Introduction

The use of plant parts as a source of medicine to treat infectious diseases predates history as a result of which nearly all cultures and civilizations from ancient times to the present day have used herbal medicines to cure infections¹. Ethno-pharmacological use of plants prevails among most African countries where plants are used in treating malaria, diarrhoea, burns, gonorrhoea, stomach disorders and other infectious diseases². These plants which are easily available and cheaper than the conventional drugs include garlic, ginger and lime fruit which in their natural state are widely used in West Africa as herbal medicines³. A report from the World Health Organisation revealed that 80% of the world’s population relies on traditional therapies which involve the use of plants extracts or their active substances⁴ whilst actively encouraging national governments of member countries to utilize their traditional systems of medicines with regulations suitable to their national health care systems⁵. Extraction refers to processes for the isolation of the active ingredients from drug material and this may be by physical means or by dissolving in a suitable menstruum (liquid solvent e.g. water or alcohol)⁶. The extracts (antimicrobial agents) when isolated are contained in the phytochemical constituents (Alkaloids, Saponins, Tannins etc.) of the plants⁷. Over the years much effort has been devoted to the search for new antimicrobial (antifungal) agents from natural sources such as plants and others for treatments and for food preservation ^{3, 8-13}.

The antimicrobial activities of such plant extracts have been linked to the presence of bioactive compounds which sometimes serve to protect the plants themselves against bacteria, fungi and viral infections as well as exhibiting their antimicrobial properties on these organisms¹⁴. Allicin in garlic extracts have been found to be effective as an antifungal, antibacterial, antiviral and anti-parasitic agent¹⁵. Ginger contains the compound caprylic acid, which has potent antifungal properties and is popularly used to treat conditions like indigestion, nausea, vomiting, sore throats, the common cold, headache, fevers, flu symptoms, rheumatism, and motion sickness¹⁶. Lime is also an essential ingredient in the preparation of most herbal concoction which is sometimes used to suppress stomach ache and possess antimicrobial activities in conjunction with other extracts³.

Aspergillus species are the most common fungal species which are able to produce mycotoxins in food and feedstuffs and these mycotoxins are known to be potent hepatocarcinogens in animals and humans¹¹. The presence and growth of fungi may cause spoilage and result in a reduction in quality and quantity of foods^{9-10, 17-19}. Most Cladosporium species are causative agents of skin lesions, keratitis, onychomycosis, sinusitis and pulmonary infections²⁰ with the ability to reproduce on frozen meat²¹. The present study seeks to confirm and compares the antifungal effectiveness of garlic, ginger and lime as both antimicrobial and preservation agents as well as to determine the most suitable extractive technique for effective activity on the test species of A. flavus, A. niger and C. herbarum.

Materials and Methods

Materials, Laboratory Location and Period of Research

The research material ginger, onion and garlic were obtained from Pedu Market in the Cape Coast Metropolis of Ghana whilst laboratory activity was undertaken at the Laboratories of Department of Laboratory Technology and Molecular and Biotechnology of the University of Cape Coast, in Cape Coast, Ghana between December, 2009 to May, 2010.

Plant Extraction: 100g of cleaned, air dried plant extracts of ginger, garlic and lime obtained were blended separately and individually soaked in 100mls of ethanol for 24hrs in a sterile glass container. The pulp obtained was shaken vigorously to allow for proper extraction of active ingredients. The crude extract was then filtered using sterile Whatmans No. 1 filter paper. The filtered extracts were then stored in the refrigerator at 4⁰C. The process was repeated using sterile distilled water as the extractive medium.

Fungal isolation: Test fungi (A. flavus, A. niger and C. herbarum) were obtained from the School of Biological Sciences, University of Cape Coast, Cape Coast, Ghana.

Antifungal Screening Test: Six each of washed, dried and sterilized plates were labeled for each ethanol base plant extracts with two each of the six labeled for each fungi. Six plates were also labeled as positive controls using ethanol only with two plates each corresponding to each test fungi. A further six plates were labeled as negative controls without any antimicrobial agent (plant extract) nor ethanol with two plates corresponding to each test fungi. This makes a total of thirty (30) plates for ethanol extract medium sensitivity testing. 1ml of ethanol base plant extract was dispensed using separate sterilized micropipettes into each extract labeled Petri dish. 1ml of ethanol only was dispensed using another sterile micropipette into ethanol only labeled Petri dishes. Sterilized molten Potato Dextrose Agar (PDA) cooled at 45 ºC was dispensed separately into each of the plates including the plates without plant extract and ethanol. Each plate was swirled gently in a clockwise and anti-clockwise motion to mix the extracts with the PDA and allowed to solidify. A streak of the pure cultures of the test fungi were then transferred unto the Petri dishes containing the PDA and the plant extract using a sterile inoculation needle. The plates were covered and incubated at room temperature at 28±3 ºC for two days after which diameter growth of the test fungi were measured horizontally, vertically and diagonally and mean values calculated. Measurement of diameter was made at daily intervals for six days. The above procedure was repeated for water base plant extraction using distilled water only as the positive control and PDA only without plant extract and water as the negative control. Incubation and measurement were undertaken as described above.

Statistical analysis: Data obtained in the study were descriptively analyzed using Statview from SAS version 5 and the means separated using double-tailed Paired Means Comparison. Microsoft Excel was used in plotting the graphs.

Results

In organic ethanolic extraction, results from the study showed that lime had the greatest inhibition of diameter growth on A. flavus (1.500cm), followed by ginger (2.484cm) and garlic (3.766cm) on day seven of incubation (Fig. 1) resulting in a marked significant difference (P<0.01) between all three extracts and PDA control (Table 1). There was a significant difference between garlic and ginger and ethanol positive control (P-0.162) but not in lime. The growth of A. niger on PDA was inhibited most by ginger (4.200cm), followed by lime (4.450cm) and garlic (4.750cm) at day seven of incubation (Fig.2) with (P<0.01) between all three extracts and PDA control (Table 1). A comparison of growth inhibition of ginger and ethanol was insignificant (P-0.037) unlike that of garlic and lime (P>0.05). Comparatively the growth of C. herbarum on PDA was the least in lime inoculated medium (1.850cm), followed by garlic (1.883cm) and ginger (1.984cm) on the seventh day of incubation (Fig. 3). However, there was no significant difference between lime extract and PDA control (P>0.05) compared with a marked significant difference (P<0.01) between extracts of garlic and ginger with PDA (Table 3). Ginger and lime had comparable inhibition effect with ethanol (P>0.05) unlike garlic which was significant (P-0.016)

With inorganic water base extraction medium, Garlic had the greatest inhibition in diameter growth (0.934cm), followed by lime (1.150cm) on A. flavus resulting in a marked significant difference (P<0.01) (Table 2) with least growth inhibition in ginger (7.783cm) on the seventh day not significant (Fig. 4). All three extracts significantly inhibit growth of fungi compared with water control (P<0.01). In A. niger, garlic had the greatest inhibition in diameter growth (0.884cm), followed by lime (2.900cm) leading to (P<0.01) whilst ginger (8.617cm) on day seven of incubation resulted in (P>0.05) (Fig. 5). Lime had the greatest inhibition in diameter growth (0.933cm), followed by garlic (0.934cm) and garlic (5.750cm) on C. herbarum at day seven of incubation (Fig. 6). There was was a marked significant difference (P<0.01) between all three extracts and PDA controls. There was significant difference between all extracts and water control.

Figure 1

Fig. 1: Growth of on PDA Inoculated with Plant Extracts over a Seven Day Incubation Period using Ethanol Base Extraction Medium

Figure 2

Fig. 2: Growth of on PDA Inoculated with Plant Extracts over a Seven Day Incubation Period using Ethanol Base Extraction Medium

Figure 3

Fig. 3: Growth of on PDA Inoculated with Plant Extracts over a Seven Day Incubation Period using Ethanol Base Extraction Medium

Figure 4

Fig. 4: Growth of on PDA Inoculated with Plant Extracts over a Seven Day Incubation Period using Water Base Extraction Medium

Figure 5

Fig. 5: Growth of on PDA Inoculated with Plant Extracts over a Seven Day Incubation Period using Water Base Extraction Medium

Figure 6

Fig. 6: Growth of on PDA Inoculated with Plant Extracts over a Seven Day Incubation Period using Water Base Extraction Medium

Figure 7

Table 1: Paired Mean Comparison of Growth Diameter of Test Fungi between Ethanolic

Figure 8

Table 2: Paired Mean Comparison of Growth Diameter of Test Fungi between Water base

Discussions

The phytochemical constituents of medicinal plants such as garlic, ginger, lime, onion etc have longed been known and their antimicrobial properties have been widely studied and reported^22-23.

In the ethanol base extraction medium, all three ethanolic extracts of garlic, ginger and lime significantly inhibited growth of A. flavus and A. niger (P<0.01) over the incubation period. This conforms to works by³ on studies on the antimicrobial effectiveness of garlic (Allium sativum Linn), ginger (Zingiber officinale Roscoe) and lime (Citrus aurantifolia Linn) against some gram positive bacteria species and fungal species such A. flavus, A. niger and others. The strong inhibition potential of ginger is attributed to fact that it contains over 400 different compounds a mixture of both volatile and non-volatile chemical constituents such zingerone, shogaols and gingerols, sesquiterpenoids (β-sesquiphellandrene, bisabolene and farnesene) and a small monoterpenoid fraction (β-phelladrene, cineol, and citral)^24-25. The antifungal activity of garlic is in agreement with results of²⁶ who found its extracts very effective in inhibiting the growth of Aspergillus species. The insignificant effect of ethanolic extract of lime on C. herbarum compared with garlic and ginger is in agreement with studies by²⁷ who found that lime is effective against some gram positive bacteria and some fungi such as A. niger and A. flavus but slightly or not effective against C. herbarum. The reduced effectiveness of lime could be attributed to the solubility and volatility of its phytochemical components such as monoterpenes, sesqui-terpenes, oxygenated hydrocarbons, monoterpenes, o-cyme (major component), some of which tend to be lost during the process of extraction especially in organic solvents as explained by²⁸. However water extract of lime showed a marked significant difference in inhibiting all three test fungi species (P<0.01) when compared with PDA control. The effectiveness of water (aqueous) extract of lime in inhibiting all three fungi species could be attributed to the fact that water is a polar solvent and the phytochemical constituents of lime such as oxygenated hydrocarbons, monoterpenes, o-cyme (major component) are very soluble in it hence retaining most of the antimicrobial (antifungal) properties during the extraction process as reported by²⁹.

In the water base extraction medium, there was a marked significant difference in inhibition of A. flavus and A. niger (P<0.01) between extracts of garlic and lime against PDA control but there was not a significant difference in inhibition of A. flavus and A. niger between ginger extract and PDA control. This results conform to works by³⁰ on the invitro antibacterial activities of crude extracts of essential oils such as is in ginger reporting that most of these oils give better antimicrobial effectiveness in organic extract than aqueous extracts. This could be due to the phytochemical constituents of these plants extracts and their different reactivity, solubility and volatility with organic and aqueous medium.

Finally, it could be observed that both ethanolic and water extract of garlic exhibited marked growth inhibiting of all the test fungi (P<0.01) when compared with PDA control. This can be attributed to the fact that allicin, the main phytochemical constituent of garlic, although volatile is very soluble with ethanol and water thereby maintaining it antimicrobial property in inhibiting some gram positive bacteria and some fungi species^{3, 30}.

The significant growth inhibitions of the test fungi by the plant extracts suggest their possible use in controlling these fungi in disease causing situations and food spoilage. Furthermore, the easy means of obtaining these extracts especially using water base extraction provides an alternative to antibiotics and artificial preservatives both of which can be toxic at certain concentrations.

Conclusion

All three medicinal plant extracts exhibited high but varied antifungal activity and can be used in controlling infection, preventing cold meat spoilage and preserve meat for longer periods against fungal contamination. Different extractive techniques correspond to different antimicrobial (antifungal) effectiveness. However, ethanol and water base extraction of garlic produces similarly strong antifungal activity against all test fungi.

Acknowledgement

We thank the technical staff of the Department of Laboratory Technology and Molecular Biology and Biotechnology.