Background: Nigeria is plagued with a variety of socioeconomic problems mainly poverty, poor access to quality healthcare and poor hygienic conditions resulting in the myriad of fungal infections that are frequently encountered in clinical practice. Method: The antifungal activity of aqueous, methanolic, propanolic and benzyl alcohol extracts of Capsicum annuum fruits and seeds, Capsicum chinense fruits and seeds, Aframomum melegueta pods and seeds, Allium sativum bulbs, Allium cepa bulbs and Zingiber officinale rhizomes on Candida albicans (yeast), Aspergillus niger (mould) and Trichophyton rubrum (dermatophyte) were evaluated by the agar well diffusion method. The aqueous and alcoholic filtered plant extracts were obtained by maceration, and also methanolic extracts were also obtained by Soxhlet extraction. The results were obtained by measuring the inhibition zone diameter in millimeters and were presented by subtracting the activity of the control. Results: Aqueous and methanolic extracts of Allium sativum gave the highest inhibition of the growth of Candida albicans (22 mm), followed closely by its propanolic extract with inhibition zone diameter of 15 mm, and also, propanolic extracts of Aframomum melegueta and Allium cepa gave inhibition zone diameters of 12 mm each. Soxhlet methanolic extract of Allium sativum had the highest inhibition of the growth of Aspergillus niger with an inhibition zone diameter of 25 mm, followed closely by Zingiber officinale Soxhlet methanolic extract with an inhibition zone diameter of 22 mm, also, the propanolic extract of Allium sativum gave an inhibition zone diameter of 21 mm, whereas Soxhlet methanolic extracts of Aframomum melegueta and Allium cepa gave an inhibition zone diameter of 19 mm each. The highest activity against Trichophyton rubrum was obtained with the Soxhlet methanolic extract of Allium sativum (39 mm), followed closely by its propanolic extract with an inhibition zone diameter of 27 mm. An inhibition zone diameter of 22 mm was recorded with the benzyl alcohol extract of Allium cepa, 22 mm with the Soxhlet methanolic extract of Aframomum melegueta and 19mm with the aqueous extract of Capsicum chinense seeds. Conclusion: The in-vitro inhibitory effects of these spice extracts indicated that the test spices could serve as potential candidates for developing new systemic and topical antifungal drugs against the wide range of pathogenic fungal strains, and they could also serve as natural prophylaxis against the fungal infections.
People all over the world are still affected by quite a large number of microbial infections with fungi causing a good number of them. It has since been discovered that active medicinal substances are present in plants and this has encouraged the inclusion of herbal remedies in the delivery of health care [
Spices are aromatic or pungent plant parts used for enhancing the taste of foods. Although spices are commonly used to improve the taste of foods, they have also been exploited for their medicinal as well as their antimicrobial activities [
Capsicum annuum (red pepper) is a fruit spice of the Solanaceae family, rich in proteins, lipids, vitamins, carbohydrates, and health phytochemicals such as carotenoids, flavonoids and capsaicinoids known to prevent diseases such as asthma, coughs, sore throats etc. [
Aframomum melegueta (Alligator pepper) is from the family Zingiberaceae together with Zingiber officinale (ginger). Alligator pepper seeds have been shown to possess phytoconstituents that have potent antimicrobial effect [
Allium cepa (onions) is from the family Amaryllidaceae and contains water, sugar, protein, fat and fibre [
Fungal pathogens of humans are grouped as: Yeasts (Candida albicans), Moulds (Aspergillus niger), and Dermatophytes (Trichophyton rubrum). C. albicans is responsible for a wide range of superficial and systemic infections. 75% of women are affected with genital Candidiasis in their lifetime while in men, alcoholics and diabetics are prone to genital Candidiasis [
The aim of this study is to investigate the inhibitory effects of six commonly used spices on the growth of three pathogenic fungi to determine whether they can serve as natural prophylactics and the possibility of developing systemic and topical antifungal agents with them using the agar well diffusion technique.
Three microorganisms were tested in this study: Candida albicans, Trichophyton rubrum, Aspergillus niger. They were characterized strains obtained from the Laboratory of Pharmaceutical Microbiology and Biotechnology, Nnamdi Azikiwe University, Awka, Nigeria.
In this study, six commonly used spices were selected to analyze their antifungal activity based on previous literature and their popularity in the Nigerian markets. The fresh bulbs of A. cepa and A. sativum, fresh rhizomes of Z. officinale, fresh fruits of C. annuum, dried fruits of C. chinense and pods and seeds of A. melegueta were purchased from Eke Awka and Ose market in Onitsha, Anambra State in the month of April. They were identified and authenticated by Mrs Oduche Anthonia in the Department of Pharmacognosy and Traditional medicine, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University Awka.
The fresh fruits of C. annuum were dried in a hot air oven at 60˚C for 8 hours. Seeds from some of the dried fruits of C. annuum and C. chinense were separated, along with the seeds of A. melegueta and were powdered separately. The dried materials including the whole fruits of C. annuum and C. chinense and pods of A. melegueta and the separated seeds were reduced to fine powder with a mechanical grinder. The bulbs of A. cepa and A. sativum, and the Z. officinale rhizome were washed thoroughly using tap water, a clean kitchen knife was used to manually peel the outer coverings, and then the fleshy parts were washed and rinsed with distilled water again. A sterile ceramic mortar and pestle was used to crush the parts into smaller marshy parts. Four solvents were used for the preparation of the extracts namely: cold water, methanol, propanol and benzyl alcohol.
The aqueous extracts were prepared by weighing out 2 g of the powdered seeds and whole fruits of C. annuum, C. chinense and A. melegueta, and 5 g of the crushed bulbs of A. cepa and A. sativum and rhizome of Z. officinale, and macerating in 40 ml of cold water in a glass jar. The combination was allowed to stand for 24 hours at room temperature (32˚C ± 2˚C) with occasional agitation. The alcoholic extracts; methanol 99.5%, propanol 99.5% and benzyl alcohol 99% were obtained by weighing out same fraction (2 g) of the powdered whole fruits and separated seeds of C. annum, C. chinense and A. melegueta, and 5 g of the crushed bulbs of A. cepa and A. sativum and rhizome of Z. officinale and macerating in 40 ml of the 99.5% methanol, 99.5% propanol and 99% benzyl alcohol in a covered glass jar. The combination was allowed to stand for 24 hours with occasional agitation at room temperature (32˚C ± 2˚C). The extracts were then filtered using a Whatman no. 1 filter paper into plastic sample bottles and stored in a refrigerator at 2˚C - 8˚C. There was altogether nine aqueous extracts, nine methanolic, nine propanolic and nine benzyl alcohol extracts.
The Soxhlet extractor was also used to afford nine different methanolic extracts of the whole fruits and separated seeds of C.annum, A. melegueta and C. chinense, and the bulbs of A. cepa and A. sativum and rhizome of Z. officinale. The extracts were then stored in a refrigerator at 2˚C - 8˚C. Altogether, 45 extracts was tested.
Three test tubes of 5 ml sterile Sabouraud dextrose broth was prepared and labeled appropriately and loopfuls of organisms were collected from the stock cultures of C. albicans, A. niger and T. rubrum and placed in the individual test tubes. The organisms were identified using morphological and cultural characteristics.
The morphological and cultural characteristics of the fungi isolates were used to identify them. The method that was used is the direct observation of plates. Colony morphology includes the type of pigment (if present), size of colony, texture (opaque, translucent, or transparent), adherence to agar and undulating/round/dentate edge. The plates were observed daily for the rate of growth of each of the isolates. The colour and morphology of the colonies were noted. The base of the plate, odour, were noted.
Standard suspensions containing 105 cfu/ml of the test organism was made by transferring colonies from the subculture into 5 mls of sterile water and then adjusting and comparing with McFarland’s 0.5 standard.
The antifungal activity of these crude extracts was determined against a yeast (C. albicans), mold (A. niger) and dermatophyte (T. rubrum). Agar well diffusion technique was used to determine the antifungal activities of the extracts. 25 mls of molten sterilized Sabouraud’s dextrose agar, fortified with 0.05 mg/ml of chloramphenicol in a McCartney bottle was seeded with 0.2 mls of 0.5 McFarland standard suspension of each fungal isolate and poured into appropriately labeled sterile Petri dishes aseptically. The Petri dishes were rotated gently to achieve a uniform distribution of the fungal isolate and was then allowed to set. A standard sterile cork borer of 8 mm diameter was used to cut uniform wells on the surface of the solidified seeded agar. The wells were then filled with 0.1 ml of each extract with the aid of a sterile syringe and needle. One of the wells in each SDA agar plate was filled with 0.1 mls of sterile water, 99.5% methanol, 99.5% propanol or 99% benzyl alcohol as a control. The plates were then incubated at room temperature (32˚C ± 2˚C) on the laboratory bench for 24 hours (C. albicans), 48 hours (A. niger), and 72 hours (T. rubrum) respectively and observed for zones of inhibition. A zone of clearance round each well signifies inhibition and the diameter of the zones were measured in millimeter (mm) and presented by subtracting the activity of the control. This was performed in triplicates for each fungal isolate and extracts and the average of the three readings was taken.
The fungal isolates were identified by their morphological characteristics (
The spice extracts differed significantly in their potential to inhibit the growth of C. albicans, A. niger and T. rubrum. Even some of the aqueous extracts had inhibition of the growth of the fungal isolates. The fruits of C. annuum and C. chinense had ten (10) seeds each. This seeds were separated, milled and tested alone for their inhibitory effects on the test fungi. The seeds of A. melegueta were also purchased from the market, different from the pods, milled, and also tested for antifungal activity.
The highest activity on C. albicans was obtained with the methanolic and propanolic extracts, moderate activity was obtained with the aqueous extracts and the lowest activity was with the benzyl alcohol and Soxhlet methanolic extraction extracts. There was no growth on the C. albicans agar plates filled with the propanol and benzyl alcohol extracts for three and four days respectively. The table below shows the IZD of tested spice extracts on Candida albicans.
Soxhlet methanol extraction gave the best activity on A. niger, propanol gave moderate activity while aqueous and benzyl alcohol extracts gave the lowest activity. There was no growth on the A. niger agar plates filled with the propanol and benzyl alcohol extracts for four days. The table below shows the inhibition zone diameter of the tested spice extracts on Aspergillus niger.
Methanolic extracts gave the highest activity against T. rubrum; aqueous extracts gave intermediate activity, while the lowest activity was obtained with the propanolic extracts. There was no growth on the T. rubrum agar plates filled with the propanol and benzyl alcohol extracts for seven days. The table below shows the inhibition zone diameter of the tested spice extracts against T. rubrum.
Significant antifungal activities were evident with extracts of selected spices from members of Solanaceae, Zingiberaceae and Amaryllidaceae families. The result obtained from this study indicated that aqueous or methanolic, propanolic or even benzyl alcohol extracts of dried fruits of C. annuum, its seeds, C. chinense, its seeds, A. melegueta pods and seeds, A. cepa and A. sativum bulbs and Z. officinale rhizomes inhibited the growth of C. albicans, A. niger and T. rubrum.
From the Solanaceae family, C. annuum and C. chinense dried fruits and seeds were studied. Only the aqueous extracts of the seeds of C. annuum and C. chinense inhibited the growth of C. albicans. Only the aqueous extract of C. annuum seeds inhibited the growth of A. niger whereas the aqueous extract of C. annum fruit and C. chinense seeds inhibited the growth of T. rubrum. This indicates that these peppers have active constituents that inhibit the growth of these fungi. Benzyl alcohol extracts of C. annuum seeds and fruits had no activity on C. albicans indicating that the active constituents might be insoluble in the alcohol. Methanolic and
Isolate Identification Code | Cultural Characteristics | Organism |
---|---|---|
A | White to cream coloured opaque colonies, with fast rate of growth, produced in less than 24 hours. | Candida albicans |
B | Fast growing, white, yellow, yellow-brown to black opaque colonies, arranged in whorls. | Aspergillus niger |
C | The colonies are opaque, flat to slightly raised, white to cream with a pale brown reverse pigment and a slow rate of growth. | Trichophyton rubrum |
benzyl alcohol extracts of C. annuum and C. chinense fruits and seeds had no activity on A. niger but the propanol extracts did indicating that their active constituents for antifungal activity are best extracted with propanol. The aqueous and propanol extracts of C. annuum seeds had the same inhibition zone diameter (15 mm) on A. niger. The aqueous and methanolic extracts of C. chinense seeds inhibited the growth of T. rubrum with IZD of 19 mm and 16 mm respectively. The aqueous extract had more activity than its methanol and benzyl alcohol extracts on T. rubrum. The propanol extract had no activity.
In the Zingiberaceae family, A. melegueta seeds and pods, and fresh rhizomes of Z. officinale was studied. Their aqueous extracts had no activity on C. albicans (
Only the methanolic extract of ginger had activity on T. rubrum. Also, only the methanolic and benzyl alcohol extract of ginger had activity on A. niger. An inhibition zone diameter of 11 mm was obtained with ethanolic extracts of ginger against C. albicans in a study conducted by Atai et al., 2009. Similarly, an IZD of 16 mm was
Spices | Aqueous Extracts (mm) | Methanolic Extracts (mm) | Soxhlet Methanolic Extracts (mm) | Propanol Extracts (mm) | Benzyl Alcohol Extracts (mm) |
---|---|---|---|---|---|
C. annuum | - | 8 mm | - | - | - |
C. annuum seeds | 5 mm | 5 mm | - | 3 mm | |
C. chinense | - | 8 mm | - | 3 mm | 7 mm |
C. chinense seeds | 6 mm | 6 mm | 7 mm | 4 mm | 6 mm |
A.melegueta | - | - | 10 mm | 12 mm | 1 mm |
A.melegueta seeds | - | 8 mm | 2 mm | ||
A .cepa | - | 10 mm | 12 mm | ||
Z. officinale | - | 9 mm | 1 mm | 5 mm | |
A. Sativum | 22 mm | 22 mm | 17 mm | 15 mm | 4 mm |
Spices | Aqueous Extracts (mm) | Methanolic Extracts (mm) | Soxhlet Methanolic Extracts (mm) | Propanol Extracts (mm) | Benzyl Alcohol Extracts (mm) |
---|---|---|---|---|---|
C. annuum | - | - | - | 10 mm | - |
C. annuum seeds | 15 mm | - | - | 15 mm | - |
C. chinense | - | - | 14 mm | 12 mm | - |
C. chinense seeds | - | - | 17 mm | 6 mm | - |
A.melegueta | - | 15 mm | 19 mm | 13 mm | - |
A. melegueta seeds | - | 10 mm | 12 mm | 7 mm | - |
A. cepa | - | 11 mm | 19 mm | - | 11 mm |
Z. officinale | - | 5 mm | 22 mm | - | 7 mm |
A. sativum | 20 mm | 14 mm | 25 mm | 21 mm | 7 mm |
obtained with ethanolic extracts of ginger against C. albicans in a study conducted by Supreetha et al., 2011 [
A. sativum and A. cepa are members of the Amaryllidaceae family studied. The aqueous and methanolic extracts of garlic had the same activity (22 mm) on C. albicans. Similarly, in a study conducted by Iwalokun et al., 2004 [
The activity of these spice extracts is comparable to the activity of some synthetic antifungal agents’ invitro. In a study conducted by Atai et al., 2009 [
Spices | Aqueous Extracts (mm) | Methanolic Extracts (mm) | Soxhlet Methanolic Extracts (mm) | Propanol Extracts (mm) | Benzyl Alcohol Extracts (mm) |
---|---|---|---|---|---|
C. annuum | 10 mm | 4 mm | - | - | - |
C. annuum seeds | - | - | - | - | - |
C. chinense | 19 mm | - | - | - | - |
C. chinense seeds | 18 mm | 16 mm | 17 mm | - | 10 mm |
A. melegueta | - | 15 mm | 22 mm | - | 12 mm |
A. melegueta seeds | 17 mm | 18 mm | - | - | |
A. cepa | 12 mm | 10 mm | - | - | 22 mm |
Z. officinale | - | 9 mm | 17 mm | - | - |
A. sativum | 21 mm | 19 mm | 39 mm | 27 mm | - |
30 mm when used as positive control whereas aqueous garlic extracts produced an IZD of 27 mm against C. albicans in a study conducted by Iwalokun et al., 2004 [
Soxhlet extraction is a better extraction method than maceration but its high cost could be the reason while it is not usually employed. Macerated methanol extracts of C. chinense seeds, A. melegueta pods, its seeds, Z. officinale and A. sativum gave inhibition diameters of 16 mm, 15 mm, 17 mm, 9 mm, and 19 mm respectively against T. rubrum whereas their Soxhlet methanol extracts gave IZD of 17 mm, 22 mm, 18 mm, 17 mm, and 39 mm respectively. Also, macerated methanol extracts of C. chinense, its seeds, A. melegueta pods and seeds, A. cepa, Z. officinale and A. sativum gave inhibition zone diameters of 0 mm, 0 mm, 15 mm, 10 mm, 11 mm, 5 mm, and 14 mm respectively against A. niger whereas their Soxhlet methanol extracts gave IZD of 14 mm, 17 mm, 19 mm, 12 mm, 19 mm, 22 mm, and 25 mm respectively as shown in
The zones of inhibition obtained from extracts of these spices shows that they have great potential as remedies for diseases caused by the test fungi and can even serve as natural prophylactics.
The study demonstrated that the six spices used had activity on the yeast (Candida albicans), the mould (Aspergillus niger) and the dermatophyte (Trichophyton rubrum). Allium sativum had the greatest activity on all the test organisms signifying that it had greater promise as an antifungal agent. Active constituents with potent activity against tested fungi were also present in the separated seeds of C. annuum, C. chinense and A. melegueta. This encouraged the use of these spices for controlling fungal infections and as natural prophylactics. They offered the advantage of being used indefinitely in large amounts.
MosesIkegbunam,MaduagwuUkamaka,OsazuwaEmmanuel, (2016) Evaluation of the Antifungal Activity of Aqueous and Alcoholic Extracts of Six Spices. American Journal of Plant Sciences,07,118-125. doi: 10.4236/ajps.2016.71013