Abstract:
The present invention refers to the application of extracts of  Cymbopogon nardus  in the preparation of pharmaceutical compositions with antifungal activity and to the process of obtaining the referred to extracts as well as their use as active component of the referred to compositions employed in the treatment of mycoses. 
     The invention is also with respect to the compositions containing the vegetal extract obtained from the aerial parts of a plant of the Graminae family with antifungal activity. Preferably the vegetal extract is obtained from the leaves of the plant. Even more preferably the plant used is  Cymbopogon nardus  (L.) Rendle.

Description:
[0001]    This application is a Continuation-in-Part of PCT International Application No. PCT/BR2008/000073 which has an International filing date of Mar. 14, 2008, which in turn claims priority under 35 U.S.C. 119(a)-(d) of Brazilian application PI0702622-6, filed Mar. 16, 2007. The entire contents of all applications listed above are hereby incorporated in their entirety by reference. 
     
    
     FIELD OF INVENTION 
       [0002]    The present invention refers to the application of extracts of  Cymbopogon nardus  in the preparation of pharmaceutical compositions with antifungal activity and in the process of obtaining the referred to extracts as well as their use as active component of the referred to compositions employed in the treatment of mycoses. 
         [0003]    The invention is also with respect to the compositions containing vegetal extract obtained from the aerial parts of a plant of the family of the Graminae with antifungal activity. Preferentially the vegetal extract is obtained from the leaves of the plant. Even more preferentially the plant employed is the  Cymbopogon nardus  (L.) Rendle. 
         [0004]    The invention is with respect also to the use of compositions containing vegetal extracts and/or isolated substances from the vegetal parts of the plant  Cymbopogon nardus  (L.) Rendle, for the treatment of mycoses (onicomycoses, dermatomycoses and candidiasis vulvovaginal). 
       STATE OF THE ART 
       [0005]    The dermatomycoses are most common affections that compromise the skin and attachments such as the nails of the hands and feet and hair that can affect the whole population. Although these disorders are not important in relation to the morbidity and mortality, they affect the quality of life of the patient. They have significant clinical consequences due to their infectious nature and, above all aesthetic prejudice which reflects in self-esteem, vanity and social discrimination. It is important to highlight the significant increase of the prevalence of these affections which seem to be a world tendency, the chronic nature and the therapeutic difficulty of these mycoses which are also aggravating factors. 
         [0006]    Classically the agents of the dermatomycoses are the dermatophytes: fungi belonging to the three  Trichophyton  spp species,  Microsporum  spp and  Epidermophytom floccosun . However, yeasts of the  Candida  spp species,  Trichosporon  spp and  Geotrichum  spp and filamentous fungi not dermatophytes like  Fusarium  spp,  Scitalidium  spp and  Scopulariopsis  spp have been diagnosed with certain frequency. 
         [0007]    The onicomycoses (OM) are most common affections that compromise the nails of the hands and feet in human beings and can affect everyone indistinctly. The OM have significant clinical consequences due to their infectious nature and above all aesthetic prejudice which reflects in self-esteem, vanity and social discrimination, being significant causes of medical consultations and even of missing work. They represent 20% of the illnesses of the nails and it is one of the most frequent causes of onicopathies in the whole world. The majority of the authors diagnose the dermatophytes as most frequent etiological agents, (80 to 90%), followed by yeasts (5 to 17) and finally non dermatophyte filamentous fungi (2 to 12%). 
         [0008]    In function of the fungi remaining restricted to the most external layer of the nail, some authors recommend topical treatment as first choice. The topical treatment is indicated in located infections and of little extension; in cases of failure, drugs of systemic administration must be prescribed. Nevertheless, the systemic treatment is more effective above all for the chronic infections. 
         [0009]    The treatment of onicomycoses continues to be a problem in spite of the undeniable advances in the development of new antifungal agents. 
         [0010]    Another infection of fungal origin of great relevance is candidiasis vulvovaginal (CVV) also known as vaginal moniliasis, which is an inflammation of the genital mucous which principally compromises the vulva and vagina. The etiological agent most common is  Candida albicans , a yeast that presents dimorphic characteristics and can be found in 20% of healthy and asymptomatic women. The principal symptoms that characterize CVV are itching and vulva-vaginal erythema, white and thick discharge with caseose aspect, burning in urination (disury), pain in sexual relations (disparunia), edema e fissures in the vulva region (Garcia &amp; Svidzinski, 2002). The diagnosis of this infection can be established by the characteristic symptoms, by the vaginal pH which in the range of normality is found between 4 and 4.5, by the aspect and odor of the secretion and by the identification of yeasts and hyphae in the microbiological examination (Daniel &amp; Robinson, 2005). 
         [0011]    It is amongst the principal gynecological problems that affect women in productive age reaching thousands of persons in the whole world, its prevalence seems to have increased in the last few years. It is estimated that close on 75% of the adult women present at least one episode of fungal vulva-vaginitis in their life being that of these, 40 to 50% experienced new outbreaks and 5% become recurrent (CVVR). 80 to 90% of the cases of CVV are due to the  Candida albicans  species and 10 to 20% to other species called  C. non - albicans  ( C. tropicalis, C. glabrata, C. krusei, C. parapsilosis ). However an increase in the frequency of isolation of yeasts non- C. albicans  in some populations has been observed. The major preoccupation resides in the fact that these other species in general tend to be more resistance to the antifungals. 
         [0012]    The resistance of the pathogens in face of the usual therapeutic agents has increased in the last years. Currently drugs are available such as Terbinafine and the azolic derivates effective in the classic treatment of the onicomycoses, however with excessive cost, turning the therapeutic choice and its success limiting. 
         [0013]    CVV is one of the most frequent diagnoses in the daily practice of gynecology and has increased in the last few years, however the treatment is still reason for worry of doctors and patients, principally in function of the symptoms and the recurrence. 
         [0014]    The treatment of CVV depends on variables relating to the agent, to the host and apparently to environmental factors. The therapeutics available include medicaments of topical use (nistatine and miconazol) and oral, principally the azolic derivates such as fluconazol, itraconazol and cetoconazol. 
         [0015]    In spite of the progress observed in the last decades in the development of new antifungals for the treatment of CVV, it still represents a significant problem. The best options available currently are limited and represent treatment of high cost to the patient. 
         [0016]    Trichomoniasis, also called as vaginitis by  Trichomonas vaginalis  is a disease sexually transmitted caused by a protozoan, unicelullar microscopic called  Trichomonas vaginalis . Infectious disease of the men genitourinary system and woman genitourinary tract. In the man it causes urethritis with demonstrations in general discreet (ardor and/or itch urethral and secretion white, yellowish or greenish yellow), being able to, eventually to be absentees in some and very intense in other. It is one of the main vaginitis causes or the adult woman&#39;s vulvovaginitis being able to however, to pass with little or any clinical demonstration. 
         [0017]    When present, is revealed in the woman as a yellow greenish or grayish vaginal discharge, foamy and with strong characteristic odor. It is not uncommon also to happen irritation in the genital region as well as symptoms miccionais that can simulate a cystitis (pain when urinating and frequent urinations), the symptoms can be worse during the menstrual period.  Trichomonas vaginalis  causes inflammation of the vagina, of the urethra and lap of the uterus, in pregnant women, the infections for the  Trichomonas vaginalis  can also increase the risk of premature rupture of the membranes and childbirth pre-term. 
         [0018]    The World Organization of the Health (OMS) esteems in 170 million the cases of trichomoniasis in the world annually in people between 15 and 49 years, with most happening (92%) in women. The impact of the trichomoniasis is not limited to vaginitises or urethritis, because other pathogenic microorganisms as micoplasmas and  Neisseria gonorrhoeae  are phagocyted for  Trichomonas vaginalis , and segments of viral RNA have been found in certain lineages of the parasite, therefore, it is possible, that this protozoan can also act as vector for other pathogens. 
         [0019]    In the chronic infection, the symptoms are light, with scarce vaginal secretion. That form is particularly important of the epidemic point of view, because those individuals are the largest source of transmission of the parasite. 
         [0020]    The treatment of the trichomoniasis is specific, the oral metronidazole or others derived nitroimidazoles (tinidazole, nimorazole, secnidazole, carnidazole). To prevent the reverse-infection, a person&#39;s sexual partner infected with  Trichomonas vaginalis  should be treated also. Although the infection for  T. vaginalis  is common among the pregnant, the metronidazole should not be used by pregnant women during the first quarter and there are no reports of use of natural products for treatment of the trichomoniasis. Besides, there are neonates&#39; reports with “vaginal discharge” infected by  T. vaginalis  and this microorganism has been observed in cultures of having aspirated tracheal in children with breathing illness as pneumonia. 
         [0021]    Innumerable are the researches done in search of compounds with biological activities from natural products. A considerable number of studies have been done to evaluate the evolution of anti-microbial activities in extracts and essential oils from medicinal plants. Many plants are resistant to different pathogens and this resistance can be related to the presence of fungistatic compounds naturally produced. 
         [0022]    The search for therapeutic alternatives directed to the phytotherapies passes through the knowledge of the vegetal drug to be used, by the optimization of the extract, by the obtaining of pharmaceutical forms adequate for the treatment and with the necessary quality to obtain the efficiency in the treatment. Allied to these requirements there is also the need for validation of the analytical technique and standardizing of the vegetal extract. 
         [0023]    There exists an enormous diffusion and popularity of the therapies of vegetal origin in the whole world, which are, in general, much less onerous and are indicated as complementary in the health services. They can be the first choice for diverse affections before resorting to other more aggressive medicaments. 
         [0024]    The medicinal plants produce a variety of components with innumerable properties that can inhibit the growth of pathogens or kill them and for this they are considered optimum options for the development of new anti-microbial drugs. 
         [0025]    The species  Cymbopogon nardus  (L.) Rendle, popularly known as citronella, is a plant of the Graminae family, common in tropical or subtropical climates, it is original from Ceylon and south of India and its essential oil, rich in citronella, isopulegol and geraniol is popularly used as an insect repellent and disinfectant 
         [0026]    The repellent activity against insects of the  Cymbopogon nardus  has been widely disclosed and is associated to the presence of the essential oils: citronella and isopulegol. Recently the action of  Cymbopogon nardus  over the  Aedes aegypti  associated with D-trans-aletrine was proved, with mortality of the insect of close on 88.9% when the concentration of 0.1% of the volatile oil was used. 
         [0027]    Examples relating to the insecticide activity of  Cymbopogon nardus , separately or in associations of the state of the art can be seen in the Brazilian patent document PI9106328-0, which is with respect to an insect repellant in the form of a spray or lotion, containing turpeniol, citronella, extract of rodenol and geraniol, which are synergistically effective against ticks that transmit the Lyme disease as well as insects that sting and triatomas (Chagas insects). 
         [0028]    In the same way the PI9610350-7 refers to an insect repellant mixture that contains mentanodiol and at least two selected components of citronella, geraniol, terpineol and rodinol, useful for repelling mosquitoes and ticks. 
         [0029]    Also preliminary studies made with plants of the Graminae family have demonstrated important fungicidal activity over fungi coming from diverse types of infections in human beings. 
         [0030]    The antifungal activity in vitro of the essential oil of  C. nardus  or of other species of the  Cymbopogon  genus over fungi of medical interest have been reported by various authors 
         [0031]    From such studies, however, there are no reports proving the identification of similar activity in the extract of  Cymbopogon nardus , as well as the existence of phytotherapeutic compositions as much as the use of them in the treatment of onychomycosis, candidiasis vulvovaginal and trichomoniasis. 
         [0032]    The use of such extracts would much facilitate the process of obtaining the phytotherapeutic pharmaceutical product, would significantly reduce the cost of the referred to product and would facilitate the incorporation of the active principle in different pharmaceutical forms. 
         [0033]    Thus it is that the search for new compounds biologically active obtained from natural products, principally of vegetal origin has become the object of great interest due to the need for new drugs effective in the combat of innumerable diseases. 
         [0034]    With relation to the anti-parasitic activity of citronella, the PI0106192-5 claims a phytotherapeutic product based on citronella oil which presents great effectiveness in the general therapy and prophylaxis for endo and ectoparasites which attack bovines, ovines, caprines and equines in general. 
         [0035]    With relation to the antimicrobial activity, PI9804814 is also known which describes compositions of oral hygiene that include an antimicrobial agent selected from: cedar oil, cloranfenicol, lemon grass oil, citronella oil, extract of  Glycyrrhizin glabra , basilicão fruit oil with juice, basilicão oil ( Ocimum  sp), of lemon and oil of  Rosmarinus officinalis.    
         [0036]    The PI0106903-9 refers to pharmaceutical compositions for the treatment of oral and vaginal candidiase covering 1 to 5% in weight of hydro-alcoholic, alcoholic and ethereal extracts and/or essential oils of the aerial parts of  Cymbopogon citratus  stapf (gramineae) separately or in mixtures of different proportions amongst themselves or with other natural or synthetic products (drugs, vitamins, salt, sugar, etc). The pharmaceutical preparations can be presented in the form of tinctures, emulsions A/O and O/A, creams, gels, aerosols, pastes, soaps, shampoos and similar for topical use in the treatment of infections caused by fungi and bacteria, especially oral and vaginal candidiase. 
         [0037]    The PI0203521-9 considers pharmaceutical compositions caused by  Candida  spp and dermatofite fungi, such as:  Epidermophyton floccusum, Microsporum canis  and  trichophyton rubrum , which contain an active pharmacological quantity of volatile oil of  Cymbopogon citratus  (lemon grass). In the said compositions the volatile oil was extracted from leaves of lemon grass for distillation by steam stripping, having as principal components citral (60 to 80′ v/v), mircene and geraniol. 
         [0038]    Patent JP7061918 reveals a cosmetic product containing at least an extract selected from  Vetiveria zizanoides, Hemidesmus indicus, Cymbopogon nardus, Piper longum, Piper chaba, Herpestris monnies, Cardiospermum halicacabum, Tinospora cordifolia, Desmodium gangeticum, Michelia champaca  and  Melaleuca leucadendroncom , with strong antioxidant action and capable of keeping the skin without cracks and with glow. 
         [0039]    One also sees that from the state of the technique, the use of volatile oil extracted from  Cymbopogon nardus  with repellant activity against insects, sanitizing and disinfecting products as well as about the species  Cymbopogon citratus  (lemon grass) is known, both about the antimicrobial pharmacological effectiveness against  Candida  spp and dermatophyte fungi. However none of the documents previously pointed out refer or suggest compositions containing extracts of the plant  Cymbopogon nardus  with activity against dermatophyte fungi and against  Candida albicans.    
         [0040]    Therefore, the state of the technique did not describe or suggest the use of extracts of  Cymbopogon nardus  as antifungal and trichomonicid. 
         [0041]    As one sees, the treatment of the onychomycosis, dermatomycosis and candidiasis continues to be a problem in spite of the undeniable advances achieved in the development of new antifungal agents. Therefore the use of products of vegetal origin with fungicidal properties can bring great benefits to the combat of onychomycosis, candidiasis vulvovaginal and dermatomycosis, which has still not been explored. 
         [0042]    Thus, as the  Cymbopogon nardus  is a plant that is also born and grows easily in all the regions of Brazil, without special requirements for cultivation, its extract can therefore be obtained with quite low production cost. Apart from this the obtaining of its extract shows good yield and its incorporation in pharmaceutical forms is quite viable, presenting facility of industrialization of the substitutes of the leaves and stalks of this plant. The biological and trichomonicid results of the present invention show high antifungal activity causing the death of the microorganisms tested (fungicide) and trichomonicid, important advantage over the majority of the antifungals available in the market which are in their great majority only fungistatics and, still, when they are fungicidal, are not effective on  Trichomonas vaginalis.    
         [0043]    In spite of the existence of compositions and medicaments obtained by extracts from plants reported in the literature, no mention or suggestion was seen as to the use or the phytotherapeutic compositions containing alcoholic and standardized extracts of  Cymbopogon nardus , as well as the process of extraction of them, with antifungal and trichomonicid activities. Such process of extraction, phytotherapeutic compositions and the use of them are found described and claimed here in the present application. 
       SUMMARY OF THE INVENTION 
       [0044]    The invention foresees a pharmaceutical composition adequate to treat onychomycosis and candidiasis vulvovaginal and trichomoniasis, which employs a pharmacologically active quantity of hydro-alcoholic and dry extracts of  Cymbopogon nardus , apart from pharmaceutically acceptable excipients. 
         [0045]    The present invention additionally covers a process of obtaining hydro-alcoholic extracts, dry extracts and standardized of a plant of the family of Gramineas, more particularly hydro-alcoholic and dry extracts of  Cymbopogon nardus.    
         [0046]    Also the present invention involves the use of a composition containing vegetal extract obtained from the aerial parts of a plant of the Gramínea family, more particularly  Cymbopogon nardus , with antifungal activity for onicomycosis, against dermatophyte fungi and candidiasis as well as with trichomonicid activity against trichomoniasis. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0047]    In order to better demonstrate the reach of the invention diverse illustrative figures of the activities and properties identified both in the plant used in the present invention— Cymbopogon nardus , and in the extract obtained from it are presented ahead, in which: 
           [0048]      FIG. 1  illustrates the distribution of the values of minimum inhibiting concentration (mg/ml) of extract of  C. nardus  for index of inhibition of 100% in isolates of dermatophytes and yeasts. 
           [0049]      FIG. 2  illustrates the minimum fungicidal concentration (mg/ml) of extract of  C. nardus  capable of killing 100% of the isolates of dermatophytes and yeasts. 
           [0050]      FIG. 3  illustrates the methodology of analysis of the antifungal activity of the  C. nardus.    
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0051]    The first aspect is that the invention deals with a pharmaceutical composition directed to the treatment of onychomycosis, dermatomycosis and candidiasis, containing hydro-alcoholic extracts of  Cymbopogon nardus.    
         [0052]    The pharmaceutical compositions of the present invention can contain hydro-alcoholic extracts of  Cymbopogon nardus  in concentrations varying from 5 to 40% (p/p). 
         [0053]    More preferably the pharmaceutical compositions can contain hydro-alcoholic extracts of  Cymbopogon nardus  in a range of concentration from 10 to 30% (p/p). 
         [0054]    Pharmaceutical excipients are used adequate for the pharmaceutical form chosen for each one of the affections to be treated. 
         [0055]    A liquid form of the pharmaceutical composition uses hydro-alcoholic solvents from C1 to C10 atoms of carbon, such as for example: ethanol and dipropylene glycol. The dipropylene glycol can be used in the proportion from 0.1 to 20 ml to each 10 mg of the dry extract, more preferably that each 1 ml contain 10 mg of  Cymbopogon nardus  dry extract. The ethanol is used diluted in water in the range from 20 to 96% (v/v), preferably used in the dilution of 50% (v/v) in water, and it can contain  Cymbopogon nardus  dry extract in the range of 0.1 to 30%, more preferably 1:10. 
         [0056]    Additionally the invention also deals with a process of hydro-alcoholic extracts of the aerial parts of a plant of the Graminae family, such as, preferably,  Cymbopogon nardus.    
         [0057]    The process of obtaining of extracts of  Cymbopogon nardus  starts from the process of vegetal extraction with the employment of physical procedures for the breaking down of the vegetal tissues in the presence of an organic solvent that will make the extraction of a liquid consisting of the gross vegetal extract of the aerial parts of a plant of the Graminae family. 
         [0058]    For the obtaining of the extract preferably are used the leaves of the plant  Cymbopogon nardus ; the physical procedures employed for the breaking down of the vegetal parts used for the turbo-extraction and the organic solvent used can be an alcohol containing between 2 and 6 atoms of carbon, such as ethanol, isopropanol, butanol and pentanol. Even more preferably the turbo-extraction is the physical process chosen and the organic solvent is ethanol. The ethanol can be used in different dilutions however the alcohol is preferably used at 50, 70 and 94.6% (weight/weight). 
         [0059]    The gross vegetal extract is then concentrated by the partial removal of the organic solvent. The concentration of the extract occurs by evaporation under controlled temperature and pressure conditions, in a temperature range of 35-55° C. and under reduced pressure (vacuum). 
         [0060]    Preferably a rotating evaporator can be used under sufficient temperature to generate the evaporation of the organic solvent used. The gross vegetal concentrated extract of the leaves was then lyophilized. The following stage consists of the evaluation as to the technological parameters and fungicidal and trichomonicid therapeutic activities. 
         [0061]    The evaluation of the antifungal activity in vitro of the extracts of  C. nardus  over agents of onychomycosis was determined through the determination of the Minimum Inhibiting Concentration (CIM) and of the Minimum Fungicidal Concentration (CFM). 
         [0062]    The Minimum Inhibiting Concentration (CIM) was determined by the method of micro-dilution in juice, following the norms of standardization proclaimed by the National Committee for Clinical Laboratory Standards published in document M-27A10 with some modifications. 
         [0063]    The test was done in sterilized plastic micro-plaques (Nunclon, Delta, Nunc A/S, Roskilde, Denmark) containing 96 wells organized in eight series identified from A to H, each one with twelve wells numbered from 1 to 12. Each line (A-H) corresponded to a fungal species which received 100 μl of the inoculate determined and each column received the extract of  C. nardus , diluted in a series way in the proportion of 2 in YNBG juice up to the dilution of 1/128. On each plaque negative, positive controls and a yeast of reference  Candida parapsilosis  (ATCC 22019) were included. The concentration of inoculate was adjusted in spectrophotometer (Baush&amp;Lomb) to correspond to the turbidity of the tube 0.5 of the Mac Farland scale in wavelength of 530 nm so that the volume of 100 ml of this suspension, added to each well of the plaque contained between 0.5 and 2.5×103 UFC/ml. The plaques thus assembled were incubated in a sterilizer at 35° C. for 48 h with daily monitoring, in the case of yeasts and incubated at ambient temperature for seven days in the case of the dermatophytes. After the adequate incubation time for each fungus, reading of the test was taken through visual comparison by reflection in mirror. 
         [0064]    The CIM was considered the least concentration of the liophilized extract of  C. nardus  capable of inhibiting 100% the growth of each fungal isolate, having as reference its respective positive control ( FIG. 3 ). For the determination of the CFM, aliquots of the cultivations which did not present growth in the test for determination of the CIM, were transferred to a Sabouraud Dextrose Agar medium against the medium exempt of drug. The least concentration that prevented the growth of the fungi was considered the CFM. 
         [0065]    The present invention also provides the use of pharmaceutical compositions containing hydro-alcoholic extracts of  Cymbopogon nardus  for the inhibition of the growth or death of dermatophyte fungi such as:  Trichophyton  spp,  Microsporum  spp and  Epidermophytom floccosun , as well as affections caused by yeasts such as Candidas, and conditions caused by protozoa, just as  Trichomonas vaginalis.    
         [0000]    Determination of the Minimum Inhibitory Concentration (CIM) for  Trichomonas vaginalis.    
         [0066]    They were prepared previous cultures of  Trichomonas vaginalis  with growth of 48 h in TYM medium (Trypticase-Yeast extract-maltose). After the growth, the tubes were centrifuged for the separation of the protozoa of the culture medium. With Trichomonas, the inoculant was prepared, containing from 1.0×10 5  to 5.0×10 5  trophozoites/ml, counted with aid of Neubauer Chamber. 
         [0067]    The CIM was determined by the method of micro-dilution in juice using a liquid nutrient medium, 1.5 ml of medium was distributed in 11 hemolysis tubes. A first tube didn&#39;t receive culture medium. In the sequence, it was put in the first and second tubes, 1.5 ml of the  Cymbopogon nardus  extract and, starting from the second tube, they were made serial dilutions until the tenth tube. After the preparation of the dilutions 1.5 ml of  Trichomonas vaginalis  inoculant already standardized was added. The tubes were incubated to 35° C. by 24 h. 
         [0068]    After the incubation, brackets of the liquid were removed and accomplished the count of the alive trophozoites in the Neubauer Chamber. CIM was considered as, the dilution in that didn&#39;t remain trophozoites alive. The eleventh tube was the negative control because it only received culture medium. The twelfth was the positive control because didn&#39;t receive the extract, just the culture medium plus the inoculant. 
         [0069]    For the  Trichomonas vaginalis , CIM was considered the smallest  C. nardus  lyophilized extract concentration capable to inhibit 100% the growth of each isolated of the parasite, tends as reference its respective positive control. 
         [0070]    The pharmaceutical preparation can be presented in form of tincture, lotions, gels, ointments, creams, vaginal ovals and tablets for topical use; or tincture, granulates, capsules and tablets for oral use. 
         [0071]    For better comprehension of the objects claimed, illustrative examples follow ahead which must not be considered delimiting of the rights of the applicant. 
         [0072]    Example of one pharmaceutical composition preferred for each one of the fungal affections affected by the invention can contain at least a quantity of 1250 μg/ml of alcoholic extracts of  Cymbopogon nardus  for yeasts of onychomycosis; at least 625 μg/ml of alcoholic extracts of  Cymbopogon nardus  for dermatofites and at least 625 μg/ml of alcoholic extracts of  Cymbopogon nardus  to inhibit vaginal yeasts ( Candida  albicans) and at least 625 μg/ml of  Cymbopogon nardus  dry extract to inhibit vaginal protozoa ( Trichomonas vaginalis ), apart from excipients and/or diluents, eluents and other acceptable pharmaceutical adjuvants. 
       Obtaining of the Extracts Cymbopogon Nardus 
     Example 1 A 
     Collecting of the Botanical Material to Start 
       [0073]    The leaves of  C. nardus  were collected for use as start material of the extracts to be used in the compositions of the present invention, in the medicinal plant nursery of the State University of Maringá (UEM), during the month of May of 2005. The plant was identified by botanical analysis and confirmed by gaseous chromatography coupled to the mass spectrometry. An exsiccate of the species was deposited in the herbarium of UEM under no. 11747. 
       Example 1 B 
     Characterization of Technological Parameters for the Vegetal Start Material 
       [0074]    Technological parameters were established for the vegetal material  Cymbopogon nardus  (n=3) such as: loss by drying (PS), loss by desiccation (PD), tenor of total flavinoids (FT), tenor of total Polyphenols (PT) and tenor of volatile oils (OV). The results are contained in Table 1. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Values obtained of total flavinoids (FT), total Polyphenols 
               
               
                 (PT), Volatile oils (OV) loss by dessication (PD) and Loss by 
               
               
                 Drying (PS) for the start material  C. nardus  (n = 3). 
               
             
          
           
               
                   
                 Parameters 
                 Average 
                 s 
                 CV % 
               
               
                   
                   
               
             
          
           
               
                   
                 FT (%) 
                 0.30 
                 0.0092 
                 3.10 
               
               
                   
                 PT (%) 
                 2.35 
                 0.12659 
                 5.38 
               
               
                   
                 PD (%) 
                 10.57 
                 0.2591 
                 2.45 
               
               
                   
                 OV (%) 
                 1.82 
                 0.4531 
                 24.85 
               
               
                   
                 PS (%) 
                 30.59 
                 0.1202 
                 0.39 
               
               
                   
                   
               
             
          
         
       
     
       Example 1.C 
     Preparation of the Extracts 
       [0075]    The fresh leaves of  C. nardus  were cleaned with compressed air, cut in small pieces and submitted to the turbo-extraction for 15 minutes with 70% (p/p) alcohol in the proportion of 20% (p/p) in relation to the dry extract of  Cymbopogon nardus  (n=3). The extract was filtered, concentrated in a rotary evaporator and later lyophylized. Technological parameters were established in these extracts before the drying, such as: dry residual (RS) and p pH and after drying, the tenors of total flavinoids (FT), total Polyphenols (PT), and antifungal activity were obtained. The results are contained in Table 2. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Values of pH, dry residual (RS), total Flavinoids (FT) 
               
               
                 and total Polyphenols obtained (PT) from the extracts of 
               
               
                   C. nardus , dry leaves (n = 3). 
               
             
          
           
               
                   
                 Parameters 
                 Average 
                 s 
                 CV % 
               
               
                   
                   
               
             
          
           
               
                   
                 FT (%) 
                 0.85 
                 0.0260 
                 3.04 
               
               
                   
                 PT (%) 
                 10.87 
                 0.2082 
                 1.92 
               
               
                   
                 RS (%) 
                 1.98 
                 0.0299 
                 1.58 
               
               
                   
                 pH 
                 6.02 
                 0.0635 
                 1.06 
               
               
                   
                   
               
             
          
         
       
     
       Example 2 
     Analysis for the Determination of the Biological Activity 
       [0076]    For the determination of the antifungal activity of the extract of  Cymbopogon nardus  in face of the different fungal isolates of clinical origin, the detailed methodology ahead was used. 
         [0077]    The fungi were reactivated in Sabouraud Dextrose Agar (SDA) culture medium for 24/48 h at 30° C., from this growth an inoculate was prepared in sterile saline, adjusting the cellular density by means of Bauch &amp; Lomb spectrophotometer in 530 nm with 90+/−2% of transmittance. This turvation resulted in 1.0 to 5.0×10 6  ufc/ml from which new dilutions were prepared in Yeast Nitrogen base glucose (YNBG) to obtain the final inoculate desired between 0.5 to 2.5×10 3  ufc/ml. 
         [0078]    The minimum inhibitory concentration (CIM) was determined by the method of micro-dilution in juice, following the norms of standardization proclaimed by the NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS (INCCLS, 1997) published in document M-27A. 
         [0079]    The test was done in sterilized plastic plaques (Nunclon, Delta, Nunc A/S, Roskilde, Denmark) containing 96 wells organized in eight series identified from A to H, each one with twelve wells numbered from 1 to 12. Each line (A-H) corresponded to a fungal isolate (100 μl of the inoculate determined) and each column received the extract of  C. nardus , diluted in a series way in the proportion of 2 in YNBG juice up to the dilution 1/1024 which corresponds to the final concentration of 9 μg/ml In each plaque negative and positive controls of the diluent and a yeast of reference  Candida  parapsilosis (ATCC 22019) were included. 
         [0080]    The plaques thus assembled were incubated in a sterilizer at 35° C. for 72 h with daily monitoring After 72 hs reading of the test was taken through visual comparison by reflection in mirror. The CIM was considered the least concentration of the dry extract of  C. nardus  capable of inhibiting 100% of the growth of each yeast, having its respective positive control as reference. For the determination of the minimum fungicidal concentration (CFM) of the  C. nardus  aliquots from the wells of the CIM were transferred, where growth was not observed, for comparison with the culture medium free from drug. The least dilution that impeded the growth of the yeasts was considered the CFM. 
         [0081]    For the analysis of the results of the minimum fungicidal inhibitory concentrations and minimum fungicidals (CIMs) and (CFMs) obtained through the method chosen were analyzed: 
         [0082]    a) variation of the values representing the limits: lower and upper of the CIMs and CFMs of the extract of  C. nardus , referent to the different species of yeasts tested. 
         [0083]    b) CIM 50  and CIM 90  defined as the minimum inhibitory concentration of the extract of  C. nardus  capable of inhibiting the growth of 50% and 90% of the samples tested respectively. 
         [0084]    c) CFM 50  and CFM 90  defined as the minimum fungicidal concentration of extract of  C. nardus  capable of preventing growth of 50% and 90% of the samples tested respectively. 
       Example 2.1 
     Facing the Yeasts in Onicomycoses 
       [0085]    19 yeasts isolated from the nails of the hands and the feet of ambulatory patients were used. The yeasts causers of onicomycoses cover the species:  Candida albicans, C. tropicalis, C. glabrata  and  C. parapsilosis.    
         [0086]    The results show that the minimum inhibitory concentration used in the ratio of 10 mg of the extract of  C. nardus  to 1 mg of diluent, varied from 0.6-1.25 mg/ml of the total liquid medium. The values of the CFM were identical to those of the CIM, presenting the same behavior in all the species, according to Table 3. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Variation of the Minimum inhibitory concentration 
               
               
                 (CIM) and Minimum fungicidal concentration (CFM) in 
               
               
                 mg/ml of the extract of  C. nardus  over Yeasts. 
               
             
          
           
               
                   
                   
                   
                   
                 CIM - 
                 CFM - 
               
               
                   
                   
                   
                   
                 Interval 
                 Interval 
               
               
                   
                 Yeasts 
                 % 
                 N 
                 mg/ml 
                 mg/ml 
               
               
                   
                   
               
             
          
           
               
                   
                 
                   C. albicans 
                 
                 42.1 
                 08 
                 0.6-1.25 
                 0.6-1.25 
               
               
                   
                 
                   C. glabrata 
                 
                 5.2 
                 01 
                 0.6 
                 0.6 
               
               
                   
                 
                   C. parapsilosis 
                 
                 31.6 
                 06 
                 0.6 
                 0.6 
               
               
                   
                 
                   C. tropicalis 
                 
                 21.1 
                 04 
                 1.25 
                 1.25 
               
               
                   
                 TOTAL 
                 100.0 
                 19 
               
               
                   
                   
               
             
          
         
       
     
       Example 2.2 
     In Face of the Dermatofite Fungi in Onicomycoses 
       [0087]    20 dermatophyte fungi isolated from the nails of the hands and feet of ambulatory patients were used. The fungi causers of onicomycoses cover the species:  Trichophyton mentagrophytes, T. tonsurans T. raubitscheki, Microsporum canis, M. gypseum, M. ferrugineum.    
         [0088]    The results contained in Table 4 show an antifungal potential from the extract of  C. nardus  for the dermatofites and the values of the CMIs oscillated between 0.075 and 0.6 mg/ml. The action of the extract showed itself to be fungicidal for all the dermatofites with the identical variation of the CMI, 0.075 to 0.6 mg/ml. The dermatofites most sensitive to the extract of  C. nardus  were the  M. canis  and the  T. tonsurans , with 0.075 mg/ml.  T. tonsurans  presented the greatest index of variation of CIM and CFM, with 0.075 and 0.6 mg/ml. The other dermatofites presented very little variation for CMI ( M. canis, M. Gypseum and T. mentagrophytes ) or no variation ( M. ferrugineum and T. raubistscheki ) 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Variation of the Minimum inhibitory concentration (CIM) 
               
               
                 and Minimum fungicidal concentration (CFM) in mg/ml of 
               
               
                 the extract of  C. nardus  over dermatofites 
               
             
          
           
               
                   
                   
                   
                 MIC - 
                 MFC - 
               
               
                   
                   
                   
                 Interval 
                 Interval 
               
               
                 Dermatophytes 
                 % 
                 N 
                 (mg/ML) 
                 (mg/ML) 
               
               
                   
               
             
          
           
               
                 
                   Mycrosporum canis 
                 
                 25 
                 05 
                 0.075-0.15 
                 0.075-0.3 
               
               
                 
                   Mycrosporum ferrugineum 
                 
                 5 
                 01 
                 0.15 
                  0.15 
               
               
                 
                   Mycrosporum gypseum 
                 
                 10 
                 02 
                 0.15-0.3 
                 0.3 
               
               
                 
                   Trichosporum mentagrophytes 
                 
                 20 
                 04 
                 0.15-0.3 
                  0.15-0.3 
               
               
                 
                   Trichosporum raubistscheki 
                 
                 15 
                 03 
                 0.3  
                 0.3 
               
               
                 
                   Trichosporum tonsurans 
                 
                 25 
                 05 
                 0.075-0.6  
                 0.075-0.6 
               
               
                 Total 
                 100 
                 20 
               
               
                   
               
             
          
         
       
     
       Example 2.3 
     In Face of Yeasts in Candidiase Vulvovaginal 
       [0089]    23 vulva-vaginal isolates of  Candida albicans  from ambulatory patients were used. 
         [0090]    The results of Table 5 show that the minimum inhibitory concentration of the extract of  C. nardus  varied from 0.018 to 0.62 mg/ml, according to the data contained in Table 6. The values of the CFM were identical to those of the CIM, presenting the same behavior for all the yeasts. 
         [0091]    Thus when we submit in diverse concentrations in face of the 19 yeasts from patients with onicomycose, 20 dermatofites and 23 vaginal yeasts and 1 standard sample (ATCC) to obtain the CIM and CFM of the extract, it was possible to note that the extract of  C. nardus  in the range of 0.018 to 1.25 mg/ml was capable of inhibiting the totality of fungi tested. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Variation of the Minimum inhibitory concentration (CIM) and 
               
               
                 Minimum fungicidal concentration (CFM) in mg/ml of the extract 
               
               
                 of  C. nardus  over vaginal isolates of  Candida albicans   
               
             
          
           
               
                   
                 Minimum inhibitory concentration of the 
               
               
                   
                 Extract of  C. nardus  (mg/ml) 
               
             
          
           
               
                 Fungus 
                 N 
                 5.0 
                 2.5 
                 1.25 
                 0.62 
                 0.31 
                 0.15 
                 0.075 
                 0.036 
                 0.018 
                 0.009 
               
               
                   
               
               
                 
                   C. albicans 
                 
                 23 
                   
                   
                   
                 08 
                 1 
                 3 
                 1 
                 7 
                 3 
                 + 
               
               
                   
               
             
          
         
       
     
         [0092]    According to the results of the biological activity these prove that the extract of  C. nardus  presents excellent performance in tests in vitro in face of isolated fungi of human clinical situations. And that this activity is not only fungistatic but also fungicidal, even in small concentrations and does not suffer alteration in function of the extractor liquid used. 
       Example 2.4 
     In Face of  Trichomonas vaginalis  Isolated of Vaginitis 
       [0093]    03 strains of the protozoan isolated were used from patients with vaginitis assisted in ambulatory services. 
         [0094]    The results show that the minimum inhibitory concentration employed in the ratio of 10 mg of the  C. nardus  extract for 1 ml of diluent, varied from 0.6-1.25 mg/ml of the liquid medium total. In agreement with the results of the biological activity, these prove that the  C. nardus  extract presents excellent performance in tests in vitro in face of isolated  Trichomonas vaginalis  of human clinical presentations. And that activity is trichomonicid, starting from 625 μg/ml, while smaller amounts varying from 10 μg/ml up to 320 μg/ml the extract provoked death of the protozoa being dependent dose, according to Table 6. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Determination of the minimum inhibitory concentration 
               
               
                 of the  C. nardus  extract capable to inhibit 
               
               
                 the growth of  Trichomonas vaginalis   
               
             
          
           
               
                 Tubes 
                 mg/ml 
                 Extract - strain CAR 
               
               
                   
               
             
          
           
               
                 1 
                 5.0 
                 — 
               
               
                 2 
                 2.5 
                 — 
               
               
                 3 
                 1.25 
                 — 
               
               
                 4 
                 0.625 
                 — 
               
             
          
           
               
                 5 
                 0.320 
                 5 × 10 3   
                 cel/ml 
               
               
                 6 
                 0.15 
                 7.75 × 10 4   
                 cel/ml 
               
               
                 7 
                 0.07 
                 5.25 × 10 4   
                 cel/ml 
               
               
                 8 
                 0.04 
                 9.25 × 10 4   
                 cel/ml 
               
               
                 9 
                 0.02 
                 1.275 × 10 5   
                 cel/ml 
               
               
                 10 
                 0.01 
                 2.275 × 10 5   
                 cel/ml 
               
               
                 C+ 
                   
                 2.575 × 10 5   
                 cel/ml 
               
               
                   
               
             
          
         
       
     
         [0095]    In accordance with the results of the biological activity, these prove that the  C. nardus  extract presents excellent performance in tests in vitro inface of isolated  Trichomonas vaginalis  from human clinical presentations. And that activity is trichomonicide, starting from 625 μg/ml, while smaller amounts varying from 10 μg/ml up to 320 μg/ml the extract provoked death of the protozoa being dose-dependent. 
       Example 3 
     Optimization of the Extraction 
       [0096]    Extracts were obtained using three dilutions of ethyl alcohol 50, 70 and 94.6% (p/p) and containing concentrations of  C. nardus  between 10, 20 and 30% (p/p). The extracts were filtered, concentrated in rotary evaporator and later lyophylized. They were submitted to the determination of the dry residual and antimicrobial evaluation for the optimization of the extraction. For the accompanying of the extractive process the following parameters were established: values of pH in aqueous solution 1%, organoleptic characteristics (color, odor and taste), dry residual, tenor of active substances and chromatography over thin layer (CCD). 
         [0097]    The results of dry residual are contained in Tables 7 and 8. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Values obtained from dry residual (RS) from the 
               
               
                 extracts of  C. nardus  (n = 12) fresh leaves 
               
             
          
           
               
                   
                 Condition 
                 Parameter 
               
             
          
           
               
                   
                 of Extraction 
                 RS (%) 
                 s 
                 CV % 
               
               
                   
                   
               
               
                   
                 Alcohol 50%-10% of 
                 1.40 
                 0.0251 
                 1.80 
               
               
                   
                 vegetal start material 
               
               
                   
                 Alcohol 70%-10% of 
                 1.32 
                 0.0623 
                 4.71 
               
               
                   
                 vegetal start material 
               
               
                   
                 Alcohol 94.6%-10% of 
                 1.13 
                 0.0335 
                 2.97 
               
               
                   
                 start material 
               
               
                   
                 Alcohol 50%-10% of 
                 0.58 
                 0.0208 
                 3.59 
               
               
                   
                 vegetal start material 
               
               
                   
                 Alcohol 50%-20% of 
                 1.05 
                 0.0392 
                 3.73 
               
               
                   
                 vegetal start material 
               
               
                   
                 Alcohol 50%-30% of 
                 1.59 
                 0.0707 
                 4.45 
               
               
                   
                 vegetal start material 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Values obtained of dry residual (RS) of the producer in 
               
               
                 comparison with the nursery of UEM (n = 9), dry leaves 
               
             
          
           
               
                   
                 Parameter 
                 RS 
                 s 
                 CV % 
               
               
                   
                   
               
               
                   
                 Nursery 
                 1.92 
                 0.0842 
                 4.39 
               
               
                   
                 Producer 
                 1.98 
                 0.0299 
                 1.58 
               
               
                   
                   
               
               
                   
                 Obs: S = standard deviation and CV % = coefficient of variance 
               
             
          
         
       
     
         [0098]    For proof of the best extract all were submitted to the evaluation of the antifungal activity. The same experiment was done varying fresh and dry leaves and between producers. The results are contained in Table 8. This process of optimization versus antifungal activity showed that the hydro-alcoholic extractor liquids in the dilutions 50, 70 and 94.6% (w/w) presented themselves as adequate in the obtaining of the extract of  C. nardus  and did not interfere in its anti-fungicidal activity, in vitro, in face of the isolated yeasts of the patients and the standard yeast (ATCC). The preferred concentrations of plant; the best response was between 10 and 20% (p/p). 
         [0000]                                      TABLE 9                   Variation of the Minimum inhibitory concentration (CIM) and Minimum       fungicidal concentration (CFM) in mg/ml of the extracts of  C. nardus         over yeasts ( C. Albicans  and  C. tropicalis )                    CIM -   CFM -               Interval   Interval       Extract   Yeasts   mg/ml   mg/ml               Alcohol 50% (p/p)   Genus   0.039-0.156   0.039-0.156       10%  C. nardus       Candida         Alcohol 50% (p/p)   Genus   0.312-0.156   0.312-0.156       20%  C. nardus       Candida         Alcohol 50% (p/p)   Genus   0.312-1.25    0.312-1.25        30%  C. nardus       Candida         Alcohol 50% (p/p)   Genus   0.62-1.25    0.6-1.25       10% vegetal     Candida         start material       Alcohol 70% (p/p)   Genus   0.62-1.25   0.62-1.25       10% vegetal start     Candida         material       Alcohol 94.6%   Genus   0.62-1.25   0.62-1.25       (p/p)     Candida         10% vegetal start       material                    
The examples presented above are not limitative of the technique and methodology used in the obtaining and preparation of the hydro-alcoholic and dry extracts of the present invention, employed as active antifungal agents in the preparation of pharmaceutical compositions that can be presented in the liquid, gel, ointment, cream, oval, capsules and tablet forms for oral or local use as well as for topical use, useful in the combat of onychomycosis, dermatomycosis and candidiasis and in the parasitoses as in the trichomoniasis.