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Clinical trial to compare the effectiveness of two concentrations of the Ageratina pichinchensis extract in the topical treatment of onychomycosis
Journal of Ethnopharmacology 126 (2009) 74–78
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Clinical trial to compare the effectiveness of two concentrations of the Ageratina pichinchensis extract in the topical treatment of onychomycosis Ofelia Romero-Cerecero a,b , Rubén Román-Ramos b , Alejandro Zamilpa a , Jesús Enrique Jiménez-Ferrer a , Gabriela Rojas-Bribiesca a , Jaime Tortoriello a,∗ a b
Centro de Investigación Biomédica del Sur, Instituto Mexicano del Seguro Social (IMSS), Argentina No. 1, 62790 Xochitepec, Morelos, Mexico Programa de Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, Mexico City, Mexico
a r t i c l e
i n f o
Article history: Received 14 May 2009 Received in revised form 6 August 2009 Accepted 6 August 2009 Available online 13 August 2009 Keywords: Onychomycosis Ageratina pichinchensis Topical treatment Asteraceae Trychophyton rubrum
a b s t r a c t The plant species Ageratina pichinchensis has been used, for many years, in Mexican traditional medicine for the treatment of superficial mycosis. Aim of the study: This study compared the therapeutic effectiveness and tolerability of two concentrations of the standardized extract from Ageratina pichinchensis (12.6 and 16.8%) on patients with clinical and mycological diagnosis of mild and moderate onychomycosis. Materials and methods: Two identical phytopharmaceuticals (containing the standardized extract from Ageratina pichinchensis) in nail lacquer solution for topical administration were evaluated in a doubleblind clinical trial. Treatments were administered for 6 months to patients distributed in two groups. Results and discussion: Of 122 patients who agreed to participate in the study, 103 (84.4%) concluded the treatment. The therapeutic effectiveness exhibited by the 12.6% Ageratina pichinchensis extract was 67.2%, while that of the 16.8% Ageratina pichinchensis extract was 79.1%. Regarding clinical evolution, analysis of results at the end of treatment evidenced that the 16.8% concentration possesses higher therapeutic effectiveness with a significant statistical difference (p = 0.010). No treatment produced side effects. Conclusion: Both concentrations of phytopharmaceuticals possess high rates of effectiveness on patients with mild and moderate onychomycosis, and the formulation with a 16.8% concentration possesses higher effectiveness. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Onychomycosis is a disease involving very difficult treatment mainly because it presents a poor response to available drugs; thus, treatment is frequently discontinued by patients. This disease is a therapeutic challenge determined by nail-substratum anatomical characteristics and due to disease chronicity (Gupta et al., 2004a,b; Rigopoulos et al., 2003). Onychomycosis not only represents an aesthetic problem; but also affects millions of persons who require adequate treatment to avoid serious complications and an important expenditure for public health services. Superficial mycoses have been recognized as a health problem of worldwide importance. In Mexico, dermatomycoses are among the 20 main causes for visiting a Physician. There are available drugs for treating toenail mycosis, but these are generally administered systemically; nevertheless, they present side effects (Lecha et al., 2005; Gupta and Cooper, 2008). Topical administration is utilized for mild or moderate cases in which the toenail lesion does not include the nail
∗ Corresponding author. Tel.: +52 777 361 2155; fax: +52 777 361 2155. E-mail address: [email protected] (J. Tortoriello). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.08.007
root or lune. Therefore, in order to select the prescription treatment, it is important to consider the severity of the lesions (Poulin et al., 2006; Gupta et al., 2005). The principal advantages of local therapy comprise safety, tolerability, and cost of treatment, which also represents greater accessibility (Gupta et al., 2006; Murdan, 2008). The treatment of onychomycosis is complicated; the infection entertains a high index of contagion, and relapses are frequent. The drugs available have limitations, are expensive, and produce side effects; thus, different scientific works report the analysis of the cost of drugs employed for treating onychomycosis with the purpose of identifying the drugs with the best costeffectiveness relationship (Casciano et al., 2003; Warshaw et al., 2005). Patients affected by these diseases considered onychomycosis to be an important problem that reduces the physical, mental, and social well-being of the individual due to social stigmatization (Szepietowski and Reich, in press; Nunley and Cornelius, 2008). In Mexican traditional medicine, the use of plants for treating skin diseases is very frequent. In an ethnobotanical approach, 200 plant species were identified which, empirically, are used as a treatment of skin diseases. The pathologies most frequently
O. Romero-Cerecero et al. / Journal of Ethnopharmacology 126 (2009) 74–78
treated by traditional practitioners included scabies, pimples, nacidos, swelling, cancer, disípela, and mazamorra (the latter identified as superficial mycosis) (Zurita and Zolla, 1986). Ageratina pichinchensis (Asteraceae) plant species originating in Mexico have been utilized in Mexican traditional medicine for the treatment of dermatophytosis (Argueta et al., 1994; Avilés and ˜ 2000). For this Suárez, 1994; Monroy-Ortiz and Castillo-Espana, purpose, fresh or dried leaves are extracted with alcohol, and the product is administered topically onto the damaged skin. The extract obtained from the plant showed, in vitro, an important capability for inhibiting the growth of different fungi, especially Trychophyton rubrum, which is the fungus that most frequently affects the nails (Navarro et al., 2003; Rippon, 1990). Previous studies have reported higher antifungal activity in lower polarity extracts such as hexane and ethyl acetate and the presence of encecalin in these extracts, which has been previously used for standardizing the Ageratina pichinchensis extract (RomeroCerecero et al., 2008). Previous works reported the development of a phytopharmaceutical formulated in a cream that contains the standardized extract of Ageratina pichinchensis. This product, evaluated by means of a double-blind clinical trial, showed therapeutic effectiveness of 80.3% in patients with a clinical and mycological diagnosis of tinea pedis (Romero-Cerecero et al., 2006). A similar extract, but depigmented and possessing a higher concentration of the active compound, was used for elaborating a phytopharmaceutical in a nail lacquer presentation for local administration in patients with a diagnosis of onychomycosis. Topical administration of this medicament employed solely one concentration of the plant extract (10%) was evaluated by means of a double-blind clinical trial to compare it with a similar drug elaborated with ciclopirox. In this study, the phytopharmaceutical demonstrated therapeutic effectiveness of 59.1%, achieving a very similar effect in patients and without differences from that produced by 8% ciclopirox (Romero-Cerecero et al., 2008). The present study shows the results of the double-blind clinical trial to compare the therapeutic effectiveness and tolerability of two concentrations (12.6 and 16.8%) of Ageratina pichinchensis standardized extract (in a nail lacquer solution) for topical treatment in patients with mild and moderate onychomycosis. 2. Materials and methods 2.1. Plant material Ageratina pichinchensis (Kunt) R.M. King & Ho. Rob (Asteraceae) was collected by local healers in Cuernavaca, Morelos, Mexico (2007, July). A voucher sample was submitted for identification and storage at the Herbarium of the Instituto de Antropología e Historia (INAHM) and was registered with the number INAH-2050. 2.2. Extract preparation Aerial parts of the plant were dried at environmental temperature under dark conditions. Dried material was extracted by maceration with a mixture of hexane and ethyl acetate (7:3). The extract was concentrated and then submitted to a chemical depigmentation procedure by means of a gravitational chromatography column with activated charcoal as support. The extract obtained was analyzed by HPLC with the aim of standardizing the content of the active compound. Microbiological tests were used to evaluate biological activity. By means of the agar dilution method the Minimal Inhibitory Concentration (MIC) was determined for the extract against Trychophyton rubrum (125 g/mL) and Trychophyton mentagrophytes (250 g/mL).
75
Fig. 1. Chemical structure of encecalin.
2.3. Quantification of the active compound in the extract In order to standardize the phytopharmaceuticals, encecalin (Fig. 1) was used as marker compound. Identity of the standard was confirmed by comparison with spectroscopy data and by cochromatography with an authentic sample. HPLC methodology was utilized for measuring the encecalin concentration in the extract and in the phytopharmaceuticals. The method was developed and implemented in a 2695 separation equipment (Waters) with a 2996 diode array detector and the Empower Chromatography Manager version 1.0 operative system (Waters). Analysis was developed with a Lichrospher column (Merck) with a stainless steel cartridge and the following characteristics: Si 60 (5 m), 250 mm × 4 mm. Elution system consisted of an isocratic solution of n-hexane/ethyl acetate, 80:20 and a flow rate of 1 mL/min. Analysis was performed employing = 254 nm; the total time run was 20 min. A control curve was built with ascending concentrations (50, 100, 200, and 400 g/mL) of encecalin. Each concentration was injected three times (20 L, R2 = 0.99). The retention time of the standard was 6.76 min. This method was used in all cases, i.e., for the production of extracts that were employed as raw material for the elaboration of phytopharmaceuticals and quality control of the final product. The encecalin concentration in the extract was 143.9 mg/g. 2.4. Phytopharmaceutical production A cosmetic nail lacquer (Perlamex 59, Mexico) was used as the base of the medicament design. As described by Romero-Cerecero et al. (2008), Ageratina pichinchensis extract (previously diluted in ethyl acetate) was added until obtaining, in one case, a 12.6% concentration, and in the remaining other case, one of 16.8%. With the aim of blinding the experimental procedure, the medicaments (in a 3-mL flask presentation with a spiral cover that contained a brush for administration on nails) were packed and labeled in identical form. 2.5. Subjects The clinical trial followed the guidelines of the Declaration of Helsinki and Tokyo for research in humans and was approved by the Institutional Ethics Committee. All patients received information concerning the study and were required to sign an informed consent letter to be included in the study. In this study, patients who were pregnant or breastfeeding, with diabetes mellitus or another serious disease, as well as patients who received treatment for this disease at least 1 month prior to the date of study inclusion, or with a history of sensitivity to topically administrated drugs, were not accepted for participation in the study. In the study, 122 patients with a diagnosis of mild or moderate onychomycosis aged 19–65 years were included. Based on a table with randomized numbers, two groups were organized: patients of group 1 were treated with the 12.6% Ageratina pichinchensis extract, and group 2 received treatment with the 16.8% Ageratina pichinchensis extract.
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A mycological diagnosis was performed by direct microscopy before and after treatment. For this purpose, a sample from the affected nails was obtained and direct microscopy was conducted for identifying fungi. At the end of treatment, a second sample was obtained from nails that maintained the pathology, as well as from nails exhibiting improvement or that were cured. All patients were followed clinically by means of monthly appointments at our Physician’s office or at the patient’s home, and comprised evaluation of clinical evolution, therapeutic effectiveness, and tolerability.
Table 2 Degree of severity of clinical manifestations in patients with onychomycosis before and after 6 months of treatment with two different concentrations (group 1 = 12.6%, group 2 = 16.8%) of the standardized extract of Ageratina pichinchensis. Treatment
2 p
Frequency/% Healthy
Mild
Moderate
Before Group 1 n = 62 Group 2 n = 60
0/0 0/0
13/20.9 18/30
49/79 42/70
0.25
After Group 1 n = 55 Group 2 n = 48
37/67.2 38/79.1
3/5.4 7/14.5
15/27.2 3/6.3
0.010
2.6. Statistical analysis Central tendency measurements and dispersion were used for analyzing study results. ANOVA and 2 tests were employed to identify differences between groups. p < 0.05 was considered significant. 3. Results 3.1. Demographic aspects Women predominated in the study with 77% (94), age of patients in the entire sample ranged between 19 and 65 years, and median age was 47 years. Distribution of these variables between both groups was similar, and no significant differences were detected. 3.2. Clinical evolution In all patients included in the study, the clinical disease form was distal onychomycosis, and of primary origin. There were 62 patients in group 1, and in terms of degree of severity, 79% (49) were classified as having moderate onychomycosis. In group 2, 60 patients were included; of these, 70% (42) were classified as suffering from moderate onychomycosis (p = 0.25). With respect to the number of affected toenails, in both groups there were patients with 1–10 affected toenails. The number of toenails affected in both groups at the beginning and at the end of treatment is shown in Table 1. Predominating signs among patients were hyperqueratosis, opacity, and modification in toenail color. The proportion of hyperqueratosis in patients included in group 1 was 98.3% (61), while in group 2, this was 96.6% (58). In terms of opacity and toenail color changes, the proportion was simi-
Table 1 Frequency and percentage of the number of affected toenails with mild and moderate onychomycosis before and after receiving treatment with two different concentrations of the standardized extract of Ageratina pichinchensis. Number of toenails affected
0 1 2 3 4 5 6 7 8 9 10
Ageratina pichinchensis extract 12.6%
Ageratina pichinchensis extract 16.8%
Number of patients Before/after 62/55 Frequency (%)
Number of patients Before/after 60/48 Frequency (%)
0 (0)/37 (67.2) 7 (11.2)/1 (1.9) 5 (8)/1 (1.9) 6 (9.6)/3 (5.4) 16 (25.8)/3 (5.4) 5 (8.4)/1 (1.9) 3 (4.8)/2 (3.6) 3 (4.8)/3 (5.4) 6 (9.6)/3 (5.4) 7 (11.2)/0 (0) 4 (6.4)/1 (1.9)
0 (0)/38 (79.1) 5 (8.3)/1 (2) 5 (8.3)/0 (0) 4 (6.7)/0 (0) 10 (16.6)/2 (4.3) 9 (15)/3 (6.2) 12 (20)/2 (4.3) 4 (6.6)/0 (0) 3 (5)/1 (2) 4 (6.6)/1 (2) 4 (6.6)/0 (0)
2 p
lar to that of hyperqueratosis, with a slight variation in group 2, in which toenail color modification was observed in 59 patients (98%). After 6 months of treatment, the total number of patients was reduced to 103 (84.4%): 55 in group 1 and 48 in group 2, demonstrating a patient withdrawal of 15.6% from the study. Therapeutic effectiveness was determined once the treatment was concluded: 67.2% (37) of patients included in group 1 and 79.1% (38) of patients from group 2 presented no clinical manifestation in the toenails and were considered as healthy (Table 2). Fig. 2 shows the clinical evolution of a patient treated with the 16.8% Ageratina pichinchensis extract. Regarding clinical evolution of patients under treatment (Table 2), at the end of the treatment there was evidence that 27.2% of patients treated with the 12.6% Ageratina pichinchensis extract showed a moderate degree of severity, while only 6.3% of patients treated with the 16.8% Ageratina pichinchensis extract experienced this condition. Results analysis considered therapeutic improvement when the patient exhibited a reduction in the number of toenails affected. In this analysis, we included patients who did not achieve the healthy stage, but who did have a reduced number of toenails. In this manner, the proportion of patients with therapeutic improvement among individuals treated with the 12.6% Ageratina pichinchensis extract was 74.5% (41 patients), while patients treated with the higher concentration (16.8%) showed a therapeutic improvement of 89.5% (43 patients), demonstrating a significantly (p = 0.048) better evolution in patients treated with the more concentrated product. After 6 months of treatment administration, there were 19 (18.44%) patients without improvement; nevertheless, after analyzing the cases, it was observed that the cause of the treatment failure was due (in 11 patients, 10.6%) to lack of compliance with the treatment, while the remainder (8 patients, 7.7%) were individuals who demonstrated a moderate degree of severity at the beginning of the study. Direct examination by microscopy demonstrated that all patients without signs and symptoms at the end of the study were negative for the presence of fungus. Both treatments were tolerated satisfactorily because the total number of patients included in both groups manifested no side effects during the study (p = 1). 4. Discussion
0.37
The phytopharmaceutical developed (from the plant species Ageratina pichinchensis) is elaborated with the standardized extract of the aerial parts of the plant with a known concentration of the active compound (encecalin). This compound is active against the dermatophytes most frequently found in tinea ungueum and tinea pedis. The phytopharmaceutical elaborated with the Ageratina pichinchensis extract constitutes an innovative medical alternative that, by means of double-blind clinical trials and compared with
O. Romero-Cerecero et al. / Journal of Ethnopharmacology 126 (2009) 74–78
77
Fig. 2. Photography that shows the clinical evolution of a patient before and after being treated topically for 6 months with a nail lacquer containing 16.8% Ageratina pichinchensis extract.
ciclopirox, a leading drug on the market (Gupta, 2000), has demonstrated its effectiveness, tolerability, and safety in patients with onychomycosis (Romero-Cerecero et al., 2008). Once the effectiveness of the phytopharmaceutical was confirmed, it was necessary to continue with the development of this study, which allowed to identify extract concentration that offers greatest effectiveness for topical treatment of mild to moderate onychomycosis, in addition to maintaining good tolerability and offering the patient a therapeutic resource that avoids evolution of the disease that could produce nail dystrophy and, consequently, affect the patient’s quality of life. Regarding the severity of clinical manifestations, we observed no differences (in the number of patients with mild and moderate onychomycosis) between both groups of patients prior to treatment initiation. Nevertheless, after 6 months of treatment with the phytopharmaceuticals, despite that the number of patients of both groups who achieved a healthy condition was similar, an important difference was observed in patients who were found at a moderate stage of severity (15 and 3 patients, for 12.6 and 16.8%, respectively). This result supports that higher concentration (16.8%) of the plant extract possesses higher effectiveness. When the number of affected toenails was analyzed (therapeutic improvement), it was also observed that the higher concentration reached higher rates of therapeutic improvement. In this study, 15.6% of patient withdrawal was observed; all cases were due to non-medical causes. This percentage could be considered normal in a study consisting of 6 months of tracking, in which changes in different personal aspects could occur (in workplace or place of residence, etc.). No side effects were exhibited by patients throughout the study period, resulting in 100% tolerability for both treatments. Considering as therapeutic success when the patient achieved (a) therapeutic effectiveness, (b) absence of fungus with direct microscopy examination, and (c) tolerability, the therapeutic success value was 67.2 and 79.1% for the 12.6 and the 16.8% extract, respectively. Previously, Romero-Cerecero et al. (2008) reported the evaluation of a similar product elaborated in a lacquer solution containing the standardized extract of Ageratina pichinchensis, but in this case, with an extract concentration of 10%. This product, also administered topically for 6 months, showed a therapeutic success of 55.1%. This 10% formulation was compared with a similar product containing 8% ciclopirox, which produced a 63.8% therapeutic success rate without evidencing significant differences. It is noteworthy that the concentration of the active compound contained in the ciclopirox nail lacquer (8%) could be considered lower than that used with the plant extract of the experimental treatment (12.6 and 16.8%). However, when the concentration of the active compound contained in the experimental treatments is considered, both phytopharmaceuticals contain considerably lower concentrations (1.81 and 2.41% of encecalin in the 12.6 and 16.8% concentrations of Ageratina pichinchensis extract, respectively).
5. Conclusion It is possible to conclude that both concentrations (those of 12.6 and 16.8%) of the Ageratina pichinchensis extract formulated in a nail lacquer presentation and administered topically on the nails of patients with mild and moderate onychomycosis were therapeutically effective, while the higher concentration (16.8%) offers a statistically higher effectiveness. Both treatments exhibited 100% tolerability. Acknowledgment This study was financially supported by a grant (CONACyTSALUD/2007CO1/071029) from CONACyT-México. References Argueta, A., Cano, L., Rodarte, M., 1994. Atlas de La Medicina Tradicional Mexicana, Tomo 1-3. Instituto Nacional Indigenista, México, D.F, p. 1786. Avilés, M., Suárez, G., 1994. Catálogo de Plantas Medicinales Jardín Etnobotánico. Centro INAH, Cuernavaca, Morelos, México, p. 47. Casciano, J., Amaya, K., Doyle, J., Arikian, S., Shear, N., Haspel, M., Kahler, K., 2003. Economic analysis of oral and topical therapies for onychomycosis of the toenails and fingernails. Managed Care 12, 47–54. Gupta, A.K., 2000. Pharmacoeconomic analysis of ciclopirox nail lacquer solution 8% and the new oral antifungal agents used to treat dermatophyte toe onychomycosis in the United States. Journal of the American Academy of Dermatology 43, S81–S95. Gupta, A.K., Cooper, E.A., Ryder, J.E., Nicol, K.A., Chow, M., Chaudhry, M.M., 2004a. Optimal management of fungal infections of the skin, hair, and nails. American Journal of Clinical Dermatology 5, 225–237. Gupta, A.K., Cooper, E.A., 2008. Update in antifungal therapy of dermatophytosis. Mycopathologia 166, 353–367. Gupta, A.K., Ryder, J.E., Chow, M., Cooper, E.A., 2005. Dermatophytosis: the management of fungal infections. Skinmed 4, 305–310. Gupta, A.K., Ryder, J.E., Johnson, A.M., 2004b. Cumulative meta-analysis of systemic antifungal agents for the treatment of onychomycosis. The British Journal of Dermatology 150, 537–544. Gupta, A.K., Poulin, Y., Lynde, C.W., 2006. Canadian perspectives on antifungal treatment for onychomycosis. Journal of Cutaneous Medicine and Surgery 10 (Suppl. 2), S34–S38. Lecha, M., Effendy, I., Feuilhade de Chauvin, M., Di Chiacchio, N., Baran, R., 2005. Taskforce on Onychomycosis Education. Treatment options—development of consensus guidelines. Journal of the European Academy of Dermatology and Venereology 19 (Suppl. 1), 25–33. ˜ P., 2000. Plantas medicinales utilizadas en MoreMonroy-Ortiz, C., Castillo-Espana, los. Centro de Investigaciones Biológicas, Universidad Autónoma del Estado de Morelos, México, pp. 61–62. Murdan, S., 2008. Enhancing the nail permeability of topically applied drugs. Expert Opinion on Drug Delivery 5, 1267–1282. Navarro, V.M., García, A., González, M., Fuentes, A., Avilés, M., Ríos, Y., Zepeda, G., Rojas, M.G., 2003. Antifungal activities of nine traditional Mexican medicinal plants. Journal of Ethnopharmacology 87, 85–88. Nunley, K.S., Cornelius, L., 2008. Current management of onychomycosis. The Journal of Hand Surgery 33, 1211–1214. Poulin, Y., Thomas, R., Gupta, A.K., 2006. Brief treatment guide for onychomycosis. Journal of Cutaneous Medicine and Surgery 10 (Suppl. 2), S39–S43. Rigopoulos, D., Katoulis, A.C., Ioannides, D., Georgala, S., Kalogeromitros, D., Bolbasis, I., Karistinou, A., Christofidou, E., Polydorou, D., Balkou, P., Fragouli, E., Katsambas, A.D., 2003. A randomized trial of amorolfine 5% solution nail lacquer in association with itraconazole pulse therapy compared with itraconazole alone
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in the treatment of Candida fingernail onychomycosis. The British Journal of Dermatology 149, 151–156. Rippon, J.W., 1990. Tratado de Micología Médica, 3ra ed. Interamericana McGrawHill, México, Capítulo 8, pp. 186–202. Romero-Cerecero, O., Rojas-Bribiesca, M.G., Navarro, V.M., Herrera-Arellano, A., Zamilpa-Álvarez, A., Tortoriello, J., 2006. Effectiveness and tolerability of a standardized extract from Ageratina pichinchensis on patients with tinea pedis. A double blind clinical trial. Planta Medica 72, 1257–1261. Romero-Cerecero, O., Zamilpa, A., Jiménez-Ferrer, J.E., Rojas-Bribiesca, M.G., RománRamos, R., Tortoriello, J., 2008. Double-blind clinical trial for evaluating the effectiveness and tolerability of Ageratina pichinchensis extract on patients with
mild to moderate onychomycosis. A comparative study with ciclopirox. Planta Medica 74, 1430–1435. Szepietowski, J.C., Reich, A., in press. For the National Quality of Life in Dermatology Group. Stigmatisation in onychomycosis patients: a population-based study. Mycoses. Warshaw, E.M., Fett, D.D., Bloomfield, H.E., Grill, J.P., Nelson, D.B., Quintero, V., Carver, S.M., Zielke, G.R., Lederle, F.A., 2005. Pulse versus continuous terbinafine for onychomycosis: a randomized, double-blind, controlled trial. Journal of the American Academy of Dermatology 43, 578–584. Zurita, E.M., Zolla, C., 1986. Enfermedades dermatológicas en la medicina tradicional de México. Boletín de la Oficina Sanitaria Panamericana 101, 339–344.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Clinical trial to compare the effectiveness of two concentrations of the Ageratina pichinchensis extract in the topical treatment of onychomycosis Ofelia Romero-Cerecero a,b , Rubén Román-Ramos b , Alejandro Zamilpa a , Jesús Enrique Jiménez-Ferrer a , Gabriela Rojas-Bribiesca a , Jaime Tortoriello a,∗ a b
Centro de Investigación Biomédica del Sur, Instituto Mexicano del Seguro Social (IMSS), Argentina No. 1, 62790 Xochitepec, Morelos, Mexico Programa de Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, Mexico City, Mexico
a r t i c l e
i n f o
Article history: Received 14 May 2009 Received in revised form 6 August 2009 Accepted 6 August 2009 Available online 13 August 2009 Keywords: Onychomycosis Ageratina pichinchensis Topical treatment Asteraceae Trychophyton rubrum
a b s t r a c t The plant species Ageratina pichinchensis has been used, for many years, in Mexican traditional medicine for the treatment of superficial mycosis. Aim of the study: This study compared the therapeutic effectiveness and tolerability of two concentrations of the standardized extract from Ageratina pichinchensis (12.6 and 16.8%) on patients with clinical and mycological diagnosis of mild and moderate onychomycosis. Materials and methods: Two identical phytopharmaceuticals (containing the standardized extract from Ageratina pichinchensis) in nail lacquer solution for topical administration were evaluated in a doubleblind clinical trial. Treatments were administered for 6 months to patients distributed in two groups. Results and discussion: Of 122 patients who agreed to participate in the study, 103 (84.4%) concluded the treatment. The therapeutic effectiveness exhibited by the 12.6% Ageratina pichinchensis extract was 67.2%, while that of the 16.8% Ageratina pichinchensis extract was 79.1%. Regarding clinical evolution, analysis of results at the end of treatment evidenced that the 16.8% concentration possesses higher therapeutic effectiveness with a significant statistical difference (p = 0.010). No treatment produced side effects. Conclusion: Both concentrations of phytopharmaceuticals possess high rates of effectiveness on patients with mild and moderate onychomycosis, and the formulation with a 16.8% concentration possesses higher effectiveness. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Onychomycosis is a disease involving very difficult treatment mainly because it presents a poor response to available drugs; thus, treatment is frequently discontinued by patients. This disease is a therapeutic challenge determined by nail-substratum anatomical characteristics and due to disease chronicity (Gupta et al., 2004a,b; Rigopoulos et al., 2003). Onychomycosis not only represents an aesthetic problem; but also affects millions of persons who require adequate treatment to avoid serious complications and an important expenditure for public health services. Superficial mycoses have been recognized as a health problem of worldwide importance. In Mexico, dermatomycoses are among the 20 main causes for visiting a Physician. There are available drugs for treating toenail mycosis, but these are generally administered systemically; nevertheless, they present side effects (Lecha et al., 2005; Gupta and Cooper, 2008). Topical administration is utilized for mild or moderate cases in which the toenail lesion does not include the nail
∗ Corresponding author. Tel.: +52 777 361 2155; fax: +52 777 361 2155. E-mail address: [email protected] (J. Tortoriello). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.08.007
root or lune. Therefore, in order to select the prescription treatment, it is important to consider the severity of the lesions (Poulin et al., 2006; Gupta et al., 2005). The principal advantages of local therapy comprise safety, tolerability, and cost of treatment, which also represents greater accessibility (Gupta et al., 2006; Murdan, 2008). The treatment of onychomycosis is complicated; the infection entertains a high index of contagion, and relapses are frequent. The drugs available have limitations, are expensive, and produce side effects; thus, different scientific works report the analysis of the cost of drugs employed for treating onychomycosis with the purpose of identifying the drugs with the best costeffectiveness relationship (Casciano et al., 2003; Warshaw et al., 2005). Patients affected by these diseases considered onychomycosis to be an important problem that reduces the physical, mental, and social well-being of the individual due to social stigmatization (Szepietowski and Reich, in press; Nunley and Cornelius, 2008). In Mexican traditional medicine, the use of plants for treating skin diseases is very frequent. In an ethnobotanical approach, 200 plant species were identified which, empirically, are used as a treatment of skin diseases. The pathologies most frequently
O. Romero-Cerecero et al. / Journal of Ethnopharmacology 126 (2009) 74–78
treated by traditional practitioners included scabies, pimples, nacidos, swelling, cancer, disípela, and mazamorra (the latter identified as superficial mycosis) (Zurita and Zolla, 1986). Ageratina pichinchensis (Asteraceae) plant species originating in Mexico have been utilized in Mexican traditional medicine for the treatment of dermatophytosis (Argueta et al., 1994; Avilés and ˜ 2000). For this Suárez, 1994; Monroy-Ortiz and Castillo-Espana, purpose, fresh or dried leaves are extracted with alcohol, and the product is administered topically onto the damaged skin. The extract obtained from the plant showed, in vitro, an important capability for inhibiting the growth of different fungi, especially Trychophyton rubrum, which is the fungus that most frequently affects the nails (Navarro et al., 2003; Rippon, 1990). Previous studies have reported higher antifungal activity in lower polarity extracts such as hexane and ethyl acetate and the presence of encecalin in these extracts, which has been previously used for standardizing the Ageratina pichinchensis extract (RomeroCerecero et al., 2008). Previous works reported the development of a phytopharmaceutical formulated in a cream that contains the standardized extract of Ageratina pichinchensis. This product, evaluated by means of a double-blind clinical trial, showed therapeutic effectiveness of 80.3% in patients with a clinical and mycological diagnosis of tinea pedis (Romero-Cerecero et al., 2006). A similar extract, but depigmented and possessing a higher concentration of the active compound, was used for elaborating a phytopharmaceutical in a nail lacquer presentation for local administration in patients with a diagnosis of onychomycosis. Topical administration of this medicament employed solely one concentration of the plant extract (10%) was evaluated by means of a double-blind clinical trial to compare it with a similar drug elaborated with ciclopirox. In this study, the phytopharmaceutical demonstrated therapeutic effectiveness of 59.1%, achieving a very similar effect in patients and without differences from that produced by 8% ciclopirox (Romero-Cerecero et al., 2008). The present study shows the results of the double-blind clinical trial to compare the therapeutic effectiveness and tolerability of two concentrations (12.6 and 16.8%) of Ageratina pichinchensis standardized extract (in a nail lacquer solution) for topical treatment in patients with mild and moderate onychomycosis. 2. Materials and methods 2.1. Plant material Ageratina pichinchensis (Kunt) R.M. King & Ho. Rob (Asteraceae) was collected by local healers in Cuernavaca, Morelos, Mexico (2007, July). A voucher sample was submitted for identification and storage at the Herbarium of the Instituto de Antropología e Historia (INAHM) and was registered with the number INAH-2050. 2.2. Extract preparation Aerial parts of the plant were dried at environmental temperature under dark conditions. Dried material was extracted by maceration with a mixture of hexane and ethyl acetate (7:3). The extract was concentrated and then submitted to a chemical depigmentation procedure by means of a gravitational chromatography column with activated charcoal as support. The extract obtained was analyzed by HPLC with the aim of standardizing the content of the active compound. Microbiological tests were used to evaluate biological activity. By means of the agar dilution method the Minimal Inhibitory Concentration (MIC) was determined for the extract against Trychophyton rubrum (125 g/mL) and Trychophyton mentagrophytes (250 g/mL).
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Fig. 1. Chemical structure of encecalin.
2.3. Quantification of the active compound in the extract In order to standardize the phytopharmaceuticals, encecalin (Fig. 1) was used as marker compound. Identity of the standard was confirmed by comparison with spectroscopy data and by cochromatography with an authentic sample. HPLC methodology was utilized for measuring the encecalin concentration in the extract and in the phytopharmaceuticals. The method was developed and implemented in a 2695 separation equipment (Waters) with a 2996 diode array detector and the Empower Chromatography Manager version 1.0 operative system (Waters). Analysis was developed with a Lichrospher column (Merck) with a stainless steel cartridge and the following characteristics: Si 60 (5 m), 250 mm × 4 mm. Elution system consisted of an isocratic solution of n-hexane/ethyl acetate, 80:20 and a flow rate of 1 mL/min. Analysis was performed employing = 254 nm; the total time run was 20 min. A control curve was built with ascending concentrations (50, 100, 200, and 400 g/mL) of encecalin. Each concentration was injected three times (20 L, R2 = 0.99). The retention time of the standard was 6.76 min. This method was used in all cases, i.e., for the production of extracts that were employed as raw material for the elaboration of phytopharmaceuticals and quality control of the final product. The encecalin concentration in the extract was 143.9 mg/g. 2.4. Phytopharmaceutical production A cosmetic nail lacquer (Perlamex 59, Mexico) was used as the base of the medicament design. As described by Romero-Cerecero et al. (2008), Ageratina pichinchensis extract (previously diluted in ethyl acetate) was added until obtaining, in one case, a 12.6% concentration, and in the remaining other case, one of 16.8%. With the aim of blinding the experimental procedure, the medicaments (in a 3-mL flask presentation with a spiral cover that contained a brush for administration on nails) were packed and labeled in identical form. 2.5. Subjects The clinical trial followed the guidelines of the Declaration of Helsinki and Tokyo for research in humans and was approved by the Institutional Ethics Committee. All patients received information concerning the study and were required to sign an informed consent letter to be included in the study. In this study, patients who were pregnant or breastfeeding, with diabetes mellitus or another serious disease, as well as patients who received treatment for this disease at least 1 month prior to the date of study inclusion, or with a history of sensitivity to topically administrated drugs, were not accepted for participation in the study. In the study, 122 patients with a diagnosis of mild or moderate onychomycosis aged 19–65 years were included. Based on a table with randomized numbers, two groups were organized: patients of group 1 were treated with the 12.6% Ageratina pichinchensis extract, and group 2 received treatment with the 16.8% Ageratina pichinchensis extract.
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A mycological diagnosis was performed by direct microscopy before and after treatment. For this purpose, a sample from the affected nails was obtained and direct microscopy was conducted for identifying fungi. At the end of treatment, a second sample was obtained from nails that maintained the pathology, as well as from nails exhibiting improvement or that were cured. All patients were followed clinically by means of monthly appointments at our Physician’s office or at the patient’s home, and comprised evaluation of clinical evolution, therapeutic effectiveness, and tolerability.
Table 2 Degree of severity of clinical manifestations in patients with onychomycosis before and after 6 months of treatment with two different concentrations (group 1 = 12.6%, group 2 = 16.8%) of the standardized extract of Ageratina pichinchensis. Treatment
2 p
Frequency/% Healthy
Mild
Moderate
Before Group 1 n = 62 Group 2 n = 60
0/0 0/0
13/20.9 18/30
49/79 42/70
0.25
After Group 1 n = 55 Group 2 n = 48
37/67.2 38/79.1
3/5.4 7/14.5
15/27.2 3/6.3
0.010
2.6. Statistical analysis Central tendency measurements and dispersion were used for analyzing study results. ANOVA and 2 tests were employed to identify differences between groups. p < 0.05 was considered significant. 3. Results 3.1. Demographic aspects Women predominated in the study with 77% (94), age of patients in the entire sample ranged between 19 and 65 years, and median age was 47 years. Distribution of these variables between both groups was similar, and no significant differences were detected. 3.2. Clinical evolution In all patients included in the study, the clinical disease form was distal onychomycosis, and of primary origin. There were 62 patients in group 1, and in terms of degree of severity, 79% (49) were classified as having moderate onychomycosis. In group 2, 60 patients were included; of these, 70% (42) were classified as suffering from moderate onychomycosis (p = 0.25). With respect to the number of affected toenails, in both groups there were patients with 1–10 affected toenails. The number of toenails affected in both groups at the beginning and at the end of treatment is shown in Table 1. Predominating signs among patients were hyperqueratosis, opacity, and modification in toenail color. The proportion of hyperqueratosis in patients included in group 1 was 98.3% (61), while in group 2, this was 96.6% (58). In terms of opacity and toenail color changes, the proportion was simi-
Table 1 Frequency and percentage of the number of affected toenails with mild and moderate onychomycosis before and after receiving treatment with two different concentrations of the standardized extract of Ageratina pichinchensis. Number of toenails affected
0 1 2 3 4 5 6 7 8 9 10
Ageratina pichinchensis extract 12.6%
Ageratina pichinchensis extract 16.8%
Number of patients Before/after 62/55 Frequency (%)
Number of patients Before/after 60/48 Frequency (%)
0 (0)/37 (67.2) 7 (11.2)/1 (1.9) 5 (8)/1 (1.9) 6 (9.6)/3 (5.4) 16 (25.8)/3 (5.4) 5 (8.4)/1 (1.9) 3 (4.8)/2 (3.6) 3 (4.8)/3 (5.4) 6 (9.6)/3 (5.4) 7 (11.2)/0 (0) 4 (6.4)/1 (1.9)
0 (0)/38 (79.1) 5 (8.3)/1 (2) 5 (8.3)/0 (0) 4 (6.7)/0 (0) 10 (16.6)/2 (4.3) 9 (15)/3 (6.2) 12 (20)/2 (4.3) 4 (6.6)/0 (0) 3 (5)/1 (2) 4 (6.6)/1 (2) 4 (6.6)/0 (0)
2 p
lar to that of hyperqueratosis, with a slight variation in group 2, in which toenail color modification was observed in 59 patients (98%). After 6 months of treatment, the total number of patients was reduced to 103 (84.4%): 55 in group 1 and 48 in group 2, demonstrating a patient withdrawal of 15.6% from the study. Therapeutic effectiveness was determined once the treatment was concluded: 67.2% (37) of patients included in group 1 and 79.1% (38) of patients from group 2 presented no clinical manifestation in the toenails and were considered as healthy (Table 2). Fig. 2 shows the clinical evolution of a patient treated with the 16.8% Ageratina pichinchensis extract. Regarding clinical evolution of patients under treatment (Table 2), at the end of the treatment there was evidence that 27.2% of patients treated with the 12.6% Ageratina pichinchensis extract showed a moderate degree of severity, while only 6.3% of patients treated with the 16.8% Ageratina pichinchensis extract experienced this condition. Results analysis considered therapeutic improvement when the patient exhibited a reduction in the number of toenails affected. In this analysis, we included patients who did not achieve the healthy stage, but who did have a reduced number of toenails. In this manner, the proportion of patients with therapeutic improvement among individuals treated with the 12.6% Ageratina pichinchensis extract was 74.5% (41 patients), while patients treated with the higher concentration (16.8%) showed a therapeutic improvement of 89.5% (43 patients), demonstrating a significantly (p = 0.048) better evolution in patients treated with the more concentrated product. After 6 months of treatment administration, there were 19 (18.44%) patients without improvement; nevertheless, after analyzing the cases, it was observed that the cause of the treatment failure was due (in 11 patients, 10.6%) to lack of compliance with the treatment, while the remainder (8 patients, 7.7%) were individuals who demonstrated a moderate degree of severity at the beginning of the study. Direct examination by microscopy demonstrated that all patients without signs and symptoms at the end of the study were negative for the presence of fungus. Both treatments were tolerated satisfactorily because the total number of patients included in both groups manifested no side effects during the study (p = 1). 4. Discussion
0.37
The phytopharmaceutical developed (from the plant species Ageratina pichinchensis) is elaborated with the standardized extract of the aerial parts of the plant with a known concentration of the active compound (encecalin). This compound is active against the dermatophytes most frequently found in tinea ungueum and tinea pedis. The phytopharmaceutical elaborated with the Ageratina pichinchensis extract constitutes an innovative medical alternative that, by means of double-blind clinical trials and compared with
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Fig. 2. Photography that shows the clinical evolution of a patient before and after being treated topically for 6 months with a nail lacquer containing 16.8% Ageratina pichinchensis extract.
ciclopirox, a leading drug on the market (Gupta, 2000), has demonstrated its effectiveness, tolerability, and safety in patients with onychomycosis (Romero-Cerecero et al., 2008). Once the effectiveness of the phytopharmaceutical was confirmed, it was necessary to continue with the development of this study, which allowed to identify extract concentration that offers greatest effectiveness for topical treatment of mild to moderate onychomycosis, in addition to maintaining good tolerability and offering the patient a therapeutic resource that avoids evolution of the disease that could produce nail dystrophy and, consequently, affect the patient’s quality of life. Regarding the severity of clinical manifestations, we observed no differences (in the number of patients with mild and moderate onychomycosis) between both groups of patients prior to treatment initiation. Nevertheless, after 6 months of treatment with the phytopharmaceuticals, despite that the number of patients of both groups who achieved a healthy condition was similar, an important difference was observed in patients who were found at a moderate stage of severity (15 and 3 patients, for 12.6 and 16.8%, respectively). This result supports that higher concentration (16.8%) of the plant extract possesses higher effectiveness. When the number of affected toenails was analyzed (therapeutic improvement), it was also observed that the higher concentration reached higher rates of therapeutic improvement. In this study, 15.6% of patient withdrawal was observed; all cases were due to non-medical causes. This percentage could be considered normal in a study consisting of 6 months of tracking, in which changes in different personal aspects could occur (in workplace or place of residence, etc.). No side effects were exhibited by patients throughout the study period, resulting in 100% tolerability for both treatments. Considering as therapeutic success when the patient achieved (a) therapeutic effectiveness, (b) absence of fungus with direct microscopy examination, and (c) tolerability, the therapeutic success value was 67.2 and 79.1% for the 12.6 and the 16.8% extract, respectively. Previously, Romero-Cerecero et al. (2008) reported the evaluation of a similar product elaborated in a lacquer solution containing the standardized extract of Ageratina pichinchensis, but in this case, with an extract concentration of 10%. This product, also administered topically for 6 months, showed a therapeutic success of 55.1%. This 10% formulation was compared with a similar product containing 8% ciclopirox, which produced a 63.8% therapeutic success rate without evidencing significant differences. It is noteworthy that the concentration of the active compound contained in the ciclopirox nail lacquer (8%) could be considered lower than that used with the plant extract of the experimental treatment (12.6 and 16.8%). However, when the concentration of the active compound contained in the experimental treatments is considered, both phytopharmaceuticals contain considerably lower concentrations (1.81 and 2.41% of encecalin in the 12.6 and 16.8% concentrations of Ageratina pichinchensis extract, respectively).
5. Conclusion It is possible to conclude that both concentrations (those of 12.6 and 16.8%) of the Ageratina pichinchensis extract formulated in a nail lacquer presentation and administered topically on the nails of patients with mild and moderate onychomycosis were therapeutically effective, while the higher concentration (16.8%) offers a statistically higher effectiveness. Both treatments exhibited 100% tolerability. Acknowledgment This study was financially supported by a grant (CONACyTSALUD/2007CO1/071029) from CONACyT-México. References Argueta, A., Cano, L., Rodarte, M., 1994. Atlas de La Medicina Tradicional Mexicana, Tomo 1-3. Instituto Nacional Indigenista, México, D.F, p. 1786. Avilés, M., Suárez, G., 1994. Catálogo de Plantas Medicinales Jardín Etnobotánico. Centro INAH, Cuernavaca, Morelos, México, p. 47. Casciano, J., Amaya, K., Doyle, J., Arikian, S., Shear, N., Haspel, M., Kahler, K., 2003. Economic analysis of oral and topical therapies for onychomycosis of the toenails and fingernails. Managed Care 12, 47–54. Gupta, A.K., 2000. Pharmacoeconomic analysis of ciclopirox nail lacquer solution 8% and the new oral antifungal agents used to treat dermatophyte toe onychomycosis in the United States. Journal of the American Academy of Dermatology 43, S81–S95. Gupta, A.K., Cooper, E.A., Ryder, J.E., Nicol, K.A., Chow, M., Chaudhry, M.M., 2004a. Optimal management of fungal infections of the skin, hair, and nails. American Journal of Clinical Dermatology 5, 225–237. Gupta, A.K., Cooper, E.A., 2008. Update in antifungal therapy of dermatophytosis. Mycopathologia 166, 353–367. Gupta, A.K., Ryder, J.E., Chow, M., Cooper, E.A., 2005. Dermatophytosis: the management of fungal infections. Skinmed 4, 305–310. Gupta, A.K., Ryder, J.E., Johnson, A.M., 2004b. Cumulative meta-analysis of systemic antifungal agents for the treatment of onychomycosis. The British Journal of Dermatology 150, 537–544. Gupta, A.K., Poulin, Y., Lynde, C.W., 2006. Canadian perspectives on antifungal treatment for onychomycosis. Journal of Cutaneous Medicine and Surgery 10 (Suppl. 2), S34–S38. Lecha, M., Effendy, I., Feuilhade de Chauvin, M., Di Chiacchio, N., Baran, R., 2005. Taskforce on Onychomycosis Education. Treatment options—development of consensus guidelines. Journal of the European Academy of Dermatology and Venereology 19 (Suppl. 1), 25–33. ˜ P., 2000. Plantas medicinales utilizadas en MoreMonroy-Ortiz, C., Castillo-Espana, los. Centro de Investigaciones Biológicas, Universidad Autónoma del Estado de Morelos, México, pp. 61–62. Murdan, S., 2008. Enhancing the nail permeability of topically applied drugs. Expert Opinion on Drug Delivery 5, 1267–1282. Navarro, V.M., García, A., González, M., Fuentes, A., Avilés, M., Ríos, Y., Zepeda, G., Rojas, M.G., 2003. Antifungal activities of nine traditional Mexican medicinal plants. Journal of Ethnopharmacology 87, 85–88. Nunley, K.S., Cornelius, L., 2008. Current management of onychomycosis. The Journal of Hand Surgery 33, 1211–1214. Poulin, Y., Thomas, R., Gupta, A.K., 2006. Brief treatment guide for onychomycosis. Journal of Cutaneous Medicine and Surgery 10 (Suppl. 2), S39–S43. Rigopoulos, D., Katoulis, A.C., Ioannides, D., Georgala, S., Kalogeromitros, D., Bolbasis, I., Karistinou, A., Christofidou, E., Polydorou, D., Balkou, P., Fragouli, E., Katsambas, A.D., 2003. A randomized trial of amorolfine 5% solution nail lacquer in association with itraconazole pulse therapy compared with itraconazole alone
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