Anti-fungal activity of traditional spices against dermatophytes and opportunistic fungi. Comparison of combinatorial effects of clove, cinnamon and kacholam

Contents

List of Figures

List of Tables

Abbreviations

Abstract

1. Introduction
1.1. Objective of the study

2. Review of literature
2.1. Spices
2.3. Opportunistic Fungi- C. albicans

3. Materials and Methods
3.1 Preparation of Raw material
3.2 Extraction process
3.3 Preparation of Media
3.4 Fungal strains and cultural conditions
3.5 Haemocytometer counts
3.6 Anti-fungal screening of extracts

4. Results
4.1 Screening of DMSO (Negative control) and standard drugs (Positive control)
4.2 Determination of antifungal effects of clove, cinnamon, kacholam against T. mentagrophytes
4.3 Determination of antifungal effects of clove, cinnamon, kacholam against T. rubrum
4.4 Determination of antifungal effects of clove, cinnamon, kacholam against C. albicans
4.5 Determination of combinatorial effects of clove, cinnamon, kacholam against pathogenic fungi

5. Discussion

6. Summary and Conclusion

References

Ratheesh Mohanan

1 Department of Biochemistry, St. Thomas College, Palai, Kottayam, Kerala, India
2 Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore, Karnataka, India.
3 Department of Zoology, Milad-E-Sherif Memorial College, Kayamkulam, Alappuzha, Kerala, India.

List of Figures

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List of Tables

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Abbreviations

CDC Control and Prevention

CFU Colony Forming Unit

DMSO Dimethyl sulphoxide

DNA Deoxyribonucleic acid

Flc Fluconazole

Ket Ketoconazole

mg Milligram

OI Opportunistic infections

R Resistance

RNA Ribonucleic acid

S Sensitive

Spp Species

SDA Sabouraud Dextrose Agar

Abstract

Spices have been used as food and flavouring agent since the ancient times, and as a medicine in the recent decades. Now it is widely used across the globe as they possess great potential in the treatment of various diseases. It can serve as a better alternative to the modern synthetic drugs due to its lack of side effects and possible role in a wide range of therapeutic application. The present study aims to determine the anti-fungal activity of combined and individual effect of Cinnamon (Cinnamomum verum), Clove (Syzygium aromaticum) and Kacholam (Kaempferia galangal) against Candida albicans, Trichophyton rubrum, and Trichophyton mentagrophytes by agar well disc diffusion method. Extract from these spices showed remarkable antifungal activity against T. mentagrophytes, T. rubrum and C. albicans. Clove exhibits significant antifungal activity against all the microorganisms tested. Cinnamon showed good inhibitory effect against T. mentagrophytes and C. albicans whereas kacholam showed an inhibitory effect against T. mentagrophytes and T. rubrum. Thus, the study concluded that individual effect of spices is better than the combinatorial effect against all the fungus tested.

Keywords: Spices; Dermatophytes; Opportunistic fungi; Agar well diffusion method

1. Introduction

The history of medicinal plants is similar to that of the advancement of human history. Since ancient times, medicinal plants have been found to be the strength and backbone of traditional herbal medicine across the worldwide. Natural products are the fundamental part of different ancient medicinal systems like Ayurveda, Chinese, Greek- Unani (Sarker & Nahar, 2007). It is estimated that 40% of the world’s population depending directly on plant-based medicine for their health care. India has a rich medicinal plant flora consist of more than 25,000 species, of which 150 are commercially used for the extraction of medicines or for the formulation of drugs (Lucy & Edgar, 1999).

Over the past decades, researchers have sought to identify and validate plant-derived substances for the treatment of various diseases. Natural medicine comprises of spices, herbs and shrubs. The spices and herbs have been considered as a part of the food in daily life. In the dietary system of humans, the addition of spices is essential for the enhancement of the taste and aroma of food items. Besides being used as flavouring agent, it is also having lots of therapeutical properties such as antioxidant, antimicrobial and anti-inflammatory activity. Spices are one of the common active ingredient used in traditional system of medicine or naturopathy because of its effectiveness against certain diseases (Langner et al, 1998). Moreover, Essential oils and extracts derived from spices are capable of controlling microorganisms related to skin diseases, dental caries and food spoilage. (Chaieb et al, 2007). A number of studies have been conducted in recent years on the use of spiceuticals as antimicrobial agents. In most cases, reports on the antifungal activity of some extracts or essential oils exposed directly to fungi have been also published (Baratta et al, 1998). Therefore, the use of spices could be an effective mode of treatment against superficial mycosis.

Superficial mycosis or cutaneous mycosis is a chronic disease condition which is commonly caused by certain dermatophytes. In human beings, T. mentagrophytes and T. rubrum are the most common pathogens which is causing skin infections. Dermatophytosis leads to the formation of cutaneous lesions which is characterized by desquamation, erythema of the edges and circular disposition (Hainer, 2003). Along with these dermatophytes, C. albicans an opportunistic fungus which can also cause mucosal and cutaneous infections along with subcutaneous infectious diseases and systemic mycoses. The previous studies showed that the active compounds present in certain spiceuticals or their extracts exhibit fungicidal activity without affecting the host.

The present study is focusing on the medicinal properties of traditional spices such as clove, kacholam and cinnamon against T. mentagrophytes, T. rubrum and C. albicans. A comparative analysis of the antifungal properties of these spices with that of different antibiotics will provide a valuable information regarding the antifungal activity of spices and its therapeutic potential targeting the replacement of common conventional drugs.

1.1. Objective of the study

Fungal infections are one of the common type of infection, but it can cause many serious clinical manifestations. Opportunistic infections (OIs) are the main leading causes of morbidity and mortality in immune-compromised patients. The discovery of antifungal agents was a major milestone in tackling the fungal infections, either as a cure or to manage the symptoms. But, the prolonged use of these agents can cause various side effects. Moreover, the human dermatophytic fungus will acquire resistance and tolerance against these antifungal agents. Recently, the opportunistic fungi have shown azole resistance and this might be the reason for the drastic emergence of Non- C. albicans species, as a cause of refractory mucosal candidiasis (Rex et al, 1995; Martins et al, 1997). Thus, in search of new and better alternatives, natural products are found to be very efficient because they have no detrimental effects on human beings but it could be deleterious to the microbial pathogens.

Therefore, the aim of the present study:

- To determine the anti-fungal effect of Clove, Cinnamon and Kacholam against Trichophyton mentagrophytes, Trichophyton rubrum and Candida albicans.
- To evaluate the combinatorial effect of Clove, Cinnamon and Kacholam, against Trichophyton rubrum, Trichophyton rubrum and Candida albicans

2. Review of literature

2.1. Spices

The Spice is a seed, fruit, root, bark or other plant substance primarily used to flavor, color or preserve food items. Spice is defined as a “strongly flavored or fragrant substance of natural origin, derived from tropical plants, frequently used it as a condiment”.

International Organization for Standardization (ISO), defines the term Spices and Condiments are natural or vegetable ingredients or mixtures of the same, in whole or ground form, used for flavoring, flavoring and spicing of fruit and for seasoning of fruit. Spices are also considered to have many positive effects. The researchers shown that widely used herbs and spices such as garlic, black cumin, cloves, cinnamon, ginger, thyme, all-spice, bay leaves, mustard and rosemary have antimicrobial properties that can be used therapeutically in certain situations.

Clove is one of the most valuable spices that has been used as food preservatives for many centuries and has been used for many medicinal purposes and it is known as ‘champion spice’. Cloves are native to Indonesia but are now cultivated in different parts of the world (Cortés-Rojas et al, 2014). Clove has many medicinal properties, including antiviral, antimicrobial, general antifungal stimulation, hypertensive aphrodisiac, light stomach, carmin and anesthetic. (Di Paoli et al, 2007).

Clove is known to have antibacterial properties and is used for cleansing bacteria in various dental creams, tooth pastes, mouth washings, and throat sprays. This is also used to relieve chronic gum pain and to improve overall dental health. Clove is an anodyne (an agent that relieves or soothes pain) in dental emergencies. It is used as an anti-inflammatory agent, because of its high flavonoids content. Clove and clove oil has the ability to strengthen the immune system by purifying the blood and helping to combat various diseases (Parle & Khanna, 2011).

Kacholam is a small monocotyledonous herb of Zingiberaceae that has been known for decades for its medicinal properties. The plant is native to southern China, Indochina, Thailand, Taiwan, Malaysia and India. Phytochemical isolated from kacholam, ethyl-p-methoxycinnamate has significant activity against Mycobactrium tuberculosis and Candida albicans. More recently, the resazurin microtitre assay has shown that this ethyl-p-methoxycinnamate will inhibit drug susceptible and multidrug resistant clinical isolates of M. tuberculosis. Kacholam extracts have also been found to exhibit antimicrobial activity against a number of organisms including both Gram positive and Gram negative organism.

Cinnamon is an important spice and aromatic crop with a wide range of applications in flavorings, perfumes, beverages and medicines. Cinnamon, also known as true cinnamon or ceylon cinnamon, is an evergreen tree with a strong aromatic bark and leaves.

Cinnamon is Native to Sri Lanka the largest producer and exporters. As well as being used as a spice and flavouring agent, cinnamon is also adding to the flavor of chewing gum because of its refreshing effects on the mouth and the ability to remove bad breath (Jakhetia et al, 2010). It improves colon health and thus reduces the risk of colon cancer (Wondrak et al, 2010). Cinnamon increases uterine blood circulation and promotes tissue regeneration (Minich et al, 2008). Cinnamon plays a vital role as a spice, but it also has significant activities in its essential oils and other constituents, including antimicrobial, antifungal, antioxidant and antidiabetic (Chang et al, 2001).

2.2.1 Dermatophytes

In general, dermatophytes are classified in three anamorphic genera, Epidermophyton, Microsporum, and Trichophyton belonging to the category of the Deuteromycota (Fungi Imperfecti) (Irene et al, 1995). This classification is based morphology of conidia and the conidial spore formation. Dermatophytes are among the few fungi that cause transmissible diseases, that is, diseases acquired from infected animals or birds or from the fomites they have endangered.

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Figure 2.1. General Classifications of Dermatophytes¹

Dermatophytes are aerobic fungi which will invade and infect keratinized layers of the skin, hair, and nails, mediated by both keratinases and proteases.

2.2.2 Epidermophyton spp.

Epidermophyton spp. have macroconidia which is narrowly clavate with usually smooth, thin to moderately thick walls and one to nine septa, 20 to 60 by 4 to 13 mm. They commonly abundant and are borne individually or in clusters. Microconidia are absent. To date, this genus includes only two known species, and only Epidermophyton floccosum can be pathogenic.

2.2.3 Microsporum spp.

Macroconidia of Microsporum spp. is characterized by rough walls that may be asperulated, echinulated, or verrucose. Emmons originally described macroconidia as spindle shaped or fusiform but the discovery of new species extended the range from obovate (egg shaped) as in Microsporum nanum (Fuentes et al, 1956) to cylindrofusiform as in Microsporum vanbreuseghemii (Georg et al, 1962). Microconidia are sessile or stalked and clavate and usually arranged singly along the hyphae or in racemes as in Microsporum racemosum, a rare pathogen (Borelli et al, 1965). The type species is M. audouinii.

2.2.4 Trichophyton spp.

In Trichophyton spp., macroconidia has smooth, usually thin walls and one to twelve septa, are borne individually or in clusters, and may be elongated and shaped like a pencil, clavate, fusiform or cylindrical. Microconidia, typically more numerous than macroconidia which can be globose, pyriform or clavate, or sessile or stalked, borne alone on the hyphae sides or in grape-like clusters. The type species is T. tonsurans.

2.2.5 Trichophyton rubrum

T. rubrum is an anthropophilic fungus that has become the most widely distributed dermatophyte on humans. It frequently causes chronic infections on skin, nails and infrequently on scalp. Granulomatous lesions may sometimes occur. Infected hairs will not fluoresce under Wood’s ultraviolet technique, and it may show endothrix or ectothrix sort of invasion.

Approximately 80–93% of chronic dermatophytic infections reported in many parts of the world are thought to be caused by T. rubrum including cases of tinea pedis, tinea unguium, tinea manuum, tinea cruris, and tinea corporis, as well as some cases of tinea barbae. It has also been known to cause folliculitis which is characterized by fungal element in follicles and giant cells in the dermis. T. rubrum infection may also form a granuloma. Extensive granuloma formations may occur in patients with immune deficiencies (e.g. Cushing syndrome). Immunodeficient neonates are highly susceptible to systemic T. rubrum infection.

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Figure 2.2. Culture of T. rubrum and its microscopic view²

2.2.6 Trichophyton mentagrophytes

T. mentagrophytes is a fungus that is part of a group known as dermatophytes. This fungus is known to cause skin infection known as Dermatophytosis or Ringworm which appears on a person's skin as an inflamed circular pattern. The invasion of this organism to skin, hair, and nails can cause diseases such as tines pedis or athlete’s foot. Spores produced by this fungus are found to be difficult to remove by disinfection. Disinfection is especially important for environments where Ringworm infections can occur and spread rapidly such as athletic facilities or schools. T. mentagrophytes is zoonotic, meaning that it can be transferred from animals to humans (Martins et al, 2014).

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Figure 2.3. Culture of T. mentagrophytes and its microscopic view³

2.3. Opportunistic Fungi- C. albicans

Opportunistic fungi can be defined as fungi that would not normally cause infections in otherwise healthy people but they are capable of causing infection under certain circumstances, such as immunodeficiency, cancer, organ transplantation, neutropenic patients, diabetes, weakened patients and long-term antibiotic patients. It is generally a harmless commensal fungus that can turn into an opportunistic organism in immunocompromised or immunologically deficient individuals as in HIV patients . C. albicans is a member of the human microflora as diploid polymorphic yeast of mucosal surfaces and is commonly found in the human gastrointestinal (GI), respiratory, and genitourinary tracts. C. albicans and other emerging NAC species, including C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis represent an important source of systemic infections worldwide. These organisms are the most common cause of superficial vaginal or mucosal oral infections and may also, under propitious conditions, enter the bloodstream leading to deep-tissue infections (Conti et al, 2014). With a variety of host cells, including Th17 cell, this microorganism can interact during the disease manifestations (Kashem et al, 2015). An abnormal growth in C. albicans due to environmental imbalance like reduction of pH may result in candidiasis. It has been demonstrated that C. albicans systemic infections lead to a mortality rate of -40%.

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Figure 2.4. Culture of C. albicans and its microscopic view⁴

3. Materials and Methods

3.1 Preparation of Raw material

Dried clove, cinnamon and kacholam were purchased from local shop in Idukki. The samples were air-dried, grinded in a Wiley Mill to fine uniform texture, and stored in glass jars until use.

3.2 Extraction process

100g of powdered samples was taken in a conical flask and added required volume of 70% ethanol. The mouth of the conical flask was covered with aluminum foil and kept in a reciprocating shaker for 24 h for continuous agitation at 150 rev/min for thorough mixing and also complete elucidation of active materials to dissolve in the respective solvent. Then, extract was filtered by using muslin cloth followed by Whatman no 1 filter paper. The solvent from the extract was removed by using rotary vacuum evaporator with the water bath temperature of 60°C. Finally, the residues were collected and refrigerated at 4 ºC until further analysis.

3.3 Preparation of Media

All ingredients/media were purchased from M/S Hi-Media Laboratories Pvt. Ltd. Mumbai, India.

- Sabouraud Dextrose Agar
- Mycological Peptone - 10 gms
- Dextrose - 40 gms .
- Agar agar - 15 gms
- Distilled water - 1000 ml
- pH - 5.4

Medium was mixed thoroughly and heated with frequent agitation. Boiled for 1 min and sterilized by autoclaving at 15 lbs/in' pressure for 15 min. About 25 ml medium poured into 90 mm diameter sterile glass plates to a depth of 4 ± 0.5 mm. Pouring was done on a level surface to obtain uniform depth of the medium.

3.4 Fungal strains and cultural conditions

All fungal strains used in this study including C. albicans, T. rubrum and T. mentagrophytes were procured from microbial culture lab. Fungal strains were cultured on Sabouraud Dextrose Agar (HiMedia Laboratories) slants for 7-14 days at 280C.

Prepare a spore suspension by pouring 5–7 mL of sterile 0.9 % NaCl (w/v) + 0.01 % Tween 80 on a SDA plate containing a premade fungal culture. It was then scrapped by using sterol glass rod for about 2 min. It was then filtered using sterile cheese cloth and filtrate was used as stock for spore solution. A diminutive amount of the stock was diluted serially and spore counts were taken using haemocytometer (Neubauer chamber, Germany).

3.5 Haemocytometer counts

The haemocytometer and the coverslip were cleaned thoroughly in soap water followed by tap water. It was then rinsed in alcohol and wiped with a tissue paper. The cover slip was placed on the slide exactly over the depression in the counting chamber. One ml of spore suspension was drawn using the pipette and expelled into the depression below the cover slip. After 3-5 minutes, spore count was taken under phase at 300-400 times magnification. For each dilution three replicates were maintained.

The number of spore ml -1 was calculated using the following formula

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These plates were incubated for 24-72 hours at 25ºC. The diameter of the zones of inhibition around each of the disc was taken as measure of the anti-fungal activity. Each experiment was carried out in triplicate and mean diameter of the inhibition zone was measured in millimeter (Kohner et al, 1994; Mathabe et al, 2006).

3.6 Anti-fungal screening of extracts

In vitro antifungal and anti-bacterial activity were determined by agar well diffusion method (Murray et al, 1995). For susceptibility testing, ethanolic extracts of spice were made into a suspension using DMSO (Merck). Fungal isolates were grown on SDA (HiMedia Laboratories). Extracts of each spices at different doses (6.25mg, 12.5mg, 18.75mg and 25mg) were introduced into the medium along with standard drug of ketoconazole (KT 50 mcg, HiMedia) and fluconazole (FLC 10 mcg, HiMedia). Then, a hole with a diameter of 6 to 8 mm is punched aseptically with a sterile cork borer or a tip, and a volume (50–200 µL) of the antifungal agent or extract solution at desired concentration is introduced into the well.

4. Results

4.1 Screening of DMSO (Negative control) and standard drugs (Positive control)

For the present study, negative and positive controls were examined for their antifungal activity against three fungal isolates by the agar well diffusion method. These are the fungal isolates were frequently encountered with human infections. The negative control DMSO 10% did not show any antifungal activity while the positive control KT, FLC showed antifungal activity.

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¹ Author’s own creation

² Author’s own creation

³ Author’s own creation

⁴ Author’s own creation