Additive potential of ginger starch on antifungal potency of honey against Candida albicans

Abstract

Objective

To evaluate the additive action of ginger starch on the antifungal activity of honey against Candida albicans (C. albicans).

Methods

C. albicans was used to determine the minimum inhibitory concentration (MIC) of four varieties of Algerian honey. Lower concentrations of honey than the MIC were incubated with a set of concentrations of starch and then added to media to determine the minimum additive inhibitory concentration (MAIC).

Results

The MIC for the four varieties of honey without starch against C. albicans ranged between 38% and 42% (v/v). When starch was incubated with honey and then added to media, a MIC drop was noticed with each variety. MAIC of the four varieties ranged between 32% honey (v/v) with 4% starch and 36% honey (v/v) with 2% starch.

Conclusions

The use of ginger starch allows honey benefit and will constitute an alternative way against the resistance to antifungal agents.

1. Introduction

Fungal diseases represent a critical problem to health and they are one of the main causes of morbidity and mortality worldwide[1],[2]. Candida albicans (C. albicans) is the most common species associated with candidiasis and is the most frequently recovered species from hospitalized patients[3]–[5]. The treatment of mycoses has lagged behind bacterial chemotherapy and fewer antifungal than antibacterial substances are available[6],[7]. Therefore, a search for new antifungal drugs is extremely necessary[8]–[10]. Different natural substances are responsible for antifungal action[11]–[13]. Among the possible alternatives, products from the hive such as honey, which is considered as natural, non toxic and with a broad spectrum of action, mainly because of its antimicrobial role, are used[14]. This could be promising alternative to substitute synthetic antifungal agent but in several countries the cost of honey is quite expensive which limits its use. Starch is a principal constituent of many foods and it does not only constitute a major energy source, but also is essential to the gross texture or consistency of many food preparations[15]. Therefore, the study was aimed to evaluate the additive action of ginger starch on the antifungal activity of honey against C. albicans.

2. Materials and methods

2.1. Honey sample and plant

From the 2009 harvest, four varieties of honeys of different botanical origin, namely: citrus (V1), jujube (V2), orange (V3) and multi floral (V4) were collected from hives located in western Algeria. All honeys were kept in glass vials and protected from light at temperature of 4 °C.

Rhizome of ginger (Zingiber officinale) purchased from local market of Tiaret (western of Algeria) was peeled and crushed using a hammer mill and then diluted in water. Obtained milk was sieved and sediment was separated from the supernatant and washed several times. The deposit obtained was spread out on aluminum foil and dried at 45 °C for 48 h. Obtained product was crushed to get starch powder[16] from which various concentrations were prepared and expressed as percentage (%).

2.2. Fungal strain and inoculums standardization

C. albicans (Institut Pasteur of Algiers) was maintained by subculture in specific media (Sabouraud agar). The inoculum suspension was obtained by taking five colonies (>1 mm diameter) from 24 old cultures grown on Sabouraud agar. The colonies were suspended in 5 mL of sterile saline water (0.85%). The inoculum suspensions were shaken for 15 sec and density was adjusted to the turbidity of a 0.5 McFarland standard (equivalent to 1−5 × 10⁶ cfu/mL).

2.3. Minimum inhibitory concentration (MIC)

Increased concentrations (10%–50% v/v) were incorporated into media to test their efficiency against C. albicans. Each plate with final volume of honey and media of 5 mL was inoculated and incubated at 37 °C for 48 h. The MIC was determined by finding the plates with the lowest concentration of honey on which the strain would not grow. All MIC values were expressed in % (v/v).

2.4. Minimum additive inhibitory concentration (MAIC)

To evaluate the effect of starch on the antifungal action of honey, 1% starch solution was prepared using sterile water. Different volumes from the stock solution were added to a range of honey concentrations lower than the MIC. The same volume of starch solution that has given inhibition with honey was added alone to media as control to check whether or not starch alone has an inhibition effect against C. albicans. An equivalent volume of water was added to honey instead of starch solution to confirm that additive inhibition is not due to the dilution of honey. The final volume in each plate was 5 mL. Starch content in media ranged between 1% and 8% (w/v). Honey and starch as well as honey and water were incubated for 24 h at 37 °C before being incorporated into media. Plates were inoculated and incubated at 37 °C for 48 h. All inoculations were carried out in duplicates.

3. Results

The inhibitory action was seen neither in the media containing starch only nor in media with water and starch. All varieties of honey were effective against the tested strain. Without starch of ginger, the MIC of the four varieties ranged between 38% and 42% (v/v). When starch was incubated with honey and added to media, a MIC drop was noticed with each variety and the MAIC of the four varieties ranged between 32% and 36% (v/v) (Table 1).

Table 1. MIC, MAIC and MIC drop of tested honeys.

Honey varieties	MIC values [% (v/v)]			MAIC values [% (v/v)] honey and starch								MIC drop (%)
				Starch solution
	Honey	Starch	Water and starch	1%	2%	3%	4%	5%	6%	7%	8%
V1	42	0	0	42	36	36	36	36	36	36	36	14.28
V2	41	0	0	41	41	41	35	35	35	35	35	14.63
V3	38	0	0	38	38	38	32	32	32	32	32	15.78
V4	39	0	0	39	39	39	33	33	33	39	39	15.38

4. Discussion

In humans, fungal infections range from superficial to deeply invasive or disseminated, and have increased dramatically in recent years[6]. The application of honey in medicine has recently been rediscovered and it is gaining acceptance as an antibacterial agent for the treatement of ulcers, wounds, and other surface infection[17].

Using corn starch and four varieties of Algerian honeys[18] has shown an additive effect against C. albicans with the MIC values of 38% and 28%, respectively, with a starch concentration of 3.6%. In a previous study, Boukraa et al[19] used other five varieties of honey and the same strain of C. albicans used in present study, and obtained a MIC value ranging between 30% (starch 2.6%) and 39% (starch 2.4%). Amylases present in honey were expected to split starch chains to randomly produce dextrin and maltose and probably increase the osmotic effect in the media by increasing the amount of sugars and consequently increase the antifungal activity. As a paradox, the variety with the lowest diastase number has shown the highest MIC drop and the variety with the highest diastase number has shown the lowest MIC drop. Resistant starch has received much attention for both its potential health benefits and functional properties. It has properties similar to fiber and shows promising physiological benefits in humans, which may result in disease prevention[20]. Eerlingen et al[21] reported that an increase in resistant starch yield was observed with high-amylose corn starch. As the final purpose of our future studies is the use of honey and starch to manage superficial mycoses by an increase in osmotic pressure, the use of resistant starch is not adequate in this case. In previous studies, the same varieties of honey and ginger were used.

We obtained a MIC drop with Aspergillus niger ranging between 10.5% and 11.5%[22]. By using 1% ginger starch solution instead of 10% corn starch used in the above mentioned studies, we obtained better results. But it must be mentioned that the honey varieties used by Boukraa[18] and Boukraa et al[19] were different from ours. It then seems that ginger starch for a reason or another is more effective than corn starch, perhaps in regard to its lesser resistance to hydrolysis by amylases. In other hand, Torley et al[23] reported that, honeys from different yields were observed with high-amylose corn starch. The experiments showed that the differences in gelatinization temperature, lipid content, and apparent amylose content of the two starches were not the main causes of the different impact of sugars on resistant starch yields. Sources show a varied effect on starch gelatinization with starch viscosity increasing with addition level for some honeys, but decreasing with increasing addition level for other samples. Neither honey nor starch has adverse effects on tissues, so they can be safely used in wounds and inserted in cavities and sinuses to clear infection. A clinical trial would be carried out to validate these findings. The results will enable a systematic study of many varieties of honey on pathogens yeast with increased resistance opposite conventional anticandidiasis.