectively; T. rubrum (n: 44) 15 to 30 mm, 24.54 4.67, T. mentagrophytes (n: 8) 20 to 40 mm, 22.50 5.97, M. canis (n: 6) 15 to 40 mm, 32.50 9.35, T. tonsurans (n: 6) 20 to 40 mm, 29.16 8.01, E. floccosum (n: 4) 33 to 60 mm, 47.00 11.22, T. violaceum (n: 2) 15 and 30 mm Table 4.
Discussion Dermatophytes are specialized group of fungi, which effect keratinous tissue of humans and other vertebrates, causing superficial infections. The organisms belong to three genera, Trichophyton, Epidermophyton, and Microsporum. Infections caused by these fungi are among the most prevalent cutaneous infections globally and the recent increase in the number of patients with immunocompromised states, such as AIDS, diabetes mellitus, cancer and organ transplantation have given these infections more prominence [5,13,23]. The treatment of dermatophytosis is based on the use of topical and systemic antifungal agents. In recent years, a number of safe and highly effective antifungal agents have been introduced into clinical practice [5,13,23]. The increasing number of problems, such as antibiotic resistance and toxicity due to long-term treatment pose a real challenge to their clinical treatment. These challenges prompt scientists to find out new broad-spectrum antifungals for the effective management of dermatomycosis. Despite great advances in modern medicine, plants play an important role and make a major contribution to health care. Since early times, a majority of the medicinal preparations were of plant origin, either in the simple form of raw plant material or in the more refined forms of the crude extract, mixtures. This resulted from the recognition of the value of traditional medicinal practices; these traditional medical practices have since been adopted in many countries as an integral part of their culture [25].
Table 3 In vitro antifungal activity of henna paste against dermatophytes species. ´ antifongique in vitro de la pa ˆ te de henne ´ contre les espe ` ces de dermatophytes. Activite Species (n)
Inhibitory diameter zone (mm) 20 mm
T. rubrum (44) T. mentagrophytes (8) M. canis (6) T. tonsurans (6) E. floccosum (4) T. violaceum (2) Total (70)
25 mm
30 mm
40 mm
45 mm
50 mm
n
%
n
%
n
%
n
%
n
%
n
%
3 1 — — — 2 6
6.8 12.5 — — — 100 8.6
4 1 5 — — — 10
9.1 12.5 83.3 — — — 14.3
8 2 1 1 — — 12
18.2 25.0 16.7 16.7 — — 17.2
23 1 — 5 4 — 33
52.3 12.5 — 83.3 100 — 47.1
3 1 — — — — 4
6.8 12.5 — — — — 5.7
3 2 — — — — 5
6.8 25.0 — — — — 7.1
Please cite this article in press as: Gozubuyuk GS, et al. An ancient plant Lawsonia inermis (henna): Determination of in vitro antifungal activity against dermatophytes species. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.07.002
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In vitro antifungal activity of henna against dermatophytes species
Figure 1 Inhibition zone of henna paste against T. rubrum. ˆ te de henne ´ contre T. rubrum. Zone d’inhibition de la pa
L. inermis is a tropical and subtropical shrub, cultivated in the Middle East, along the African coast of the Mediterranean Sea. The powder made of dried crushed leaves is called henna. When applies in a form of paste onto hair or skin, it imparts a reddish brown coloration lasting for up to twelve weeks. It was used as a hair dye as early as ancient times; for instance, the hair and nails of Egyptian mummies was dyed with henna and is one of the oldest cosmetics still in use [11,29]. Besides its use in cosmetics, henna was also used in Medieval Persian, Arab, Turkish and Jewish medicine to treat headaches, skin and teeth diseases, as well as animal bites. In many countries, it is still used in folk medicine to treat different skin disorders [29]. Use of henna as a natural dye is related with cultural practices, rituals, arts and crafts, fabrics and for personal embodiment. It has been in use as hair colour and for the decoration of soles of the feet and palm of hands. Henna is widely used in cosmetology for its dyeing properties, due to thr strong binding of lawsone to hair and skin, which probably can be attributed to the reaction of thiol group with keratin. It is also used in the form shampoo and hair lotion. L. inermis is a natural colorant with antimicrobial effect. It is environmental friendly as it can exhibit better biodegradability and have a higher compatibility with the environment [24]. Quinones, and particularly naphthoquinones are widespread among the secondary metabolites of henna plant. They
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can also be prepared synthetically and are widely produced by the chemical industry as organic dyes. The interest of naphthoquinones is not restricted to the chemistry of dyes; a wide spectrum of biological activities is described for them, including antitumor, antibacterial, anti-inflammatory, antiparasitic, cytotoxic activities and antifungal activity [27]. In Turkey, L. inermis are not cultivated. Henna is imported from other countries as henna powder, which is made by drying the henna leaves and milling them to powder, and then the powder is sifted. This powder is purchased from the local herbalist and is mixed with usually water, in less lemon juice or tea. This is mixed to a toothpaste consistency (henna paste) and applied with traditional tools. Henna is used in the treatment of various skin diseases, which is made by applying the henna paste on the affected skin. In present study, are investigated antifungal activity of henna paste against clinical dermatophytes species by using agar diffusion methods and the inhibition zones were calculated. Henna was used as a paste form according to traditional use in our study. Henna paste showed the high antifungal activity against all dermatophytes species in this study. Henna paste exhibited widely inhibition zone (40 to 50 mm inhibition zone) against 29 (65.9%) T. rubrum strains, 4 (50.0%) T. mentagrophytes strains, 5 (83.3%) T. tonsurans strains, 4 (100%) E. floccosum strains and showed inhibition zone against; 15 (34.1%) T. rubrum strains (20 to 30 mm), 4 (50.0%) T. mentagrophytes strains (20 to 30 mm), 5 (83.3%) M. canis strains (25 mm), 1 (16.7%) M. canis strain (30 mm), 1 (16.7%) T. tonsurans strain (30 mm) and 2 (100%) T. violaceum strains (20 mm). Henna has been used to treat skin infections, such as tinea and it is known to have antimicrobial properties which have been attributed to naphthoquinones, including lawson [6]. During antifungal screening of higher plants, the leaves of L. inermis were found to exhibit strong fungitoxicity where naphthoquinones were found to be the active factor. L. inermis exhibited absolute toxicity against ringworm causing fungal species, such as M. gypseum and T. mentagrophytes. The ethanol extract of the whole plant of L. inermis showed antifungal activity against T. mantagrophytes, Candida albicans, C. neoformans, Aspergillus niger and M. canis [4,7,16,20,25,31,32,36]. Bark extract of L. inermis showed fungitoxic effect against ringworm fungi [35]. In a study realized against the strains T. mentagrophytes, T. rubrum, T. tonsurans, T. violaceum, Trichophyton verrucosum, Trichophyton schoenleinii, E. floccosum, Microsporum ferrugineum, M. canis, Sporothrix schenckii, sensitivity toward henna was strong [22]. The present study identifies L. inermis (henna) as a potential source of biological antimicrobial, since it showed
Table 4 In vitro antifungal activity of miconazole against dermatophytes species. ´ ces de dermatophytes. ´ antifongique in vitro de miconazole contre les espe Activite Species (n)
Antifungal agent
IZD range (mm)
Mean IZD SE (mm)
T. rubrum (44) T. mentagrophytes (8) M. canis (6) T. tonsurans (6) E. floccosum (4) T. violaceum (2)
Miconazole Miconazole Miconazole Miconazole Miconazole Miconazole
15—30 20—40 15—40 20—40 33—60 15 and 20
24.54 4.67 22.50 5.97 32.50 9.35 29.16 8.01 47.00 11.22 —
Please cite this article in press as: Gozubuyuk GS, et al. An ancient plant Lawsonia inermis (henna): Determination of in vitro antifungal activity against dermatophytes species. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.07.002
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MYCMED-490; No. of Pages 6
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high activity against wide spectrum of dermatophytes species. These findings have also been mentioned in literature [7,16,20,22,25,31,32,35,36]. It is important later to reinforce further our investigation in more than one model of fungal species in order to establish a basis for taking an extract or preparation into in vivo or clinical tests.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
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Please cite this article in press as: Gozubuyuk GS, et al. An ancient plant Lawsonia inermis (henna): Determination of in vitro antifungal activity against dermatophytes species. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.07.002