EVALUATION OF ANTIFUNGAL ACTIVITIES OF SOME MEDICINAL PLANT EXTRACTS AND FRACTIONS AGAINST CLINICAL ISOLATES OF CANDIDA ALBICANS

ABSTRACT

The development of drug resistance by pathogenic yeast especially Candida albicans, has resulted in waste of financial resources globally. This study was aimed at evaluating the antifungal activities of some medicinal plant extracts and fractions against clinical isolates of Candida albicans. Four medicinal plants namely: Vernonia amygdalina, Azardirachta indica, Moringa oleifera, and Vitellaria paradoxa were used in this study. The leaves, stembark and root of each medicinal plants were successively extracted using reflux method to obtain crude extracts, the fractions were obtained by column and thin layer chromatography. Clinical samples used were obtained from high vaginal swab (25) and urine(5). All samples were cultured in Sabouraud Dextrose broth (SDB) and then on Sabouraud Dextrose Agar (SDA). Isolates were identified using standard microbiological and molecular techniques. Antifungal activities of the crude extracts and fractions were done by Agar Well Diffusion Method. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the extracts and fractions were also determined. Gas chromatography and mass spectrophotometer analysis (GCMS) were used to determine bioactive compounds in fractions. Acute and sub-acute toxicity studies were done using the method of Lorkes and Organization for Economic Corporation and Development (OECD) respectively. Antifungal activities of the crude extracts were assayed at varying concentrations of 40, 60, 80 and 100 mg/mL against eight strains of Candida albicans. The highest mean zone of inhibition (MZI) 21.00± 0.30 mm was observed  for  methanol  leaf  extract  of  Vernonia  amygdalina  against  strain  SC5314 (isolate B7). The lowest results for the MIC and MFC of the crude extracts were 6.25 and 25.0 mg/mL, respectively. The n-hexane, ethyl acetate and methanol crude extract of Vernonia  amygdalina  yielded  4,  6  and  6  fractions  respectively while  the  n-hexane, ethylacetate and methanol crude extracts of Azardirachta indica gave 7 fractions each. Antifungal activity of the fractions against the strains were determined at a concentration of 5 and 10 mg/mL. The highest mean zone of inhibition (MZI) for the fractions was 16.00±0.80 mm which was recorded for fraction (F4) of Vernonia amygdalina leaf of n- hexane extract against strain P37005. The lowest value for MIC and MFC results for the active  fractions   were   1.25   and   3.125   mg/mL,   respectively.   The  standard  drug (fluconazole 1mg/mL) used had a higher inhibitory activity that ranged from 33.66±1.00- 42.00±0.49 mm compared to the activity of the crude extracts and the fractions. Results of the GCMS revealed probable compounds that may have enhanced the activity of the extracts  and  fractions.  Acute  toxicity  profile  of  the  crude  extracts  of  Vernonia amygdalina and Azardirachta indica on experimental rats showed no mortality for all groups exposed to the ,extract and as well no group had signs of toxicity. The sub-acute toxicity results on haematological and kidney parameters showed for both extracts were within the control value. Although, liver parameters and lipid profile showed significant increase (P<0.05) especially at doses of 600 mg/kgbw. Haematological and biochemical parameters for the active fractions were all within the control. The crude extracts of Vernonia amygdalina and Azardirachta indica leaf were the two active plants parts that showed significant activity.

CHAPTER ONE

1.0      INTRODUCTION

1.1      Background to the Study

Plants are used as valuable sources of food and medicine for the prevention of illness and maintenance of human health. In Nigeria, many indigenous plants are widely consumed as food or home remedies especially in the treatment or management of common diseases. The importance of plants in medicine remains even of greater relevance with the current global shift to obtain drugs from plant sources, as a result of which attention has been given to the medicinal value of herbal remedies for safety, efficacy and economy (Glombitza et al., 1993). Medicinal plants contains bioactive constituents with high therapeutic potentials; which also enable them to function as antioxidant, anti- inflammatory, antimicrobial and antipyretic agent . They have been used as a source for centuries as an alternative remedy for treating human diseases (Abayomi et al., 2013). Medicinal plants such as Anchomanes difformis, Anisopus mannii Vernonia blumeoides and Pavetta crassipes etc are continually being utilized as therapeutic agents in formulations for treating diseases in the traditional ethnomedicinal system (Abayomi et al., 2013).

Medicinal plants are a source of great economic value in Africa continent and the world (Iwu, 1993; Sofowora, 2013). The ethnomedicinal resources of Africa remain largely unexploited. Medicinal plants have been used as natural chemotherapeutic agents that can be described as nature pharmacy for nearly 80% of people living in Africa (Tan et al., 2010). Nature has bestowed on us a very rich botanical wealth and a large number of diverse plants growing in different parts of the continent. In Nigeria, thousands of species are known to have medicinal value (Sofowora, 2013) and the use of different parts to cure specific ailments has been in vogue since ancient times (Sofowora, 2013). Herbal medicine still remain the mainstay of 75-80 % of the whole population in the developing countries. Plant based medicines have been used for decades especially in the rural areas to prevent or even eliminate diseases worldwide and have proven to be promising in their actions (Robinson, 2006). Herbal medicines otherwise called herbal drugs are generally of natural plant parts such as the leaves, stem, flowers, stem bark, seeds and bulb (Robinson, 2006).

Candida albicans is a dimorphic fungus that grows both as yeast and filamentous cells and one of the few species of Candida that cause the infection candidiasis in humans (Zadik et al., 2010). Candida albicans have emerged as important cause of morbidity and mortality in immunocompomised patients (e.g., AIDS, cancer chemotherapy, organ or bone marrow transplantation).  In  addition,  hospital-acquired  infections  by  Candida  albicans  have become a major cause of public health concern (Tartorano and Gerald, 2010). Candida albicans is a common member of the human gut flora and is detectable in the gastrointestinal tract in 40% of healthy adults (Martins et al., 2014).

It is usually a commensal organism, but can become pathogenic in immunocompromise individuals under a variety of conditions. (Martins  et al., 2014). Candidiasis is often observed in immunocompromised individuals, including HIV-infected patients. It commonly occurs on mucous membranes in the mouth or vagina but may affect a number of other regions of the body (Zadik et al., 2010).

In immunocompromised individuals, Candida infections in the esophagus occur more frequently than in healthy individuals and have a higher potential of becoming systemic, causing  a much more serious  condition,  a  fungemia  called  candidemia (Fidel,  2006; Pappas,  2006).  Symptoms  of  esophageal  candidiasis  include  difficulty  swallowing, painful swallowing, abdominal pain, nausea, and vomiting (Yamada and Alpers, 2009).

1.2      Statement of the Research Problem

The development of drug resistance by pathogenic yeast especially Candida albicans has resulted in wastage of financial resources globally (Moen et al., 2009). Candida albicans represent the most common fungal pathogens that affect humans and produce a wide spectrum of disease ranging from superficial mucocutaneous to invasive illnesses such as hepatosplenic candidiasis. More so, it is a serious public health challenge with increasing medical and economic importance due to the high mortality rates and increased cost of care (Lim et al., 2012). Several drugs commonly used for the treatment of these infections tend to have toxic effect on the body (Pfaller et al ., 2012). The abuse of these antimycotic drugs that include: Nystatin, Itraconazole and Amphotericin B during treatment (Pfaller et al., 2012) has resulted to the development of drug resistance hence a significant drop in their efficacies.

1.3      Justification for the Study

Medicinal plant have played a major role in the production of biological compounds some of which can be used in drug development (Martins et al., 2014). Inspite of the influence of modern technology and orthodox medicine, plant-based medicine have been used for decades especially in rural areas to prevent or even eliminates disease worldwide and have prove to be promising in their actions (Bonjor and Farrokhi, 2004). Efforts in this regard have focused on plant because of their history and the fact that they are cheap and a good portion of the world population particularly in developing countries rely on plants for the treatment of infectious and non-infectious disease (Martins et al., 2014). Some of this medicinal plant include Vernonia aqmygdalina , Azadirachta indica, Moringa oleifera and  Vitellaria  paradoxa.  They  have  also  being  reported  to  possess  antimicrobial properties which have aid their uses in the treatment of some ailment such as stomach disorder, fever symptoms, skin infection , cough and other respiratory disease (Martins et al., 2014).

The impact of these medicinal plants based and their relevance in treating disease could serve as a good source of antimycotic agent. The economic predicament of most developing countries like Nigeria necessitate the search for new herbal drugs that are safe, less toxic and can serve as an alternative source of medicine. Therefore the above reasonings are partly what prompted this investigation.

1.4   Aim and Objectives of the Study

The aim of this study was to  evaluate antifungal  activities of  Vernonia amygdalina, Azadirachta indica, Moringa oleifera, and Vitellaria paradoxa extracts and fractions against clinical isolates of Candida albicans. This was with a view to elucidating some of their pharmacologic roles as medicinal plants.

The objectives of the study were to:

i.         isolate and identify Candida albicans from clinical samples

ii.         screen quantitatively for phytochemical components of the leaf, stembark and roots of the selected medicinal plants

iii.        determine antifungal activities of the crude extracts and fractions of the plants

iv.        determine the minimum inhibitory/minimum fungicidal concentrations of the active crude extracts and fractions of the above mentioned plant components.

v.         identify and elucidate the most active fractions using GCMS.

vi.        evaluate the toxicological characteristics of the active plant extracts and fractions of the plants

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