CHAPTER -2

AIMS & OBJECTIVES

AIM OF THE STUDY

The aim of the study was to evaluate the antifungal activity of leaf extracts of nine tree

species selected based on their use in respiratory and other disorders in traditional systems of

medicine and then to isolate and characterize the bioactive compound from the most active

plant species.

OBJECTIVES

The present study was undertaken with the following objectives:

Screening of various extracts of plants to evaluate their in vitro antifungal activity.

Isolation and purification of antifungal molecule from plants having in vitro activity

against pathogenic fungi.

Evaluation of antifungal potential of isolated molecule.

Studies on toxicity of purified molecule.

Biochemical characterization of purified molecule.

CHAPTER -3

REVIEW OF LITERATURE......

There have been continuous efforts for the improvement of antifungal therapies over

the last 30 years, the phenomenon of antifungal resistance is still of major concern in clinical

practice. With increasing numbers of immune-compromised patients with malignancy,

hematologic disease, and HIV, as well as those receiving immunosuppressive drug regimens

for the management of organ transplantation or autoimmune inflammatory conditions, the

incidence of fungal infections has dramatically increased over recent years. Although

invasive fungal diseases are now more frequent than during the first half of the century, they

are still difficult to diagnose clinically. During the latter half of the century, particularly

during the past two decades, a number of different classes of antifungal agents have been

discovered. Although, since the discovery of amphotericin B, there has been much progress

in this field, there is still a critical need for new antifungal agents to treat life threatening

invasive mycoses. The differences between the human host cells and the fungal pathogen

cells, both of which are eukaryotic, and have same molecular processes; therefore, there is

always the risk that what is toxic to the fungal cells will be toxic to the host cells (Wilson et

al., 2002). That's why; there is a great demand for novel antifungals belonging to a wide

range of structural classes, selectively acting on new targets with fewer side effects.

Therefore, one approach might be the testing of traditionally medicinal plants for their

antifungal activities as potential sources for drug development.

The present review is a brief account of the research works of various scientific

groups about the conventional and new strategies currently undertaken to discover alternative

therapy targets and antifungals.

3.1. Fungal Infection: A Renewed Threat

There are about 300 fungal species which have been reported to be pathogenic to

humans (Hay, 2006). Disseminated fungal infections in the hospital settings are not rare any

more as approximately 200,000 cases are reported every year from developed countries

which are considered to be associated with a high mortality rate of upto more than 40 %. The

fatal infections are mainly due to species of Candida and Aspergillus. While, the

retrospective analysis of US mortality trends due to invasive mycotic diseases conducted by

McNeil et al (2001) showed a marked and steady increase in mortality due to mycoses other

than Candida and Aspergillus during the period from 1980 to 1997. Overall mortality due to

other-mycoses has been found to be increased by 329 % from 0.14 to 0.60 deaths per 100,000

people during the study period. Hence, fungi such as Fusarium, Pnemocystis, Zygomycetes

or Penicillium which were normally considered to be non pathogenic, are emerging as a

threat to public health.

Mycotic infections are classified broadly into three groups namely superficial,

subcutaneous and systemic infections. A brief description of different groups of these

infections has been given below:

3.1.1. Superficial Infections

Superficial fungal infections are among the most common skin diseases affecting

millions of people throughout the world. These infections, which occur in both healthy and

immunocompromised persons, are caused by dermatophytes, yeasts and nondermatophyte

molds. The common dermatophytes that cause superficial skin infections in humans include

Trichophyton, Epidermophyton and Microsporum species. The disorders caused by

dermatophytes are commonly known as tinea corporis, tinea pedis, tinea cruris, tinea

unguum and tinea capitis, etc (Tosanger and Crutchfield, 2004).

In infected individuals, the yeast usually occurs in moist, occluded sites e.g. under

breasts, axillae or between buttocks. The skin folds between fingers of hands and toes of feet

are other very common sites of infections (Mandell et al., 1994). The Candida albicans is

common yeast that has been found to cause superficial infections. Candidal skin infections

often present with erythema, cracking or maceration (Hay, 1996). Malassezia is yeast which

can cause superficial infections. Malassezia furfur (Pityrosporum orbiculare and

Pityrosporum ovale) are reported to cause pityrosporum folliculitis and pityriasis (tinea)

versicolor which is a chronic, asymptomatic condition with well marked depigmented scaling

patches found mainly on the trunk (Inamadar and Palit, 2003).

Although the skin and nail infections due to molds are less common, they often cause

indolent infections in healthy or immunocompromised individuals, especially the elderly

people. The soil contaminated with infective organisms such as Scopulariopsis brevicaulis,

Fusarium, Aspergillus, Alternaria, Acremonium, Scytalidinum dimidiatum (Hendersonula

toruloides), Scytalidinum hyalinum has been considered to be the important source of

infections (Escobar and Carmona-Fonseca, 2003). Molds may cause ophthalmologic diseases

such as keratitis. The keratitis caused due to infections with Fusarium, Aspergillus and

Alternaria species have been common problems in India and other parts of the world (Guarro

et al., 2003).

3.1.2. Subcutaneous Fungal Infections

There have been three general types of subcutaneous fungal infections:

chromoblastomycosis, mycetoma and sporotrichosis. All these infections appear to be caused

by traumatic inoculation of the etiological fungi in to the subcutaneous tissue.

Chromoblastomycosis has been subcutaneous mycoses characterized by verrucoid lesions of

the skin, usually of the lower extremities. Histological examination of lesions revealed

muriform cells with perpendicular septations or so-called "copper pennies" that are

characteristic of this infection (Sayal et al., 2002). The most common causes of

chromoblastomycosis are Fonsecaea pedrosoi, F. compacta, Cladosporium carionii and

Phialophora verrucosa. These infections generally remain limited to the subcutaneous tissue

with no involvement of bones, tendons or muscle cells. Many of the fungi causing mycetoma

are pigmented brown to black. The causes of mycetoma have been quite diverse but could be

classified as eumycotic and actinomycotic mycetomas. The most common causative agents of

eumycotic mycetoma and actinomycotic mycetoma in United States have been reported to be

Pseudallescheria boydii and Nocardia brasiliensis respectively (Mariat et al., 1977). These

organisms are known as dematiaceous (melanized) fungi. The melanin pigment is deposited

in the cell walls of these organisms. These fungi may produce a range of infections from

superficial to subcutaneous to deep (visceral) infection characterized by the presence of

dematiaceous hyphal and/or yeast-like cells in tissues (Rippon, 1988). Sporotrichosis has

been the third general class of subcutaneous mycoses. The main causative agent of

sporotrichosis has been the Sporothrix schenckii which necrotizes the subcutaneous tissues at

the site of traumatic inoculation and finally causes nodular lymphangitis (Tomimori-

Yamashita et al., 1998). The infection usually spreads along cutaneous lymphatic channels of

the extremities involved.

3.1.3. Systemic Fungal Infections

The systemic fungal infections can either be worldwide in distribution or restricted to

some geographic region of globe. Histoplasma capsulatum, Cryptococcus neoformans and

Aspergillus sp. are worldwide in distribution. However, some of the pathogenic fungi such as

Coccidioides and Paracoccidioides have been found in the American continent only.

Penicilliosis marneffei is so far known only from Southeast Asia. Blastomyces dermatitis has

been found in India, America, Africa and other Asian countries. It has been reported that

endemic zone of blastomycosis has extended during the last 15 years from American and

Africa to Asia.

Pathogenic fungal organisms might gain entry through lungs, gastrointestinal tract or

intravenous route to cause invasive infections. Immunocompromised patients generally

remain at risk of systemic diseases; however, healthy individuals also may develop invasive

infections. Moreover, invasive fungal pathogens can be divided into two general classes,

primary pathogens and opportunistic pathogens (Pisseri et al., 2009). Fungi in the former

class usually have an environmental reservoir and infect individuals who have either been

exposed to a large dose or who are immunologically naive to the fungus. Opportunistic

pathogens take advantage of debilitated or immunocompromised hosts to cause infection.

They may have an environmental reservoir e.g., Cryptococcus neoformans, Aspergillus

fumigatus or exist as commensals in healthy organisms e.g., Candida species (Morgan et al.,

2005).

The opportunistic mycoses constitute a large category of fungal diseases. There are

etiological agents, which are ordinarily incapable of causing diseases in healthy individuals

but turn potential pathogens in immunocompromised or debilitated patients. Species of

Candida and Aspergillus are classical examples of opportunistic fungi, which are reported

from world over being very common in India. The number of opportunistic fungal diseases is

very large and ever increasing. Opportunistic human fungal pathogens have become

increasingly important over the past 20 years, paradoxically because the success of modern

medical practice has led to the survival of debilitated and immunosuppressed patients (Hay,

2006). Such patients are highly susceptible to infections by opportunistic pathogens such as

Candida species, C. neoformans, A. fumigatus and other Aspergillus species, and the

zygomycetes.

3.2. Fungal Pathogens: An Emerging Problem Agent

Fungi have emerged as the most common pathogens in causing infections particularly

in patients with a reduced defence such as those with haematological malignancies, transplant

recipients, cancer, diabetes, HIV and other immunodeficiencies (Pisseri et al., 2009). Since

opportunistic mycoses pose a serious threat to such patients, it is anticipated that these

infections may break out in epidemic proportions. While aspergillosis, candidiasis,

zygomycosis and infections with Fusarium species may be encountered with greater

frequency in neutropenics or in organ/bone marrow transplant recipients, cryptococcosis and

histoplasmosis are likely to be found often in patients with AIDS (Rhiannon, 2002). Besides,

the infections due to pathogenic fungi under immunocompromised conditions often become

invasive which are generally untreatable and fatal. Invasive fungal infections, particularly

in immunosuppressed patients, have continued to increase in incidence during the

past 30 years and invasive aspergillosis causes approximately 30% of invasive fungal

infections in patients. The overall mortality due to invasive aspergillosis has been around 85

% which may fall to 50 % if aggressively treated (Denning, 1996). The new drugs in trial

may further reduce the mortality but only moderately and still a very large portion of patients

remain at risk of fatal consequences (Ullmann, 2003). Moreover, invasive aspergillosis has

overtaken candidiasis as the most frequent fungal pathogen detected post mortem in tertiary

care hospitals in Europe (Vogeser et al., 1999). It was observed on autopsy that 4 % of all

patients died, had invasive aspergillosis as compared to about 2 % with invasive candidiasis

(Groll et al., 1996). Despite a better understanding of the epidemiology of Aspergillus

infections, diagnostic limitations persist. The Aspergillus has more than 260 species (Samson

and Varga, 2009), however, only 4 of them, namely A. fumigatus, A. flavus, A. niger and A.

terreus have been considered mainly to be pathogenic to humans. A. nidulans has been

isolated as the less common opportunistic mould among pathogenic species of Aspergillus.

Of these species, the A. fumigatus has been reported to be responsible for more than 90% of

Aspergillus induced infections and the marked predominance of A. fumigatus on clinical

samples reflects its environmental preponderance over other pathogenic species of

Aspergillus (Latge, 2001).

3.2.1. Aspergillus fumigatus: Ecological Significance

A. fumigatus has been a saprophytic fungus that plays an essential role in recycling

environmental carbon and nitrogen (Gugnani, 2003). The soil has been considered as the

natural ecological niche of A. fumigatus, where it survives and grows on organic debris. The

A. fumigatus has been very frequently found in the human habitats (Hirsch et al., 2000)

including pillows (Woodcock et al., 2006) and in the epicentre of vegetable matter compost

(Ryckeboer et al., 2003). The hyphae of A. fumigatus develop conidiophores and each of

which may produce large number of tiny (2.0 to 3.0 μm) green brown conidia (Levdansky et

al., 2007). The conidia released into the atmosphere become airborne as micro particles both

indoors and outdoors. The A. fumigatus does not have an elaborated mechanism for releasing

its conidia into the air and their dispersal depends simply on disturbances in the environment

and the air currents. Environmental surveys have indicated that all humans will inhale at least

several hundred A. fumigatus conidia every day and they counter with the immune system of

the host. In susceptible individuals, the germination of A. fumigatus conidia in the airways

causes a wide spectrum of diseases.

3.2.2. A. fumigatus as a Human Pathogen

Among the human pathogenic species of Aspergillus, the A. fumigatus has been found

to be primary causative agent of human infections, followed by A. flavus, A. terreus, A. niger

and A. nidulans (Morgan et al., 2005). Though A. fumigatus can establish infection in any

organ of the body, the involvement of lung has been the most common due to uptake of

conidia by inhalation (O'Gorman et al., 2009) (Figure 3.1). Repeated exposure to conidia of

A. fumigatus may cause allergic conditions, including asthma, allergic sinusitis, and

alveolitis. The A. fumigatus may invade the lung tissues and disseminate to the deeper body

parts to cause invasive aspergillosis. The invasive aspergillosis has been considered to be the

most devastating form of aspergillosis which very frequently occurs in severely

immunocompromised patients (Post et al., 2007). The mortality rate in invasive aspergillosis

has been reported up to 90% in high-risk populations and to be dependent on factors such as

host immune status, the site of infection, and the treatment regimen applied (Nicolle et al.,

2011).

Figure 3.1- Life cycle of pathogenic A. fumigatus showing infection of human host by

conidia.

3.3. Biochemical Targets for Antifungal Chemotherapy

Fungal cells are complex organisms that share many biochemical targets with other

eukaryotic cells. Thus fungal and human cells are similar at the molecular level. This makes

it more difficult to find or design drugs that target fungi without affecting human cells. As a

consequence, many antifungal drugs cause side-effects (Neely and Ghannoum, 2000).

Therefore, agents that interact with fungal targets (Figure 3.2) not found toxic in eukaryotic

cells are needed. The fungal cell wall is a unique organelle that fulfills the criteria for

selective toxicity. The fungal cell wall differs greatly from the bacterial cell wall and is not

affected by antibacterial cell wall inhibitors such as the β-lactams or vancomycin (Sable et

al., 2008). Arrangement of the biomolecular components of the cell wall accounts for the

individual identity of the organism. Although, each organism has a different biochemical

composition, their gross cell wall structure is similar. There are three general mechanisms of

action for the antifungal agents: cell membrane disruption, inhibition of cell division and

inhibition of cell wall formation.

Figure 3.2- Biochemical targets for various antifungal agents used in current chemotherapy.

Interference with fungal cell wall biosynthesis has not been as successful and

effective as penicilins and cephalosporins against bacteria. Many chemicals have been

discovered that interfere with various steps in fungal cell wall synthesis with excellent

antifungal activity in vitro (Neely and Ghannoum, 2000). Unfortunately, development of

these agents into useful drugs has proven very difficult. Many of these agents are developed

to target β-glucan synthesis. Moreover, antifungal agents that disrupt the cell membrane do so

by targeting ergosterol, either by binding to the sterol, forming pores and causing the

membrane to become leaky (as with polyene antifungals), or inhibiting ergosterol

biosynthesis (as seen with azole antifungal agents) (Milhaud et al., 2002). Ergosterol is

similar to mammalian cholesterol, thus agents binding ergosterol may have a cytotoxic effect

in the host tissue. Ergosterol has two conjugated double bonds that are lacking in mammalian

sterols. Furthermore, Nucleoside antifungal agents affect cell division by targeting the

microtubule effects in forming the mitotic spindle.

3.4. Conventional Antifungal Drugs and their Clinical Limitations

Until the 1940s, relatively few agents were available for the treatment of systemic

fungal infections (Wingard and Leather, 2003). The development of the polyene antifungals,

nystatin in the late 1940s, and the broader spectrum, more effective amphotericin B by the

late 1950s represented a major advance in the treatment of fungal infections (Sutton et al.,

2004). Until 1990 there was only one drug useful for treatment of invasive Aspergillus

disease, amphotericin B, which has to be given intravenously (Chamilos et al., 2008).

However, its clinical use is associated with numerous adverse effects related to both the drug

(nephrotoxicity) and its administration (fever, rigors, and hypotension). Additionally,

mortality associated with systemic fungal infections remains unacceptably high despite the

use of amphotericin B. The search for newer systemic antifungals led to the discovery of the

azoles several decades later, with the release of ketoconazole in the early 1980s followed by

fluconazole and itraconazole in the early 1990s. In 1990 itraconazole capsules became

available that included Aspergillus species in the spectrum although the drug was mainly used

in the prophylactic setting due to poor bioavailability (Naschimento et al., 2003). Ten years

later an intravenous formulation of itraconazole became available allowing the drug to be

used for the empiric or pre-emptive treatment of high risk patients. With the

registration of voriconazole the arsenal of drugs available has further increased (Pastor and

Guarro, 2007). Voriconazole is the newest azole antifungal agent for the treatment of

systemic mycosis. Voriconazole was developed as part of a program designed to enhance the

potency and spectrum of activity of fluconazole (Alexander et al., 2005). These agents were

available in oral formulation and demonstrated a relatively improved safety profile compared

with that of amphotericin B. Due to the azoles fungistatic nature, the gold standard for serious

systemic infections continues to be amphotericin B; however, a clear role for the azoles has

been established in the treatment of mild to moderate and refractory infections (Kretzer et al.,

2006). However, despite antifungal therapy, mortality in patients with invasive

aspergillosis remains very high and clearly new therapeutic approaches are needed. The

various agents available to treat fungal infections include

(a) Polyenes: Amphotericin B, Nystatin.

(b) Azoles: Clotrimazole, Miconazole, Ketoconazole, Fluconazole, Itraconazole.

(c) Echinocandins: Micafungin, Caspofungin, Pneumocandins

(d) Nucleoside analogues: Flucytosine

(e) Other antifungal Agents: Cationic peptides, Sordarins, Allylamines and thiocarbamates,

Pradimicins and benanomicins, Nikkomycins.

3.4.1. Polyenes

The polyene antifungal agents form complexes with ergosterol and disrupt the

fungal plasma membrane, resulting in increased membrane permeability, leakage of

the cytoplasmic contents and death of the fungal cell (Zotchev, 2003). Thus, the

polyenes are fungicidal and have the broadest spectrum antifungal activity. They are also

somewhat less toxic to mammalian cells because the affinity of the polyenes for ergosterol

in fungal cells is higher than the affinity for cholesterol in mammalian cells (Bruheim

et al., 2004). The minimum inhibitory concentration of amphotericin B ranges from 0.03 to

30.0 µg/ml against various fungal species (Sabatelli et al., 2008). Amphotericin B was

isolated from a strain of Streptomyces nodosus recovered from a soil sample obtained at

Tembladora on the Orinoco River in Venezuela by Gold and colleagues. Brown & Hazen

discovered nystatin in 1949 in a soil sample, obtained from a farm in Virginia, USA,

containing a strain of Streptomyces noursei (Zotchev, 2003). The minimum inhibitory

concentration of Nystatin ranges from 1.56 to 6.25 ug/ml (Fjaervik and Zotchev, 2005).

3.4.2. Azoles

The initial azole compounds were the imidazoles (clotrimazole, miconazole and

ketoconazole), which were then followed by the triazoles, fluconazole and itraconazole

(Diekema et al., 2003). The azoles inhibit fungal cytochrome P450 3A-dependent C14-a-

demethylase which is responsible for the conversion of lanosterol to ergosterol

(Rodriguez–Tudela et al., 2008). This leads to the depletion of ergosterol in the fungal cell

membrane. Clotrimazole, discovered in 1969,

cannot be given parenterally, has poor

oral absorption and is used for the treatment of oral and vaginal candidosis. The

minimum inhibitory concentration of Clotrimazole ranges from 1.0 to 10.0 µg/ml.

Miconazole was also discovered in 1969 and also has poor oral bioavailability.

Miconazole has been found to be active against a wide range of fungi and its MIC against

different organisms varies from 0.25 to 8.0 µg/ml (Panackal et al., 2006). Ketoconazole was

discovered in 1978, and has good oral absorption, a broad spectrum of activity and low

toxicity, although it may be hepatotoxic and does produce endocrine abnormalities by

suppression of testosterone and ACTH-stimulated cortisol synthesis (Kretzer et al., 2006).

The minimum inhibitory concentration of Ketoconazole ranges from 0.02 to 128.0 µg/ml

against various fungal species.

Fluconazole was formulated in 1981. It is a novel bistriazole, is metabolically stable

and water soluble and has low lipophilicity and plasma protein binding (Garey et al.,

2006). It is well tolerated and has a very low incidence of side effects and a broad spectrum

of antifungal activity, except against Aspergillus spp. Itraconazole, discovered in 1986, is

another triazole antifungal agent with broad-spectrum antifungal activity, including

activity against Aspergillus spp (Wheat et al., 2006). It is very insoluble, is only available in

oral form and may be given once daily. The minimum inhibitory concentrations of both the

drugs (Itraconazole and Fluconazole) have been found to ranges from 0.12 to 2.0 µg/ml

(Sabatelli et al., 2008).

3.4.3. Echinocandins

The echinocandins are cyclic lipopeptide fungicidal agents. They act by preventing

cell wall synthesis by non-competitive inhibition of 1,3-beta-D-glucan synthase, an enzyme

which is absent in mammalian cells (Balani et al., 2000). This inhibition is highly specific

and brief exposure to these drugs leads to cell death. Micafungin is a natural antifungal

product derived from other fungi as a defense mechanism for competition of nutrients, etc.

More specifically, micafungin is produced by Coleophoma empetri. Compared to

amphotericin B, caspofungin seems to have a relatively low incidence of side effects (Balani

et al., 2000). Both are administered intravenously.

The pneumocandins are echinocandin analogues. They are cyclic hexapeptides

which inhibit 1,3-beta-D-glucan synthase which synthesizes a critical structural cell

wall component (Petersen et al., 2001). They were called pneumocandins because they

possess activity against Pneumocystis carinii. They also have activity against Candida

and Aspergillus spp.

Like other analogues of echinocandins, the pneumocandins lack

activity against cryptococci (Petersen et al., 2001).

3.4.4. Nucleoside analogues

5-Fluorocytosine (flucytosine, 5-FC) is a fluorinated pyrimidine that was

synthesized in 1957 as a cytosine analogue for the treatment of leukaemia; however, it

was ineffective because it had no cytotoxic activity. The antifungal activity of 5-

fluorocytosine was discovered later and was reported in 1963 in a murine model of

candidosis (Thomas and Katiyar, 2010). 5-Fluorocytosine had low activity against

Aspergillus species and the MIC was recorded greater than 100.0 µg/ml. It inhibits

pyrimidine metabolism by interfering with RNA and protein synthesis in the fungal cell.

Flucytosine enters fungal cells via cytosine permease and is deaminated to 5-

fluorouracil, which is incorporated into RNA. Uridine 5-monophosphate

pyrophosphorylase then converts 5-fluorouracil into fluorodeoxyuridine

monophosphate, which inhibits thymidylate synthetase and interferes with DNA

synthesis. This drug is selectively toxic to fungi because mammalian cells lack

cytosine permease and do not convert large amounts of flucytosine to 5-fluorouracil.

3.4.5. Other Antifungal Agents

The search for new antifungal agents has been expanded as progress in molecular

biology has led to a better understanding of important and essential pathways in fungal

cell growth and multiplication (Borowski, 2000). One group includes cationic peptides,

both naturally occurring and synthetic derivatives, bind to ergosterol and cholesterol in

fungal cell membranes, ultimately leading to cell lysis (Kretzer et al., 2006). These

peptides have antifungal activity against Aspergillus spp., Candida spp., C. neoformans

and Fusarium spp. Naturally occurring cationic peptides include cecropins, dermaseptins,

indolicin, histatins, bactericidal permeability-increasing factor (BPI), lactoferrin and

defensins. The sordarins are a new class of potential

antifungal agents. They inhibit protein synthesis in pathogenic fungi: the primary target for

sordarin activity has been identified recently as elongation factor 2 (Sable et al., 2008).

The allylamines and thiocarbamates are synthetic fungicidal agents that are

reversible, non-competitive inhibitors of squalene epoxidase, an enzyme which,

together with squalene cyclase, converts squalene to lanosterol. In fungal cells, if

squalene is not converted to lanosterol, the conversion of lanosterol to ergosterol is

prevented (Bruheim et al., 2004). The resulting ergosterol depletion affects fungal cell

membrane structure and function. There are two allylamine antifungal agents, naftifine and

terbinafine, and one thiocarbamate, talnaftate. Naftifine is a topical preparation whereas

terbinafine is an oral systemic agent (Borowski, 2000). The allylamine, naftifine, is

considered an effective topical agent for treatment of dermatophytes infections of the skin.

Similarly, the pradimicins and benanomicins are fungicidal compounds. They

appear to bind, in a calcium-dependent manner, to cell wall mannoproteins and this causes

osmotic lysis and leakage of intracellular contents, particularly potassium, ultimately leading to

cell death.

The nikkomycins are competitive inhibitors of fungal chitin synthase enzymes

which are necessary for fungal cell wall synthesis (Sable et al., 2008). Chitin is a

linear polymer of â-(1,4)-linked N-acetylglucosamine residues and is synthesized on

the cytoplasmic surface of the plasma membrane. Chitin synthase catalyses the

polymerization of N-acetylglucosamine in the formation of chitin.

Also, we need to learn more about combination antifungal therapy, e.g. about

the effects of sequential blockade at two or more sites, and about the combination of

antifungal agents with cytokines in an attempt to augment the inflammatory and immune

responses of patients (Baddley and Pappas, 2005).

3.4.6. Limitations of Conventional Approaches

The molecules available for treatment of aspergillosis are not only fewer in number,

they have limited efficacy. The amphotericin B which was developed in early 1960s still

remains a preferred drug inspite of the fact that it has been highly toxic to the host (Bruheim

et al., 2004). A new antifungal agent called caspofungin was launched in 2002 and included

in the existing therapeutic options for candidiasis and invasive aspergillosis in patients

refractory to, or intolerant of, treatment with other drugs. Caspofungin showed good activity

against Aspergillus and Candida, however, its intravenous route of administration remained

an unsolved problem. Other anti-Aspergillus drugs also have been found to cause toxic

effects (Howard and Arendrup, 2011). Most drugs used for treating aspergillosis have been

shown to be immunosuppressive (Steinbach et al., 2004). The development of drug resistance

in A. fumigatus has become an increasingly serious problem (Camps et al., 2012) which has

made treatment of aspergillosis almost impossible with limited available drugs. The drug

resistance can be primary (fungal pathogen inherently resistant to antifungal drug) or

secondary (development of resistance during treatment). The resistance in Aspergillus to

polyenes has been shown to be primary rather than secondary resistance. The primary

resistance to polyenes in A. fumigatus has been less frequent (Masia et al., 2002) than in

A. terreus. Most A. terreus isolates were found to be resistant to amphotericin B in vitro but

were susceptible to itraconazole or voriconazole (Sutton et al., 2004). There are reports which

have demonstrated resistance in Aspergillus species against voriconazole among stem cell

transplant recipients (Siwek et al., 2004). The development of resistance in A. fumigatus

against most of antifungal drugs makes chemotherapy of aspergillosis less efficient;

therefore, immunotherapy by using vaccine may be important for the better management of

aspergillosis.

3.5. Phytopharmaceutical Approaches and their Significance

3.5.1. Historical Perspectives

The medicinal plants have been one of the important sources of

medicines since the dawn of human civilization. According to medicinal records, in India,

the earliest mention about the use of medicinal plants is to be found in the Rig-Veda which was

written between 4500-1600 B.C (Padma, 2005). The Materia Medica of Hippocrates, who

is now referred to as the father of medicine consisted essentially of herbal recipes, some 400

simple remedies having been compiled and described by him. Theophrastus of Athens

(370-287 B.C.) was another famous biologist-botanist who produced a number of

manuscripts including the famous Historia Plantarium. About 500 plants, mostly cultivated,

were described in this manuscript. Pliny, the elder (23-79 A.D.), a Roman naturalist and

philosopher, described 1000 plants with their medicinal properties, anatomy and

horticultural practices in his book, Historia Naturalis. Dioscorides (60 A.D) wrote “De

Materia Medica” describing 600 plant species of medicinal value from Mediterranean

region. In the middle Ages, the writing of Galen (131 A.D.) becomes popular. Galen is

considered today to be the most distinguished physician of antiquity after Hippocrates. He treated

diseases essentially by the use of herbs. Allopathic as

well as homeopathic systems of medicine today are based on the doctrine explained by Galen.

In the early stages, the science of medicine developed around those plants which

had curative properties. A continued search for medicinal plants during the last several

centuries has given rise to a long list of plants which are of great use in the treatment of

diseases, and for promoting health. It can be stated, more or less truthfully, that every disease

has a cure in a plant growing in nature. In ancient times, medicinal plants were chosen for

their colour or the shape of their leaves (Park, 2008). For example, heart-shaped leaves were

used for heart problems, while plants with red flowers were used to treat bleeding disorders. The

formal study of herbs, called herbology, dates back to the ancient cultures revered the power of

nature and developed herbal remedies based on plants found in their in home

environments (Kamboj, 2000). Herbal therapy is also a major component of India’s

Ayurvedic medicine, traditional Chinese medicine, Native American medicine, homeopathy,

and naturopathy (Patwardhan et al., 2003).

3.5.2. Current Status of Herbal Medicine

A total of 122 biologically active compounds have been identified derived only from

94 species of plants. A conservative estimate of the number of flowering plants occurring on

the planet is 250,000. Of these, only about 6% have been screened for biological activity and

a reported 15% have been evaluated phytochemically (Turker and Usta, 2008). This means

that about 3.5 to 4 billion people in the world rely on plants as a source of drugs. According to

World Health Organisation (WHO), about 80% of the world’s population, primarily those of

developing countries rely on plant-derived medicines for their healthcare (Gurib-Fakim,

2006). The world Health Organization is now actively encouraging developing countries to

use herbal medicine which they have been traditionally used for centuries. They have

identified 3000 plants from the forests of India and other tropical countries which can be used as

medicine. The active ingredients from these plants are worth nearly Rs. 2000 crores of rupees for

the US market alone and nearly 8 times that for the world market.

India has a rich diversity of medicinal plants and only a relatively small number of

plants species have been scientifically validated, some of indigenous plant species may

become extinct before their potential as sources of pharmaceutical drugs is investigated and

applied (Kong et al., 2003). Some of the medicinal plants that had been used traditionally are

becoming endangered, rare, or threatened due to unsustainable harvesting methods (Ming et

al., 2003). Other factors such as exposure to modern culture and urbanization also play a

role in the loss of the traditional use of plants. If plant leaf extracts are as effective as the

bark, bulbs or roots, these endangered species can be utilized in a sustainable way. Plants

have the major advantage of still being the cheapest and most effective alternative source of

drugs. Natural products, either as pure compounds or as standardized plant extracts, provide

unlimited opportunities for new drug leads because of the unmatched availability of chemical

diversity (Cos et al., 2006). Since plants produce a variety of compounds with antifungal

properties, screening plant leaf extracts and isolation of antifungal compounds may become

the base for the development of a medicine, a natural blueprint for the development of new

drugs, or a phytomedicine to be used for the treatment of fungal infections (Kala and Sajwan,

2007).

3.5.3. Market Potential of Herbal Drugs

Herbal medicines are readily available in the market from health food stores without

prescriptions and are widely used all over the world. The utilization of herbal drugs is on the

flow and the market is growing step by step. The annual turnover of the Indian herbal

medicinal industry is about Rs. 2,300 crores as against the pharmaceutical industry’s turnover

of Rs. 14,500 crores with a growth rate of 15 percent (Sharma et al., 2008). The major

pharmaceuticals exported from India in the recent years are Isabgol, Opium alkaloids, Senna

derivatives, Vinca extract, Cinchona alkaloids, Ipecac root alkaloids, solasodine, menthol,

Gudmar herb, papian, Rauwolfia guar gum, Jasmine oil, Agar wood oil, Sandal wood oil, etc

(Kokate et al., 2005). The global market for herbal medicines currently stands at over $60

billion annually. The sale of herbal medicines is expected to get higher at 6.4% an average

annual growth rate (Inamdar et al., 2008). Herbal drugs are marketed in various forms. They

are available in both classical forms (tablets, powder, decoction, medicated oil, medicated

ghee, fermented products) and modern drug presentation forms like capsules, lotions, syrups,

ointments, creams, granules etc. There are more than 8500 manufacturers of herbal drugs in

India (Jain, 2001). Thus, plant-based therapeutic agents continue to have scientific, social,

and commercial significance and appear to be gathering a momentum in health relevant areas.

A study of the process by which the traditional or more recent plant-based molecular drugs or

the new breed of herbal drugs came to be used in present-day medicine reveals that, in over

70% of the cases, the starting point has been some reference to the use of that plant as an

indigenous cure in a folklore or traditional system of medicine of one culture or other.

3.5.4. Traditional Herbal Medicine in Medical Health Practices

During the last decade, the use of TM (traditional medicine) has expanded globally

and has gained popularity (Fabricant and Farnsworth, 2001). It has not only continued to be

used for primary health care of the poor in developing countries, but has also been used in

countries where conventional medicine is predominant in the National health care system.

The practice of traditional medicine is widespread throughout Asia including India, China,

Japan, Pakistan, Srilanka, and Thailand (Venkatasubramanian, 2007). In Japan, herbal

medicinal preparations are more in demand than mainstream pharmaceutical products. 60

to 70% of allopathic doctors in Japan prescribe TM for their patients. In Malaysia, traditional

forms of Malay, Chinese and Indian medicine are used extensively. China is the leading country

for incorporating traditional herbal medicine into a modern health care system. In this

country, TM accounts for around 40% of all health care delivered and are used to treat

roughly 200 million patients annually. According to a recent survey, almost 7,300 plants have

been used in traditional Chinese medicine (Venkatasubramanian, 2007). The traditional

system of medicine is so engrained in Indian culture that, even now 75% of the Indian

population depend on this indigenous system for relief (Ravishankar and Shukla, 2007). With

such a huge section of an ever-increasing population relying on herbal remedies, it is imperative

that the plant products which have been in use for such a long time be scientifically supported

for their efficacy.

The

potential toxicity of the traditional medicines is an important consideration when studying

their biological activities (McGaw et al., 2007). Plant extracts might be very toxic as they

contain many different compounds; therefore it is very important to investigate cytotoxicity

of both crude extracts and isolated compounds. The use of medicinal plants in the form of

crude extracts presents several difficulties. The amount of the bioactive compound(s) from

plants may vary with both the locality and the season in which they are collected. Also,

bioactive molecules of many plants are powerful poisons when taken in excess, and if the

plant extract contains a lower content of bioactive compound(s) than usual, suboptimal

dosage may not be effective. Medicinal properties of many plants are also rapidly lost on

storage, for example, Foxglove leaf’s bioactive molecules decompose on long storage, unless

dried quickly after collection. Furthermore, crude extracts from many medicinal plants may

contain, in addition to the bioactive molecules, other constituents which have harmful effects.

It is therefore important to isolate and identify the bioactive molecules from plant extracts.

The advantage of using pure drugs instead of crude plant extracts includes, amongst others,

accurately prescribed dosage. Structural modification of isolated and identified bioactive

compounds from plant extracts may allow an improvement in the efficacy and moderation of

side effects. Pure bioactive molecule can frequently be synthesized economically, thus

preventing dependence on plants as sources.

3.6. Re-emergence of Antifungal Herbs as the Treatment of Choice

Plants are the oldest source of pharmacologically active compounds, and have

provided humankind with many medically useful compounds for centuries. The primary

benefits of using plant derived medicines are that they are relatively safer than synthetic

alternatives, offering profound therapeutic benefits and more affordable treatment (Singh,

2007). It is estimated that more than two thirds of the world’s population relies on plant

derived drugs; some 7000 medicinal compounds used in the Western pharmacopoeia are

derived from plants. Several methods have been used to acquire compounds for drug

discovery including isolation from plants and other natural sources, synthetic chemistry,

combinatorial chemistry, and molecular modeling (Lombardino and Lowe, 2004). However,

natural products, and particularly medicinal plants, remain an important source of new drugs,

new drugs leads, and new chemical entities (Newman et al., 2003). The compounds have

provided the basic scaffold for medicinal chemistry modifications to expand the spectrum

and/or potency of improved analogues in subsequent years (Walsh, 2003).

3.7. Antifungal Compounds from Plants

Plants are storehouses of a wide variety of secondary metabolites, such as tannins,

terpenoids, alkaloids and flavonoids which have demonstrated their antimicrobial properties

in vitro (Rawat et al., 2008). Moreover, the plant derived fungicides are easily biodegradable

and selective in their toxicity. These antifungal metabolites can be performed in the plant, the

so-called 'constitutive antifungal substances' or they are produced by plants in response to an

infecting organism i.e., phytoalexins. In other words phytoalexins are chemical compounds

formed in plants via a metabolic sequence induced either biotically or in response to chemical

or environmental factors (Rawat et al., 2008). In many plants, a significant proportion of the

assimilated carbon and energy is diverted to the synthesis of organic molecules that may have

no obvious role in growth and development. These molecules are known as secondary

metabolites. The important secondary metabolite includes terpenoids, phenolic compounds,

saponins, cardiac glycosides, cyanogenic glycosides, glucosinolates and alkaloids.

3.7.1. Terpenoids having Antifungal Activity

A large number of studies have been done in recent years on the antifungal activity of

terpenoids of natural origin. These reports concern mainly sesquiterpenes and sesquiterpene

lactones. The fragrance of plants is carried in essential oil fraction. These oils are secondary

metabolites that are highly enriched in compounds based on an isoprene structure. They are

called terpenes, their general chemical structure is C10H16, and they occur as diterpenes,

triterpenes, and tetraterpenes (C20, C30, and C40), as well as hemiterpenes (C5) and

sesquiterpenes (C15). The mechanism of action of terpenes is not fully understood but is

speculated to involve membrane disruption by the lipophilic nature (Cheng et al., 2007). The

terpene family includes hormones, the carotenoid pigments, sterols and sterol derivatives,

latex and many of the essential oils that give plants their distinctive odours and flavours

(Adam et al., 2002). Monoterpenoid from essential oils are well known for their antifungal

activities. Iridoids are a group of monoterpenoid lactones, which usually occur as glycosides

since their aglycones tend to be highly unstable. However, antifungal activity of iridoids

appears to be associated with the few stable unglycosylated structures known. Sesquiterpene

lactones, showing a wide range of biological activities are found to be more active than

sesquiterpenes (Cheng et al., 2007) as well as many diterpenoids also show antifungal

activity. Another important source of antifungal triterpenoids is the saponins (Adam et al.,

2002). Saponins are terpene glycosides. They may be steroid glycosides, steroid alkaloid

glycosides or triterpene glycosides. The antifungal activity of saponins is usually correlated

with the sugar moiety glycosylated to the 3- hydroxy group of the triterpenoids, whereas most

other antifungal constituents tend to be strongly lipophilic and inactive in glycosidic form

(Chiang and Kuo, 2000). They are stored in plant cells as inactive precursors but are readily

converted into biological active compounds by enzymes in response to pathogen attack.

3.7.2. Nitrogenous Compounds having Antifungal Activity

Nitrogen first appears in organic form as glutamic acid, the key reaction being the

transfer of ammonia to a ketoglutarate, catalysed by glutamic dehydrogenase. The other

amino acids are subsequently synthesized from glutamic acid through the catalytic action of

nonspecific transaminases. Amino acids are involved in the biosynthesis of all other

nitrogenous plant compounds, from the proteins to alkaloids, amines, cyanogenic glycosides,

porphyrins, purines, pyrimidines and cytokiins (Bandaranayake, 2002). Alkaloids and amines

are nitrogen-containing compounds, which include representatives showing anti-fungal

activities. Heterocyclic nitrogen compounds are called alkaloids. The first medically useful

example of an alkaloid was morphine; isolated in 1805 from the Papaver somniferum,

codeine and heroin are both derivatives of morphine. The polyamines, spermidine and

spermine, which have a universal occurrence in plants inhibit spore germination of

Penicillium species. Both glucosinolates and cyanogenic glucosides are further groups of

nitrogen and sulphur containing plant compounds which occur in plants in an inactive form

called post-inhibitins. After plant damage these are transformed into the active

isothiocyanates and HCN, which are toxic to insects and microorganisms (Dewick and Paul,

2002).

3.7.3. Aromatic Compounds having Antifungal Activity

Many of the aromatic compounds are emerging as a major group of antifungal plant

compounds. They include simple and alkylated phenols, phenolic acids, phenyl propanoids,

coumarins, flavonoids, isoflavonoids, stiibenoids, quinones and xanthones (Begley and

Tadhg, 2009). Some of the simplest bioactive phytochemicals consist of a single substituted

phenolic ring. The site(s) and number of hydroxyl groups on the phenol group are thought to

be related to their relative toxicity to microorganisms, with evidence that increased

hydroxylation results in increased toxicity (Ross, 2005). In addition, it was also reported that

more highly oxidized phenols are inhibitorier. The mechanisms thought to be responsible for

phenolic toxicity to microorganisms include enzyme inhibition by the oxidized compounds,

possibly through reaction with sulfhydryl groups or through more nonspecific interactions

with the proteins. Many phenolic acids like benzoic, protocatechuic and gentisic acids have

been reported as constitutive antifungal compounds (Bandaranayake, 2002). Antifungal

phenyl propanoid or hydroxy cinnamic acids include p-coumaric, ferulic, caffeic, sinapic and

chlorogenic acids. All these compounds have a widespread distribution in plants. The

structure - activity relationship studies have proved that the aromatic group rather than the

carboxyl group seems to be necessary for the fungal inhibition.

Xanthones are a restricted group of plant polyphenols, biosynthetically related to the

flavonoids. These are planar-six carbon molecules in a conjugated ring system consisting of a

backbone molecule and various chemical groups attached to it (Dewick and Paul, 2002).

Xanthone backbone consists of two benzene rings attached through a carbonyl group and

oxygen not allowing free rotation about the carbon - carbon bonds. The unique backbone

along with type and position of the attached chemical groups defines specific properties of

xanthones. Xanthones possess numerous bioactive capability including antifungal properties.

Coumarins are phenolic substances made of fused benzene and α-pyrone rings which have a

phenyl propanoid nucleus, are another group of aromatic substances rich in antifungal

representations (Dewick and Paul, 2002). Coumarins have been reported to stimulate

macrophages which could have an indirect negative effect on infections. Examples are

coumarin, esculetin, hermiarin, scopoletin and umbelliferone all of which have a wide

distribution in higher plants. There are about twelve recognized

groups of flavanoids that differ from one another only by the oxidation state of this

heterocyclic ring. Three major groups of flavonoids are flavones, falvonols and

anthocyanidins (Begley and Tadhg, 2009). Flavones are phenolic structures containing one

carbonyl group and the addition of a 3 hydroxyl group yields a flavonol. Flavonoids are

hydroxylated phenolic substances synthesized by plants in response to microbial infection.

They have been found to be effective antimicrobial substances against a wide array of

microorganisms. Their activity is probably due to their ability to complex with extracellular

and soluble proteins and to complex with fungal cell walls. More lipophilic nature of

flavonoids may also disrupt fungal membranes (Ross, 2005).

One group of flavonoids, the isoflavonoids have become known for their

antimicrobial activities. lsoflavonoids are one of the several classes of chemicals of diverse

structures formed as phytoalexins. In addition to isoflavonoids; flavonoids, flavanones,

flavans, flavones, flavonols, certain biflavones, chalcones and dihydro chalcones are known

to be associated with antifungal activity (Dewick and Paul, 2002). Quinones are aromatic

rings with two ketone substitutions and characteristically highly reactive. They can switch

between diphenol (hydroquinone) and diketone (quinone) easily through oxidation and

reduction reactions. These compounds, being colored, are responsible for the browning

reaction in cut or injured fruits and vegetables (Ross, 2005). In addition to providing a source

of stable free radicals, quinones are known to complex irreversibly with nucleophilic amino

acids in proteins. Therefore the quinones inactivate the protein and impair their function.

Quinones bind with surface exposed adhesins, cell wall polypeptides, membrane-bound

enzymes and form complex which inactivate the enzymes.

3.7.4. Aliphatic Compounds having Antifungal Activity

These compounds form the simplest form of lipids; they contain only carbon and

hydrogen. These molecules are found mainly in petroleum but living organisms, eukaryotic

or prokaryotic, contain frequently hydrocarbons which are directly derived from fatty

acids. Several hydrocarbons (octane, nonane, dodecane, hexadecane.) belong to aroma

compounds which are found in environmental or food systems. Such as aliphatic C(17)-

polyacetylenes of the falcarinol type, which occur in common food plants of the Apiaceae

family such as Carrot, Celeriac, Parsnip and Parsley, have demonstrated interesting

bioactivities including antibacterial, antimycobacterial, and antifungal activity as well as anti-

inflammatory, anti-platelet-aggregatory, neuritogenic and serotonergic effects (Christensen,

2011). In addition, the cytotoxicity of falcarinol type polyacetylenes towards human cancer

cells, bioavailability, and their potential anticancer effect in vivo indicates that these

compounds may contribute to the health effects of certain vegetables and hence could be

important nutraceuticals (Christensen, 2011). Isolation

and characterization of pharmacologically active compounds from medicinal plants continue

even today. It has only been in the past ten years or so that interest in higher plant antifungal

agents has been reawakened worldwide, and the literature in this area is becoming

substantial. The use of medicinal plants for the treatment of fungal infections predates written

records. Thus, phytochemical screening of plants species, especially of ethnopharmaceutical

use, will provide valuable baseline information in the search for new pharmaceuticals.

Therefore, the presence of antifungal activities demonstrated in various extracts and

compounds of different plant species which used in traditional medicine practices have been

listed in Table 3.1.

Table 3.1- List of various selected plants with reported antifungal activity.

Plants

Name

Family

Plant

parts

Fraction

Used

Pathogens

References

Lantana

camara

Verbenaceae Aerial

part

β-Caryophyllene,

Geranyl acetate

A. niger, C.

albicans,

Fusarium solani,

(Deena and

Thoppil,

2000)

Allium sativum Alliaceae Bulb Cyclohexane

extract

A. niger, A. flavus,

F. oxysporum

(Kumbhar

et al., 2001)

Capsicum

annuum

Solanaceae Fruit Cyclohexane

extract

A. niger, A. flavus,

F. oxysporum

(Kumbhar

et al., 2001)

Zingiber

officinale

Rutaceae Rhizome Cyclohexane

extract

A. niger, A. flavus,

F. oxysporum

(Kumbhar

et al., 2001)

Oxalis

corniculata

Oxalidaceae Shoots Water

extract

A. niger (Iqbal et al.,

2001)

Phyllanthus

debilis

Euphorbiaceae Shoots Water

extract

Pestalotiopsis

theae

(Iqbal et al.,

2001)

Ageratum

conyzoide

Asteraceae Shoots Water

extract,

Essential oil

P. theae, A. niger,

Rhizoctonia

solani,

Didymella

bryoniae

(Iqbal et al.,

2001)

(Okunade,

2002)

Perilla

frutescens

Lamiaceae Leaves Essential oil Rhizoctonia

cerealis

(Cai et al.,

2002)

Centaurea

attica

Asteraceae Whole

plant

4-Acetoxymalaci-

Tanolide

A. niger, A. flavus,

A. ochraceus,

(Neerman,

2003)

Centaurea

thessala

Asteraceae Whole

plant

Hydroxyepi-

sonchucarpolide

A. niger, A. flavus,

A. ochraceus,

(Neerman,

2003)

Zingiber

officinale

Rutaceae Rhizome [10]-Gingerol,

[6]-Gingerdiol

A. niger, (Ficker et

al., 2003)

Polyalthia

lateriflora

Eclipta

prostrata

Annonaceae

Asteraceae

Leaves

Whole

Methanol extract

Methanol extract

C. albicans

C. albicans

(Wiart et

al., 2004)

(Wiart et

al., 2004)

Dillenia

suffruticosa

Dilleniaceae Leaves Methanol extract C. albicans (Wiart et

al., 2004)

Euphorbia

hirta

Euphorbiaceae Whole

plant

Methanol extract C. albicans (Wiart et

al., 2004)

Knema

glaucescens

Myristicaceae Leaves Methanol extract C. albicans (Wiart et

al., 2004)

Eleusine

indica

Poaceae Whole

plant

Methanol extract C. albicans (Wiart et

al., 2004)

Datura metel Solanaceae Whole

plant

Methanol extract

A. niger, A. flavus,

A. fumigatus.

(Dabur et

al.,2004)

Solanum

surattense

Solanaceae Whole

plant

Methanol extract

A. niger, A. flavus,

A. fumigatus.

(Dabur et

al.,2004)

Chenopodium

botrys

Chenopodiaceae Aerial

part

Essential oil A. niger,

C. albicans

(Maksimovi

c et

al.,2005)

Cynara

scolymus

Asteraceae Leaves,

Flower

Chloroform,

ethanol extract

A. niger,

C. albicans

(Zhu et al.,

2005)

Helichrysum

italicum

Asteraceae Whole

plant

Terpenoid,

Terpene oil.

C. albicans (Mastelic et

al., 2005)

Ailanthus

excelsa

Simaroubaceae Stem,

Bark.

Dichloromethane,

methanol extract.

C. albicans,

S. cerevisiae

(Kumar et

al., 2006)

Albizia lebbeck Mimosaceae Pod Dichloromethane,

methanol extract.

C. albicans,

S. cerevisiae

(Kumar et

al., 2006)

Ceratonia

siliqua

Mimosaceae Pod Dichloromethane,

methanol extract.

C. albicans,

S. cerevisiae

(Kumar et

al., 2006)

Gloriosa

superba

Liliaceae Root Dichloromethane,

methanol extract.

C. albicans,

S. cerevisiae

(Kumar et

al., 2006)

Ruta

graveolens

Rutaceae Leaves Dichloromethane,

methanol extract.

C. albicans,

S. cerevisiae

(Kumar et

al., 2006)

Sesbania

sesban

Fabaceae Leaves Dichloromethane,

methanol extract.

C. albicans,

S. cerevisiae

(Kumar et

al., 2006)

Solanum

indicum

Solanaceae Fruit Dichloromethane,

methanol extract.

C. albicans,

S. cerevisiae

(Kumar et

al., 2006)

Citrus

aurantifolia

Rutaceae Whole

plant

Essential oil A. niger (Verma et

al.,2007)

Citrus limon Rutaceae Whole Essential oil A. niger (Verma et

al.,2007)

Citrus

paradisi

Rutaceae Whole

plant

Essential oil A. niger (Verma et

al.,2007)

Fragaria

virginiana

Rosaceae Leaves Water extract Yeast isolates (Ducan et

al., 2008)

Acacia nilotica Fabaceae Bark,

Leaves

Methanol extract A. flavus (Mahesh

and Satish,

2008)

Ziziphus

mauritiana

Rhamnaceae Bark,

Leaves

Methanol extract Dreschlera

turcica

(Mahesh

and Satish,

2008)

Sida cordifolia Malvaceae Bark,

Leaves

Methanol extract Fusarium

verticillioides

(Mahesh

and Satish,

2008)

Thymus

vulgaris

Lamiaceae Aerial

part

Essential oil A. niger, A. flavus,

A. fumigatus

(Soković et

al., 2009)

Mentha spicata Labiatae Aerial

part

Essential oil A. niger, A. flavus,

A. fumigatus

(Soković et

al., 2009)

Piper

regnellii

Piperaceae Leaves Hexane extract Paracoccidioides

brasiliensis

(Johann et

al., 2010)

Baccharis

dracunculifolia

Asteraceae Leaves Hexane extract Paracoccidioides

brasiliensis

(Johann et

al., 2010)

Grindelia

camporum

Asteraceae Leaves Methanol extract F. oxysporum,

A. flavus,

A.fumigatus

(Zabka et

al., 2011)

Cajanus cajan Fabaceae Leaves,

Root

Ethanolic extract C. albicans,

C. tropicalis,

C. krusei,

(Brito et al.,

2012)

Arctotis

arctotoides

Asteraceae Leaves, Hexane,

Acetone extract

C. glabrata,

C.krusei,

(Otang et

al., 2012)

Gasteria

bicolor

Asphodelaceae Leaves, Hexane,

Acetone extract

C. glabrata,

C.krusei,

(Otang et

al., 2012)

Psoralea

corylifolia

Fabaceae Seeds petroleum ether

extract

F.oxysporum,

F. moniliforme,

F.graminearum,

(Srinivasan

and Sarada,

2012)

3.8. Selection of Plants Species for Screening

To available estimates, the total number of higher plants species is approximately

250,000 species. Of them, only 6% have been reportedly screened for biological activity and

about 15% have been screened for phytochemical activity (Fabricant and Farnsworth, 2001).

Initial listing of the aspirants’ species for screening of biological activity is a major task of

specific importance in itself. The selection of suitable plants could be done, including

traditional use, chemical content, toxicity, randomized selection or a combination of several

criteria. Here, the reasonable plants selection will be on the bases that have been exploited for

human use as traditional medicines in some place. The method involves a careful observation

of the use of natural resources in folk medicine in different cultures.

As mentioned above, Achyranthes aspera, Aegle marmelos, Argemona mexicana,

Callistemon lanceolatus, Capparis aphylla, Catharanthus roseus, Commelina bengalensis,

Justicia adhatoda and Syzygium cumini have been selected for present study on the basis of

traditional medicine systems, which have been used for thousands of years in India.

Moreover, the use of these plants in the traditional medicine systems of many other cultures

has been extensively documented. Therefore, a search for antifungal drugs has been

examined where the investigator determined the well-defined pharmacological activity and

performs a randomized exploration.