International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 2(12), 5-10, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 5 Comparison of the Therapeutic effects of Nano-essence of Medical herb Artemisia sieberi with the ointment of Ketoconazole in guinea pig infected by Microsporum canis Mashhady Rafie Siamak, Baradaran alizadeh Saed1* and Bayat MansourDepartment of Clinical Sciences, Science and Research Branch, Islamic Azad University, Tehran, IRAN Department of Pathobiology, Science and Research Branch, Islamic Azad University, Tehran, IRAN Available online at: www.isca.in, www.isca.me Received 7th July 2013, revised 13th August 2013, accepted 20th September 2013Abstract Essences have well known properties. Transforming a drug to nano sized particles usually adds some additional potency to that drug. In this study we used Artemisia sieberi nano-essence to treat Microsporum canis induced dermatophytosis in guinea pig model. In vivo and In vitro methods were used to investigate the antifungal properties of nano-essence. Minimum inhibitory concentration of nano-essence was 0.3% to 2%. Treatment started 5 days after infection as 12 hours regimen until 45 days post infection. Both Ketoconazole and nano-essence groups had complete cure at day 40. Results show that this nano-essence is effective drug to treat M.canis induced dermatophytosis. Keywords: Nano-essence, Artemisia sieberi, Ketoconazole, Microsporum canis, guinea-pig. Introduction Dermatophytosis is a dermatologic problem with zoonotic risk. On the basis of primary habits there are three classes of dermatophytes, geophilic, zoophilic and anthropophilic. Dermatophytosis has word wide distribution and the causative agents are Microsporum, Trichophyton and epidermaphyton spp. Microsporum canis(M.canis) is the most common cause of dermatophytosis in animals and human1,2,3. Every confirmed case of dermatophytosis should receive topical therapy. Since there is a public health problem, an aggressive therapy should be designed to treat patients3,4. Traditional topical antifungal drugs can treat dermatophytosis but there is no advantage one product over another. Besides they have variable side effects and fungal resistance is becoming common. Ketoconazole is one of these drugs. It has moderate degree of success against M.canis with systemic use. Clinicians use topical Ketoconazole to treat small lesions6,7. Nano drugs are novel therapeutic agents. Their properties improved after nano encapsulation. Chitosan is the main agent in this process and it adds many properties such as mucoadhesiveness, absorption enhancing and sustained-release characteristics to nano sized drugs8-12. Artemisia sieberi (A.sieberi) is an ancient medical herb that grows in China, Russia and desert part of Iran. Locally known as “Dermaneh”, has antifungal, vermicidal, antibacterial and anticandidal effects. Although the chemical composition of this herb varies according to growing place but main components of that is generally the same for all species those are -thujone and -thujone13-25. In present study we decided to use A.sieberi nano-essence to treat M.canis induced experimental dermatophytosis. Material and Methods Animals: In this study, 24 male Guinea pigs with the same weight (ranging from 350-400 grams) were obtained from Pasture institute (Tehran, Iran). All animals were put in separate polycarbonate cages in controlled condition (12 hours light period, relative humidity of 50±3% and temperature: 24±1°C). Animals were put in optimized condition and fed with basic diet for 1 week. Drugs: A.sieberi essence was purchased from Barij Essence Pharmaceutical Company, (Kashan,Iran) and Nano encapsulation was done by Zist Shimi Azma Roshd Company, (Tehran, Iran). 5 cc of A.sieberi essence is sufficient to produce 1liter of nano-essence. The product’s reliability was confirmed by Fourier Transform Infrared Spectrometer and screening electron microscopy (figure-1 and figure-2). Ketoconazole topical cream that was utilized in this study was purchased from Iran Najo pharmaceutical Company (Tehran, Iran). Test organism:M.canis standard isolate (PTCC: 5069) and 4 field isolates was used to measure minimum inhibitory concentration (MIC) and infection was induced by standard isolate. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(12), 5-10, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 6 MIC determination: Microdilution broth, using Clinical:Laboratory Standards Institute (CLSI) M38-A protocol was used for MIC determination in vitro. Using RPMI1640 medium, a 0.5-5x10 cells/ml suspension was gained as described before5,26,27. Figure-1 Loaded screening electron microscopy (SEM) Figure-2 Loaded Fourier Transform Infrared Spectrometer (FTIR) Animal infection: Posterior dorsal portion of every animal was shaved gently for as wide as 4 cm. Shaved area was abraded with the back of sterile scalpel blade. Suspension containing 10M.canis spores per milliliter inoculated to abraded site. The entire area was occluded with Vaseline® in order to keep the area closed just for 24 hours. Animals divided to 4 groups randomly, positive control, negative control, nano-essence (according to MIC) and Ketoconazole treatment group. All animals except negative control group were mycological positive at 5th day28,29,30. Treatment: According to statements in previous studies, 12 hours treatment regimen started at 5th day with nano-essence and Ketoconazole11. In 45 days course of treatment, nano-essence was applied with sprinkler so that drug covered all the shaved and unshaved area around it. In negative and positive control groups, saline were used as a placebo during treatment. Efficacy evaluation: Drug efficacy was evaluated by clinical lesion scoring and fungal culture. Modified lesion scoring is 5 degrees scoring system (0 to 4) indicated as: score 0, no visible lesion; score 1, only hair loss; score 2, well defined redness with few scales; score 3, well defined redness with large scale; and score 4, ulceration and scarring in addition to lesion 3. Scale and hair cultured at the day 30, 37 and 44 using scraping and plucking technique. Data analysis: Mann-Whitney -test (with SPSS for windows) was used to analyze lesion scores. Results and Discussion MIC: MIC ranges of A.sieberi nano-essence were 3 - 20 l/ml (0.3%-2%). Forasmuch as the concentration of essence used for drug production process, MIC of A.sieberi essence will range from 0.15 to 1 %. This numbers are much lesser than stated before14,21,25. For this study MIC was considered as 1%. Lesion scoring: Gross findings are shown in figure-3. All animals except negative control were mycological positive when the treatment started. Clinical lesion score average on day 5 (treatment start) for all groups were between 2 and 3. The clinical lesion score average in nano-essence treatment group was higher than positive control group when treatment had started (figure-4). Decrease in score average happens gradually until all treatment receiving groups reach score 0 on day 40 except positive control group. Nano-essence and positive control groups show significant statistical difference on day 20, 25, 30, 35 and 40 (0.05). This statistical difference is similar between Ketoconazole and positive control groups. Intra group assessments shows clinical score reduction over treatment period for animals in drug receiving and negative control groups except positive control group (Figure-5 and figure-6). Comparing Ketoconazole and nano-essence treatment groups on various dates shows significant difference just on 10thday (p0.05). Culture results: As it is shown on table-1, three consecutive culture results for all animals was negative on days 33, 40 and 47 in treatment groups and negative control group. Table-1 Number and percentage of culture positive animals in every group. PC,positive control;NC,negative control; nano, nanoessence; keto, ketoconazole Culture positive (%) Day 33 Day 40 Day 47 Group PC 6/6(100) 5/6(83.3%) 5/6(83.3) NC 0/6(100%) 0/6(0%) 0/6(0%) nano 0/6(0%) 0/6(0%) 0/6(0%) keto 0/6(0%) 0/6(0%) 0/6(0%) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(12), 5-10, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 7 Day5 Day 10 Day15 Day20 Day 25 Day30 Day35 Day40 PC NC Nano Keto Figure-3 Time manner gross findings in different groups infected with M.canis. PC, positive control; NC, negative control; Nano,nanoessence; Keto, Ketoconazole Figure-4 Clinical score average linear chart in different groups. Note the decrease in scores in day 20. PC, positive control; NC, negative control; Nano, nanoessence; Keto, Ketoconazole International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(12), 5-10, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 8 Figure-5 Ketoconazole intergroup clinical lesion score keto1-6 represents animal numbers, Note the reduction of scores from day 20 Figure-6 Nano-essence intergroup clinical lesion score. Nano1-6 represents animal numbers. Note the score at which most of animals were when treatment started and dramatic decrease at the day 20 Discussion: Different topical and systemic drugs have been used to treat dermatophytosis. From standpoint of clinical practice, topical medications are cornerstone of treatment. Various herbal extracts have been tested for their antifungal effects. In present study we decided to use A.sieberi nano-essence to treat M.canis induced dermatophytosis in guinea pigs. Microdilution broth, using CLSI M38-A protocol was used for MIC determination. This is well known and widely used method in mycology. Using this technique, nano-essence MIC was determined at the range of 0.3% to 2%. Chitosan and its properties were discussed in previous studies. In this study we decided to utilize nano-essence to add some additional properties to A.sieberi essence in treatment of M.canis induced dermatophytosis. Therapy started from 5th day after infection and lasted until day 40 after infection. All animals in day 40 were clinically cured except positive control group. Clinical score average in 5th day after infection in all groups was between 2 and 3 except negative control group. It should be noted that, there was a drastic decrease in clinical lesion score average between 10thand 15th day post infection in Ketoconazole treatment group that is accompanied by an increase at following 5 days while there was very little changes in nano-essence treatment group at these days. So that clinical score average in nano-essence treatment group at 25th day was lesser than that of Ketoconazole receiving group, although there was not any statistically significant difference. Reduction in clinical score averages continues until International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(12), 5-10, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 9 40th day after treatment in both treatment groups, although there is not any significant difference between nano-essence and Ketoconazol receiving groups. As it is shown on linear chart, there is a decreasing manner in clinical score from day 20 to day 40 after infection in positive control, Ketoconazole and nano-essence receiving groups but this reduction in nano-essence group is much more severe than these groups Because the clinical lesion scores average in nano-essence receiving animals were higher than that of animals in positive control group when treatment had started. This declining trend in clinical score average from 30th day post infection, is followed by 3 consecutive negative culture result in 100% of nano-essence and Ketoconazole receiving animals compared to 100% positive culture result in 33rd and 83.3% in 40th and 47th day in positive control group. Intra group clinical score assessments shows logical decrease over treatment period. Assessments in nano-essence group indicates that 33% of these animal were in score 4 when trial started but none of the animals in Ketoconazole receiving groups were in this level at the same day. None of the animals in Ketoconazole treatment groups reached score 4 during trial but 33% of animals in nano-essence group were in score 4 on day 20. These differences are expressed on clinical score average. Although there is a dramatic decrease between 20th and 25th day post infection in both treatment receiving groups, it should be considered that nano-essence receiving animals had higher score at the beginning of trial so their score reduction are contemplated more prominent. Generalization of results in animals and human patients needs further clinical trials. Conclusion It is concluded that nano-essence of A.sieberi could be a replacement for Ketoconazole ointment to treat dermatophytosis but generalization of results in animals and human patients needs further clinical trials. 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